title: Inflammatory pathways confer resistance to chemoradiotherapy in anal squamous cell carcinoma

city: Bad Nauheim

first: Mario

last: Looso

address: Ludwigstrasse 43

institute: Max Planck Institute for Heart and Lung Research

summary: Acantholytic squamous cell carcinoma (ASCC) is associated with immunosuppression and infection with human papillomavirus (HPV). Response to standard chemoradiotherapy (CRT) varies considerably. A comprehensive molecular characterization of CRT resistance is lacking, and little is known about the interplay between tumor immune contexture, host immunity, and immunosuppressive and/or immune activating effects of CRT. Patients with histologically confirmed, localized ASCC, treated between 1998 and 2019 at three different sites (Frankfurt, Berlin, Tübingen) of the German Cancer Consortium (DKTK) were included. All patients received 5-FU/MMC-based CRT. Patient cohorts for molecular analysis were defined based on availability and quality of samples, including baseline formalin fixed paraffin embedded biopsies for immunohistochemistry (n=130), baseline RNA sequencing (n=98), peripheral blood immune profiling by sequential multiparametric flow cytometry (n=47), and serum cytokine multiplex assays measurement (n=35). Tumor response, freedom from locoregional failure (FFLF), and freedom from distant metastasis (FFDM) were correlated with clinicopathologic and molecular findings.

overall_design: Baseline FFPE tumor samples were processed for total RNA isolation, library preparation and raw counts generation. Total RNA was isolated from FFPE samples using Qiagen RNeasy FFPE Kit (Qiagen) and depleted from ribosomal RNA (Ribozero rRNA Removal Kit (Illumina, San Diego, CA, USA)) to prepare sequencing libraries (NEBNext Ultra RNA Library Prep Kit for Illumina). Briefly, enriched RNAs were fragmented for 15 minutes at 94 °C. First strand and second strand cDNA were subsequently synthesized.  cDNA fragments were end repaired and adenylated at 3’ends, and universal adapter was ligated to cDNA fragments, followed by index addition and library enrichment with limited cycle PCR. Sequencing libraries were validated using the Agilent Tapestation 4200 (Agilent Technologies, Palo Alto, CA, USA), and quantified by using Qubit 2.0 Fluorometer (Invitrogen, Carlsbad, CA) as well as by quantitative PCR (Applied Biosystems, Carlsbad, CA, USA). Samples were sequenced using a 2 × 150 paired end configuration (HiSeq 2x150 PE HO) and quality guarantee ≥80% of bases ≥Q3.

first: Daniel

last: Martin

first: Franz

last: Rödel

last: Hehlgans

first: Paul

last: Ziegler

first: Maximilian

last: Fleischmann

first: Markus

last: Diefenhardt

first: Lea

last: Fries

first: Goda

last: Kalinauskaite

first: Ingeborg

last: Tinhofer

last: Zips

first: Cihan

last: Gani

first: Claus

first: Emmanouil

last: Fokas


title: CCR2 and CCR7 promote inflammatory monocyte recruitment through synergistic roles during encephalitic virus infection, but the monocyte response in the brain is dependent on the infecting virus

status: Public on Mar 18 2024

city: Hamilton

first: Clayton

last: Winkler

state: MT

address: 903 S. 4th St

country: USA

institute: Rocky Mountain Laboratories/NIAID/NIH

type: Expression profiling by high throughput sequencing

summary: Inflammatory monocytes (iMO) migrate from the bone marrow to the brain during viral encephalitis. For many viruses, including Herpes simplex virus type-1 (HSV), iMO recruitment is dependent on the chemokine receptor CCR2. However, La Crosse virus (LACV) induces iMO recruitment independent of CCR2. Comparison of iMOs from HSV and LACV-infected mice showed higher expression of the g protein-coupled receptor CCR7 in LACV-induced iMOs. CCR2/CCR7 double knockout mice (DKO) had reduced iMO recruitment following LACV infection compared to CCR2 or CCR7 single knockout mice indicating that each receptor regulated iMO recruitment through complimentary roles. Thus, CCR7 is a novel, synergistic pathway to CCR2-induced iMO recruitment during virus infection. Interestingly, unlike HSV-recruited iMOs, LACV-recruited iMOs did not influence disease and had higher expression of proinflammatory and proapoptotic transcripts but reduced mitotic, phagocytic and phagolysosomal pathway transcripts. These findings indicate that virus-specific activation of iMOs may affect their survival, maturation and functional capabilities.

overall_design: RNA was extracted from ½ brains, and inguinal lymph nodes following a Trizol and chloroform protocol. Brain immune cells from 3 HSV and 3 LACV (both 2 females and 1 male) infected mice were prepared and labeled for FACs isolation as described above. Isolated iMOs from were lysed in RLT lysis buffer. The lysate was combined with additional RLT buffer and beta mercaptoethanol (MilliporeSigma, St. Louis, MO) to bring the final volume to 700 µL with 1% BME. Samples were passed through QIAshredder column (Qiagen, Valencia, CA) at 21,000 x g for 2 minutes to homogenize any genomic DNA. RNA was extracted using Qiagen AllPrep DNA/RNA mini columns (Valencia, CA). RNA integrity was assessed using the Agilent 2100 Bioanalyzer using RNA 6000 Pico kit (Agilent Technologies, Santa Clara, CA). RNA was quantitated using a fluorescence assay (Quant-it RiboGreen RNA, Thermofisher Scientific, Waltham, MA) on a Tecan Spark multiplate reader (Tecan, Switzerland). Starting with 200 pg of high-quality total RNA for each sample, volumes were concentrated via speed-vacuum to 9.5 ul. Sequencing libraries were constructed following the Takara SMART-Seq v4 PLUS protocol 011820 (Takara, Mountain View, CA) beginning with oligo(dT) priming/cDNA synthesis and following the manufacturers recommendations.  Modifications during cDNA amplification included the use of SeqAmp CB PCR Buffer with 14 cycles of PCR. After AmPure XP bead purification (Agencourt Biosciences, Beverly, MA), cDNA quality was visualized and quantity assessed on a BioAnalyzer HS chip (Agilent Technologies, Inc., Santa Clara, CA). Purified cDNA for each sample was normalized to 1.5 ng in 8 ul volume going into library preparation with the Stem-Loop Adaptor mix. Following 15 cycles of library amplification using balanced SMARTer RNA unique-dual index primers from 96U SetA kit 042121 for Illumina sequencing (Takara, Mountain View, CA), individual libraries were cleaned using an 80% vol of AmPure XP beads, visualized on a BioAnalyzer HS chip, and quantified using the Kapa SYBR FAST Universal qPCR kit for Illumina sequencing (Kapa Biosystems, Wilmington, MA) on the CFX384 Real-Time PCR Detection System (Bio-Rad Laboratories, Hercules, CA). Each final library was diluted to a final concentration of 1.5 nM and pooled together in equimolar concentrations for sequencing. After an initial MiSeq paired-end 2 X 150 cycle sequencing run was completed to confirm proper index balancing, samples were run on a NextSeq2k instrument using 1000pM of manually-denatured final library pool and sequenced on a P2 flowcell following a paired-end 2 X 100 run with 300 cycle chemistry.

middle: W

first: Alyssa

middle: B

last: Evans

first: Aaron

last: Carmody

first: Justin

last: Lack

first: Tyson

middle: A

last: Woods

first: Karin

middle: E

last: Peterson


title: Effects of Apigenin and Rutaecarpine on Gene Expression Profiling of Human BMSC-derived osteoblasts [Microarray]

status: Public on Mar 20 2024

city: Riyadh

first: Meshail

last: Okla

country: Saudi Arabia

institute: King Saud University

summary: Aging causes dysfunctional changes to the bone microenvironment that lead to osteoporosis development. Osteoporosis is a serious bone health issue that is characterized by decreased bone formation and increased bone resorption.  The aim of this study is to evaluate the effects of natural compounds, Apigenin and Rutaecarpine on osteoblast differentiation and examine if Apigenin and Rutaecarpine can rescue osteogenesis in aging-context. Using human bone marrow stromal cell line (hBMSCs) and primary cells obtained from young and aged women, we showed that Apigenin and Rutaecarpine are potent in inducing osteoblast differentiation and mineralization. These compounds were able to reduce senescence along with their impacts on suppressing oxidative stress and inflammation, which both contribute to aging and bone metabolic dysfunctions. Our microarray-based transcription profiling of hBMSC-derived osteoblasts treated with either 1 µM of Apigenin or Rutaecarpine during osteoblast differentiation for 21 days revealed distinct impacts of Apigenin and Rutaecarpine on different genes including those involved in bone metabolism and skeletal system development. Additionally, Ex-vivo organotypic embryonic chick-femur culture model was used to determine the osteogenic effects of Apigenin and Rutaecarpine. The average bone volume and cortical thickness of these chick femurs were both increased significantly by Apigenin and Rutaecarpine. Collectively, our study provides a novel insight for developing therapeutic strategies using natural compounds to combat aging and its effect on bone health.

overall_design: For microarray analysis: human bone marrow stromal cell line (hBMSCs) were treated with 1uM of DMSO (as a control), Apigenin, or Rutaecarpine and subjected for osteoblast differentiation for 21 days. Fresh osteoblast differentiation media containing the testing compounds were changed every 24 h.

first: Dalia

last: Ali

first: Sara

last: Abuelreich

first: Radhakrishnan

last: Vishnubalaji

first: Nicholas

last: Ditzel

first: Rimi

last: Hamam

first: Justyna

middle: M

last: Kowal

first: Ahmed

last: Sayed

first: Abdullah

last: Aldahmash

first: Nehad

last: Alajez

first: Moustapha

last: Kassem


title: Transcription and DNA Replication Shapes DNA Break Dynamics in Long Neuronal Genes

city: Heidelberg

first: Pei-Chi

last: Wei

state: Baden-Württemberg

address: Im Neuenheim Feld 280

department: Structural and functional genomics

country: Germany

institute: German Cancer Research Center

summary: Transcription and replication conflict (TRC) are one of the main driving forces for genome instability. Yet, TRC rarely been discussed without the context of DNA:RNA entanglement, rending the role of transcription in other TRC unclear . In neural stem and progenitor cells, genes encode protein regulating neuron adhesion are hotspots for recurrent DNA break clusters (RDC). While RDC-containing genes are all actively transcribed, most RDC lack DNA:RNA entanglement. We demonstrated that, through controlled gain and loss of function genetic approaches, transcription activity is essential while not sufficient to induce RDC formation. In combination of a deep neural network and single-nucleotide resolution DNA break mapping approaches, we found RDC break densities mirror the replication fork dynamics. We demonstrated that, for the first time that, head-on TRC results in higher DNA break density than its co-direction counterparts. In summary, our results revealed that transcription has a higher-level regulatory role that has to be coordinated with DNA replication.

overall_design: Two parental lines of mouse ES cell (NXP010, NXP047) were used for CRISPR-Cas genome editing to create daughter lines that lack one allele of Ctnna2 (47-5_Ctnna2_allele-B), Ctnna2 promoter-enhanced deleted ES cell clones (38-3_Ctnna2_allele-B_promoter-A and 18-4_Ctnna2_allele-B_promoter-A), or ES cell lines that lacks one allele of Nrxn1 (22_Nrxn1_allele-B), and ), and Nrxn1 promoter-enhanced deleted ES cell clones (22-5_Nrxn1_allele-B_promoter-A and 22-37_Nrxn1_allele-B_promoter-A). The parental ES cells and the derived ES cell clones were in vitro differentiated to neural progenitor cells (ESC-NPC). To map replication stress-mediated DNA breaks, pX330-spCas9 and sgRNA expression plasmids were transfected to ESC-NPCs, allowing specific DSB to be created as "Bait" for LAM-HTGTS at five unique genomic area (Chr12_13Mb, Chr17_41Mb, Chr5_101Mb, Chr6_70Mb, Chr8_61Mb). Cells were then treated with aphidicolin (02: 0.2 uM. 03: 0.2 uM. 04: 0.4 uM. 05: 0.5 uM. 06: 0.6 uM) or veichle control DMSO for indicated time period. Genomic DNA were extracted four days after treatment and subjected to LAM-HTGTS. Nascent RNA from ESC-NPCs with or without specific genomic alteration were analyzed by GRO-seq, and the R-loop formation was determined by DRIP-seq.

first: Vivien

last: Ionasz

first: Sergej

last: Andrejev

first: Lorenzo

last: Corazzi


title: Combinational effect of the ADD domains of DNMT3A and DNMT3L on DNA methylation landscapes in mouse germ cells [WGBS]

city: Fukuoka

first: Naoki

last: Kubo

state: Fukuoka

address: 3-1-1 Maidashi, Higashi-ku

country: Japan

institute: Kyushu University

type: Methylation profiling by high throughput sequencing

summary: DNA methyltransferase 3A (DNMT3A) and DNA methyltransferase 3-Like (DNMT3L) form functional heterotetramers and ATRX-DNMT3-DNMT3L (ADD) domains, shared by both DNMT3A and DNMT3L, recognizes histone H3 tail unmethylated at lysine-4 (H3K4me0) to deposit DNA methylation properly in mammalian germ cells. However, the combinational and differential role of ADD domains of DNMT3A and DNMT3L in vivo has not been fully defined. Here we analyze both female and male germ cells derived from mouse with amino-acid substitutions in ADD domains of DNMT3A and/or DNMT3L that impair their domain function. Loss of either DNMT3A-ADD or DNMT3L-ADD domain function shows moderate reduction of global CG methylation level, but in different degree, in both germ cells. However, when both DNMT3A and DNMT3L lost their ADD domain functions, reduction of the global CG methylation level is much more severe and comparable with that of Dnmt3a/3L knockout germ cells. In contrast, such double mutant germ cells have thousands of genomic regions where non-CG methylation is aberrantly accumulated. These results highlight a critical role of the combinational function of ADD domains for the robust CG and non-CG methylation in germ cells.

overall_design: WGBS (PBAT) and mRNA-seq experiments in oocytes, sperms, and embryoes derived from DNMT3A/DNMT3L-ADD domain mutated mouse.

first: Hiroyuki

last: Sasaki


title: The functional small RNA interactome reveals targets for the vancomycin-responsive sRNA RsaOI in vancomycin tolerant Staphylococcus aureus [RNA-seq]

city: Sydney

first: Jai

middle: Justin

last: Tree

state: NSW

address: Rm s110 Bldg F25, UNSW, Gate 11 Botany St

department: School of Biotechnology and Biomolecular Sciences

country: Australia

institute: University of New South Wales

summary: Small RNAs have been found to control a broad range of bacterial phenotypes including tolerance to antibiotics. Vancomycin tolerance in multidrug resistance Staphylococcus aureus is correlated with dysregulation of small RNAs although their contribution to antibiotic tolerance in poorly understood. RNA-RNA interactome profiling techniques are expanding our understanding of sRNA-mRNA interactions in bacteria; however, determining the function of these interactions for hundreds of sRNA-mRNA pairs is a major challenge. At steady-state, protein and mRNA abundances are often highly correlated and lower than expected protein abundance may indicate translational repression of an mRNA. To identify sRNA-mRNA interactions that regulate mRNA translation, we examined the correlation between gene transcript abundance, ribosome occupancy, and protein levels. We used the machine learning technique self-organising maps (SOMS) to cluster genes with similar transcription and translation patterns and identified a cluster of mRNAs that appeared to be post-transcriptionally repressed. By integrating our clustering with sRNA-mRNA interactome data generated in vancomycin tolerant S. aureus by RNase III-CLASH, we identified sRNAs that may be mediating translational repression. We have confirmed sRNA-dependant post-transcriptional repression of several mRNAs in this cluster. Two of these interactions are mediated by RsaOI, a sRNA that is highly upregulated by vancomycin. We demonstrate regulation of HPr and the cell-wall autolysin Atl. These findings suggest RsaOI coordinates carbon metabolism and cell wall turnover during vancomycin treatment.

sample_organism: Staphylococcus aureus subsp. aureus str. JKD6008

overall_design: This study uses a metaomics approach to assess gene transcription and translation, and identify genes that are post-transcriptionally regulated. RNA-seq is used to assess mRNA abundance and is presented in this entry. Total RNA was extracted from S. aureus strain JKD6008 (VISA) in biological triplicate. Cultures were prepared with or without treatment with 8ug/ml vancomycin for 30 minutes.

first: Ignatius

last: Pang

first: Winton

last: Wu


title: Combinational effect of the ADD domains of DNMT3A and DNMT3L on DNA methylation landscapes in mouse germ cells [RNA-seq]


title: Determinant of m6A regional preference by transcriptional dynamics

status: Public on Mar 17 2024

city: Hangzhou

first: Yuanhui

last: Mao

state: Zhejiang

address: 1369 West Wenyi Road

country: China

institute: Zhejiang University

summary: N6-methyladenosine (m6A) is the most abundant chemical modification occurring on eukaryotic mRNAs, and has been reported to be involved in almost all stages of mRNA metabolism. The distribution of m6A sites is notably asymmetric along mRNAs, with a strong preference toward the 3’ terminus of the transcript. How m6A regional preference is shaped remains incompletely understood. In this study, by performing m6A-seq on chromatin-associated RNAs, we found that m6A regional preference arises during transcription. Nucleosome occupancy is remarkedly increased in the region downstream of m6A sites, suggesting an intricate interplay between m6A methylation and transcriptional dynamics. Notably, we found a remarkable slowdown of Pol-II movement around m6A sites. In addition, inhibiting Pol-II movement increases nearby m6A methylation levels. By analyzing massively parallel assays for m6A, we found that RNA secondary structures inhibit m6A methylation. Remarkably, the m6A sites associated with Pol-II pausing tend to be embedded within RNA secondary structures. These results suggest that Pol-II pausing could affect the accessibility of m6A motifs to the methyltransferase complex and subsequent m6A methylation by mediating RNA secondary structure. Overall, our study reveals a crucial role of transcriptional dynamics in the formation of m6A regional preference.

overall_design: Chromatin associated RNAs in MEF cells with or without flavopiridol treatment are subjected to m6A-seq

first: Yalan

last: Wang

first: Shen

first: Xiao-Min

last: Liu


title: Regulation by the RNA-binding protein Unkempt at its effector interface [Tail seq]

city: Riverside

first: Shiyang

last: He

state: CA

address: 3401 Watkins Drive, Boyce Hall

department: Biochemistry

institute: UCR

summary: How RNA-binding proteins (RBPs) convey regulatory instructions to the core effectors of RNA processing is unclear. Here we document the existence and functions of a multivalent RBP–effector interface. We show that the effector interface of a conserved RBP with an essential role in metazoan development, Unkempt, is mediated by a novel type of ‘dual-purpose’ peptide motifs that can contact two different surfaces of interacting proteins. Unexpectedly, we find that the multivalent contacts do not merely serve effector recruitment but are required for the accuracy of RNA recognition by Unkempt. Systems analyses reveal that multivalent RBP–effector contacts can repurpose the principal activity of an effector for a different function, as we demonstrate for reuse of the central eukaryotic mRNA decay factor CCR4-NOT in translational control. Our study establishes the molecular assembly and functional principles of an RBP–effector interface.

overall_design: We performed poly(A)tail-seq experiments to assess changes in poly(A) tail length of mRNAs upon induction of UNKWT, UNKdPAM2, or UNK3M for 24 hours in inducible HeLa cells. We processed two biological replicates of either Dox-induced or uninduced cells

first: Kriti

last: Shah

first: David

last: Turner

first: Khadija

last: Rebbani

first: Joshua

last: Corbo

first: Joseph

last: Bateman

first: Sihem

last: Cheloufi

first: Cátia

last: Igreja

first: Eugene

last: Valkov

first: Jernej

last: Murn


title: Regulation by the RNA-binding protein Unkempt at its effector interface [RNA]

overall_design: We performed RNA-seq experiments to assess changes in transcript abundance upon induction of UNKWT, UNKdPAM2, or UNK3M for 24 hours in inducible wild-type HeLa cells, and upon induction of UNKWT for 24 hours in inducible CNOT9 knockout (CNOT9KO) HeLa cells.  3 replicates in wild-type HeLa cells. Duplicates in CNOT9KO HeLa lines.


title: The functional small RNA interactome reveals targets for the vancomycin-responsive sRNA RsaOI in vancomycin tolerant Staphylococcus aureus.

first: Jai

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

platform_organism: Staphylococcus aureus subsp. aureus str. JKD6008

overall_design: Refer to individual Series


title: Regulation by the RNA-binding protein Unkempt at its effector interface  [iCLIP]

overall_design:
We mapped binding sites of ectopic UNK in HeLa cells with doxycycline (Dox)-inducible expression of UNKWT, UNKdPAM2, or UNK3M at 24 hours of incubation with Dox. We separately mapped binding sites of PABPC1 in HeLa cells with Dox-inducible expression of UNKWT, UNKdPAM2, or UNK3M at 24 hours of incubation with Dox, as well as in untreated UNKWT-inducible HeLa cells.
We processed four biological replicates for UNK iCLIP per condition. Two biological replicates for PABPC1 iCLIP


title: Combinational effect of the ADD domains of DNMT3A and DNMT3L on DNA methylation landscapes in mouse germ cells

first: Naoki

last: Kubo


title: A time-resolved multi-omics atlas of transcriptional regulation in response to high-altitude hypoxia across whole-body tissues

status: Public on Mar 16 2024

city: Beijing

first: Ze

last: Yan

address: No.2, Yuanmingyuan Road (West), HaiDian District

institute: China Agricultural University

summary: High-altitude hypoxia acclimatization requires whole-body physiological regulation in highland immigrants, but the underlying genetic mechanism has not been clarified. Here we used sheep as an animal model for low-to-high altitude translocation. We generated multi-omics data including whole-genome sequences, time-resolved bulk RNA-Seq, ATAC-Seq and single-cell RNA-Seq from multiple tissues as well as phenotypic data from 20 bio-indicators. We characterized transcriptional changes of all genes in each tissue, and examined multi-tissue temporal dynamics and transcriptional interactions among genes. In particular, we identified critical functional genes regulating the short response to hypoxia in each tissue (e.g., PARG in the cerebellum and HMOX1 in the colon). We further identified TAD-constrained cis-regulatory elements, which suppressed the transcriptional activity of most genes under hypoxia. Phenotypic and transcriptional evidence indicated that antenatal hypoxia could improve hypoxia tolerance in offspring. Furthermore, we provided time-series expression data of candidate genes associated with human mountain sickness (e.g., BMPR2) and high-altitude adaptation (e.g., HIF1A). Our study provides valuable resources and insights for future hypoxia-related studies in mammals

overall_design:
Sheep low-to-high altitude translocation experiment, and produced time-series multi-omics data (e.g., RNA-Seq).
Raw data files are deposited at SRA under the following BioProjects: PRJNA1000743, PRJNA1001016

first: Ji

last: Yang


title: ZNF469 is a novel pro-fibrotic regulator of extracellular matrix in hepatic stellate cells

status: Public on Mar 15 2024

city: Pathumwan

first: Archittapon

last: Nokkeaw

state: Bangkok

address: Padtayapatana Building, 1873, Henri Dunant Road

country: Thailand

institute: Chulalongkorn university, faculty of medicine

summary: Activation of quiescent hepatic stellate cells (HSCs) into proliferative myofibroblasts drives extracellular cellular matrix (ECM) accumulation and liver fibrosis; nevertheless, transcriptional network that promotes such the process remains elusive. ZNF469, a putative C2H2 zinc finger protein, is found to be upregulated upon HSC activation; however, the molecular function of ZNF469 is completely unknown. Here, we show that knockdown of ZNF469 in primary human HSCs impaired proliferation, migration, and collagen production. Conversely, overexpression of ZNF469 in HSCs yielded the opposite results. TGFb promoted expression of ZNF469 in a Smad3-dependent manner where the binding of Smad3 was confirmed at the ZNF469 promoter. RNA-seq data of ZNF469-knockdown HSCs revealed the ECM-receptor interaction as the top affected pathway, and significant downregulation of various collagen and proteoglycan genes was observed. To explore the function of ZNF469, we cloned a full-length open reading frae (ORF) of ZNF469 with an epitope tag and identified a nuclear localization of the protein. ChIP assays revealed the presence of ZNF469 at the promoter of ECM genes, supporting its function as a transcription factor. Analysis of human fibrotic and cirrhotic tissues showed increased expression of ZNF469 and a positive correlation between expression levels of ZNF469 and ECM genes. Moreover, this observation was similar in other fibrotic organs, including heart, lung, and skin. Together, this study is the first to reveal the roles of ZNF469 as a pro-fibrotic factor in HSCs and suggests ZNF469 as a novel target for antifibrotic therapy.

overall_design: Primary human hepatic stellate cells with control and ZNF469 knockdown with short-hairpin RNAs. Three technical replicates were performed for each group.

first: Chaiyaboot

last: Ariyachet

first: Bootsakorn

last: Boonkaew

first: Pisit

last: Tangkijvanich


title: Development of an orally bioavailable SWI/SNF ATPase degrader and acquired mechanisms of resistance [WES]

city: Ann Arbor

first: Arul

last: Chinnaiyan

state: MI

address: 1500 East Medical Center Dr

department: MCTP

institute: University of Michigan

summary: SWI/SNF ATPase degraders have been shown to be effective in enhancer-driven cancers by functioning to impede oncogenic transcription factor chromatin accessibility. Here we developed AU-24118, a first-in-class, orally bioavailable degrader of SMARCA2/4 and PBRM1. AU-24118 demonstrated tumor regression in a castration resistant model of prostate cancer which was further enhanced in combination with enzalutamide. Prostate cancer cell lines exposed to increasing doses of a SWI/SNF degrader developed SMARCA4 bromodomain mutations and ABCB1 overexpression as acquired mechanisms of resistance. While SMARCA4 mutations provided specific resistance to SWI/SNF degraders, ABCB1 overexpression provided broader resistance to other compounds as well as could be overcome with ABCB1 inhibition.

overall_design: All the AURs cell line were treated in vitro with SMARCA2/4 degrader (AU-15330, abbreviated as AU) and accquired resistance to AU.

first: Tongchen

last: He

first: Caleb

last: Cheng

first: Eleanor

last: Young

first: Hanbyul

last: Cho


title: Jurkat CRISPRi ATAC-seq [Series 3]

city: Stanford

first: Howard

middle: Y

last: Chang

address: CCSR 2130, 269 Campus Drive

department: Center for Personal Dynamic Regulomes

institute: Stanford University

type: Genome binding/occupancy profiling by high throughput sequencing

summary:
We develop a system for bidirectional epigenetic editing (CRISPRai), in which orthogonal activating (CRISPRa) and repressive (CRISPRi) perturbations are applied simultaneously to multiple loci the same cell.
We perform ATAC-seq on CRISPRi perturbed Jurkat T cells to investigate chromatin accessibility changes upon perturbation of individual regulatory elements.

overall_design:
ATAC-seq of CRISPRi perturbed Jurkat T cells
E2 - Jurkat E2 CRISPRi ATAC-seq
E4 - Jurkat E4 CRISPRi ATAC-seq
E6 - Jurkat E6 CRISPRi ATAC-seq
E9 - Jurkat E9 CRISPRi ATAC-seq
NTC - Jurkat NTC CRISPRi ATAC-seq
TSS - Jurkat TSS CRISPRi ATAC-seq

first: Naomi

last: Pacalin

first: Zachary

last: Steinhart

first: Quanming

last: Shi

first: Julia

last: Belk

first: Dmytro

last: Dorovskyi

first: Katerina

last: Kraft

first: Kevin

middle: R

last: Parker

first: Brian

last: Shy

first: Alexander

last: Marson


title: The Functional Role of L-fucose on Dendritic Cell Function and Polarization

city: Tampa

first: Dongliang

last: Du

state: FL

address: 12902 Magnolia Dr

institute: Moffitt Cancer Center

summary: Despite significant advances in the development and refinement of immunotherapies administered to combat cancer over the past decades, a number of barriers continue to limit their efficacy. One significant clinical barrier is the inability to mount initial immune responses towards the tumor. As dendritic cells are central initiators of immune responses in the body, the elucidation of mechanisms that can be therapeutically leveraged to enhance their functions to drive anti-tumor immune responses is urgently needed. Here, we report that the dietary sugar L-fucose can be used to enhance the immunostimulatory activity of dendritic cells (DCs). L-fucose polarizes immature myeloid cells towards specific DC subsets, specifically cDC1 and moDC subsets. In vitro, L-fucose treatment enhances antigen uptake and processing of DCs. Furthermore, our data suggests that L-fucose-treated DCs increase stimulation of T cell populations. Consistent with our functional assays, single-cell RNA sequencing of intratumoral DCs from breast tumor-bearing mice confirmed transcriptional regulation and antigen processing as pathways that are significantly altered by dietary L-fucose. Together, this study provides the first evidence of the ability of L-fucose to bolster DC functionality and provides rational to further investigate how L-fucose can be used to leverage DC function in order to enhance current immunotherapy.

overall_design: To investigate the effect of L-fucose on immune subsets we took intratumoral CD45+ cells treated +\- L-fucose. We then preformed gene expression profiling comparing L-fucose-treated tumors to control-treated tumors at three time points. Comparative gene expression profiling analysis of RNA-seq data for intratumoral CD45+ cells with L-fucose-treated groups compared to control-treated groups.


title: Regulation by the RNA-binding protein Unkempt at its effector interface

first: Shiyang


title: Global gene expression profile of CNS-infiltrating E2A-PBX1+/preBCR+ ALL cells

city: Kuopio

first: Merja

last: Heinäniemi

address: Yliopistonranta 1E

department: School of Medicine, Institute of Biomedicine

country: Finland

institute: University of Eastern Finland

summary:
In order to elucidate mechanism of resistance to BTKi and dasatinib in CNS-infiltrating leukemia cells and to investigate the CNS homing of E2A-PBX1/preBCR+ ALL cells, we performed global transcriptome analysis by RNA sequencing.
RNA was isolated from CNS-infiltrating cells from vehicle, dasatinib, ibrutinib, dasatinib + ibrutinib-treated mice

overall_design: Three mice per condition

first: Gaia

last: Gentile

first: Mari

last: Lahnalampi

first: Minna

last: Voutilainen

first: Jesus

last: Duque-Afonso


title: Development of an orally bioavailable SWI/SNF ATPase degrader and acquired mechanisms of resistance [RNA-seq]

first: Arul

last: Chinnaiyan

summary: SWI/SNF ATPase degraders have been shown to be effective in enhancer-driven cancers by functioning to impede oncogenic transcription factor chromatin accessibility.  Here we  developed AU-24118, a first-in-class, orally bioavailable degrader of SMARCA2/4 and PBRM1.  AU-24118 demonstrated tumor regression in a castration resistant model of prostate cancer which was further enhanced in combination with enzalutamide.  Prostate cancer cell lines exposed to increasing doses of a SWI/SNF degrader developed SMARCA4 bromodomain mutations and ABCB1 overexpression as acquired mechanisms of resistance.  While SMARCA4 mutations provided specific resistance to SWI/SNF degraders, ABCB1 overexpression provided broader resistance to other compounds as well as could be overcome with ABCB1 inhibition.

first: Tongchen

last: He

first: Caleb

last: Cheng

first: Eleanor

last: Young


city: Rochester

first: Dominik

last: Saul

state: Minnesota

address: 200 First Street SW Rochester

institute: Mayo Clinic

summary: Evolutionary insights reveal a new role of peptidyl arginine deiminase 2 in transcription regulationA systematic evolutionary analysis identified positively selected residues in the non-catalytic domain of PADI2. We established a link between the positive evolution of key residues in the PADI2 and their role in transcription. Specifically, we identified the structurally exposed loop encompassing the positively selected L162 in the PADI2 middle domain and its role in transcription and cellular proliferation. This loop contributes to the PADI2 interaction with the P-TEFb complex and cellular proliferation. Our results showcase the use of combining evolutionary and experimental approaches to dissect the dynamic of evolutionary processes.

overall_design: Hela cells expressing the wild-type (WT) and mutant (L162A/W161A/T159A) peptidyl arginine deiminase 2 (PADI2)  were used for single cell sequecing.

first: José

middle: L

last: Villanueva-Cañas

first: Narcis

last: Fernandez-Fuentes

first: Robyn

last: Kosinsky

first: Catherine

last: Teyssier

first: Malgorzata

last: Rogalska

first: Ferran

middle: P

last: Pérez

first: Baldomero

last: Oliva

first: Cedric

last: Notredame

first: Miguel

last: Beato

first: Priyanka

last: Sharma


title: Bidirectional epigenetic editing reveals hierarchies in gene regulation

status: Public on Mar 14 2024

first: Howard

last: Chang


title: Regulation by the RNA-binding protein Unkempt at its effector interface  [Ribo]

first: Shiyang

overall_design:
We performed ribosome profiling experiments to assess changes in ribosome occupancy upon induction of UNKWT, UNKdPAM2, or UNK3M for 24 hours in inducible wild-type HeLa cells, and upon induction of UNKWT for 24 hours in inducible CNOT9 knockout (CNOT9KO) HeLa cells. We processed two biological replicates from either Dox-induced or uninduced cells.
Comparisons between uninduced and WT, 3M, or dPAM2 UNK-expressing cells were performed. The log2FoldChange between the conditions were expressed as log2(Translation efficiency ratio): log2TER.

first: Kriti

last: Shah

first: David

last: Turner

first: Khadija

last: Rebbani

first: Joshua

last: Corbo

first: Joseph

last: Bateman

first: Sihem

last: Cheloufi

first: Cátia

last: Igreja

first: Eugene

last: Valkov

first: Jernej

last: Murn


title: CRISPRai Regulatory Element Screens [Series 2]

summary:
We develop a system for bidirectional epigenetic editing (CRISPRai), in which orthogonal activating (CRISPRa) and repressive (CRISPRi) perturbations are applied simultaneously to multiple loci the same cell.
We perform CRISPR gRNA enrichment screens to investigate pairwise CRISPRai perturbations of enhancers and the promoter of IL2 and IFNG genes in order to investigate enhancer-enhancer and enhancer-promoter interactions and regulatory element heirarchies.

overall_design: CRISPR gRNA enrichment

first: Naomi

last: Pacalin

first: Zachary

last: Steinhart

first: Quanming

last: Shi

first: Julia

last: Belk

first: Dmytro

last: Dorovskyi

first: Katerina

last: Kraft

first: Kevin

last: Parker

first: Brian

last: Shy

first: Alexander

last: Marson


title: Excess glucocorticoids inhibit murine bone turnover via modulating the immunometabolism of the skeletal microenvironment

status: Public on Mar 12 2024

city: Hong Kong

first: Tongzhou

last: Liang

state: Hong Kong

address: 30 Ngan Ching Street

department: Department of Orthopaedics and Traumatology

institute: The Chinese University of Hong Kong

summary: Elevated bone resorption and diminished bone formation have been recognized as the primary features of glucocorticoid-associated skeletal disorders. However, the direct effects of excess glucocorticoids on bone turnover remains unclear. Here, we explored the outcomes of exogenous glucocorticoid treatment on bone loss and delayed fracture healing in mice and found that reduced bone turnover was a dominant feature, resulting in a net loss of bone mass. We investigated the single-cell gene expression profiles of these two models. In the glucocorticoid-induced bone loss model, we sequenced the cell population digested from the bone surface. In the glucocorticoid-associated fracture healing model, we sequenced the cell population digested from the callus. Overall, the cell population on the bone surface was predominantly composed of hematopoietic cells, while only a small fraction represented the bone progenitor cells. In the fracture healing model, the callus contained a significant number of osteogenic and osteoclastic lineage cells. We primarily analyzed the gene expression of functional genes in the fracture healing model and characterized the metabolic profiles. These data provide insights into the multifactorial metabolic mechanisms by which glucocorticoids generate skeletal disorders.

overall_design: We utilized single-cell RNA-sequencing (scRNA-seq; 10x genomics) to investigate the principal cell types involved that modulate bone turnover during glucocorticoids exposure.

first: Xu

last: Li

first: Liang

last: Chang

first: Jiankun

last: Xu

first: Ling

last: Qin


title: KAT6A deficiency impairs cognitive functions through suppressing RSPO2/Wnt signaling in hippocampal CA3 [snRNA-seq]

city: Baltimore

first: Yongqing

last: Liu

state: MARYLAND

address: 725 N Wolfe Street, WBSB 216

institute: Johns Hopkins University

summary: Intellectual disability (ID) affects ~2% of the population and ID-associated genes are enriched for epigenetic factors, including those encoding the largest family of histone lysine acetyltransferases (KAT5-KAT8). Among them is KAT6A, whose mutations cause KAT6A Syndrome, with ID as a common clinical feature. However, the underlying molecular mechanism remains unknown. Here, we find that KAT6A deficiency impairs synaptic structure and plasticity in hippocampal CA3, but not in CA1 region, resulting in memory deficits in mice. We further identify a CA3-enriched gene Rspo2, encoding Wnt activator R-spondin 2, as a key transcriptional target of KAT6A. Importantly, deletion of Rspo2 in excitatory neurons impairs memory formation, and restoring RSPO2 expression in CA3 rescues the deficits in Wnt signaling and learning-associated behaviors in Kat6a mutant mice. Collectively, our results demonstrate that KAT6A-RSPO2-Wnt signaling plays a critical role in regulating hippocampal CA3 synaptic plasticity and cognitive function, providing potential therapeutic targets for KAT6A Syndrome and related neurodevelopmental diseases.

overall_design: Nuclei mRNA profiles in hippocampal tissues of adult Kat6a-f/f and NEX-cre, Kat6a-f/f (Kat6a-cKO) mice were generated by snRNA-seq, using Illumina NovaSeq 6000


title: Single-cell 3D genome structure reveals distinct human pluripotent states (RNA-Seq)

status: Public on Mar 11 2024

city: Tianjin

first: Niannian

state: Tianjin

address: No. 94 Weijin Road, Nankai District, Tianjin

department: college of Life Sciences

institute: Nankai University

summary: Pluripotent states of embryonic stem cells (ESCs) with distinct transcription profile affect their differentiation capacity and therapeutic potential. By single cell analysis of high-resolution three-dimensional (3D) genome structure, we show that remodeling genome structure is highly associated with pluripotent states of human ESCs (hESCs). Naive pluripotent state is featured with specialized 3D genome structures and clear chromatin compartmentation distinct from primed state.  Naive pluripotent state may be achieved by remodeled active euchromatin compartment and less chromatin interaction in the nuclear center. This unique genome organization is linked to elevated chromatin accessibility on enhancers and thus high expression levels of naive pluripotent genes localized in this region. On the contrary, primed state exhibits intermingled genome organization. Moreover, active euchromatin and primed pluripotent genes are distributed in the nuclear periphery and yet repressive heterochromatin densely concentrated in the nuclear center, reducing chromatin accessibility and transcription of naive genes. Thus, inversion or relocation of heterochromatin to euchromatin compartmentation is related to regulating chromatin accessibility and thus define pluripotent states and cell identity.

overall_design: Naïve hPSCs are converted from Primed hPSCs based on the expression of HERVH after the treatment with several chemicals, and we illustrate the difference by RNA-seq, ATAC-seq, and CUT&Tag-seq between Naïve and Primed hPSCs.

first: Kairang

last: Jin

first: Bin

first: Mingzhu

last: Yang

first: PanPan

first: Dai

last: Heng

first: Jichang

last: Wang

first: Lin


title: Single-cell 3D genome structure reveals distinct human pluripotent states (CUT&Tag-seq)

last: Shi


title: CRISPRai Perturb-seq [Series 1]

first: Howard

summary:
We develop a system for bidirectional epigenetic editing (CRISPRai), in which orthogonal activating (CRISPRa) and repressive (CRISPRi) perturbations are applied simultaneously to multiple loci the same cell.
We perform Perturb-seq, a method for single-cell RNA-seq paired with CRISPR guide RNA (gRNA) detection, on cells perturbed using CRISPRai for a panel of pairwise combinations of genes in K562 cells.

overall_design: Single-cell RNA-seq with paired CRISPR gRNA detection

first: Naomi

last: Pacalin

first: Zachary

last: Steinhart

first: Quanming

first: Julia

last: Belk

first: Dmytro

first: Katerina

first: Kevin


title: Microbiota of infants consuming secretors or non-secretors mothers milk impacts gut and immune system in mice.

city: Pittsburgh

first: Dhivyaa

last: Rajasundaram

state: PA

address: 4401 Penn Avenue

department: Pediatrics

institute: University of Pittsburgh

summary: Maternal secretor status is one of the determinants of human milk oligosaccharides (HMOs) composition, which in turn changes the gut microbiota composition of infants.  To understand if this change in gut microbiota impacts immune cell composition, intestinal morphology and gene expression, day 21-old germ-free mice were transplanted with fecal microbiota from infants whose mothers were either secretors (SMM) or non-secretors (NSM) or from infants consuming dairy-based formula (MFM). For each group, one set of mice was supplemented with HMOs. HMO supplementation did not significantly impact the microbiota diversity however, SMM mice had higher abundance of genus Bacteroides, Bifidobacterium, and Blautia, whereas, in the NSM group, there were higher abundance of Akkermansia, Enterocloster, and Klebsiella. In MFM, gut microbiota was represented mainly by Parabacteroides, Ruminococcaceae_unclassified, and Clostrodium_sensu_stricto. In mesenteric lymph node, Foxp3+ T cells and innate lymphoid cells type 2 (ILC2) were increased in MFM mice supplemented with HMOs while in the spleen, they were increased in SMM+HMOs mice. Similarly, serum immunoglobulin A (IgA) was also elevated in MFM+HMOs group. Distinct global gene expression of the gut was observed in each microbiota group, which was enhanced with HMOs supplementation. Overall, our data shows that distinct infant gut microbiota due to maternal secretor status or consumption of dairy-based formula and HMO supplementation impacts immune cell composition, antibody response and intestinal gene expression in a mouse model.

overall_design: To understand the impact of microbiota-derived from infants who were fed by secretor or non-secretor mothers or those consuming infant formula in mice, we transplanted fecal materials from these infants into germ-free mice on day 1 and day 7. One set of mice from each group was also supplemented with pooled Human Milk Oligosaccharides (HMOs), daily for 14 days. Total RNA from the distal small intestine (ileum) and large intestine (colon) were extracted and subjected to mRNA sequencing. Ileum and colon tissues from germ-free mice were included as the control. The following experimental groups were included: Germ -Free Control (GF), Secretor Microbiota (SMM), Non-Secretor Microbiota (NSM), Milk Formula Microbiota (MFM), SMM+HMOs, NSM+HMOs and MFM+HMOs.

first: Manoj

last: Gurung

first: Brent

middle: T

last: Schlegel

first: Renee

last: Fox

first: Lars

last: Bode

first: Tianming

last: Yao

first: Stephen

last: Lindemann

first: Tanya

last: LeRoith

first: Quentin

middle: D

last: Read

first: Christy

last: Simecka

first: Laura

last: Carrol

first: Aline

last: Andres

first: Laxmi

last: Yeruva


title: Single-cell 3D genome structure reveals distinct human pluripotent states (ATAC-Seq)

first: Niannian

last: Li

first: Kairang

last: Jin

first: Bin

last: Liu

first: Mingzhu

last: Yang

first: PanPan

last: Shi

first: Dai

last: Heng

first: Jichang

last: Wang

first: Lin


title: Single-cell 3D genome structure reveals distinct human pluripotent states


title: RNA levels in wild-type and HIF1α-mutant MCF7 cells in normoxia (21% O2), and in hypoxia (1% O2) for 3 h or 24 h

city: London

first: Dimitrios

last: Anastasiou

address: 1 Midland Road

department: Cancer Metabolism Laboratory

country: United Kingdom

institute: The Francis Crick Institute

summary: We report the generation of CRISPR/Cas9-engineered MCF7 human breast cancer cells that lack functional HIF1α. We show that the HIF1α-dependent transcriptional response to hypoxia is severely impaired in HIF1α-mutant MCF7 cells.

overall_design: RNA profiles of 2 cell lines (wild-type and HIF1α-mutant MCF7 cells ) in 3 conditions (grown at 21% oxygen, at 1% oxygen for 3 hours and at 1% oxygen for 24 hours) in triplicates, using Illumina HiSeq 2500

first: Fiona

last: Grimm


title: Neutral competition explains the clonal composition of neural organoids

city: Vienna

first: Simon

middle: Emanuel

last: Haendeler

address: Dr. Bohr Gasse 9

department: Max Perutz Labs

country: Austria

institute: University of Vienna

summary: Cerebral organoids model the development of the human brain and are an indispensable tool for studying neurodevelopment. Whole-organoid lineage tracing has revealed the number of progeny arising from each initial stem cell to be highly diverse, with lineage sizes ranging from one to more than 20,000 cells. This exceeds what can be explained by existing stochastic models of corticogenesis, indicating the existence of an additional source of stochasticity. We propose an explanation in terms of the SAN model in which this additional source of stochasticity is the survival time of a lineage within a long-lived population of symmetrically dividing cells under neutral competition. We demonstrate that this model explains the experimentally observed variability of lineage sizes and we derive a formula that captures the quantitative relationship between survival time and lineage size. Finally, we show that our model implies the existence of a regulatory mechanism to keeps the size of the symmetrically dividing cell population constant.

overall_design: Barcode-based Lineagetracing to measure the lineage size distribution in brain organoids in three replicates

first: Florian

middle: G

last: Pflug

first: Christopher

last: Esk

first: Dominik

last: Lindenhofer

first: Jürgen

last: Knoblich

first: Arndt

last: von Haeseler


title: A Novel Viral Assembly Inhibitor Blocks SARS-CoV-2 Replication in Airway Epithelial Cells

status: Public on Mar 10 2024

city: San Francisco

first: Li

last: Du

address: 360 Spear St

institute: Vitalant Research Institute

summary: The ongoing evolution of SARS-CoV-2 to evade vaccines and therapeutics underlines the need for novel therapies with high genetic barriers to resistance. The small molecule PAV-104, identified through a cell-free protein synthesis and assembly screen, was recently shown to target host protein assembly machinery in a manner specific to viral assembly. Here, we investigated the capacity of PAV-104 to inhibit SARS-CoV-2 replication in human airway epithelial cells (AECs). Our data demonstrate that PAV-104 inhibited >99% of infection with diverse SARS-CoV-2 variants in primary and immortalized human AECs. PAV-104 suppressed SARS-CoV-2 production without affecting viral entry or protein synthesis. PAV-104 interacted with SARS-CoV-2 nucleocapsid (N) and interfered with its oligomerization, blocking particle assembly. Transcriptomic analysis revealed that PAV-104 reversed SARS-CoV-2 induction of the type-I interferon response and the maturation of nucleoprotein signaling pathway known to support coronavirus replication. Our findings suggest that PAV-104 is a promising therapeutic candidate for COVID-19.

overall_design: To understand the transcriptional impact of PAV-104 in the setting of SARS-CoV-2 infection and immunopathology, we performed RNA-seq analysis on ALI-cultured primary AECs from 5 different healthy donors infected for 36 hours with SARS-CoV-2 (MOI=0.1) in the presence or absence of PAV-104. Uninfected, untreated cells (Control) were characterized as a reference for both experimental conditions. The effects of PAV-104 administration alone (in the absence of SARS-CoV-2 infection) were evaluated in three additional donors.

first: Satish

last: Pillai


title: Genomic Analysis of Human Brain Metastases Treated with Stereotactic Radiosurgery Reveals Unique Signature Based on Treatment Failure. [RNA-Seq]

status: Public on Mar 21 2024

city: Madison

first: Jack

last: Shireman

state: WI

address: 600 Highland Ave

institute: UW-Madison

summary: Stereotactic radiosurgery (SRS) has been shown to be efficacious for treatment of limited brain metastasis (BM), however, the effects of SRS on human brain metastases have yet to be studied. We performed genomic analysis of resected brain metastases from patients whose resected lesion was previously treated with SRS. Our analyses demonstrated for the first time that patients possess a distinct genomic signature based on type of treatment failure including local failure, leptomeningeal spread, and radio-necrosis. Examination of the center and peripheral edge of the tumors treated with SRS indicated differential DNA damage distribution and an enrichment for tumor suppressor mutations and DNA damage repair pathways along the peripheral edge. Furthermore, the two clinical modalities used to deliver SRS, LINAC and GK, demonstrated differential effects on the tumor landscape even between controlled primary sites. Our study provides, in humans, biological evidence of differential effects of SRS across BM’s.

overall_design: Pre-operative SRS samples analyzed during surgical reception from the center and peripheral edges of the tumor

first: Christina

last: Kendziorski

first: Mahua

last: Dey


title: KAT6A deficiency impairs cognitive functions through suppressing RSPO2/Wnt signaling in hippocampal CA3 [RNA-seq]

first: Yongqing

last: Liu

overall_design: Hippocampal tissues mRNA profiles of adult Control (wild type) and Kat6a+/- mice were generated by RNA-seq, using Illumina NovaSeq 6000


title: SPA regulation of light induced gene expressions in dark-grown Arabidopsis thaliana

city: Taipei

first: Mei-Chun

last: Cheng

address: No. 1, Roosevelt Rd

country: Taiwan

institute: National Taiwan University

summary: The total mRNA and polysomal RNA expression profiles of wild type (Col-0) and the quadruple spa mutant (spaQ) were analyzed under dark or in 4 hour light treated condition. The gene expression changed in spaQ mutant was analyzed and compared with Col-0.

overall_design: We have total of 24 samples. Twelve of them are 4 days dark-grown seedlings, Col-0 and spaQ mutant, to do the total RNA extraction and polysomal RNA extraction, each has three replicates. And the rest of them are the 4 days dark-grown seedlings treated with 4 h light treatment, Col-0 and spaQ mutant, to do the total RNA extraction and polysomal RNA extraction, each has three replicates.

first: Lin-Chen

last: Huang


title: RNA sequencing on Postnatal day (P) 7 wildtype and Zmiz1-Knockout cortex

city: new orleans

first: Rajan

last: K C

state: LA

address: 6602 Freret St

department: Cell and Molecular Biology

institute: Tulane University

summary:
Purpose: Zinc Finger MIZ-Type Containing 1 (Zmiz1) is a member of the PIAS family of protein and function as a transcriptional coactivator of Notch, Androgen Receptor (AR), p53, Estrogen Receptor (ER), and Smad3/4 . Despite Zmiz1 mutations association with neurodevelopmental disorders such as ASD, ADHD, and intellectual disability, its role in physiological and pathological neurodevelopment  is significantly unknown. Here, we use murine model to knockout Zmiz1 using Emx1Cre and performed RNA sequencing on P7 cortex to profile transcriptional changes upon Zmiz1 deletion.
Timed pregnancy was carried out. Mice was euthanized using CO2 and embryos brain were extracted at E15.5. Cortex was dissected and dissociated into single cell suspension using Neural Tissue Dissociation Kit (P) (Miltenyi Biotec, 130-092-628) following manufacture instruction. Intermediate progenitors were magnetically selected and isolated using Anti-Prominin-1 MicroBeads, mouse (Miltenyi Biotec, 130-092-333). Total RNA was extracted from wildtype and Zmiz1-KO samples. RNA concentration and RNA integrity number were determined. RNA library was prepared, quantified, and verified using TruSeq RNA Library Prep Kit v2, Qubit dsDNA High Sensitivity Assay kit and Bioanalyzer DNA1000 assay kit respectively. Verified samples were sequenced using the NextSeq1000/2000 P2 Reagents (200 Cycles) v3 on a Nextseq1000/2000. RNA-seq data analysis was performed using illumina BaseSpace Sequence Hub. Briefly, sequenced reads were aligned to mouse (mm10) reference genome with RNA-Seq alignment tool (STAR aligner) and differentially expressed genes (DEG) were determined using the RNA-Seq Differential Expression tool (version 1.0.1).
Results: We found 114 differentially expressed genes of which 35 genes were upregulated while 69 genes were downregulated. Downregulated genes were enriched in biological processes such as forebrain deveopment, axon development, neuron differentiation etc.
Conclusions: We assessed P7 cortex specific transcriptional profile changes which are potentially regulated by Zmiz1.

overall_design: P7 WT and Zmiz1-KO cortex RNA sequencing

first: Stryder

last: Meadows

first: Maria

last: Galazo


title: Development of an orally bioavailable SWI/SNF ATPase degrader and acquired mechanisms of resistance

first: Arul

last: Chinnaiyan

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

overall_design: Refer to individual Series


title: RBM5 and RBM10

status: Public on Mar 08 2024

city: LOS ANGELES

first: Chia-ho

last: Lin

address: 6577 MRL. 675 CHARLES E YOUNG DR. SOUTH

institute: UCLA


title: RNP-seq and IP-seq from cells expressing epitope-tagged RBM5, RBM10, or SF3A3

summary: We isolated snRNP complexes derived from precatalytic A or B-like spliceosomes solubilized from the chromatin pellet of lysed nuclei. These complexes contained U2 snRNP proteins and a portion of the U2 snRNA, bound with intronic branch sites prior to the first catalytic step of splicing. The complexes have similar composition, whether directly isolated from extracted material, or after selection from glycerol gradients. Here we describe sequencing libraries prepared from RNA derived from directly isolated (IP-seq), or gradient-purified complexes (RNP-seq). Libraries from deproteinized RNA were prepared after selective degradation of U2 snRNA, each in a triplicate. An additional library from RBM5-Flag RNP complex was prepared without such degradation. The majority of sequenced pre-mRNA fragments map to intronic branch sites.

overall_design: High molecular weight (HMW) nuclear extracts were obtained after enzymatic solubilization with a cocktail of RNase A+T1, DNase, and Benzonase from 293Flp-in cells expressing RBM5, RBM10, or SF3A3 protein with a C-terminal Flag tag. RBM5-Flag and SF3A3-Flag extracts were sedimented through 10-30% glycerol gradients in SW41Ti rotor (Beckman) for 17 hours at 32,000 RPM. The gradients were fractionated in 0.5 mL fractions top to bottom, fractions 11-13 containing peak RNP regions were dialyzed to reduce glycerol, and the complexes were immunopurified on anti-FLAG agarose. RBM10-Flag and SF3A3-Flag extracts were also directly incubated with anti-FLAG agarose to immunoprecipitate complexes. Following elution with 3xFLAG peptide and deproteinization, the RNA was incubated with U2-antisense oligonucleotide and RNase H to remove U2 snRNA. RNA fragments of 28-55 nt-size were selected from denaturing gels and used in construction of sequencing library with in-line barcodes. The libraries are sequenced in Illumina high-throughput sequencer at single end 100 nt.

first: Andrey

last: Damianov

first: Chia-Ho

first: Douglas

last: Black


title: Disruption of RNAPII transcription elongation links Oncogenic splicing factor mutations to replciation stress and targetable alterations in chromatin landscape.

city: NEW HAVEN

first: MANOJ

last: PILLAI

state: CT

address: 300 GEORGE STREET #786

institute: YALE UNIVERSITY

sample_organism:
Homo sapiens
Mus musculus

platform_organism:
Homo sapiens
Mus musculus


title: S100 Calcium-Binding Protein A16 Suppresses the Osteogenic Differentiation of Rat Bone Marrow Mesenchymal Stem Cells via Inhibiting SMAD Family Member 4 Signaling

city: Luohe

first: Yafei

last: Shen

state: Henan

address: 56 Renmin East Road

institute: Luohe Central Hospital

summary: Osteogenesis is a complex process of bone formation regulated by various factors, yet its underlying molecular mechanisms remain incompletely understood. This study aimed to investigate the role of S100A16, a novel member of the S100 protein family, in the osteogenic differentiation of rat bone marrow mesenchymal stem cells (BMSCs) and uncover a novel Smad4-mitogen-activated protein kinase (MAPK)/Jun N-terminal kinase (JNK) signaling axis. We herein evaluated the expression level of S100A16 in bone tissues and BMSCs from ovariectomized rats and then examined the impact of S100A16 silencing on osteogenic differentiation. Increased S100A16 expression was observed in bone tissues and BMSCs from ovariectomized rats, and S100A16 silencing promoted osteogenic differentiation. Further transcriptomic sequencing revealed that the Smad4 pathway was involved in S100A16 silencing-induced osteogenesis. The results of our Western blot analysis showed that S100A16 overexpression not only downregulated Smad4 but also activated MAPK/JNK signaling, which was validated by treatment with MAPK and JNK inhibitors U0126 and SP600125. Overall, this study elucidated the novel regulatory factors influencing osteogenic differentiation and provided mechanistic insights that could aid in the development of targeted therapeutic strategies for osteoporosis patients.

overall_design: To investigate the function S100A16 in the regulation of osteogenesis, we isolated primary bone marrow stem cells from rats. After transfection of S100A16-specific siRNA during osteogenic differentiation, we  performed gene expression profiling analysis using data obtained from RNA-seq of S100A16 downregulated cells or control cells.


title: Effect of Sulfopin and Abemaciclib treatment on gene expression in mouse TNBC

city: boston

first: Shizhong

last: Ke

state: Massachusetts

address: 3 BLACKFAN CIRCLE, CLS0428D

institute: Beth Israel Deaconess Medical Center (BIDMC)

summary: To investigate the role of combined PIN1 and CDK4 inhibition in the regulation of immune response, we performed RNA sequencing of TNBC tumors after treatment with Sulfopin, Abemaciclib or their combination

overall_design:
We then performedgene set enrichment analyses using data obtained from RNA-seq of different treatments.
Comparative analysis of immune response signatures in single-drug treatment and combination treatment using RNA-sequencing data.


title: A cluster-randomized trial of water, sanitation, handwashing and nutritional interventions on stress and epigenetic programming

city: Santa Cruz

first: Audrie

address: METX, 1156 High St.

department: Microbiology and Environmental Toxicology

institute: University of California, Santa Cruz

summary: A regulated stress response is essential for healthy child growth and development trajectories. We conducted a cluster-randomized trial in rural Bangladesh (funded by the Bill & Melinda Gates Foundation, ClinicalTrials.gov NCT01590095) to assess the effects of an integrated nutritional, water, sanitation, and handwashing intervention on child health. We previously reported on the primary outcomes of the trial, linear growth and caregiver-reported diarrhea. Here, we assessed additional prespecified outcomes: physiological stress response, oxidative stress, and DNA methylation (N = 759, ages 1-2 years). Eight neighboring pregnant women were grouped into a study cluster. Eight geographically adjacent clusters were block-randomized into the control or the combined nutrition, water, sanitation, and handwashing (N+WSH) intervention group (receiving nutritional counseling and lipid-based nutrient supplements, chlorinated drinking water, upgraded sanitation, and handwashing with soap). Participants and data collectors were not masked, but analyses were masked. There were 358 children (68 clusters) in the control group and 401 children (63 clusters) in the intervention group. We measured four F2-isoprostanes isomers (iPF(2α)-III; 2,3-dinor-iPF(2α)-III; iPF(2α)-VI; 8,12-iso-iPF(2α)-VI), salivary alpha-amylase and cortisol, and methylation of the glucocorticoid receptor (NR3C1) exon 1F promoter including the NGFI-A binding site. Compared with control, the N+WSH group had lower concentrations of F2-isoprostanes isomers (differences ranging from -0.16 to -0.19 log ng/mg of creatinine, P<0.01), elevated post-stressor cortisol (0.24 log µg/dl; P<0.01), higher cortisol residualized gain scores (0.06 µg/dl; P=0.023), and decreased methylation of the NGFI-A binding site (-0.04; P=0.037). The N+WSH intervention enhanced adaptive responses of the physiological stress system in early childhood.

overall_design: 745 samples were analyzed for DNA methylation; no replicates; there was a control and a Nutrition + WASH arm

first: Ann

last: Meyer

first: Liying

last: Yan

first: Sophia

last: Tan

first: Andrew

middle: N

last: Mertens


title: Effect of depletion of PIN1 on gene expression of breast cancer cells

summary: To investigate the role of PIN1 in the regulation of immune response, we established PIN1 knouckout cell lines

overall_design:
We then performedgene set enrichment analyses using data obtained from RNA-seq of 2 different cell lines.
Comparative analysis of immune response signatures in wild-type (WT) and PIN1 knockout (PIN1-KO) cells using RNA-sequencing data.


title: PGE2 effect on unstimulated and exhausted human CD8+ T-cells

city: Lausanne

first: Nicolas

last: Rayroux

address: Rue du Bugnon 25A

department: Oncology

country: Switzerland

institute: Centre Hospitalier Universitaire Vaudois

summary: Prostaglandin E2 (PGE2) is overexpressed in many tumors and plays an important role in suppressing T-cell function. In this study, we investigated the effect of PGE2 in human T-cells. Importantly, PGE2 caused profound transcriptomic changes both in unstimulated and exhausted T-cells with many genes and pathways altered including T-cell lipid metabolism.  Our study suggests that PGE2-induced metabolic reprogramming is detrimental to dysfunctional T-cells and TIL, and its manipulation could be used to dampen the immunosuppressive effects of PGE2.

overall_design: we adapted an in-vitro model of T-cell chronic activation by repetitively stimulating human peripheral blood lymphocytes (PBLs) with anti-CD3/CD28 beads (Dunsford et al., 2020) and low-dose IL2 (60IU). Following 10 days of stimulation, chronically activated T cells or unstimulated T-cells were treated with PGE2 for 24 hours. CD8+ T-cells were then sorted and RNA extracted.

first: Matteo

last: Morotti

first: Alizée

last: Grimm


title: Synovium and Infrapatellar fat pad are one functional tissue unit sharing common mesenchymal progenitors and undergoing coordinated changes in osteoarthritis

status: Public on Mar 07 2024

city: Shenyang

first: Lutian

last: Yao

address: 155 Nanjing st

department: Department of Orthopedic

institute: the First Affiliated Hospital of China Medical University

summary: The Infrapatellar fat pads (IFP) are two wedge shaped fatty structures situated below the kneecap (patella), lying either side of the patellar tendon, first described by Hoffa in 1904. Recent studies reveal that knee OA causes pathological fibrosis, stiffening, and inflammation of the synovium and IFP (4, 5) (6). Multiple groups, including our co-investigator Dr. Changhai Ding, reported that MRI signal intensity alteration in IFP is strongly associated with OA knee structural abnormalities and may serve as an imaging biomarker to predict OA progression in patients (7-10). However, we have limited knowledge regarding the regulation of synovium and IFP functions for joint homeostasis and OA development.

overall_design: cell atlas of mouse synovium cells during DMM induced OA process

first: Ling

last: Qin


title: Glucose Hypometabolism Prompts RAN Translation and Exacerbates C9orf72-related ALS/FTD Phenotypes

status: Public on Mar 06 2024

city: philadelphia

first: Davide

last: Trotti

state: Pennsylvania

address: 900 WALNUT ST, JEFFERSON HOSPITAL FOR NEUROSCIENCE BUILD

institute: PROFESSOR OF NEUROSCIENCE, THOMAS JEFFERSON UNIVERSITY

summary: To investigate the effect of glucose hypometabolism on the transcriptome of human neurons

overall_design: Comparative gene expression analysis of RNAseq data for human i3Neurons treated with either complete/normal media, glucose-deprived media, or 2-deoxyglucose-containing media. Two independent differentiations for two different i3Neurons lines (for a total n = 4) were used for each condition

last: Nelson


title: Identification of G-quadruplex-interacting Proteins in Living Cells using an artificial G4-targeting Biotin ligase

city: Wuhan

first: Ziang

last: Lu

address: Wuhan University, No.299, Bayi Road, Wuchang District

institute: Wuhan University

summary: We engineering a G4-targeted biotin ligase probe (G4PID), which consists of three parts, RHAU23 at the C-terminus, miniTurbo at the N-terminus, and a flexible linker in the middle to prevent the disturbance of the two functional moieties. We used eCLIP to  verified the  G4s  affinity of G4PID. We also used eCLIP method to verify the direct G4 interaction of Splicing factor 3B subunit 4 (SF3B4) in cellular transcriptome.

overall_design: Haiyan, Huang

first: Shengjie

last: Xie

first: Haomiao

last: Su

first: Shao

first: Xiang

last: Zhou


title: Muscle stem cell niche localization, quiescence, and maintenance are cadherin-dependent but separable functions

city: New York

first: Robert

middle: S.

last: Krauss

state: NY

address: One Gustave L. Levy Place

department: Cell, Developmental, and Regenerative Biology

institute: Icahn School of Medicine at Mount Sinai

summary: Adhesion between stem cells and their niche promotes stable niche localization and provides signaling information to sustain properties such as quiescence. Skeletal muscle stem cells (MuSCs) are localized between an adjacent myofiber and an enwrapping basal lamina. The most abundant cadherin in MuSCs, M-cadherin, is dispensable for MuSC function, whereas N-cadherin is required to prevent MuSCs from breaking quiescence in the absence of injury. Loss of both cadherins exacerbates this phenotype, yet the MuSCs remain in the niche and maintain regenerative function. These findings raise the possibility that cadherins are dispensable for anchorage of MuSCs to their niche. To address this question, we conditionally removed from MuSCs b- and g-catenin and, separately, aE- and aT-catenin, factors essential for cadherin-dependent adhesion. Catenin-deficient MuSCs break quiescence similarly to N-/M-cadherin-deficient MuSCs, but exit the niche, and are depleted via a single fate: precocious differentiation, reentry to the niche, and fusion to myofibers. These findings indicate that separable, cadherin-dependent functions are required in MuSCs for niche localization, quiescence, and stem cell maintenance.

overall_design: Skeletal muscle stem cells (MuSCs)of Ctnna1fl/fl;Ctnna3fl/fl;Pax7CreERT2/+;Rosa26-tdTomato+ and Pax7CreERT2/+;Rosa26-tdTomato+ mice were isolated by Fluorescence-activated cell sorting (FACS) according to the presence or absence of tdTomato signal and analyzed using scRNAseq.

first: Margaret

last: Hung

first: Hsiao-Fan

last: Lo

first: Aviva

last: Beckmann

first: Deniz

last: Demircioglu

first: Gargi

last: Damle

first: Dan

last: Hasson

first: Glenn

last: Radice

middle: S


title: Analysing the effect of cellular energy levels on codon-specific ribosome occupancy in vivo

city: Solna

first: Vicent

last: Pelechano

address: Nobels väg 16

department: MTC

country: Sweden

institute: ScilifeLab - Karolinska Institutet

summary: The aim of this study is to evaluate how cellular energy levels shape codon-specific decoding and affect codon-mediated mRNA degradation. To this end we made use of HT5P-seq (Zhang & Pelechano, 2021, PMID:35474692)  in S. cerevisiae to footprint the ribosome of co-translationally degraded mRNAs in vivo upon swift changes in the concentration of intracellular ATP and other energy metabolites.

overall_design: HT5P-seq was performed in 5 different time points (3 replicates each time points), spanning 5 min before and 10 min after the addition of antimycin A to respiring yeast as described in Walther T et al. (PMID: 20087341)

first: Yujie

last: Zhang

first: Pedro

last: da Silva

first: Julien

last: Gagneur


title: The germline regulates longevity and somatic repair in a sex-specific manner

city: Jerusalem

first: Itamar

last: Harel

address: Givat Ram

department: Genetics

country: Israel

institute: The Hebrew University of Jerusalem

first: Eitan

last: Moses

first: Tehila

last: Atlan

first: Xue

last: Sun

first: Roman

last: Franěk

first: Atif

last: Siddiqui

first: Georgi

middle: K

last: Marinov

first: Sagiv

last: Shifman

first: David

last: Zucker

first: Adi

last: Oron-Gottesman

first: William

middle: J

last: Greenleaf

first: Ehud

last: Cohen

first: Oren

last: Ram


title: Endocardial cells function as antigen-presenting cells to promote zebrafish heart regeneration [dst93_scrnaseq]

city: Bad Nauheim

first: Carsten

last: Kuenne

address: Ludwigstrasse 43

department: Bioinformatics

institute: Max Planck Institute for Heart and Lung Research

summary: In contrast to adult mammals, adult zebrafish are able to fully regenerate injured cardiac tissue, and this regeneration process requires an adequate and tightly controlled immune response.  However, which immune aspects drive particular aspects of the regenerative response are unclear.  Here, we report the participation and requirement of the antigen presentation-adaptive immunity axis during zebrafish cardiac regeneration.  We found that, in addition to immune cells, endocardial cells start expressing antigen presentation genes following the initial innate immune response.  Consistent with this finding, we observed that helper T cells, a.k.a. Cd4+ T cells, are closely associated with phosphoERK+ (pERK+) (i.e., activated) endocardial cells at these stages.  We inactivated major histocompatibility complex (MHC) class II antigen presentation by generating cd74a; cd74b double mutants, which display a defective immune response.  In these mutants, both Cd4+ T cells and pERK+ endocardial cells fail to efficiently infiltrate the injured tissue.  Notably, this model of compromised antigen presentation exhibits additional defects in cardiac regeneration including reduced cardiomyocyte dedifferentiation and proliferation.  Altogether, these findings reveal a necessary role for antigen presentation during zebrafish cardiac regeneration and point to an immune crosstalk between T cells and endocardial cells, thereby further establishing a link between the adaptive immune response and tissue regeneration.

overall_design: scRNA-Seq of FACS-sorted zebrafish Tg(mhc2dab:EGFP)+ and/or Tg(ptprc:DsRed)+  immune cells from uninjured ventricles and ventricles at various time points post-cryoinjury [i.e., 6, 24, 72 hours post-cryoinjury (hpci) and 7, 14 and 30 days post-cryoinjury (dpci)]. For each time point, three ventricles (from 2 females and 1 male, or 1 female and 2 males) were pooled.

first: João

last: Cardeira-da-Silva

first: Qianchen

last: Wang

first: Stephan

last: Latting

first: Stefan

last: Günther

first: Pooja

last: Sagvekar

first: Radhan

last: Ramadass

first: Michail

last: Yekelchyk

first: Janita

last: Mintcheva

first: Jens

last: Preussner

first: Mario

last: Looso

first: Jan

last: Junker

first: Didier

last: Stainie


title: Endocardial cells function as antigen-presenting cells to promote zebrafish heart regeneration

city: bad nauheim

address: ludwigtr. 43

institute: MPI for heart and lung research

overall_design: Comparative gene expression analysis between wild-type zebrafish cryoinjured ventricles (7 days post-cryoinjury) and cd74a;cd74b mutant ventricles (duplicates).


title: Neddylation orchestrates the complex transcriptional and post-transcriptional program that drives axonal myelination

city: Derio

first: Ana

middle: Maria

last: Aransay

state: BIZKAIA

address: Parque tecnologico de Bizkaia, Building 801-A

department: Genome Analysis Platform

country: Spain

institute: CIC bioGUNE

summary: Myelination is essential for neuronal function and health. In peripheral nerves, >100 causative mutations have been identified that cause Charcot-Marie-Tooth disease, a disorder that can affect myelin sheaths. Among these, a number of mutations are related to essential targets of the post-translation modification neddylation, although how these lead to myelin defects is unclear. Here, we demonstrate that inhibiting neddylation leads to a striking absence of peripheral myelin and axonal loss both in developing and regenerating mouse nerves. Importantly, our data indicate that neddylation exerts a global influence on the complex transcriptional and post-transcriptional program by simultaneously regulating the expression and function of multiple essential myelination signals, including the master transcription factor EGR2 and the negative regulators c-Jun and Sox2, and inducing global secondary changes in downstream pathways, including the mTOR and YAP/TAZ signalling pathways. This places neddylation as a critical regulator of myelination and delineates the potential pathogenic mechanisms involved in CMT mutations related to neddylation.

overall_design: RNA was isolated from 7 days old mice sciatic nerves from WT (n=4) and Nae1 cKO mice (n=4). Total RNA was treated with DNAse I (Invitrogen) for 30 min at 37 ºC. Then the samples were cleaned with Genejet RNA cleanup and Concentration kit (Thermo Scientific) (the columns were eluted with 20-30µl ultra-pure water), and samples processed for RNA sequencing.

first: Paula

last: Ayuso-García

first: Alejandro

last: Sánchez-Rueda

first: Sergio

last: Velasco-Avilés

first: Miguel

last: Tamayo-Caro

first: Cristina

last: Riobello

first: Aroa

last: Ferrer-Pinos

first: Cecilia

last: Huarte-Sebastian

first: Selene

last: Jiménez-Vega

first: Izaskun

last: Buendia

first: Jorge

last: Cañas-Martin

first: Héctor

last: Fernández-Susavila

first: Romane

last: Guhl

first: Nicolas

last: Laville

first: Encarni

last: Pérez-Andrés

first: José

last: Lavín

first: Monika

last: Gonzalez-Lopez

first: Nuria

last: Cámara

first: James

last: Sutherland

first: Rosa

last: Barrio

first: María

last: Martinez-Chantar

first: Mikel

last: Azkargorta

first: Félix

last: Elortza

last: Soriano-Navarro

first: Carlos

last: Matute

middle: V

last: Sánchez-Gómez

first: Alberto

last: Pérez-Samartín

first: Susana

middle: B

last: Bravo

first: Thimo

last: Kurz

first: Tomas

last: Lama-Díaz

last: Blanco

first: Saif

last: Haddad

first: Alexander

last: Rossor

first: Christopher

last: Record

first: Mary

last: Reilly

first: Marta

last: Varela-Rey

first: Ashwin

last: Woodhoo

first: Vanesa

last: Alvarez

first: Adrián

last: Aparicio-Rey

first: Eva


first: Carsten

last: Kuenne


title: Endocardial cells function as antigen-presenting cells to promote zebrafish heart regeneration [dst173_scrnaseq]

overall_design: scRNA-Seq of all cells from the injured and border zone tissues of wild-type and cd74a;cd74b mutant zebrafish ventricles at 120 hours post-cryoinjury. Each sample is a pool of cells from 4 ventricles (2 females and 2 males).

first: João

last: Cardeira-da-Silva

first: Qianchen

last: Wang

first: Stephan

last: Latting

first: Stefan

last: Günther

first: Pooja

last: Sagvekar

first: Radhan

last: Ramadass

first: Michail

last: Yekelchyk

first: Janita

last: Mintcheva

first: Jens

last: Preussner

first: Mario

last: Looso

first: Jan

last: Junker

first: Didier

last: Stainie


title: Modular regulation of Myc transcription by a topologically defined enhancer cluster dedicated to pluripotency and early embryonic expression [4C-seq]

city: Madrid

first: Carlos

last: Torroja

state: Madrid

address: Melchor Fernandez Almagro, 3

department: Bioinformatics Unit

institute: Centro Nacional de Investigaciones Cardiovasculares (CNIC)

summary: The transcription factor MYC plays essential roles in pluripotent stem cells, including the promotion of somatic cell reprogramming to pluripotency and the regulation of cell competition and embryonic diapause; however, how Myc expression is regulated in this context remains unknown. The Myc gene lies within a ~3-megabase gene desert that contains multiple cisregulatory elements. We used large-scale genomic deletions/inversions, BAC transgenesis and nested deletion of regulatory regions to characterize the cis-regulation of Myc transcription in the early mouse embryo and Embryonic Stem Cells (ESCs). We identified an Early Embryonic Expression (EEE) topologically-associated region that homes enhancers dedicated to Myc transcriptional regulation in stem cells of the early embryo, including the pluripotent cells of the inner cell mass, pre-implantation and post-implantation epiblast and other early embryonic stem cell populations. Within this region, we identified elements exclusively dedicated to Myc regulation in pluripotent cells, with distinct enhancers that sequentially regulate Myc ranscription during naive and formative pluripotency. Deletion of these pluripotency-specific enhancers dampens the competitive ability of ESCs. These results identify a topologically defined enhancer cluster dedicated to early embryonic expression and uncover a modular mechanism for the regulation of Myc expression in different states of pluripotency.

overall_design: Transgenic mice carrying a BAC generated by knock in of Turquoise Fluorescent Protein (TFP) to generate a TFP-MYC fussion protein in a 250kb  BAC RP24-78D24 that covers 28kb upstream of Myc TSS and 203kb downstream. The transgenic insertion has been characterize by whole-genome sequencing. And the regulatory interactions have been compared with the GFP-MYC endogenous knockin by 4C-Seq.

first: Lin

last: Bao

first: Covadonga

last: Diaz-Diaz

first: Rocio

last: Sierra

first: Francisco

last: Moya

first: Sandra

last: Rodriguez-Perales

first: Giovanna

last: Giovinazzo

first: Tore

last: Bleckwehl

first: Alvaro

last: Rada-Iglesias

first: Francois

last: Spitz

first: Miguel

last: Torres


title: Modular regulation of Myc transcription by a topologically defined enhancer cluster dedicated to pluripotency and early embryonic expression

overall_design: Refer to individual Series


title: An FXR-induced novel lncRNA, FincoR

status: Public on Mar 05 2024

city: HOUSTON

first: Ruoyu

last: Wang

state: Texas

address: 6431 Fannin St

institute: UTHealth McGovern Medical School

summary: The nuclear receptor, Farnesoid X Receptor (FXR, NR1H4), is a key transcriptional regulator of metabolism and a promising drug target for non-alcoholic fatty liver disease (NAFLD), a leading cause of liver failure and death. Protein coding genes regulated by FXR are known, but it remains unknown whether FXR mediates its function through regulating expression of long non-coding RNA (lncRNA) genes. Utilizing global RNA-seq and Gro-seq analyses, we identify an FXR-induced novel long non-coding RNA (lncRNA), termed FincoR.

overall_design: Transcriptome analyses and nascent transcriptiome profiing in both FXR agnosit treated and untreated mice livers

first: Wenbo

last: Li


title: Deletion of FNDC5/Irisin modifies murine osteocyte function in a sex-specific manner

city: Indianapolis

first: Anika

last: Shimonty

state: Indiana

address: 635 Barnhill Dr

department: Indiana Center for Musculoskeletal Health

institute: Indiana University

summary: Irisin, released from exercised muscle, has been shown to have beneficial effects on numerous tissues but its effects on bone are unclear. We found significant sex and genotype differences in bone from wildtype (WT) mice compared to mice lacking Fndc5 (KO), with and without calcium deficiency. Despite their bone being 2 indistinguishable from WT females, KO female mice were partially protected from osteocytic osteolysis and osteoclastic bone resorption when allowed to lactate or when placed on a low-calcium diet. Male KO mice have more but weaker bone compared to WT males, and when challenged with a low-calcium diet lost more bone than WT males. To begin to understand responsible molecular mechanisms, osteocyte transcriptomics was performed. Osteocytes from WT females had greater expression of genes associated with osteocytic osteolysis and osteoclastic bone resorption compared to WT males which had greater expression of genes associated with steroid and fatty acid metabolism. Few differences were observed between female KO and WT osteocytes, but with a low calcium diet, the KO females had lower expression of genes responsible for osteocytic osteolysis and osteoclastic resorption than the WT females. Male KO osteocytes had lower expression of genes associated with steroid and fatty acid metabolism, but higher expression of genes associated with bone resorption compared to male WT. In conclusion, irisin plays a critical role in the development of the male but not the female skeleton and protects male but not female bone from calcium deficiency. We propose irisin ensures the survival of offspring by targeting the osteocyte to provide calcium in lactating females, a novel function for this myokine.

overall_design: 5-month-old mice long bone osteocyte total RNA was analyzed. The groups are: WT female control, WT female low-calcium-diet, KO female control, KO female low-calcium-diet, WT male control, WT male low-calcium-diet, KO male control, KO male low-calcium-diet. There are 2/3 samples per group.

first: Fabrizio

last: Pin

first: Matt

last: Prideaux

first: Gang

last: Peng

first: Joshua

last: Huot

first: Hyeonwoo

last: Kim

first: Clifford

last: Rosen

first: Bruce

middle: M

last: Spiegelman

first: Lynda

middle: F

last: Bonewald


title: EZH2 gain-of-function mutations distort  H3K27me3 landscapes and transform transcriptional responses to PRC2 inhibition.

city: Paris

first: Laia

last: Richart

address: 13 Rue Pierre et Marie Curie

department: Developmental Biology

country: France

institute: Institut Curie

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


title: Modular regulation of Myc transcription by a topologically defined enhancer cluster dedicated to pluripotency and early embryonic expression [WGS]

first: Carlos

last: Torroja

first: Lin

last: Bao

first: Covadonga

last: Diaz-Diaz

first: Rocio

last: Sierra

first: Francisco

last: Moya

first: Sandra

last: Rodriguez-Perales

first: Giovanna

last: Giovinazzo


title: Airway Hillocks are Injury-Resistant Reservoirs of Plastic Stem Cells

city: Boston

first: Brian

last: Lin

state: MA

address: 136 Harrison Avenue

department: DMCB

institute: Tufts University

summary: Airway hillocks are newly described discrete epithelial structures of unknown function that are distinct from the classic pseudostratified epithelium lining the airway lumen. We show that hillocks are islands of stratified epithelia harboring migratory stem cells that are capable of extensive clonal expansion. Hillocks resist a broad range of injuries, preserving a basal stem cell population that ultimately resurfaces the airway lining. The fully resurfaced hillock epithelium then exhibits plasticity and converts into a normal pseudostratified epithelium indistinguishable from its healthy, uninjured counterpart.

overall_design: We performed plate-based SMART-Seq2 on hillock vs pseudostratified epithelium that was microdissected from a p63-CreER;LSL-TdTomato mouse

first: Jayaraj

last: Rajagopal


title: Systematic identification and characterization of Exon-intron circRNAs

status: Public on Mar 04 2024

city: Hefei

first: Yan

last: Yang

state: Anhui

address: No.373 Huangshan Road

institute: University of Science and Technology of China

summary: Exon-intron circRNA (EIciRNA) is a subclass of backsplicing-generated circRNAs featured with intron retention, among which some play roles in transcriptional regulation. We aim to characterize EIciRNAs by developing pipeline and investigate the factors involved in regulating EIciRNA biogenesis using CRISPR-Cas9 screen, as well as the physiology functions of EIciRNAs during neuronal differentiation.

overall_design: To detect EIciRNAs and intron retention in the human transcriptome, we performed both RNase R-treated and poly(A)+ RNA-seq in HEK293 cells. To investigate the regulation of SRSF1 on EIciRNA biogenesis, SRSF1 knockdown cell line was established and both the two types of RNA-seq were performed. To investigate the functional roles of EIciRNAs during neuronal differentiation, differentiated neuronal cell line was established by inducing SH-SY5Y cells using Retinoic acid (RA).

first: Yinchun

last: Zhong

first: Ge

last: Shan

first: Liang

last: Chen


title: EZH2 gain-of-function mutations distort  H3K27me3 landscapes and transform transcriptional responses to PRC2 inhibition [ChIP-Seq/Cut&Run-Seq]

summary:
We have modelled several tumor-associated PRC2 mutations in a single isogenic system in order to reveal their comparative impacts on chromatin and transcription.
We have also explored the link between EZH2 mutational status and abnormal H3K27 methylation in a unique longitudinal cohort of follicular lymphoma (FL) patient samples.

overall_design: Differential analyses assessing the impact of PRC2 mutations on H3K27me3 genome-wide binding patterns in MEFs and follicular lymphoma samples.

first: Pierre

last: Romero

first: Michel

last: Wassef

first: Daniel

last: Holoch

first: Raphael

last: Margueron


title: EZH2 gain-of-function mutations distort  H3K27me3 landscapes and transform transcriptional responses to PRC2 inhibition [RNA-seq]

first: Laia

last: Richart

overall_design:
Differential analysis comparing gene expression profiles of WT vs PRC2 mutant immortalized mouse embryonic fibroblasts (iMEFs). The panel of PRC2 mutations encompasses: Ezh2-KO, Eed-KO, H3.3K27M, and the gain-of-function Y641F Ezh2 mutant (Ezh2Y641F).
RNA-Seq was also used to profile gene expression changes in WT and Ezh2Y641F iMEFs treated with two doses of the EZH2 inhibitor UNC1999.
Gene expression differential analysis between WT and EZH2 mutant FL samples, as measured by RNA-Seq.


title: Systematic identification and characterization of Exon-intron circRNAs in the human transcriptome


title: Systematic identification and characterization of Exon-intron circRNAs (RNA-Seq)


title: Systematic identification and characterization of Exon-intron circRNAs in the human transcriptome (CRISPR)

overall_design: To screen for potential regulators of EIciRNA biogenesis, two EIciGFP reporter cell lines were established. We performed genome-wide CRISPR knock-out screen using these two reporter cell lines.


title: Mettl16 promotes hematopoietic stem and progenitor cell expansion in embryonic hematopoiesis [RNA-seq]

first: Yunqiao

last: Han

address: Luoyu Road 1037

institute: Huazhong University of Science and Technology

summary: The RNA N6-methyladenosine (m6A) modification has emerged as an essential regulator of vertebrate embryogenesis and malignancies. However, its functions and underlying molecular mechanisms in the expansion of hematopoietic stem and progenitor cells (HSPCs) during early embryonic development remain elusive. Here we show that Mettl16, an RNA methyltransferase identified recently, is specifically required for HSPC expansion during zebrafish early embryonic development in an m6A-dependent manner. In mettl16 deficient embryos, HSPCs exhibit defective proliferation capacity due to G0/G1 arrest. Mechanistically, HSPC proliferation is blocked by impaired methyltransferase function of Mettl16 in vivo. We identify cell cycle gene mybl2b as a novel direct m6A target of Mettl16, and Mettl16 deficiency destabilizes mybl2b mRNA, which is mediated by m6A reader Igf2bp1. Moreover, we revealed that the METTL16-m6A-MYBL2-IGF2BP1 signaling axis in G1/S progression is conserved in humans. Collectively, our findings demonstrate the critical function of m6A modification deposited by Mettl16 in HSPC expansion during early embryonic development.

overall_design: RNA-seq profiles of sibling and  mettl16-/- zebrafish embryos at 3 days post fertilization for triplicates

first: Kui

last: Sun

first: Shanshan

last: Yu

first: Mugen

last: Liu


title: COVID-19 vaccination is associated with attenuation of inflammatory responses during breakthrough disease [long-read RNA-Seq]

city: Oxford

first: Irina

last: Chelysheva

address: Roosevelt Dr

department: NDM

institute: University of Oxford

summary: A subset of individualsenrolledina randomised controlled trial ofChAdOx1 nCoV-19 and received either ChAdOx1 COVID-19 or control MenACWY vaccine whoexperienced COVID-19-associated symptoms were evaluated using multi-omictechnologies. Participants were selected to represent symptomatic individuals with and without COVID-19 as determined by a nucleic acid amplification test (NAAT).

overall_design: Blood samples taken on theday of vaccination were compared with samples taken at onset of COVID-19 associated symptoms.Long-read Nanopore PCR-cDNA sequencing technology was utilised.

first: Ruth

last: Drury

last: O'Connor

first: Susana

last: Camara


title: H2A.Z is required to initiate DNA repair to prevent premature aging

city: ILLKIRCH-GRAFFENSTADEN

first: Christophe

last: Papin

address: 1 rue Laurent Fries

institute: IGBMC - CNRS UMR 7104 - Inserm U 964

summary: Histone variants are key epigenetic players, but their functional and physiological roles remain poorly understood. Here, we show that depletion of the histone variant H2A.Z in mouse skeletal muscle causes oxidative stress, oxidation of proteins, accumulation of DNA damages, and both neuromuscular junction and mitochondria lesions that consequently lead to premature muscle aging and reduced life span. Investigation of the molecular mechanisms involved shows that H2A.Z is required to initiate DNA double strand break repair by recruiting Ku80 at DNA lesions. This is achieved via specific interactions of Ku80 vWA domain with H2A.Z. Taken as a whole, our data reveal that H2A.Z containing nucleosomes act as a molecular platform to bring together the proteins required to initiate and process DNA double strand break repair.

overall_design: To adress the effect of H2A.Z on transcription, we have used skeletal muscles isolated from WT and H2A.Z null 12 months-old animals

first: Edwige

last: Belotti

first: Nicolas

last: Lacoste

first: Arslan

last: Iftikhar

first: Thomas

last: Simonet

first: Nathalie

last: Streichenberger

first: Pierre-Olivier

last: Mari

first: Emmanuelle

last: Girard

first: Alexis

last: Osseni

first: Guiseppina

last: Giglia-Mari

first: Stefan

last: Dimitrov

first: Ali

last: Hamiche

first: Laurent

last: Schaeffer


title: COVID-19 vaccination is associated with attenuation of inflammatory responses during breakthrough disease [Illumina RNA-Seq]

last: Chelysheva

summary: A subset of individualsenrolledina randomised controlled trial ofChAdOx1 nCoV-19 and received either ChAdOx1 COVID-19 or control MenACWY vaccine whoexperienced COVID-19-associated symptoms were evaluated using multi-omictechnologies. Participants were selected to represent symptomatic individuals with and without COVID-19 as determined by a nucleic acid amplification test (NAAT) for stage 1 and only NAAT+ve for stage 2 of the study.

overall_design: Blood samples taken on theday of vaccination were compared with samples taken at onset of COVID-19 associated symptoms and 7 days later.Short-read RNA Illumina sequencing technology was utilised.

first: Daniel


title: Single cell RNA sequencing and single cell TCR sequencing of Type 2 lymphocytes in human nasal polyps

status: Public on Mar 01 2024

first: Patrick

last: Brennan

address: 75 Francis St

institute: Brigham & Women's Hospital

summary: We explored the heterogeneity of lymphocytes in primary human tissues with type 2 inflammation through single cell RNA sequencing. TCRs were sequenced through single cell TCR sequencing and used as barcodes to probe lineage relationships. Comparisons of transcriptional profiles were also made with  CD8+ T cells and non-type 2 lymphocytes.

overall_design:
We performed scRNA-Seq of CD4+ T cells, CD8+ T cells, and innate lymphoid cells (ILC) from human nasal polyps to define the heterogeneity of type 2 lymphocytes in diseases characterized by type 2 inflammation. We FACS sorted several subsets including: CD4+CD8-CCR4+CXCR3-CRTh2+/-CD45+ T cells; CD4-CD8+CD45+ T cells; CD8+CD4-CD45+ T cells; and ILCs (CD45+lineage-CD127+). For donors 1-3, paired samples from peripheral blood were obtained and CD45+CD4+CCR4+CRTh2+/- cells were sorted. Blood cells were hash labeled with Biolegend CD45 antibody (clone 2D1) to differentiate from polyp cells. All cells were then labeled with Biolegend CITESeq antibodies (TotalSeq C kit). For donors 4-7, cells were hash labeled with Biolegend hashtag antibodies. Cells were then loaded on the droplet-based Chromium system (10X Genomics). Single cell gene expression and TCR libraries were prepared using the 5’ 10X Genomics kit.

***Submitter declares that raw sequencing files will be submitted to dbGap due to privacy concerns.***

first: Radomir

last: Kratchmarov


title: Formation of Memory Assemblies through the DNA Sensing Tlr9 Pathway

city: Bronx

first: Jelena

last: Radulovic

address: 1410 Pelham Parkway South

department: Neuroscience

institute: Albert Einstein College of Medicine

summary: As hippocampal neurons respond to diverse types of information a subset assembles into stable microcircuits representing a memory. Those neurons typically undergo energy-intensive molecular adaptations, which occasionally results in transient DNA damage. We found, however, discrete clusters of excitatory hippocampal CA1 neurons showing persistent dsDNA breaks, nuclear envelope ruptures, and perinuclear release of histone and dsDNA fragments for hours after learning. Following these early events, some neurons acquired an inflammatory phenotype involving activation of Toll-like receptor 9 (Tlr9) signaling and accumulation of centrosomal DNA damage repair (DDR) complexes. Neuron-specific knockdown of Tlr9 impaired memory while blunting CFC-induced changes of gene expression in specific clusters of excitatory CA1 neurons. Most notably, Tlr9 played an essential role in centrosome function, including DDR, ciliogenesis, and build-up of perineuronal nets (PNN). We demonstrate a novel cascade of learning-induced molecular events in discrete neuronal clusters undergoing dsDNA damage and Tlr9-mediated repair, resulting in their recruitment to memory circuits. With compromised Tlr9 function, this fundamental memory mechanism becomes a gateway to genomic instability and cognitive impairments, which have been implicated in accelerated senescence, psychiatric disorders, and neurodegenerative disorders. Maintaining the integrity of Tlr9 inflammatory signaling thus emerges as a promising preventive strategy for neurocognitive deficits.

overall_design:
To investigate the effect of contextual fear conditioning (CFC) and the role of Tlr9 signaling in CFC, we knocked out Tlr9 in neurons of the dorsal hippocampus  (Tlr9 KO) and performed snRNA-seq to determine cell type specifc effects of CFC, Tlr9 KO, or both.
To determine gene expression changes following fear conditioning, we compared mice that underwent CFC to naive mice (home cage controls). To determine Tlr9 driven gene expression changes, we compared fear conditioned Tlr9 KO mice to control mice (viral injection lacking Cre). Lastly, to ensure expression changes were due to Tlr9 KO and CFC rather than the KO alone, we compared naive Tlr9 KO mice to naive control mice.

first: Wood

last: Elizabeth

first: Xusheng

last: Zhang

first: Parker

last: Kendra

first: Reynolds

last: David

first: Radulovic

last: Jelena


title: Mettl16 promotes hematopoietic stem and progenitor cell expansion in embryonic hematopoiesis [m6A-Seq]

first: Yunqiao

last: Han

overall_design: m6A-seq profiles of sibling and  mettl16-/- zebrafish embryos at 3 days post fertilization for triplicates

first: Kui

last: Sun

first: Shanshan

last: Yu

first: Mugen

last: Liu


title: COVID-19 vaccination is associated with attenuation of inflammatory responses during breakthrough disease [small RNA-Seq]

first: Irina

type: Non-coding RNA profiling by high throughput sequencing

overall_design: Blood samples taken on theday of vaccination were compared with samples taken at onset of COVID-19 associated symptoms and 7 days later.Small RNA sequencing technology was utilised.

first: Ruth

last: Drury

last: O'Connor

first: Susana

last: Camara


title: Mettl16 promotes hematopoietic stem and progenitor cell expansion in embryonic hematopoiesis

status: Public on Mar 03 2024


title: COVID-19 vaccination is associated with attenuation of inflammatory responses during breakthrough disease