title: CD8α and CD70 mark human natural killer cell populations which differ in cytotoxicity

status: Public on Feb 11 2025

city: London

first: Daniel

last: Davis

address: Exhibition Road

department: Life Sciences

country: United Kingdom

institute: Imperial College London

summary: Natural Killer (NK) cells are innate immune cells that can directly detect and kill cancer cells. Understanding the molecular determinants regulating human NK cell cytotoxicity could help harness these cells for cancer therapies. To this end, we compared the transcriptome of NK cell clones derived from human peripheral blood, which were strongly or weakly cytotoxic against 721.221 and other target cells. After one month of culture, potent NK cell clones showed a significant upregulation in genes involved in cell cycle progression, suggesting that proliferating NK cells were particularly cytotoxic. Beyond two months of culture, NK cell clones which were strongly cytotoxic varied in their expression of 28 genes, including CD8Α and CD70; NK cells with high levels of CD70 expression were weakly cytotoxic while high CD8Α correlated with strong cytotoxicity. Thus, NK cells were cultured and sorted for expression of CD70 and CD8α, and in accordance with the transcriptomic data, CD70 + NK cells showed low cytotoxicity against 721.221 and K562 target cells. Cytotoxicity of CD70 + NK cells could be enhanced using blocking antibodies against CD70, indicating a direct role for CD70 in mediating low cytotoxicity. Furthermore, time-lapse microscopy of NK cell-target cell interactions revealed that CD8α + NK cells have an increased propensity to sequentially engage and kill multiple target cells. Thus, these two markers relate to NK cell populations which are capable of potent killing (CD70 -) or serial killing (CD8α + ).

overall_design: RNA-seq profiling of NK cell clones that have been tested for their killing capacity

first: Camille

last: Rey

first: Katie

last: Jones

first: Kevin

last: Stacey

first: Alicia

last: Evans

first: Jonathan

last: Worboys

first: Gareth

last: Howell


title: Stepwise activation of primary human memory CD4+ T cells

city: Edinburgh

first: Jose

middle: Ignacio

last: de las Heras

address: Max Born Crescent

department: Wellcome Trust Centre

institute: University of Edinburgh

type: Expression profiling by high throughput sequencing

summary: CD4+ T cells are a highly differentiated cell type that maintain enough transcriptomic plasticity to cycle between activated and  memory statuses. We generated RNA-seq libraries from T-cells harvested from three independent blood donors under three separate conditions: unstimulated fresh ex vivo, IL-2 only stimulated and TCR+CD28+IL-2 stimulated, conferring increasingly stronger activation signals.   .

overall_design: RNA-seq profiling of primary CD4+ memory T-cells, isolated from three blood donors and purified via FACS. Cells were treated with either IL-2, IL2+TCR or untreated. Donor 1 experiments were performed in triplicate, while donors 2 and 3 were performed in duplicate.

first: Alex

middle: I

last: Ward

first: Eric

middle: C

last: Schirmer

first: Ariberto

last: Fassati


title: TET2 Deficiency Increases the Competitive Advantage of Hematopoietic Stem Cells through Upregulation of Thrombopoietin Receptor Signaling

city: Toronto

first: Yitong

last: Yang

address: 101 College st

country: Canada

institute: University of Toronto

type: Genome binding/occupancy profiling by high throughput sequencing

summary: Ten-Eleven Translocation-2 (TET2) mutations drive the expansion of mutant hematopoietic stem cells (HSCs) in clonal hematopoiesis (CH). However, the precise mechanisms by which TET2 mutations confer a competitive advantage to HSCs remain unclear. Here, through an epigenetic drug screen, we discovered that inhibition of disruptor of telomeric silencing 1-like (DOT1L), a H3K79 methyltransferase, selectively reduced the fitness of Tet2 knockout (Tet2KO) hematopoietic stem and progenitor cells (HSPCs). Mechanistically, we found that TET2 deficiency increased H3K79 dimethylation and expression of Mpl, which encodes the thrombopoietin receptor (TPO-R). Correspondingly, TET2 deficiency was associated with a higher proportion of primitive Mpl-expressing (Mpl+) cells in the HSC compartment. Importantly, inhibition of Mpl expression or the signaling downstream of TPO-R was sufficient to reduce the competitive advantage of murine and human TET2-deficient HSPCs. Our findings demonstrate a critical role for aberrant TPO-R signaling in TET2 mutation-driven CH and uncover potential therapeutic strategies against this condition.

overall_design:
For RNA-seq, wildtype and Tet2 knockout (KO) HPCHOXB4 cells were cultured in growth medium supplemented with 0.01% DMSO or SGC0946 at 1uM for seven days prior to harvest. Total RNA was extracted from cells using the Qiagen RNeasy plus kit (Qiagen, #74134) and quantified using a Nanodrop spectrophotometer. All samples had a RIN value greater than 9. Samples were submitted to Novogene Corporation for sequencing analysis. Library construction, sequencing, and processing of sequencing data were done by Novogene following the standard pipeline.

For H3K79me2 ChIP-seq, wildtype and Tet2 knockout (KO) HPCHOXB4 were cultured in growth medium supplemented with DMSO (0.01%) or SGC0946 (1uM) for seven days prior to harvest. Cells were fixed with 1% formaldehyde (Thermo Scientific, #410730050) according to the Active Motif ChIP cell fixation protocol. Fixed cell pellets containing 5 millon cells per sample were submitted to Active Motif for Ch-IP reactions and downstream data processing and analyses.

For scRNA-seq, Whole bone marrow cells were isolated from 15 to 20-week-old Tet2WT and Tet2KO male littermates following red blood cell depletion. Cells from each donor were transplanted into four lethally irradiated 8-week-old CD45.2 Tet2WT recipients at 1x106 cells per animal. Following four weeks of engraftment, mice were subject to pinometostat (60mg/kg) or vehicle (10% DMSO, 40% PEG300, 5% Tween-80, and 45% saline) treatment through subcutaneous injection twice a day (2 animals per cell type per treatment group) for 21 days.

first: Severine

last: Cathelin

last: Liu

first: Amit

last: Subedi

first: Abdula

last: Maher

first: Mohsen

last: Hosseini

first: Dhanoop

last: Manikoth Ayyathan

first: Robert

last: Vanner

first: Steven

middle: M

last: Chan


title: A p53-CCF circuit integrates genome integrity with senescence phenotypes [WGS 2]

city: La Jolla

first: Xue

last: Lei

state: CA

address: 10901 N Torrey Pines Rd

country: USA

institute: SBP medical discovery institute

summary: Genomic instability and inflammation are distinct hallmarks of aging, but the connection between them is poorly understood. Understanding their interrelationship will help unravel new mechanisms and therapeutic targets of aging and age-associated diseases. Here we report a novel mechanism directly linking genomic instability and inflammation in senescent cells, through a mitochondria-regulated molecular circuit that connects the p53 tumor suppressor and cytoplasmic chromatin fragments (CCF), a driver of inflammation through the cGAS-STING pathway. Activation or inactivation of p53 by genetic and pharmacologic approaches showed that p53 suppresses CCF accumulation and the downstream inflammatory senescence-associated secretory phenotype (SASP), independent of its effects on cell cycle arrest. p53 activation suppressed CCF formation by promoting DNA repair, reflected in maintenance of genomic integrity, particularly in subtelomeric regions, as shown by single cell genome resequencing. Activation of p53 by pharmacological inhibition of MDM2 in old mice decreased features of SASP in liver, indicating a senomorphic role in vivo. Remarkably, mitochondria in senescent cells suppressed p53 activity by promoting CCF formation and thereby restricting ATM-dependent nuclear DNA damage signaling. These data provide evidence for a mitochondria-regulated p53-CCF circuit in senescent cells that controls DNA repair, genome integrity and inflammatory SASP, and is a potential target for senomorphic healthy aging interventions.

overall_design: To better understand the genomic structure of senescent cells, as well as identify potential genomic deletions associated with CCF formation, we performed single-cell genome resequencing of irradiation-induced senescent cells.

first: Karl

middle: N

last: Miller

first: Hannah

middle: R

last: Pierce-Hoffman

first: Peter

middle: D

last: Adams


title: A p53-CCF circuit integrates genome integrity with senescence phenotypes [WGS 1]

overall_design: To confirm genomic copy number variations in senescent cells with an alternative approach, and to and test whetherMDM2 inhibitor-associated DNA repair improves genome integrity, we treated irradiation-induced senescent IMR90 with the MDM2i RG7388 and performed single-cell genome resequencing.

first: Yoav

last: Altman


title: A p53-CCF circuit integrates genome integrity with senescence phenotypes

summary: This SuperSeries is composed of the SubSeries listed below.

sample_organism:
Homo sapiens
Mus musculus

platform_organism:
Homo sapiens
Mus musculus

overall_design: Refer to individual Series


title: A p53-CCF circuit integrates genome integrity with senescence phenotypes  [RNA-Seq 2]

overall_design: To test the effect of p53 activation on senescence-associated inflammation, we treatedirradiation-induced senescent IMR90 cells with MDM2 inhibitor (RG7388 or HDM201) or DMSO control for 7 days.


title: A p53-CCF circuit integrates genome integrity with senescence phenotypes [RNA-Seq 3]

overall_design: To test whether the effects of MDM2 inhibitors we observed in culture translated to an in vivo model, we treated aged C57BL6  mice (20 month female, 24 month male) with HDM201 at 10 mg/kg once daily by oral gavage for 14 days, or vehicle control.  An additional young (5 month) vehicle control for each sex was included to assess the effect of age.  We assessed gene expression in whole liver.

first: Aaron

middle: P

last: Havas


title: A p53-CCF circuit integrates genome integrity with senescence phenotypes [RNA-Seq 1]

overall_design: To test the ability of the DNA repair regulator 53BP1 to suppress senescence-associated inflammation, we  exogenously expressed 53BP1 on a modest PGK promoter or empty vector in IMR90 human fibroblasts by lentivirus, then induced senescence by 20 Gy irradiation, and analyzed gene expression 10 days after irradiation.

last: N Miller


title: Blockade of glucagon receptor induces α-cell hypersecretion by hyperaminoacidemia in mice

city: Xiamen, Fujian

first: jianxin

last: jia

address: Xiamen University, Xiamen, Fujian

country: China

institute: Xiamen University

summary: Blockade of the glucagon receptor (GCGR) has been shown to improve glycemic control. However, this therapeutic approach also brings side effects, such as α-cell hyperplasia and hyperglucagonemia, and the mechanisms underlying these side effects remain elusive. Here, we conduct single-cell transcriptomic sequencing of islets from male GCGR knockout (GCGR-KO) mice. Our analysis confirms the elevated expression of Gcg in GCGR-KO mice, along with enhanced glucagon secretion at single-cell level. Notably, Vgf (nerve growth factor inducible) is specifically upregulated in α cells of GCGR-KO mice. Inhibition of VGF impairs the formation of glucagon immature secretory granules and compromises glucagon maturation, lead to reduced α-cell hypersecretion of glucagon. We further demonstrate that activation of both mTOR-STAT3 and ERK-CREB pathways, induced by elevated circulation amino acids, is responsible for upregulation of Vgf and Gcg expression following glucagon receptor blockade. Thus, our findings elucidate a previously unappreciated molecular mechanism underlying hyperglucagonemia in GCGR blockade.

overall_design: The pancreatic islets were isolated from 8-12-week-old male C57BL/6J mice.  To prepare the single-cell suspension, the purified islets were digested with 0.25% trypsin-EDTA and filtered through a 40 μm cell strainer. Cell viability was evaluated by trypan blue staining, followed by microscopy to ensure that cell viability was greater than 90%, and over 90% of cells were singlets.

first: Jianxin

last: Jia

first: Xuanxuan

last: Bai

first: Qi

last: Kang

first: Fuquan

last: Jiang

last: Wong

first: Quanwen

last: Jin

first: Mingyu

last: Li


title: Transposable elements silencing by the histone demethylase dLSD1 is required to achieve normal wing size

city: Toulouse

first: Luisa

last: Di Stefano

address: 118 Route de Narbonne

country: France

institute: Centre de Biologie Intégrative (CBI)

summary: Determination of tissue size depends on the proper response of cells to a wide array of external and internal signals. Chromatin acts as a platform/substrate of signal integration and storage. Signal transduction pathways converge upon chromatin modifying enzymes and transcription factors to reprogram gene expression. However, little is known about the role of chromatin modifying factors in determining tissue size. Here, we show that loss of function of the histone demethylase dLSD1 results in a significant reduction of Drosophila wing size due to a decreased cell number. We show that dLSD1 depletion affects proliferation and induces DNA damage in larval wing imaginal discs. Through transcriptomics analysis, we find that dLSD1 controls the expression of multiple gene networks in wing imaginal discs. We then show that normal dLsd1 function contributes to repressing Transposable Element (TE) transcription and mobility. Additionally, our results suggest that increased TE mobility due to dLsd1 depletion contributes directly to altered organ size.

overall_design: To determine dLsd1 targets in the drosophila  wing discs. We dissected wing discs from wild type and dLSD1 loss of function L3 larvae and performed RNASeq experiments.

first: Manuela

last: Texier

first: Ines

last: Selmi

first: Marion

last: Aguirrebengoa


title: Cryptic mitochondrial DNA mutations coincide with mid-late life and are pathophysiologically informative in single cells across tissues and species

status: Public on Feb 10 2025

first: Alistair

last: Green

address: Imperial College London

summary: Ageing is associated with a range of chronic diseases and has diverse hallmarks. Mitochondrial dysfunction is implicated in ageing, and mouse-models with artificially enhanced mitochondrial DNA (mtDNA) mutation rates show accelerated ageing. A scarcely studied aspect of ageing, because it is invisible in aggregate analyses, is the accumulation of somatic mtDNA mutations which are unique to single cells (cryptic mutations). We find evidence of cryptic mtDNA mutations from diverse single-cell datasets, from three species, and discover: cryptic mutations constitute the vast majority of mtDNA mutations in aged post-mitotic tissues, that they can avoid selection, that their accumulation is consonant with theory we develop, hitting high levels coinciding with species specific mid-late life, and that their presence covaries with a majority of the hallmarks of ageing including protein misfolding and ER stress. We identify mechanistic links to ER stress experimentally and further give an indication that aged brain cells with high levels of cryptic mutations show markers of neurodegeneration and that calorie restriction slows the accumulation of cryptic mutations.

overall_design: RNA-seq of human cybrid cell lines on the same osteosarcoma 143B rho0 nuclear genetic background.

first: Florian

last: Klimm

first: Aidan

middle: S

last: Marshall

first: Rein

last: Leetmaa

first: Juvid

last: Aryaman

first: Aurora

last: Gómez-Durán

first: Patrick

middle: F

last: Chinnery

first: Nick

last: Jones


title: Mast cell - tumor cell interaction related gene and microRNA expression profiles in oral squamous cell carcinoma [miRNA-seq]

status: Public on Feb 13 2025

city: Goettingen

first: Gabriela

last: Salinas

state: Lower-Saxony

address: Kreuzbergring 57

department: Department of Pathology

country: Germany

institute: Universitaetsmedizin Goettingen

type: Non-coding RNA profiling by high throughput sequencing

summary: In the tumor microenvironment (TME) of oral squamous cell carcinoma (OSCC), mast cells have a prognostic impact on overall patient survival. In addition, these cells interact with OSCC tumor cells and alter their cellular properties such as proliferation and invasion. To gain further insight into the gene and miRNA expression profiles of both cell types, a transcriptome and miRNA sequencing analysis of both cell populations in monoculture and co-culture was performed.

overall_design: Three biological replicates were performed in each case. The samples were three OSCC tumor cell lysates (PCI-13) from monoculture and three OSCC tumor cell lysates (PCI-13) after 48h co-culture with mast cells (LUVA).  In addition, three mast cell lysates (LUVA) from monoculture and three mast cell lysates (LUVA) after 48h co-culture with OSCC tumor cells (PCI-13) were examined.

first: Tatjana

last: Khromov

first: Maren

last: Sitte

first: Boris

last: Schminke

first: Andreas

last: Fischer

first: Henning

last: Schliephake

first: Phillipp

last: Brockmeyer


title: Pre-Transplant T-Cell Clonal Analysis Identifies CD8+ Donor Reactive Clones That Contribute To Kidney Transplant Rejection Following Two Different Induction Modalities

city: Chicago

first: James

last: Mathew

state: IL

address: 300 E Superior

department: Department of Surgery, Division of Organ Transplantation, Comprehensive Transplant Center

summary: Introduction: Responses to allogeneic human leukocyte antigen (HLA) molecules limit the survival of transplanted organs. The changes in T-cell alloreactivity that contribute to this process, however, are not fully understood. We defined a set of donor reactive T-cell clones (DRTC) with the goal to elucidate signatures of kidney allograft rejection. Methods: DRTC were identified pretransplant using an anti-donor mixed lymphocyte reaction assay: CFSE-diluting CD4+ and CD8+ DRTC were flow-sorted, and the TCR sequences were identified using Adaptive Immunosequencing. DRTC were then tracked in post-transplant biopsies, blood, and urine samples in a cohort of kidney transplant recipients. Results: In patients with an abnormal biopsy, the majority of CD8+ DRTC found within the allograft were detected in the circulating pre-transplant repertoire. Circulating CD8+ DRTC were more abundant pre- and post-transplant in patients that received non-lymphodepletional induction and developed an abnormal biopsy when compared to stable patients. Additionally, DRTC were detected as early as two weeks post-transplant in the urine of some patients, with some of these clones subsequently identified in follow-up kidney biopsy samples. Discussion: The findings of our study add to our understanding of T-cell alloreactivity following kidney transplantation and provide evidence for the role of pre-defined alloreactive T-cells in the development of allograft rejection.

overall_design:
Study Subjects: This was a single-center, non-randomized prospective observational study performed at the Comprehensive Transplant Center at Northwestern University Feinberg School of Medicine. Kidney transplant recipients were enrolled between January 2018 and December 2019. Enrolled subjects were followed for one year with sample collections obtained at designated protocol visits. Clinical laboratory values, post-transplant complications, and outcomes were assessed at 3 and 12 months post-transplant. Informed consent was obtained from all subjects. The study protocol was approved by the Northwestern Institutional Review Board (IRB number: STU00206157), and no organs/tissues were procured from prisoners. Informed consent was obtained from all subjects after the nature and possible consequences of the study were explained. Samples: Blood samples were obtained from donors and recipients prior to transplantation, and peripheral blood mononuclear cells (PBMCs) were isolated using Ficoll-Hypaque gradient centrifugation. During the post-transplant period, recipient blood samples were serially collected at 3-months, 6-months, 12-months and any potential rejection episode(s). 50 to 100 milliliters of urine were collected from recipients at 2 weeks post-transplant (as a baseline post-transplant), 3-months, 6-months and 12-months post-transplant, and any possible rejection episode(s). Cell components were isolated by centrifugation at 300g and were frozen as cell pellets at -80oC for DNA isolation and Adaptive immunosequencing. Protocol allograft biopsies were acquired at the back-table before implantation, 3-months and 12-months post-transplant, as well as any potential rejection episode (i.e., “for-cause” biopsy). The decision to perform a “for-cause” biopsy was left up to the treating physician based upon clinical presentation and laboratory values. A second pass biopsy was also obtained, which was frozen dry at -80oC for DNA isolation and Adaptive immunosequencing. Mixed Lymphocyte Reaction: PBMCs were isolated from recipient and donor blood samples obtained before transplantation. Recipient PBMCs were labeled with CFSE, and donor cells were labeled with PKH26 and irradiated (3000 rad). The labeled recipient and irradiated donor cells were then cultured in bulk in RPMI-1640 supplemented with 15% AB serum, at 37oC, 5% CO2, and a concentration of 1x106 cells/mL. After 7 days of culture, cells were harvested and labeled with anti-CD3, anti-CD4, and anti-CD8 monoclonal antibodies (Beckman-Coulter, Miami, FL, or Becton-Dickinson [BD], San Jose, CA). CD8+ and CD4+ donor reactive T-cells were then sorted on FACSAria (BD, San Jose, CA) by gating on the CFSE dim population (i.e., proliferating responder cells), after gating out both CFSE high cells (i.e., recipient non-proliferating cells) and residual PKH26+ donor stimulator cells. Flow-sorted T-cells were frozen as cell pellets at -80oC for future DNA isolation and Adaptive immunosequencing. DRTC Identification and Monitoring: Donor reactive T cells clones (DRTC) were identified utilizing differential abundance analyses generated for each subject. In brief, each rearrangement with a combined total count of at least 5 is treated as a fixed number of “trials” (Bernoulli experiments) in a two-sided binomial test. The probability, p, of the observed template counts in each sample is calculated under the null hypothesis that these templates are evenly distributed between the two samples, relative to their respective repertoire sizes (i.e., the total productive template count of each sample). Rearrangements that are more unequally distributed relative to this expected proportion will result lower probabilities. A rearrangement is considered differentially abundant if it satisfies two criteria: 1) p < 0.01 after applying the Benjamini-Hochberg adjustment procedure to control false discovery rate (FDR); and 2) the rearrangement has a frequency at least 2-fold higher in one sample than in the other. In the context of DRTC, the proliferated CD8+ or CD4+ sorted cell populations were compared against the unstimulated, pre-transplant PBMC sample. DRTC were then defined as clones that were significantly, and at least 2-fold, more abundant in the MLR sort relative to the unstimulated pre-transplant sample in order to identify those that may be most biologically relevant (i.e., expand more readily post-transplant). Once DRTC were identified, they were then monitored over time in the blood, urine, and allograft biopsy samples obtained from recipients (described above). DRTC metrics used for analysis included the absolute number of DRTC, the frequency of these clones within the entire T cell repertoire (i.e., depth), and proportion of all unique TCR clones detected that were DRTC (i.e., breadth). The Morisita Index (MI) and Jaccard Index (JI) were used to compare the similarities of the bulk repertoire between two, independent samples. Both indices are scored 0-1, with 0 being completely divergent repertoires and 1 being identical.

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Adaptive Biotechnologies data
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first: Jes

last: Sanders

first: Barb

middle: L

last: Banbury

first: Erika

last: Schumacher

first: J

last: He

first: Y

last: Sambandam

first: Paul

last: Fields

first: L

last: Gallon

first: Joseph

last: Leventhal


title: ACSS2 drives senescence-associated secretory phenotype by limiting purine biosynthesis through PAICS acetylation

city: Shanghai

first: Pingyu

last: Liu

address: 2005 Songhu Rd

institute: Human Phenome Institute, Fudan University

summary: Senescence-associated secretory phenotype (SASP), an established feature of cellular senescence, mediates the biological effects of senescent cell on tissue microenvironment and contributes to aging-associated disease progression. The metabolic enzyme ACSS2 produces acetyl-CoA from acetate and epigenetically regulates gene expression through histone acetylation. Whether and how ACSS2 regulates cellular senescence through non-histone acetylation mechanisms remain unclear. Here we show that ACSS2 drives SASP by promoting PAlCS acetylation to restrain purine metabolism. Genetic and pharmacological inhibition, as well as deletion of Acss2 in mice blunts SASP gene expression and abrogates the pro-tumorigenic and immune surveillance functions of senescent cells. Mechanistically, ACSS2 directly interacts and promotes acetylation of PAlCS, a key enzyme for purine metabolism. The acetylation of PAlCS promotes autophagy-mediated degradation of PAlCS to limit purine metabolism and reduces dNTP pools for DNA repair, which exacerbates cytoplasmic chromatin fragment (CCF) accumulation and SASP. Altogether, our work links ACSS2-mediated local acetyl-CoA generation to purine metabolism through PAlCS acetylation that dictates the functionality of SASP, and identify ACSS2 as a potential senomorphic target for healthy aging and to prevent senescence-associated diseases.

overall_design: To investigate how ACSS2 regulates cellular senescence, we established oncogene-induced senescent IMR90 cells in which ACSS2 has been knocked down by shRNA. We then performed transcriptomic profiling using RNA-seq data of 3 groups of cells including proliferating, senescent cells with and without ACSS2 knockdown.

first: Li

last: Yang

first: Xincheng

first: Jie

last: Xu


summary: Senescence-associated secretory phenotype (SASP), an established feature of cellular senescence, mediates the biological effects of senescent cell on tissue microenvironment and contributes to aging-associated disease progression. The metabolic enzyme ACSS2 produces acetyl-CoA from acetate and epigenetically regulates gene expression through histone acetylation. Whether and how ACSS2 regulates cellular senescence through non-histone acetylation mechanisms remain unclear. Here we show that ACSS2 drives SASP by promoting PAlCS acetylation to restrain purine metabolism. Genetic and pharmacological inhibition, as well as deletion of Acss2 in mice blunts SASP gene expression and abrogates the pro-tumorigenic and immune surveillance functions of senescent cells. Mechanistically, ACSS2 directly interacts and promotes acetylation of PAlCS, a key enzyme for purine metabolism. The acetylation of PAlCS promotes autophagy-mediated degradation of PAlCS to limit purine metabolism and reduces dNTP pools for DNA repair, which exacerbates cytoplasmic chromatin fragment (CCF) accumulation and SASP. Altogether, our work links ACSS2-mediated local acetyI-CoA generation to purine metabolism through PAlCS acetylation that dictates the functionality of SASP, and identify ACSS2 as a potential senomorphic target for healthy aging and to prevent senescence-associated diseases.

overall_design: Human IMR90 ER:RAS cell were established and induced to senesce with 4-hydroxytamoxifen (4-OHT). Chromatin immunoprecipitation DNA sequencing (ChIP-seq) for histone modification H3K27ac in proliferating and oncogene induced senescent cells (OIS) with and without ACSS2 knockdown.


title: Single-nuclei sequencing of skeletal muscle reveals subsynaptic-specific transcripts involved in neuromuscular junction maintenance

city: Basel

first: Alexander

last: Ham

address: Spitalstrasse 41

department: Biozentrum

country: Switzerland

institute: University of Basel


title: Single-nuclei sequencing of skeletal muscle reveals subsynaptic-specific transcripts involved in neuromuscular junction maintenance (snRNA-Seq)

summary: The neuromuscular junction (NMJ) is the synapse formed between motor neurons and skeletal muscle fibers. Its stability relies on the continued expression of genes in a subset of myonuclei, called NMJ myonuclei. Here, we use single-nuclei RNA-sequencing (snRNA-seq) to identify numerous undescribed NMJ-specific transcripts. To elucidate how the NMJ transcriptome is regulated, we also performed snRNA-seq on sciatic nerve transected, botulinum toxin injected and Musk knockout muscles. These data show that NMJ gene expression is not only driven by agrin-Lrp4/MuSK signaling, but is also affected by electrical activity and trophic factors other than agrin. By selecting three previously undescribed NMJ genes Etv4, Lrtm1 and Pdzrn4, we further characterize novel contributors to NMJ stability and function. AAV-mediated overexpression and AAV-CRISPR/Cas9-mediated knockout show that Etv4 is sufficient to upregulate expression of ~50% of the NMJ genes in non-synaptic myonuclei, while muscle-specific knockout of Pdzrn4 induces NMJ fragmentation. Further investigation of Pdzrn4 revealed that it localizes to the Golgi apparatus and interacts with MuSK protein. Collectively, our data provide a rich resource of NMJ transcripts, highlight the importance of ETS transcription factors at the NMJ and suggest a novel pathway for NMJ post-translational modifications.

overall_design: Single-nuclei RNA seq and bulk RNA seq data of mouse skeletal muscles

first: A

first: M

middle: A

last: Rüegg


title: Single-nuclei sequencing of skeletal muscle reveals subsynaptic-specific transcripts involved in neuromuscular junction maintenance (bulk RNA-Seq)


title: Rescuing DNMT1 Fails to Fully Reverse the Molecular and Functional Repercussions of Its Loss in Mouse Embryonic Stem Cells.

city: Montreal

first: Serge

last: McGraw

state: Qc

address: 3175 Côte Sainte-Catherine, office 5.17.005

institute: CHU Ste-Justine Research Center/Université de Montréal

type: Methylation profiling by high throughput sequencing


title: Rescuing DNMT1 Fails to Fully Reverse the Molecular and Functional Repercussions of Its Loss in Mouse Embryonic Stem Cells [ChIP-seq]

summary: Epigenetic mechanisms are crucial for developmental programming and can be disrupted by environmental stressors, increasing susceptibility to disease. This has sparked interest in therapies for restoring epigenetic balance, but it remains uncertain whether disordered epigenetic mechanisms can be fully corrected. Disruption of DNA methyltransferase 1 (DNMT1), responsible for DNA methylation maintenance, has particularly devastating biological consequences. Therefore, here we explored if rescuing DNMT1 activity is sufficient to reverse the effects of its loss utilizing mouse embryonic stem cells. However, only partial reversal could be achieved. Extensive changes in DNA methylation, histone modifications and gene expression were detected, along with transposable element de-repression and genomic instability. Reduction of cellular size, complexity and proliferation rate were observed, as well as lasting effects in early embryonic lineages and embryoid bodies. Interestingly, by analyzing the impact on imprinted regions, we uncovered 20 regions exhibiting imprinted-like signatures. Notably, while many permanent effects persisted throughout Dnmt1 inactivation and rescue, others arose from the rescue intervention. Lastly, rescuing DNMT1 after differentiation initiation worsened outcomes, reinforcing the need for early intervention. Our findings highlight the far-reaching functions of DNMT1 and provide valuable perspectives on the repercussions of epigenetic perturbations during early development and the challenges of rescue interventions.

overall_design: Dnmt1 was inactivated and rescued in mouse embryonic stem cells (mESCs) using the Tet-Off system, i.e. Dnmt1tet/tet mESCs developed and published by Borowczyk et al. (Borowczyk et al. Proc Natl Acad Sci U S A. 2009. doi:10.1073/pnas.0905668106). Samples were collected prior to Dnmt1 inactivation (i.e. Dnmt1 control, CTL), after Dnmt1 inactivation (i.e. Dnmt1 inactive, INV) and after rescuing Dnmt1 (i.e. Dnmt1 rescue, RES) and various epigenomic sequencing experiments were conducted for assessing the importance of sustained DNMT1 activity for epigenomic regulation. DNA methylation profiles were assessed by EM-seq, histone modification (H3K4me3, H3K27ac, H3K4me1, H3K27me3, H3K9me3) landscapes by ChIP-seq, gene expression levels by mRNA-seq and transposable element transcription levels by RNA-seq. The impact on gene expression in germ layers was also assessed by mRNA-seq; CTL and RES mESCs were differentiated towards endoderm, mesoderm, and ectoderm lineages. Finally, we investigated whether rescuing Dnmt1 after differentiation initiation during embryoid body formation may worsen outcomes; embryoid bodies were derived from CTL, INV and RES mESCs and grown for 10 days, reactivating Dnmt1 on day 5 in a subset of INV embryoid bodies for post-differention rescue.

first: Elizabeth

last: Elder

first: Anthony

last: Lemieux

first: Lisa-Marie

last: Legault

first: Maxime

last: Caron

first: Virginie

last: Bertrand-Lehouillier

first: Thomas

last: Dupas

first: Noël

last: Raynal

first: Guillaume

last: Bourque

first: Daniel

last: Sinnett

first: Nicolas

last: Gévry


title: Endometrial zinc transporter Slc39a10/Zip10 is indispensable for progesterone responsiveness and successful pregnancy in mice

city: Sagamihara

first: Junya

last: Ito

state: Kanagawa

address: 1-17-71 Fuchinobe, Chuo-ku

department: School of Veterinary Medicine

country: Japan

institute: Azabu university

summary: To investigate the molecular mechanism underlying zinc transporter in the endometrium during embryo implantation, we generated mice with uterine-specific deletion of one of zinc transporter, Zip10. We report the sequencing of the uterine transcriptome of control and uterine-specific Zip10-deficient mice at day 4 (1600h) of pregnancy.

overall_design: Comparative gene expression profiling analysis of RNA-seq data for murine endometrium at day 4 (1600h) of pregnancy between control and uterine-specific Zip10-deficient mice.

first: Yui

last: Kawata

first: Jumpei

last: Terakawa

first: Hironobu

last: Murakami


title: Inhibiting EZH2 complements steroid effects in Duchenne muscular dystrophy [mouse]

status: Public on Feb 09 2025

city: Seoul

first: Murim

last: Choi

address: 103 Daehakro, Jongrogu

country: South Korea

institute: Seoul National University

summary: Duchenne muscular dystrophy (DMD) is a devastating X-linked disorder caused by mutations in the dystrophin gene. Despite recent advances in understanding the disease etiology and applying emerging treatment methodologies, glucocorticoid derivatives remain the only general therapeutic option that can slow disease development. However, the precise molecular mechanism of glucocorticoid action remains unclear, and there is still need for additional remedies to complement the treatment. Here, using single-nucleus RNA-sequencing and spatial transcriptome analyses of human and mouse muscles, we investigated pathogenic features in DMD patients and palliative effects of glucocorticoids. Our approach further illuminated the importance of proliferating satellite cells, and revealed increased activity of a signal transduction pathway involving EZH2 in the patient cells. Subsequent administration of EZH2 inhibitors to Dmd mutant mice resulted in improved muscle phenotype through maintaining the immune-suppressing effect but overriding the muscle weakness and fibrogenic effects exerted by glucocorticoids. Our analysis reveals pathogenic mechanisms that can be readily targeted by extant therapeutic options for DMD.

overall_design: DBA/2J and DBA/2J-mdx mice (D2.B10-Dmdmdx/J), hereafter denoted as D2-mdx mice, were purchased from the Jackson Laboratory (JAX ID: 013141; Bar Harbor, ME). All experiments were approved by the Institutional Animal Care and Use Committee in Seoul National University Hospital (#20-0216-S1A0) and animals were maintained in a facility accredited by AAALAC International (#001169) in accordance with the Guide for the Care and Use of Laboratory Animals 8th edition, NRC. Muscle samples were taken from the hindlimb and quadriceps and frozen with liquid nitrogen. snRNA-seq was performed on fresh frozen muscle cells from two male wild-type DBA/2J mice, two male D2-mdx mice, and two male D2-mdx mice treated with deflazacort. Each genotype group included one 7-week-old and one 28-week-old mouse. Deflazacort (SML0123-10MG; Sigma, St. Louis, MO) was formulated as a 0.2 mg/ml suspension in a solution comprised of DMSO (10%), PEG300 (40%), Tween-80 (5%), and saline (45%). This formulation was administered at a dose of 1 mg/kg by intraperitoneal injection once a week for 28 days to four week-old mice. GSK126 (S7061; Selleck chemicals, Houston, TX) was administered to four-week-old mice by intraperitoneal injection once every two days for 28 days at a dose of 50 mg/kg in 20% Captisol.

first: Eun Young

last: Jeon

first: Yejin

last: Kwak

first: Hyeji

last: Kang

first: Hanbyeol

last: Kim

first: Se Young

last: Jin

first: Soojin

last: Park

first: Ryeo Gyeong

first: Dayoung

last: Ko

first: Jae-Kyung

last: Won

first: Anna

last: Cho

first: Inkyung

last: Jung

first: Chul-Hwan

last: Lee

first: Jeongbin

first: Hyun-Young

first: Jong-Hee

last: Chae


title: Rescuing DNMT1 Fails to Fully Reverse the Molecular and Functional Repercussions of Its Loss in Mouse Embryonic Stem Cells [EM-seq]

first: Serge

last: McGraw

first: Elizabeth

last: Elder

first: Anthony

last: Lemieux

first: Lisa-Marie

last: Legault

first: Maxime

last: Caron

first: Virginie

last: Bertrand-Lehouillier

first: Thomas

last: Dupas

first: Noël

last: Raynal

first: Guillaume

last: Bourque

first: Daniel

last: Sinnett

first: Nicolas

last: Gévry


title: Rescuing DNMT1 Fails to Fully Reverse the Molecular and Functional Repercussions of Its Loss in Mouse Embryonic Stem Cells [mRNA-seq]

summary: Epigenetic mechanisms are crucial for developmental programming and can be disrupted by environmental stressors, increasing susceptibility to disease. This has sparked interest in therapies for restoring epigenetic balance, but it remains uncertain whether disordered epigenetic mechanisms can be fully corrected. Disruption of DNA methyltransferase 1 (DNMT1), responsible for DNA methylation maintenance, has particularly devastating biological consequences. Therefore, here we explored if rescuing DNMT1 activity is sufficient to reverse the effects of its loss utilizing mouse embryonic stem cells. However, only partial reversal could be achieved. Extensive changes in DNA methylation, histone modifications and gene expression were detected, along with transposable element de-repression and genomic instability. Reduction of cellular size, complexity and proliferation rate were observed, as well as lasting effects in germ layer lineages and embryoid bodies. Interestingly, by analyzing the impact on imprinted regions, we uncovered 20 regions exhibiting imprinted-like signatures. Notably, while many permanent effects persisted throughout Dnmt1 inactivation and rescue, others arose from the rescue intervention. Lastly, rescuing DNMT1 after differentiation initiation worsened outcomes, reinforcing the need for early intervention. Our findings highlight the far-reaching functions of DNMT1 and provide valuable perspectives on the repercussions of epigenetic perturbations during early development and the challenges of rescue interventions.

overall_design: Dnmt1 was inactivated and rescued in mouse embryonic stem cells (mESCs) using the Tet-Off system, i.e. Dnmt1tet/tet mESCs developed and published by Borowczyk et al. (Borowczyk et al. Proc Natl Acad Sci U S A. 2009. doi:10.1073/pnas.0905668106). Samples were collected prior to Dnmt1 inactivation (i.e. Dnmt1 control, CTL), after Dnmt1 inactivation (i.e. Dnmt1 inactive, INV) and after rescuing Dnmt1 (i.e. Dnmt1 rescue, RES). Various sequencing experiments were then conducted. DNA methylation profiles were assessed by EM-seq, histone modification (H3K4me3, H3K27ac, H3K4me1, H3K27me3, H3K9me3) landscapes by ChIP-seq, gene expression levels by mRNA-seq and transposable element transcription levels by RNA-seq. The impact of inactivation and rescue of Dnmt1 in mESCs on gene expression profiles in germ layers was also assessed by mRNA-seq; CTL and RES mESCs were differentiated towards endoderm, mesoderm, and ectoderm lineages. Finally, we investigated whether rescuing Dnmt1 after differentiation initiation during embryoid body formation would worsen outcomes; embryoid bodies were derived from CTL, INV and RES mESCs and grown for 10 days, reactivating Dnmt1 on day 5 in a subset of INV embryoid bodies for post-differention rescue (PostDiff). Embryoid bodies were harvested and pooled on day 5 and day 10 for mRNA sequencing.


title: Rescuing DNMT1 Fails to Fully Reverse the Molecular and Functional Repercussions of Its Loss in Mouse Embryonic Stem Cells [RNA-seq]


title: Analysis of placenta-derived factor-treated human iPSC-liver organoid.

city: Minato-ku

first: Yoshiki

last: Kuse

address: Shirokanedai 4-6-1

institute: The University of Tokyo

summary: Organoid derived from human induced pluripotent stem cells (hiPSC) is potentially applicable for regenerative medicine. However, the applications have been hampered by limited organoid size and function as a consequence of a lack of progenitor expansion. Here, we report the recapitulation of the in vivo progenitor expansion in hiPSC-liver organoid based on the analysis of mouse development. Visualization of blood perfusion and oxygen levels in mouse embryos revealed a transient hypoxic environment despite blood flow while hepatoblast expansion. During this specific stage, the placenta was seen to express various growth factors. Human and mouse placenta-liver interaction analysis identified various placenta-derived factors. Among them, IL1α efficiently induced the growth in hiPSC-liver organoids as well as mouse fetal livers following progenitor expansion under hypoxia. Subsequent oxygenation demonstrated that expanded progenitors by IL1α contributed to hiPSC-liver organoid size and function. Taken together, treatment of placenta-derived factor under hypoxia is a crucial organoid culture technique that efficiently induces progenitor expansion.

overall_design: RNA expression profiles of HE, EC, MC derived from iPSC-organoid treated with IL1α

first: Kiyoshi

last: Yamaguchi


title: Inhibiting EZH2 complements steroid effects in Duchenne muscular dystrophy

first: Murim

last: Choi

overall_design: Muscle biopsies from patients were performed in accordance with informed written consent from Seoul National Hospital Children’s Hospital institutional review board (IRB)-approved protocols (#1009-030-331). The patients did not take any medication at the time of the biopsy. Muscle samples were taken from the quadriceps femoris and frozen with isopentane cooled in liquid nitrogen. Healthy muscle samples were acquired from the quadriceps or abdomen of subjects undergoing surgery for non-muscular symptoms. snRNA-seq was performed on fresh frozen quadriceps femoris obtained from three male individuals with DMD, three male individuals with BMD, and five healthy control subjects. Among the control samples, three were derived from abdominal muscle tissue undergoing surgery for non-muscular symptoms. All participants were under the age of 17, and each group was composed of individuals of similar ages.

first: Eun Young

last: Jeon

first: Yejin

last: Kwak

first: Hyeji

last: Kang

first: Hanbyeol

last: Kim

first: Se Young

last: Jin

first: Soojin

last: Park

first: Ryeo Gyeong

first: Dayoung

last: Ko

first: Jae-Kyung

last: Won

first: Anna

last: Cho

first: Inkyung

first: Chul-Hwan


title: The SSUP-72/PINN-1 module is required for efficient 3’end processing and coordinates developmental gene expression during exit from diapause in C. elegans [RNAseq-suppression]

status: Public on Feb 08 2025

city: Genève

first: François-Xavier

last: Stubbe

state: Genève

address: 30, quai Ernest-Ansermet

department: MOCEL

institute: University of Geneva

summary: During the exit from the developmental diapause of Caenorhabditis elegans (C. elegans), a network of growth and developmental genes is activated. These genes are organized into operons, where transcriptional termination is uncoupled from mRNA 3’-end processing. Although the Pol II CTD-S2P mark deposited by CDK-12 is unnecessary during embryogenesis, it plays a critical role in the timely expression of most operonic genes by enhancing SL2 trans-splicing at position 2 and above. This process protects mRNA from degradation and prevents termination. Here, a genetic screen has identified the SSUP-72/PINN-1 module as an effective suppressor of CDK-12-induced defects. Our data show that the SSUP-72/PINN-1 module regulates intra-operon termination and affects 3’ pausing genome-wide, coordinating growth and developmental gene expression during post-embryonic development in C. elegans.

overall_design: To investigate the phenotypical suppression of cdk-12as inhibition by ssup-72[E22K]; we conducted RNA-seq on synchronyzed L1 worms (N2, cdk-12as, ssup-72[E22K], cdk-12as;ssup-72[E22K]) grown in presence or absence of 3MB-PP1 (3MB-PP1). Worms were L1 synchornized and grown for 4H with the inhibitor or DMSO.


title: The SSUP-72/PINN-1 module is required for efficient 3’end processing and coordinates developmental gene expression during exit from diapause in C. elegans [GROseq]

overall_design: Global Run-On  sequencing (GRO-seq) for CDK-12as (analogue-sensitive) and its suppressive mutation ssup-72[E22K]. Worms were L1 synchronized by hypochlorite treatment and grown 4H in presence of 3MB-PP1 (2 uM) prior to nuclei extraction.


title: The SSUP-72/PINN-1 module is required for efficient 3’end processing and coordinates developmental gene expression during exit from diapause in C. elegans [RNAseq-SSUP72]

overall_design:
SSUP-72[E22K] is a potent suppressor of the L1 arrest induced by the inhibition of CDK-12as. However, little is known about its cellular role. To investigate the implication of the SSUP-72/PINN-1 module in transcription, we conducted RNA-seq on various SSUP-72/PINN-1 mutants.
Worms were L1 synchronized and fed for 4H (liquid culture) prior to RNA extraction.


title: RNA seq analysis from the kidney neutrophils of pycard mutant adult zebrafish in Mycobacterium marinum infection

status: Public on Feb 07 2025

city: Tampere

first: Sanna-Kaisa

middle: Emilia

last: Harjula

address: Arvo Ylpön katu 34

country: Finland

institute: University of Tampere

summary: Inflammasome regulates the host response to intracellular pathogens including mycobacteria. We have previously shown that the course of M. marinum infection in adult zebrafish (Danio rerio) has similar features than the course of tuberculosis in human. Here we have investigated the role of the inflammasome adaptor pycard in M. marinum infection in zebrafish with two CRISPR/Cas9 mutagenesis generated zebrafish lines. Adult zebrafish devoid of pycard showed impaired survival and increased bacterial burden in M. marinum infection. Transcriptome analysis with RNA sequencing of a kidney derived neutrophils of M. marinum infected fish homozygous for pycardptu5 mutation revealed differentially expressed genes compared to WT controls. Genes associated with neutrophil degranulation, haematopoiesis and PI3K signalling are differentially expressed in the pycard deficient neutrophils when compared to wild type controls.

overall_design: Identification of differentially expressed genes in Mycobacterium marinum infection in kidney derived neutrophils of zebrafish devoid of inflammasome adaptor pycard at four weeks post infection. 12 samples were analyzed, six biological replicates in two groups, fish homozygous for pycardtpu5 mutation and wild type (WT) control.

first: Meri

last: Uusi-Mäkelä

middle: E

first: Maiju

last: Junno

first: Alina

last: Sillanpää

first: Reetta

last: Nätkin

first: Mirja

middle: T

last: Niskanen

first: Anni

middle: K

last: Saralahti

first: Matti

last: Nykter

first: Mika

last: Rämet


title: Identifying phosphate deficiency responsive genes depend on SlNSP1 and SlNSP2 in tomato

city: Beijing

first: Ba

middle: Xin

last: Wei

address: No.1 West Beichen Road, Chaoyang District

institute: Institute of Genetics and Developmental Biology CAS

summary: The goal of this study is to idetify phosphate deficiency induced genes depedent on SlNSP1 and SlNSP2 in tomato, by treating seedlings cultured in half-strength Hoagland solution with or without phosphate.

overall_design: Transcriptome profiling of WT, Slnsp1 and Slnsp1/2 roots treated with  or without phosphate for 12 days in triplicate.

first: Jiayang

last: Li

first: Bing

last: Wang

first: Xinwei

last: Ban


title: Emergence of inflammatory fibroblasts with aging in Hermansky-Pudlak syndrome associated pulmonary fibrosis

city: Philadelphia

first: Sylvia

last: Michki

state: PA

address: 3615 Civic Center Blvd

department: Pulmonary & Sleep Medicine

institute: Children's Hospital of Philadelphia

summary: The longitudinal cellular interactions that drive pulmonary fibrosis are not well understood. To investigate the disease underpinnings associated with fibrosis onset and progression, we generated a scRNAseq atlas of lungs from young and aged mouse models of multiple subtypes of Hermansky-Pudlak syndrome (HPS), a collection of rare autosomal recessive diseases associated with albinism, platelet dysfunction, and pulmonary fibrosis. We identified an age-dependent increase in SAA3+ inflammatory lung fibroblasts in HPS mice, including in double-mutant HPS1-2 mice which develop spontaneous fibrosis. HPS1 fibroblasts showed increased expression of IL-1R1, whereas alveolar type II epithelial cells from HPS2 mice induced the inflammatory gene signature in co-cultured fibroblasts. scRNAseq of lung tissue from three HPS1 patients similarly showed the presence of inflammatory fibroblasts and increased IL1R1 expression on fibroblasts. These data posit complex interactions between dysfunctional epithelial cells, inflammatory fibroblasts, and recruited immune cells, suggesting potential opportunities for mitigation of the fibrotic cascade.

overall_design: To investigate age and genotype-dependent mRNA expression differences in multiple lung cell types using scRNA-seq.

first: Brandon

middle: J

last: Banaschewski

first: Sneha

last: Sitaraman

first: Joanna

middle: Y

first: Ruby

last: Pan

first: Dominique

last: Stewart

first: Mary

last: Goldy

first: Susan

last: Lin

first: Edward

last: Cantu

first: Jeremy

middle: B

last: Katzen

first: Maria

last: Basil

first: Amir

last: Emiatzjoo

first: Jamie

last: Todd

first: Jason

last: Gokey

first: Jonathan

last: Kropski

first: David

last: Frank

first: Jarod

last: Zepp

first: Lisa

last: Young


title: Argonaute CSR-1A promotes H3K9me3 maintenance to protect somatic development in offspring

first: DI

last: RAO

address: Westlake University(Yungu), Hangzhou, Zhejiang Province, China

institute: Westlake University

summary: Paternal stress can be encoded into altered epigenetic information to influence their offspring. How environment-induced perturbation in sperm epigenome is reprogrammed to protect offspring is less well understood. In the nematode Caenorhabditis elegans, parental Argonaute and small RNA pathway defend offspring against viruses, starvation, and pathogenic bacteria. Here we report that, when challenged by universal heat-shock stress, paternal Argonaute CSR-1A potentiates recovery of spermic H3K9me3 to secure the late somatic developmental competence of offspring. CSR-1A employs its repetitive RG motif to engage with SET-25/32, putative histone 3 lysine 9 (H3K9) methyltransferases, and helps germ cell to rescue suppressive chromatin marks H3K9me3 following stress, protecting the late development of somatic cells in the progeny. Finally, among the genes regulated by CSR-1A, we identified dim-1, at which decreased H3K9me3 persists in the progeny, and RNAi of dim-1 mitigates the somatic defects associated with csr-1a loss under stress. Thus, CSR-1A coordinates the epigenetic machinery to shield the latter stages of a progeny's development from the influences of the paternal environment. We speculate that, driven by both natural environmental stressors and the unique characteristics of spermatogenic chromatin, the emergence of multiple RG motif-featured and spermatogenesis-specific CSR-1A and small RNA serves as an adaptive strategy to safeguard against variability in the orchestration of inherited developmental programs from the paternal lineage.

overall_design: To investigate how the csr-1a mutation causes morphological changes in the tails of C. elegans, we used fluorescence-activated cell sorting (FACS) to isolate rectal valve and rectal gland (abbreviations: RVRG or vir & rep) cells from 10 million offspring whose parents had been subjected to heat shock in wild type and csr-1a mutant strains.

first: Di

last: Rao

first: Dengfeng

last: Li

first: Lili


overall_design: To gain insight into the genome-wide changes in H3K9me3 occupancy induced by a csr-1a mutation in response to heat-stress, we isolated C. elegans round spermatids 8-10 hours after heat stress


title: Transcriptome analysis of Prdm10 maternal  knock out mouse oocytes

city: Copenhagen N

first: Daniel

middle: Martin

last: Messerschmidt

state: Zealand

address: 3C Blegdamsvej, Building 18

department: Faculty of Health and Medical Sciences

country: Denmark

institute: University of Copenhagen

summary: PRDM proteins are metazoan specific transcriptional regulators that play diverse roles in mammalian development and disease. Several members such as PRDM1, PRDM14 and PRDM9, have been implicated in germ cell formation and homeostasis and are essential to fertility-related processes. Others, such as PRDM14, PRDM15 and PRDM10 play a role in early embryogenesis and embryonic stem cell maintenance. Here, we reveal an additional maternal requirement for PRDM10 . Absence of maternal Prdm10 results in catastrophic failure of oocyte-to-embryo transition and complete arrest at the 2-cell stage. We describe multiple defects in oocytes, zygotes and 2-cell stage embryos relating to the failure to accumulate PRDM10 target gene transcripts in the egg. Transcriptomic analysis and integration of genome-wide chromatin-binding data reveals novel, essential PRDM10 targets, including the cytoskeletal protein SEPTIN11. We demonstrate that the failure to express maternal Septin11, in the absence of maternal PRDM10, disrupts Septin-complex assembly at the polar body extrusion site in MII oocytes. Our study sheds light into the essentiality of maternal PRDM10, the requirement of the maternal Septin-complex and the likely evolutionary conservation of this regulatory axis in human female germ cells.

overall_design: This dataset contains RNA-seq analysis of wildtype and maternal Prdm10 knock out GV oocytes generated by Zp3-cre mediated deletion of floxed Exon5 of Prdm10.

middle: M

first: Michelle

last: Seah

first: Ernesto

last: Guccione


title: Unravelling Stargardt Disease (STGD1): Modelling Genotype-Phenotype Correlations and Unresolved Genetic Variants in iPSC-Derived Retinal Organoids

city: Newcastle Upon Tyne

first: Majlinda

last: Lako

address: International Centre for Life, Central Parkway

department: Institute of Genetic Medicine and Institute for Ageing

institute: Newcastle University

summary:
Stargardt disease (STGD1) is an inherited retinopathy affecting approximately 1:8,000 individuals. It is characterised by biallelic mutations in ABCA4, and encodes a vital protein for the recycling of retinaldehyde in the retina. Despite its prevalence and impact, there are currently no treatments available for this condition. Furthermore, 35% of STGD1 cases remain genetically unsolved. Efforts have been directed towards comprehending the fundamental disease mechanisms and identifying all disease-causing variants in the extensive 150kb gene, to better aid with the development of effective therapeutic strategies.
In this study, we generated iPSC lines from two monoallelic (PT1 & PT2), late onset STGD1 cases with the heterozygous complex allele - c.[5461-10T>C;5603A>T]. We differentiated these cells alongside a biallelic affected control (AC) - c.4892T>C, and c.4539+2001G>A, to retinal organoids (ROs) allowing us to investigate cellular and molecular characteristics associated with STGD1. We hypothesized that the missing inheritance in our monoallelic cases is due to an RNA defect. Consequently, we utilised a myriad of sequencing strategies including WGS, single-cell RNA sequencing (scRNAseq) and long-read RNA sequencing (LRS) to address this.
ROs were grown for 230 days and developed all key retinal neurons with photoreceptor outer segments capable of ABCA4 expression. We observed patient-specific disruption to lamination with OPN1MW/LW+ cone photoreceptor retention in the RO centre during differentiation. Retention was more severe in the AC case affecting both cones and rods, suggesting a genotype/phenotype correlation. scRNAseq suggests retention may be due to the induction of apoptosis in photoreceptors. WGS successfully identified the missing alleles in both cases; PT1 reported c. 5603A>T in homozygous state and PT2 uncovered a rare hypomorph - c.4865T>C. Furthermore, ROs were able to recapitulate the retina-specific splicing defect in PT1 as shown by LRS data.
Collectively, these results highlight the suitability of ROs in STGD1 modelling. Their ability to display genotype-phenotype correlations enhances their utility as a platform for therapeutic development. Importantly, both PT1 and PT2 cases were genetically resolved in this study, providing two more individuals their confirmed genetic diagnosis.

overall_design: Comparative gene expression profiling analysis of RNA-seq data for RNA isolated from Day 200 retinal organoids - unaffected control (BMP4-activated protocol), unaffected control (IGF-dependent protocol), 2C7 (BMP4-activated protocol) and AC (IGF1-dependent protocol).

first: Avril

last: Watson

first: Birthe

last: Hilgen

first: Rachel

last: Queen

first: Joseph

last: Collin

first: Andrew

last: Nelson

first: Michael

last: McCorkindale

first: Valeria

last: Chichagova

first: Rafiqul

last: Hussain

last: Coxhead

first: Frans

last: Cremers

first: Lyle

last: Armstrong


title: Combinatorial mapping of E3 ubiquitin ligases to their target substrates

city: Seattle

first: Jay

last: Shendure

state: WA

address: 3720 15th Ave NE

department: Genome Sciences

institute: University of Washington

summary: E3 ubiquitin ligases (E3s) confer specificity of protein degradation through the recognition of specific target substrates for ubiquitination and subsequent degradation, making E3s attractive candidates for drug development. There are over 600 annotated E3s in the human genome, yet the vast majority have no known substrate proteins, due in part to the limited scalability of methods to evaluate the vast number of potential E3-substrate interactions. To address this, we developed COMET (Combinatorial Mapping of E3 Targets), a combinatorial framework that can test the role of many specific E3s in regulating the abundance of many specific substrates, within the context of a single experiment. As a proof-of-concept, we applied COMET to identify specific E3 subunits of the SCF ubiquitin ligase complex that mediate the degradation of target substrates, including short-lived transcription factors (TFs). Our data suggest that many E3-substrate relationships are complex rather than simple 1:1 associations. Further, we provide computational models of E3-substrate interactions from our screen. Looking forward, we anticipate that COMET can be further scaled to enable comprehensive mapping of regulators of protein stability to their target substrates

sample_organism:
Escherichia coli
Homo sapiens

platform_organism:
Escherichia coli
Homo sapiens

overall_design: Combinatorial plasmid libraries of E3 ubiquitin ligase gRNAs and potential E3 ubiquitin ligase substrates were cloned. These libraries were integrated into K562-Cas9-rtTA cells, which were then sorted into four FACS bins based on the GFP:mCherry ratio. Sequencing libraries were prepared from each bin with PCR replicates.

first: Chase

middle: C

last: Suiter


title: Hippocampal RNA-Seq Data and Behavioral Data from an F2 intercross of Rats Selectively-Bred to Show High or Low Levels of Locomotor Activity in a Novel Environment (bred High Responders vs. bred Low Responders)

city: Ann Arbor

first: Elaine

last: Hebda-Bauer

state: Michigan

address: 205 Zina Pitcher Place

department: Michigan Neuroscience Institute

institute: University of Michigan

summary: Using selective breeding, we have produced two rat lines with highly contrasting heritable behaviors: bred Low Responders (bLR) are extremely inhibited and anxious in a novel environment, whereas bred High Responders (bHR) are exploratory and sensation-seeking. These traits map onto temperamental extremes predictive of internalizing vs. externalizing psychiatric disorders in humans and can model two divergent paths to drug abuse. To elucidate gene expression underlying these behavioral extremes, bHRxbLR were bred to produce a F0-F1-F2 cross. We then measured exploratory locomotion, anxiety-like behavior, and sensitivity to reward cues (Pavlovian Conditioned Approach) and performed hippocampal RNA sequencing in both F0s (n=24, n=6/phenotype/sex) and F2s (n=250, n=125/sex).   The data included in this GEO upload is the data from the F2s from this experiment.

overall_design: Selectively breeding rats for a high or low propensity to explore a novel environment produced the bHR and bLR lines, respectively, at the University of Michigan (Stead et al., 2006). After 37 generations, an initial set (“F0”) of bHRs and bLRs were chosen from 24 distinct families (12 bHR, 12 bLR) to cross-breed for creating 12 intercross families. Their “F1” offspring were screened for novelty-induced locomotion, and individuals with similarly high or low LocoScores were bred with each other to produce a re-emergence of diverse behavioral phenotypes in the “F2” generation. These 48 F2 litters generated 540 rats, 323 of which were phenotyped as young adults (2-4 months old). Our current study used a subset of these animals (F0: n=24, n=6/phenotype per sex; F2: n=250, n=125/sex).     Behavioral data analysis: All F0 and F2 rats were tested for locomotor activity in a novel environment (“LocoScore,” protocol: (Stead et al., 2006)). Exploratory and anxiety-related behaviors were measured on an Elevated Plus Maze (protocol: (Chitre et al., 2022)), including distance traveled (cm), time immobile (sec), and percent time in the open arms. A subset of F2s (n=209) was subsequently tested for Sign Tracking vs. Goal Tracking behavior during the last two days of a week-long Pavlovian Conditioned Approach task (protocol: (Meyer et al., 2012)) using a combined score (“PavCA Index”) summarizing behaviors indicating preference for the conditioned stimuli (lever) vs. unconditioned stimuli (food magazine).    Tissue dissection, RNA extraction, and sequencing: Adults (postnatal days 113-132) were decapitated without anesthesia and brains extracted within two minutes. For the F0s, whole HPC was immediately dissected and flash frozen. For the F2s, whole brains were flash frozen, and then later hole punches from the dorsal and ventral HPC were pooled from four hemisected coronal slabs (-2.12 to -6.04 mm Bregma; (Paxinos & Watson, 2013)) for each animal. RNA was extracted using the Qiagen RNeasy Plus Mini Kit. A NEB PolyA RNA-seq library was produced and then sequenced using a NovaSeq S4 101PE flowcell (targeting 25 million reads/sample).    The data included in this GEO upload is the data from the F2s from this experiment.

first: Megan

middle: H

last: Hagenauer

first: Huzefa

last: Khalil

first: Stanley

last: Watson Jr

first: Huda

last: Akil


title: Hippocampal RNA-Seq Data and Behavioral Data from Rats Selectively-Bred to Show High or Low Levels of Locomotor Activity in a Novel Environment (bred High Responders vs. bred Low Responders)

summary: Using selective breeding, we have produced two rat lines with highly contrasting heritable behaviors: bred Low Responders (bLR) are extremely inhibited and anxious in a novel environment, whereas bred High Responders (bHR) are exploratory and sensation-seeking. These traits map onto temperamental extremes predictive of internalizing vs. externalizing psychiatric disorders in humans and can model two divergent paths to drug abuse. To elucidate gene expression underlying these behavioral extremes, bHRxbLR were bred to produce a F0-F1-F2 cross. We then measured exploratory locomotion, anxiety-like behavior, and sensitivity to reward cues (Pavlovian Conditioned Approach) and performed hippocampal RNA sequencing in both F0s (n=24, n=6/phenotype/sex) and F2s (n=250, n=125/sex).   The data included in this GEO upload is the data from the F0s from this experiment.

overall_design: Selectively breeding rats for a high or low propensity to explore a novel environment produced the bHR and bLR lines, respectively, at the University of Michigan (Stead et al., 2006). After 37 generations, an initial set (“F0”) of bHRs and bLRs were chosen from 24 distinct families (12 bHR, 12 bLR) to cross-breed for creating 12 intercross families. Their “F1” offspring were screened for novelty-induced locomotion, and individuals with similarly high or low LocoScores were bred with each other to produce a re-emergence of diverse behavioral phenotypes in the “F2” generation. These 48 F2 litters generated 540 rats, 323 of which were phenotyped as young adults (2-4 months old). Our current study used a subset of these animals (F0: n=24, n=6/phenotype per sex; F2: n=250, n=125/sex).     Behavioral data analysis: All F0 and F2 rats were tested for locomotor activity in a novel environment (“LocoScore,” protocol: (Stead et al., 2006)). Exploratory and anxiety-related behaviors were measured on an Elevated Plus Maze (protocol: (Chitre et al., 2022)), including distance traveled (cm), time immobile (sec), and percent time in the open arms. A subset of F2s (n=209) was subsequently tested for Sign Tracking vs. Goal Tracking behavior during the last two days of a week-long Pavlovian Conditioned Approach task (protocol: (Meyer et al., 2012)) using a combined score (“PavCA Index”) summarizing behaviors indicating preference for the conditioned stimuli (lever) vs. unconditioned stimuli (food magazine).    Tissue dissection, RNA extraction, and sequencing: Adults (postnatal days 113-132) were decapitated without anesthesia and brains extracted within two minutes. For the F0s, whole HPC was immediately dissected and flash frozen. For the F2s, whole brains were flash frozen, and then later hole punches from the dorsal and ventral HPC were pooled from four hemisected coronal slabs (-2.12 to -6.04 mm Bregma; (Paxinos & Watson, 2013)) for each animal. RNA was extracted using the Qiagen RNeasy Plus Mini Kit. A NEB PolyA RNA-seq library was produced and then sequenced using a NovaSeq S4 101PE flowcell (targeting 25 million reads/sample).    The data included in this GEO upload is the data from the F0s from this experiment.


title: The immune response differences of rats induced using RBD3-Fc mRNA vaccines inoculated intradermally by NFIS and  intramuscularly by needles

status: Public on Feb 06 2025

city: Changchun city

first: Cenrong

last: Wang

state: Jilin Province

address: 吉林省长春市朝阳区前进大街吉林大学前卫南区

institute: Jilin University

summary: We have employed a single cell sequencing approach using 10× chromium microfluidic system to further illustrate the effect of inoculation routes on T cell immune responses induced by the mRNA-LNP vaccine and naked mRNA vaccine. Rat splenocytes were harvested at week 6. CD45+ splenocytes were separated and captured as immune cells using flow cytometry, then analyzed using scRNA-seq. THis study revealed the advantages of NIFS delivery, which evoked potent T cell responses.

overall_design: Following RBD3-Fc vaccines' injecton, splenocytes were gained at 4 weeks after boost. CD45+ splenocytes were sorted using flow cytometry and analyzed by scRNA-seq.


title: CTCF-mediated insulation and chromatin environment modulate Car5b escape from X inactivation

first: Xinxian

last: Deng

address: 1959 NE Pacific St.

department: Laboratory Medicine and Pathology

summary: This SuperSeries is composed of the SubSeries listed below.

sample_organism:
Mus musculus
Mus spretus

platform_organism:
Mus musculus
Mus spretus

overall_design: Refer to individual Series


title: Apolipoprotein E is a marker of all chondrocytes in the growth plate resting zone

city: Baltimore

first: Satoru

last: Otsuru

state: MD

address: 670 W Baltimore Street

institute: UMB Department of Orthopaedics, Otsuru Lab

summary: The resting zone (RZ) in mammalian growth plates is critical for maintaining and regulating chondrocyte turnover during longitudinal bone growth as a control tower and stem cell reservoir. Although recent lineage tracing studies have identified several markers for stem cells in the RZ, these markers only partially label chondrocytes in the RZ, suggesting that the resting chondrocytes (RCs) are a heterogeneous population with different types of stem cells. Since a comprehensive marker for RCs is still lacking, the RZ is generally determined based on ambiguous histological criteria, such as small and round chondrocytes without columnar formation, which may lead to inconsistencies among researchers. Therefore, in this study, we used single-cell RNA sequencing (scRNAseq) of growth plate chondrocytes followed by validation by fluorescence in situ hybridization (FISH) to precisely annotate cell clusters in scRNAseq and search for a marker of RCs. The scRNAseq analysis revealed that apolipoprotein E (Apoe) was the top-hit gene, which was ubiquitously expressed in the RC cluster. FISH confirmed that Apoe was exclusively localized to the histologically defined RZ. In newly generated ApoemCherry knock-in mice, we further confirmed that mCherry expression mirrored the distribution of Apoe-expressing chondrocytes in the RZ particularly after the formation of the secondary ossification center. These mCherry+ RCs were slow cycling in vivo and exhibited stem cell properties in vitro. Moreover, APOE was detected in human growth plate RCs. These findings suggest that apolipoprotein E is a novel pan-RC marker in both mouse and human growth plates.

overall_design: Single-cell RNA sequencing of digested epiphyses tissues of hindlimbs from a 4-week-old female C57BL/6J mouse.


title: DNA lesions can frequently precede DNA:RNA hybrid accumulation [DRIP-seq]

city: Paris

first: Benoit

last: Palancade

address: 15 rue Hélène Brion

department: Institut Jacques Monod

institute: Université Paris Cité, CNRS

summary: While DNA:RNA hybrids contribute to multiple genomic transactions, their unscheduled formation is a recognized source of DNA lesions. Here, through a suite of systematic screens, we rather observed that a wide range of yeast mutant situations primarily triggering DNA damage actually leads to hybrid accumulation. Focusing on Okazaki fragment processing, we established that genic hybrids can actually form as a consequence of replication-born discontinuities such as unprocessed flaps or unligated Okazaki fragments. Strikingly, such “post-lesion” DNA:RNA hybrids neither detectably contribute to genetic instability, nor disturb gene expression, as opposed to “pre-lesion” hybrids formed upon defective mRNA biogenesis, e.g., THO complex mutants. Post-lesion hybrids similarly arise in distinct genomic instability situations, triggered by pharmacological or genetic manipulation of DNA-dependent processes, both in yeast and human cells. Altogether, our data establish that the accumulation of transcription-born DNA:RNA hybrids can occur as a consequence of various types of natural or pathological DNA lesions, yet do not necessarily aggravate their genotoxicity.

overall_design: Stranded DNA:RNA hybrid ImmunoPrecipitation-sequencing (DRIP-seq) in RAD27-AID yeast cells treated or not with Auxin, and/or synchronized in G1.

first: Raphael

last: Mangione


title: DNA lesions can frequently precede DNA:RNA hybrid accumulation [RNA-seq]

overall_design: RNA sequencing (RNA-seq) with rRNA removal, in RAD27-AID yeast cells treated or not with Auxin.


title: Single, but not mixed dietary fibers suppress body weight gain and adiposity in high fat-fed mice.

status: Public on Feb 05 2025

city: Aberdeen

first: Morven

last: Cruickshank

state: Aberdeenshire

address: Ashgrove Road West

department: Rowett Institute

institute: University of Aberdeen

summary: Dietary fiber can suppress excess adipose tissue and weight gain in rodents and humans when fed high fat diets. The gut microbiome is thought to have a key role, although exactly how remains unclear. In a tightly controlled murine study, we explored how different types of dietary fiber and doses affect the gut microbiota and gut epithelial gene expression.   We show that 10% pectin and 10% FOS suppress high fat diet (HFD)-induced weight gain, effects not seen at 2% doses. Furthermore, 2 and 10% mixtures of dietary fiber were also without effect. Distinct gut microbiota profiles were stimulated at the family and operational taxonomic unit (OTU) level by the different fiber treatments. Plasma levels of the gut hormone PYY were elevated by 10% pectin and FOS, but not 10% mixed fibers, and similarly RNA seq revealed some distinct effects of the 10% single fibers on gut epithelial gene expression. These data show how the ability of dietary fiber to suppress HFD-induced weight gain is dependent upon both fiber type and dose. It also shows that the microbial response to dietary fiber is distinct and that there is not a single microbial response associated with the inhibition of adiposity and weight gain.  PYY seems key to the latter response, although the role of other factors such as Reg3g and CCK needs to be explored.

overall_design: RNAseq profiling of mouse gut epithelial cell differential gene expression in response to single versus mixed fibre diets on a high fat background.

first: Swang

last: Shallangwa

first: Alexander

middle: W

last: Ross

first: Peter

last: Morgan


title: Mutation in Wdr45 leads to early motor dysfunction and widespread aberrant axon terminals in a beta-propeller protein associated neurodegeneration (BPAN) patient-inspired mouse model

city: Sioux Falls

first: Louis-Jan

last: Pilaz

state: SD

address: 2301 East 60th St N

institute: Sanford Research

summary: Beta-propeller Protein Associated Neurodegeneration (BPAN) is a devastating neurodevelopmental and neurodegenerative disease linked to variants in WDR45. Currently, there is no cure or disease altering treatment for this disease. This is, in part, due to a lack of insight into early phenotypes of BPAN progression and WDR45’s role in establishing and maintaining neurological function. Here we generated and characterized a mouse model bearing a c52C>T BPAN patient variant in Wdr45. We show this mutation ablates WDR45 protein expression and alters autophagy in the brain. Behavioral analysis of these mice revealed characteristic signs of BPAN including cognitive impairment, hyperactivity, and motor decline. We show these behaviors coincide with widespread glial activation and development of axonal spheroids in multiple neuron subclasses throughout the brain. Several lines of evidence suggest these spheroids arise from axon terminals. Transcriptomic analysis uncovered multiple disrupted pathways in the cortex including genes associated with synapses, neurites, endosomes, endoplasmic reticulum, and ferroptosis. This is supported by accumulation of the iron regulating transferrin receptor 1 (TFRC) and the endoplasmic reticulum resident calreticulin (CALR) in the cortex as these animals age. CALR forms spheroid structures similar to the axonal spheroids seen in these animals. Taken together, our data suggest that WDR45 is necessary for healthy brain function and maintenance of axon terminals. This model opens the door to therapeutics targeting BPAN and further exploration of the role of WDR45 in neuronal function.

overall_design: Cortical samples from 3-month-old Wdr45 c52C>T mice and wildtype mice were collected and RNA was isolated using a TRIzol – Chloroform-Isopropanol protocol. RNA sequencing experiments were then performed with analysis comparing transcriptomes between Wdr45 c52C>T mice and wildtype mice.

first: Brandon

last: Meyerink


title: Heparan sulfate determines myofibroblast identity during alveolar morphogenesis (scRNA-seq)

city: Chengdu

first: Hua

last: He

state: Sichuan

address: 2222 Xinchuan Rd

institute: West China Second University Hospital Sichuan University

summary: Postnatal respiration requires the bulk formation of alveoli that produces extensive surface area for gas diffusion from epithelium to the circulatory system. Alveolar morphogenesis initiates at late gestation or postnatal stage during mammalian development and is mediated by coordination among epithelial, mesenchymal and endothelial cells. Here we show that stromal cells produce Heparan Sulfate Glycosaminoglycan (HS-GAG) to maintain a niche for alveolar development by modulating both the biophysical and biochemical cues. HS is predominantly enriched in myofibroblasts in postnatal murine lungs, and genetic ablation of HS synthase gene Ext1 in fibroblast populations results in enlarged and simplified alveolar structures. HS is selectively required for the cell identity of a subset of alveolar myofibroblasts (PDGFRαhi αSMA+) residing in the distal alveolar region, which exhibit contractile properties and maintain WNT signaling activity to support normal proliferation and differentiation of alveolar epithelial cells. HS is essential to prevent precocious apoptosis of alveolar myofibroblasts. We show that these processes are dependent upon FGF/MAPK signaling and forced activation of MAPK/ERK signaling partially corrected alveolar simplification along with restoration of alveolar myofibroblast identity and AT2 cell proliferation in HS deficient mice. These data reveal HS as an essential orchestrator for developing alveolar niche critical for generation of gas exchange units.

overall_design: We performed 10X genomics single-cell RNA-seq of single cell suspension of lungs from Ext1 flox/flox, Gli1CreER  mice and their littermate controls. Deletion was induced by treating mice with tamoxifen on P0 and P1, and lungs were harvested on postnatal day 8 (P8). Library was constructed using the 10X Chromium Next GEM Single Cell 3' Reagent Kits v3.1. Reads were aligned with standard Cell Ranger pipeline.


title: Heparan sulfate regulates amphiregulin programming of tissue reparative lung mesenchymal cells during influenza A virus infection in mice

city: New York

first: Nicholas

last: Arpaia

state: NY

department: Microbiology & Immunology

institute: Columbia University

summary: Amphiregulin is an immune cell-derived growth factor that plays critical roles in tissue repair. After influenza A infection, Treg cells in the lung produce amphiregulin, which signals to a specific Col14a1-expressing subpopulation of lung mesenchymal cells (Col14-LMC) to coordinate their repair activities towards the damaged lung epithelium. Heparan sulfate is a subtype of proteoglycan that can be produced by all cells in the body, and has been implicated in altering growth factor signaling in numerous contexts. Amphiregulin has a heparan sulfate-binding domain; we sought to investigate how the inhibition of heparan sulfate on primary Col14-LMC affects their response to amphiregulin at the transcriptional level.

overall_design: We isolated Col14-LMC, a reparative subset of lung mesenchymal cells, from mice by flow cytometric sorting. Cells were allowed to adhere in culture for 24h, then dead cells were washed out/media was replaced. Cells were subsequently incubated with water vehicle or 3 mg/ml sodium chlorate (NaClO3) overnight (16-18h) to inhibit heparan sulfate. Then, cells were treated with varying concentrations of recombinant mouse amphiregulin (vehicle, 200 ng/ml, 500 ng/ml, 1000 ng/ml), incubated for 4h, then lysed for total mRNA for bulk RNA-seq.

first: Lucas

last: Loffredo

first: Anmol

last: Kustagi

first: Olivia

middle: R

last: Ringham

first: Fangda

last: Li

first: Kenia

last: de los Santos-Alexis

first: Anjali

last: Saqi


title: CTDP1 and RPB7 stabilize and license Pol II termination to reinitiation [PRO-seq]

first: Xiong

last: Ji

address: Haidian District, Beijing Summer Palace Road No. 5

department: School of Life Sciences

institute: Peking University

summary: The regulations of RNA Pol II initiation, elongation, and termination have been extensively investigated in mammalian cells, but how RNA Pol II terminates and then reinitiates a new round of transcription remains elusive. Here, we found that RPB7 loss triggers the degradation of RNA Pol II. Strikingly, RPB7 degradation combines knock out of E3 ligase Cullin 3 led to the accumulation of phosphorylated Pol II in the soluble fractions and Pol II reinitiation failure. The stability of recycled Pol II is dependent on the loop domain of RPB7 and the dephosphorylation of Pol II CTD. Termination-reinitiation recycling requires RPB7, CTD phosphatase subunit 1 (CTDP1), and stabilization of Pol II. Our study demonstrates that RPB7/CTDP1 controls the dephosphorylation and stabilization of RNA Pol II to permit RNA Pol II termination to reinitiation recycle in mammalian cells.

overall_design: Precision Run-On Sequencing (PRO-Seq) in CTDP1 degron cells.

first: Haonan

last: Zheng

first: Qiqin

last: Xu


title: CTDP1 and RPB7 stabilize and license Pol II termination to reinitiation [Micro-C]

overall_design: Micro-C in CTDP1 degron cells.


title: RPB7/CTDP1 stabilizes and licenses Pol II termination to reinitiation [ChIP-Seq]

summary: The regulations of transcription process have been extensively investigated, but how RNA Pol II terminates and reinitiates a new round of transcription remains elusive. Here, we found that RPB7 loss triggers the degradation of RNA Pol II. RPB7 degradation combing knock out of E3 ligase Cullin 3 led to the accumulation of phosphorylated Pol II in the soluble fractions and Pol II reinitiation failure. The stability of recycled Pol II is dependent on the loop-region and dimerization of RPB7 and the phosphorylation state of Pol II CTD. Site-specific cancer mutations in the RPB7 loop-region caused the dysregulations of specific gene expression. Moreover, the loss of RPB7 or CTD phosphatase subunit 1 (CTDP1) result in the defects in Pol II termination-reinitiation recycle. Together, our study demonstrates that RPB7/CTDP1 controls the dephosphorylation and stabilization of RNA Pol II to permit RNA Pol II termination to reinitiation recycle in mammalian cells.

overall_design: Chromatin immunoprecipitation DNA-sequencing (ChIP-seq) for RPB1, pSer2 and RPB7 in RPB7 degron cells or Cul3KO cells under various treatments.


title: RPB7/CTDP1 stabilizes and licenses Pol II termination to reinitiation [ChAR-Seq]

overall_design: Comparative gene expression profiling analysis of chromatin-associated RNA sequencing (ChAR-seq) data in RPB7 degron cells,  Cul3KO cells or TIGRE-RPB7-K41E/F158A cells upon RPB7 degradation.


title: A torpor-like state in mice slows blood epigenetic aging and prolongs healthspan

status: Public on Feb 04 2025

city: Cambridge

first: Sinisa

last: Hrvatin

state: MA

address: 455 Main Street

department: Whitehead Institute

institute: MIT

summary: Torpor and hibernation are extreme physiological adaptations of homeotherms associated with pro-longevity effects. Yet the underlying mechanisms of how torpor affects aging, and whether hypothermic and hypometabolic states can be induced to slow aging and increase health span, remain unknown. We demonstrate that the activity of a spatially defined neuronal population in the avMLPA, which has previously been identified as a torpor-regulating brain region, is sufficient to induce a torpor like state (TLS) in mice. Prolonged induction of TLS slows epigenetic aging across multiple tissues and improves health span. We isolate the effects of decreased metabolic rate, long-term caloric restriction, and decreased core body temperature (Tb) on blood epigenetic aging and find that the decelerating effect of torpor-like states on aging is mediated by decreased Tb. Taken together, our findings provide novel mechanistic insight into the decelerating effects of torpor and hibernation on aging and support the growing body of evidence that Tb is an important mediator of aging processes.

overall_design: Tissues were collected from time-0 mice, control mice, and experimental mice after 3 months of TLS.

first: Lorna

last: Jayne

first: Julian

last: Roessler


title: Transcriptomic analysis of early stages of ‘Candidatus Liberibacter asiaticus’ infection in susceptible and resistant genotypes after inoculation by Diaphorina citri feeding on young shoots

status: Public on Feb 14 2025

city: Valencia

first: Javier

last: Forment

address: Ingeniero Fausto Elio, s/n

country: Spain

institute: Instituto de Biología Molecular y Celular de Plantas, IBMCP (Universitat Politècnica de València-CSIC)

summary: Huanglongbing (HLB) is a devastating disease of citrus plants caused by the non-culturable phloem-inhabiting bacterium Candidatus Liberibacter asiaticus (CLas). Bacteria are vectored by the psyllid Diaphorina citri and introduced into sieve cells, evading plant immunity and establishing a successful infection in citrus plants. Although no resistance has been reported in Citrus species, complete or partial resistance has been documented in the distant relatives Murraya paniculate and Bergera koenigii, providing excellent systems to investigate molecular mechanisms leading to either resistance or susceptibility. The first weeks after bacterial release into the phloem are critical for the establishment of the bacteria. In this study, a thorough transcriptomic analysis of young flushes exposed to CLas-positive or negative psyllid has been performed in Citrus x sinensis, as well as in these resistant species along the first eight weeks after exposure. Our results suggest that the resistant genotypes do not deploy a classical immunity response. Instead, transcriptome changes are scarce and only a few genes are differentially expressed. Functional analysis suggest that primary metabolism and other basic cellular functions could be rewired in the resistant genotypes to limit infection. Transcriptomes of young flushes of the three species are very different, supporting the existence of distinct biochemical landscapes. These findings suggest that both intrinsic metabolic inadequacies to CLas survival, as well as inducible reprogramming of physiological functions upon CLas recognition, could orchestrate together to restrict bacteria multiplication in these resistant hosts.

sample_organism:
Citrus sinensis
Murraya paniculata
Murraya koenigii

overall_design: Citrus x sinensis, Murraya paniculata and Bergera koenigii flushes were exposed to Clas-negative and Clas-infected Diaphorina citri psyllids. RNAseq was performed before psyllid exposure, at 48h after psyllid exposure (t0), and after 10, 20, 30 and 60 of psyllid exposure.

first: Jose

last: Gadea

first: Monica

last: Alves

first: Leandro

last: Peña


title: Effects of IFN beta treatment on gene expression in human fibrosarcoma HT1080 cells

city: Fuzhou

first: Jun

last: Lu

address: Xierhuan Road No.156

institute: The 900th Hospital of Chinese PLA Logistic Support Forces

summary: The cyclic GMP-AMP synthase (cGAS)-stimulator of interferon genes (STING) pathway is a cytosolic DNA sensing system. The pathway product interferon-β (IFNβ) can inhibit tumor cell growth, but it remains unclear whether ferroptosis is involved in IFNβ-induced cell death. We found that IFNβ can increase intracellular Fe2+ and lipid peroxidation levels while decreasing GSH levels in tumor cells. RNA sequencing data showed that IFNβ caused abnormal transcriptional expression of ferroptosis-related genes in HT1080 cells, with upregulation of multiple genes including TRIM21, PML, PARP9, PARP14, and PARP10. These results indicate that ferroptosis is involved in IFNβ-induced tumor cell ferroptosis.

overall_design: RNA sequencing analysis was conducted on HT1080 cells following a 24-hour IFNβ treatment period, with untreated cells serving as controls

first: Huiyue

last: Dong

first: Ling

last: Zhu

first: Jingjing

last: Sun

first: Qiuyan

last: Chen


title: PARP inhibitor radiosensitization enhances anti-PD-L1 immunotherapy through depression of chemokine translation in small cell lung cancer

city: Toronto

first: Benjamin

last: Lok

state: ON

address: 101 College Street, PMCRT, 13-307

institute: Princess Margaret Cancer Centre

summary: Immunotherapy (IO) is an effective therapeutic for various cancers however the benefits are modest for small cell lung cancer (SCLC). The poor response of SCLC to anti-PD-1/PD-L1 IO is due in part to the lack of cytotoxic T cells because of limited chemokine expression from SCLC tumors. Immunogenic radiosensitizers that enhance chemokine expression may be a promising strategy forward. Here, we showed that the PARP inhibitor, olaparib, in combination with radiotherapy (RT) enhanced immune activation and anti-tumor efficacy in SCLC cell lines, patient-derived xenograft (PDX) and syngeneic mouse models, that was further pronounced with continued delivery of adjuvant olaparib. The combination treatment (olaparib with RT) activated the cGAS-STING pathway and increased the mRNA levels of the T cell chemoattractants, CCL5 and CXCL10. Interestingly, we found the combination treatment significantly downregulated expression of the mRNA translational repressor EIF4E2 to further increase chemokine CXCL10 protein levels via post-transcriptional stabilization of CXCL10 mRNA in its 3’ UTR region. We demonstrated the combination treatment augmented cytotoxic CD8+ T cell tumor infiltration and upregulated PD-L1 gene expression and protein levels. The incorporation of anti-PD-L1 IO with olaparib and RT significantly improved anti-tumor efficacy by protecting T cells from exhaustion. This study identified a novel mechanism of treatment-induced chemokine regulation that provides a rationale for the combination of PARP inhibitors, RT and anti-PD-L1 IO as a novel treatment strategy for SCLC.

overall_design: To investigate the gene expressions modulated by the olaparib combined with radiation, we treated SBC5 cells with vehicle (DMSO), olaparib (1uM), radiation (4Gy) or the combination (olaparib+radiation) for 3 days. We then performed  gene expression profiling analysis using data obtained from RNA-sequencing from cells under four conditions. Comparative gene profiling analysis of RNA-seq data for combination (PARPi+4Gy) vs Vehicle (DMSO+0Gy), RT (DMSO+4Gy) vs Vehicle (DMSO+0Gy) and PARPi (PARPi+0Gy) vs Vehicle (DMSO+0Gy).

first: Xiaozhuo

last: Ran

first: Mona

last: Teng

first: Wail

last: Ba-alawi


title: Defining the interplay of carbon dioxide and the carbonic anhydrase CanB in regulating Mycobacterium tuberculosis PhoPR signaling and virulence

city: East Lansing

first: Robert

last: Abramovitch

state: MI

address: 2215 Biomedical And Physical Sciences

institute: Michigan State University

summary: Regulation of M. tuberculosis gene expression by carbon dioxide at acidic and neutral pH.

overall_design: M. tuberculosis CDC1551 cultures were seeded at a starting OD600 of 0.2  in 8 mL of 7H9 buffered media and grown at 37°C in standing T-25 culture flasks. Two biological replicates of the following culture conditions were examined: i)  0.5% CO2 at pH 5.7, ii)  5% CO2 at pH 5.7, , iii) 0.5% CO2 at pH 7.0, iv) 5% CO2 at pH 7.0. Cultures were incubated for six days, after which total bacterial RNA was extracted.

first: S

middle: D

last: Dechow

first: R

last: Kumar

first: B

last: Johnson


title: Bmal1 KO mouse lung versus WT under three ribosomal depletion methods

city: Philadelphia

first: Thomas

middle: G

last: Brooks

address: 3400 Civic Center Blvd

department: ITMAT

institute: University of Pennsylvania

summary: Embryonic Bmal1 knock-out (KO) mouse lungs were compared to wildtype (WT) mouse lungs. Three different library preparations were done on each sample in order to assess the impact of ribosomal depletion on differential expression.

overall_design: Lungs from six KO and six WT mice were extracted and then prepared using a polyA selection method and using a rRNA pull-down ("RiboZero") method. In addition, four of each genotype were prepared as NoSelect libraries, done in six replicates to increase mRNA reads.

first: Nicholas

middle: F

last: Lahens

first: Amruta

last: Naik

first: Gregory

last: Grant

first: Peter

middle: S

last: Choi

first: Shaon

last: Sengupta


title: RPB7/CTDP1 stabilizes and licenses Pol II termination to reinitiation

city: Beijing

first: Xiong

last: Ji

overall_design: Refer to individual Series


title: Impact of ribosomal depletion, PCR ramp rate, and fragment length on RNA-seq

summary: Coverage of short-read RNA-seq is highly non-uniform across transcripts even in genes with only one expressed isoform, contrary to biological expectation. We investigate the impact of several library preparation factors on the non-uniformity of coverage. Specifically, a mouse liver sample is prepared under varying ribosomal depletion (PolyA / rRNA digestion / rRNA pull-down / NoSelection), PCR ramp rate, and fragment length along with one heart sample without selection.

overall_design: One mouse liver sample was extracted and then prepared under several protocols. Each combination of selection method (PolyA / rRNA pull-down / rRNA digestion), PCR ramp rate (slow/medium/fast), and fragment length (short/medium/long) was prepared. In addition, libraries without any selection or depletion of rRNA were prepared ("NoSelect") in triplicate in two batches to increase the amount of mRNA reads. Finally, one heart NoSelect sample was prepared.


title: Persistent alveolar bronchiolization, interstitial fibrosis and impaired lung function post exercise are features of long-COVID-19 in SARS-CoV-2-Delta variant infected aged hamsters

city: Braunschweig

last: Geffers

address: Inhoffenstr. 7

department: Dep. Molecular Bacteriology

institute: HCI - Helmholtz Centre for Infection Research

summary: The impact of Covid-19 infection on the hamster lung shortly after infection and up to 112 days was investigated using RNA-Seq to examine both short-term effects and long-term changes in the lung environment.

overall_design: The transcriptome of the hamster lung was measured at various time points (1, 3, 6, 14, 28, 56, and 112 days post-infection (dpi)) and compared with the corresponding uninfected mock controls

first: K

last: Schughart

first: W

last: Baumgärtner


title: Study 7- RNA-seq of male KOLF2.2J hiPSC-derived differentiated cells for lines in which the original mutation to create null alleles was reverted back to wildtype

status: Public on Feb 03 2025

city: Hinxton

first: MorPhiC

last: Consortium

address: Wellcome Genome Campus

institute: EMBL-EBI

summary: This study aimed to phenotype iPSC-derived cortical brain organoids that contained corrections (reversions to wt) for homozygous null alleles for transcription factor PAX6 and to phenotype iPSC-derived extra-embryonic cell lineages that had contained homozygous null alleles for transcription factors PPARG, EPAS1, GCM1, and GRHL1. The original homozygous mutant lines were created by introducing a premature termination codon and frameshift (PTC+1) approach. Here in these clones, these mutations were reverted back to wt and restored gene function. The homozygous null alleles for the five genes studied here showed pronounced transcriptomic changes compared to the parental line, the data provided here indicate, upon reversion of the PTC+1 mutations back to wt, the normal wt transcriptomic response was largely restored indicating specificity of the engineered alleles in manipulating the respective gene’s function.

overall_design: The study involved the generation of RNA-seq on 30 samples, comprising cells differentiated into cortical brain organoids (with dorsal forebrain patterning) and differentiated into the trophoblast lineage, promoting the formation of a primitive syncytium. Three biological replicates were run for each gene KO line. The hypoxia-inducible factor (EPAS1) was differentiated under both 20% oxygen and 3% (~ level present at peri-implantation) oxygen concentrations. Additionally, three biological replicates were run of wild-type KOLF2.2J cells (the parental male cell line in which the KOs were generated) under the same differentiation conditions.

first: Juliana

middle: A

last: Diniz

first: Bill

last: Skarnes

first: Paul

last: Robson


title: Study 5- RNA-seq of male KOLF2.2J hiPSC-derived cortical brain organoids and extra-embryonic lineages homozygous null for nine different transcription factors and one RNA splicing factor

summary: This study aimed to phenotype iPSC-derived cortical brain organoids that contain homozygous null alleles for transcription factors SIX3 and NR2F1 and to phenotype iPSC-derived extra-embryonic cell lineages that contain homozygous null alleles for transcription factors HEY1, HOPX, MAFF, TWIST2, DLX4, DLX5, and DLX6 and the splicing factor MBNL2. Null alleles were generated by the insertion of a premature termination codon with frameshift (PTC+1). This analysis provided insight into the impact of loss of function for these transcription factors in the respective cell lineages.

overall_design: The study involved the generation of RNA-seq on 48 samples, comprising 12 differentiated into cortical brain organoids (with dorsal forebrain patterning) and 36 differentiated into the trophoblast lineage, promoting the formation of a primitive syncytium. Three biological replicates were run for each gene KO line. Additionally, three biological replicates were run of wild-type KOLF2.2J cells (the parental male cell line in which the KOs were generated) under the same differentiation conditions.