title: RNA-sequencing of cutaneous melanoma and non-small cell lung cancer biopsies

city: Montréal

first: Claude

last: Perreault

state: Québec

address: 2950, Chemin de Polytechnique

institute: Institute for Research in Immunology and Cancer

summary: We used a proteogenomic approach combining RNA-sequencing and mass spectrometry to study the MHC I-immunopeptidome of cutaneous melanoma and non-small cell lung cancer (NSCLC) samples. The RNA-sequencing of each sample was used to construct sample-specific databases for the mass spectrometry-based identification of MHC I-associated peptides (MAPs). MAPs were then filtered based on their RNA expression in the respective cancer types from The Cancer Genome Atlas (TCGA-SKCM for melanoma MAPs, or TCGA-LUSC and TCGA-LUAD for NSCLC MAPs) vs. benign tissues (from the Genotype-Tissue Expression (GTEx) Project, medullary thymic epithelial cells, purified blood and bone marrow cells, and normal melanocytes for melanoma or bronchial brushing samples for NSCLC). MAPs were classified as mutated tumor-specific antigens (mTSAs, derived from non-synonymous mutations expressed in the sample of origin), or unmutated tumor antigens: aberrantly expressed tumor-specific antigens (aeTSAs, no/low expression in benign tissues and at least two times higher expression in TCGA), tumor-associated antigens (TAAs, significant expression in benign tissues and at least two times higher expression in TCGA), lineage-associated antigens (LSAs, specific expression to cancer and normal tissue of origin, i.e., lung and bronchial brushing samples for NSCLC or skin and melanocytes for melanoma). The tumor antigens described here represent attractive targets for immunotherapy of melanoma and NSCLC.

overall_design: The 12 flash-frozen cutaneous melanoma specimens used in this study were purchased from Tissue Solutions, whereas the 26 flash-frozen NSCLC samples were purchased from Tissue Solution, BioIVT or Reprocell. Between 30 mg and 70 mg were used for RNA-seq. Total RNA extraction was done using TRIzol (Invitrogen) and further purification with the RNeasy Micro Kit (QIAGEN) for all primary tumor samples. The RNA quantification was performed using a QuBIT (Life Technologies), and the RNA quality was assessed using a Bioanalyzer Nano (Agilent). cDNA library preparation was done using the KAPA Hyperprep RNAseq stranded kit (KAPA) with polyA capture. The amount of RNA used for cDNA library preparation, and the number of PCR cycles used for library amplification can be found in Supplementary Table 4. Libraries were quantified by QuBit, and average library length was evaluated with the BioAnalyzer DNA1000. All libraries were diluted to 10 nM and normalized by qPCR using the KAPA library quantification kit (KAPA). Libraries were pooled to equimolar concentration. Sequencing was performed with the Illumina HiSeq2000 or the Illumina Nextseq500 using PE100 or PE75 sequencing.

first: Anca

last: Apavaloaei


title: Global transcriptional profiles of mouse intestinal organoids treated with IFN-g or/and MEK inhibitor [RNA-Seq]

status: Public on Feb 06 2025

city: Kyoto

first: Takuya

last: Yamamoto

address: 53 Kawahara-cho, Shogoin, Sakyo-ku

department: CiRA

country: Japan

institute: Kyoto University

type: Expression profiling by high throughput sequencing

summary: In this study, we found that it was possible to reflect age-associated changes of intestinal epithelium using the intestinal organoid system by modulating the balance between the IFN-g signaling and ERK/MAPK signaling pathways. To investigate the molecular mechanisms underlying the crosstalk between IFN-g and ERK/MAPK signaling pathways, we performed bulk RNA-seq on intestinal organoids treated with IFN-g and/or MEK inhibitor (iMEK).

overall_design: Mouse intestinal organoids were treated with IFN-g and/or iMEK for 1, 6, and 24 hours. RNA was isolated with and RNeasy micro kit (Qiagen). The RNAseq libraries were generated using the  Illumina Stranded mRNA Prep, Ligation Kit (Illumina). Sequencing was performed on an Illumina NextSeq 2000 platform with the paired-end mode.

first: May

last: Nakajima-Koyama


title: POISONING OF HEALTHY HEMATOPOIESIS IS AN UNANTICIPATED MECHANISM DRIVING CLONAL DOMINANCE IN VEXAS SYNDROME [scRNA-seq]

city: Segrate

first: Roberta

last: Alfieri

state: MI

address: Via Fratelli Cervi 93

department: ITB

country: Italy

institute: CNR

summary: Clonal dominance characterizes hematopoiesis during aging and increases susceptibility to blood cancers and common non-malignant disorders. VEXAS syndrome is a recently discovered adult-onset autoinflammatory disease burdened by a high mortality rate and caused by dominant hematopoietic clones bearing somatic mutations in the UBA1 gene. However, pathogenic mechanisms fueling clonal dominance are unknown. Moreover, the lack of disease models hampers the development of disease-modifying therapies. Here, we performed immunophenotypic dissection of hematopoiesis and single-cell transcriptomics in a VEXAS patient cohort revealing pervasive inflammation across all lineages. Hematopoietic stem/progenitor cells (HSPCs) in VEXAS patients proliferate less, are mostly committed toward myelopoiesis, and prone to egress from the bone marrow. Humanized xenograft models of VEXAS syndrome, generated by inserting the causative mutation in healthy HSPCs through base editing, fully recapitulated patients’ hematologic and inflammatory hallmarks. Competitive transplants of VEXAS-like and normal HSPCs showed that while mutant cells are resilient to the inflammatory milieu, wild-type ones are poisoned and progressively overwhelmed by VEXAS clones, becoming unable to support functional multilineage hematopoiesis. Our study unveils an unanticipated mechanism of clonal dominance, provides new models for preclinical investigation of therapeutic strategies, and has relevant implications for clinical management of VEXAS patients.

sample_organism:
Homo sapiens
Mus musculus

platform_organism:
Homo sapiens
Mus musculus

overall_design: scRNA-seq libraries were sequenced on an Illumina NovaSeq6000 with the standard sequencing protocol of R1 28; I1 10; I2 10; R2 90 nt read length. The dataset is composed by 14 samples: 2 samples from human BM cells from UBA1mut and UBA1wt mice harvested 14 weeks post transplantation; 2 samples from sorted human CD34+ HSPCs from the BM of UBA1mut and UBA1wt hematochimeric mice harvested at 14 weeks; 2 samples from murine lineage-negative (Lin-) cells from hematochimeric mice reconstituted by UBA1mut or UBA1wt human cells; 6 samples from BM mononuclear cells, enriched in the CD34+ HSPC fraction, from six patients (PT1, PT2, PT3, PT5, PT8, PT9); 1 samples from human BM cells (enriched for CD34+ HSPC) from VEXAS mice harvested 6 weeks post transplantation; 1 samples from human BM cells (enriched for CD34+ HSPC) from WT mice harvested 6 weeks post transplantation. Both sample are composed of female and male cells. Specifically mice were transplanted with female UBA1wt HSPCs in competition with either male UBA1mut or male UBA1wt HSPCs to distinguish mutant and wildtype cells. Thus, each sample is devided in two during the analysis by probing for XIST gene expression.

first: Raffaella

last: Molteni

first: Martina

last: Fiumara

first: Samuele

last: Ferrari

first: Ivan

last: Merelli


title: POISONING OF HEALTHY HEMATOPOIESIS IS AN UNANTICIPATED MECHANISM DRIVING CLONAL DOMINANCE IN VEXAS SYNDROME [RNA-seq]

overall_design: Whole transcriptomic analyses were performed on edited HSPCs from a pool of male healthy donors (24 hrs or 7 days after editing) and on monocytes from VEXAS patients. All conditions were performed in technical replicates.


title: Comparison of RNA quality in fresh and RAID-fixed cells with drug-induced replication stress

city: Utrecht

first: Frank

last: Riemers

address: Uppsalalaan 8

country: Netherlands

institute: Utrecht University

summary: The underlying mechanisms that influence cancer cells'  sensitivity to replication stress are impeded by the analysis of bulk-samples which neglect tumor heterogeneity and fail to accurately interpret cell cycle mediated resistance. Here, by combining intracellular immunostaining and RNA-sequencing of single cells, we characterized the transcriptomes of subsets of cells with oncogenic RAS that exhibit low levels of RS even when challenged with a CHK1 inhibitor and gemcitabine.

overall_design: All samples consist of human RPE cell lines harboring the Tet Repressor, FUCCI4 system, fluorescently tagged 53BP1 and HRASG12V, which are described in Segeren et al 2022. Cells with doxycycline-inducible oncogenic HRASG12V were treated with replication stress inducing drugs, gemcitabine + prexasertib (referred to as GP). In this experiment, cells were not treated with doxycycline, and thus have no expression of HRASG12V. Cells in S and G2 phase were selected based on expression of the FUCCI marker Geminin1-110. Cells were either freshly sorted prior to scRNA sequencing (fresh) or they were reversibly fixed, intracelularly stained with yH2AX, and decrosslinked prior to scRNA sequencing (RAID). Fresh cells and RAID-fixed cells were each sorted into 384-well plates, with half of each plate containing veh-treated cells and the other half containing GP-treated cells. After sorting, scRNA sequencing was performed to correlate the degree of yH2AX staining to transcriptional profiles of individual cells.

first: Hendrika

middle: A

last: Segeren

first: Kathryn

last: Wierenga

first: Bart

last: Westendorp

middle: M


title: Global transcriptional profiles of intestinal epithelial cells during aging [scRNA-seq]

summary: We report aging-induced transcriptional alteration of mouse intestinal epithelial cells. Our analyses revealed that age-related changes occur in a cell-type-specific manner.

overall_design: Intestinal epithelial cells were isolated from three young (3-month-old, all male) mice and three aged (22- to 26-month-old, all male) mice. After dissociating the intestinal epithelium into single cells, the libraries were generated using Chromium Next GEM Single Cell 3ʹ Kit v3.1 (10X Genomics) from randomly captured intestinal epithelial cells and sequenced using Illumina NextSeq500. We profiled 10,844 cells (5,135 and 5,709 cells from young mice and aged mice, respectively).

first: Mio

last: Kabata


title: Specific and efficient RNA A-to-I editing through cleavage of an ADAR inhibitor

city: Shanghai

first: Li

last: Yang

address: 131 Dong-An Road

department: Institutes of Biological Sciences

country: China

institute: Fudan University

summary: RNA editing can be a promising therapeutic approach. However, ectopic expression of RNA editing enzymes was found to trigger off-target editing, and the recruitment of endogenous adenosine deaminase acting on RNA (ADAR) suffers from low efficiency and fluctuating ADAR expression. Here, we identified ADAR inhibitors (ADIs) that suppressed the activity of the fused ADAR2 deaminase domain (ADAR2DD). Using ADI, we developed an RNA transformer adenine base editor (RtABE) with both high specificity and high efficiency. With the fusion of ADI to ADAR2DD, RtABE remains inactive until it binds its target site. After binding to the target site, ADI is cleaved from ADAR2DD and RtABE becomes active. RtABE induced efficient on-target editing in various cells with different ADAR expression levels. Delivering RtABE via an adeno-associated virus enabled up to a 45% RNA correction rate in Hurler syndrome mice with no significant off-target editing, and -L-iduronidase activity was restored. RtABE is a highly specific and efficient RNA editing system with broad applicability.

overall_design: Total RNAs from 293FT cells and mouse livers tissues are extracted and sequenced for off-target analysis.

first: Jia

last: Chen

first: Guangye

last: Li

first: Guo

first: Guo-Hua

last: Yuan


title: Exploration of tumor associated macrophages in patients with metastatic uveal melanoma by single cell RNAseq

status: Public on Feb 05 2025

city: Barcelona

first: Paula

last: Nieto

state: Spain

address: Barcelona Science Park, Carrer de Baldiri Reixac, 4, tower I, 08028 Barcelona

department: CNAG

country: Spain

institute: CNAG

summary: Pre- and post-treatment tumor biopsies from 8 patient with metastatic uveal melanoma patients were analysed by single cell RNAseq. Transcriptomic profiling of myeloid populations including macrophages, monocytes, and dendritic cells was carried out. M2 macrophage subsets were identified at baseline and treatment-induced M2 to M1 macrophage reprogramming was characterised.

overall_design: Single-cell RNA uveal melanoma metastases pre-treatment with one patient having also a post-treatment sample sequenced.

first: Esra

last: Güç

first: Holger

last: Heyn

first: Josep

last: Piulats

first: Adel

last: Benlahrech


title: Phagosomal RNA sensing through TLR8 controls susceptibility to tuberculosis

city: Zaragoza

first: Joaquin

last: Sanz

state: Zaragoza

address: Mariano Esquillor

department: Institute for Biocomputation & Physics of Complex Systems BIFI

institute: University of Zaragoza

summary: Genetic determinants of susceptibility to Mycobacterium tuberculosis (Mtb) remain poorly understood but could provide insights into critical pathways involved in infection, informing host-directed therapies and enabling risk stratification at individual and population levels. Through a genome-wide forward genetic screen, we identify the Toll-like Receptor 8 (TLR8), as a key regulator of intracellular killing of Mtb. Pharmacological TLR8 activation enhances killing of phylogenetically diverse clinical isolates of drug-susceptible and multidrug-resistant Mtb by macrophages and during in vivo infection in mice. TLR8 is activated by phagosomal mycobacterial RNA released by extracellular membrane vesicles, and enhances xenophagy-dependent Mtb killing. We find that the TLR8 variant, M1V, common in far eastern populations, enhances intracellular killing of Mtb through preferential signal-dependent trafficking to phagosomes. TLR8 signalling may therefore both regulate susceptibility to tuberculosis and provide novel drug targets.

overall_design: RNA extracted from Mycobacterium tuberculosis-derived extracellular vesicles was sequenced to investigate bacterial RNA content and its potential role in TLR8 activation within macrophages.

first: Charlotte

last: Maserumule

last: Passemar

first: Olivia

last: S.H. Oh

first: Kriztina

last: Hegyi

first: Karen

last: Brown

first: Aaron

last: Weimann

first: Adam

last: Dinan

first: Sonia

last: Davila

first: Catherine

last: Klapholz

first: Josephine

last: Bryant

first: Deepshikha

last: Verma

first: Jacob

last: Gadwa

first: Shivankari

last: Krishnananthasivam

first: Kridakorn

last: Vongtongsalee

first: Edward

last: Kendall

first: Andres

last: Trelles

first: Martin

last: Hibberd

first: Joaquín

first: Jorge

last: Bertol

first: Kaliappan

last: Andi

first: Siva

last: Kumar

first: Diane

last: Ordway

first: Rafael

last: Prados-Rosales

first: Paul

last: MacAry

last: Floto


title: Global transcriptional profiles of intestinal epithelial cells during aging

first: Takuya

last: Yamamoto

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

overall_design: Refer to individual Series


title: Metformin reduces the competitive advantage of Dnmt3aR878H HSPCs

status: Public on Feb 07 2025

city: Toronto

first: Mohsen

last: Hosseini

state: Ontario

address: 101 College Street

country: Canada

institute: Princess Margaret Cancer Centre

type: Methylation profiling by high throughput sequencing

first: Steven

last: Chan

first: Veronique

last: Voisin

first: Ali

last: Chegini


title: Gli2 Facilitates Tumor Immune Evasion and Immunotherapeutic Resistance by Coordinating Wnt Ligand and Prostaglandin Signaling [Cell_Line_RNAseq]

city: Durham

first: Nicholas

last: DeVito

state: NC

address: 308 Research Drive

country: USA

institute: Duke University

summary: Therapeutic resistance to immune checkpoint blockade has been commonly linked to the process of mesenchymal transformation (MT) and remains a prevalent obstacle across many cancer types. An improved mechanistic understanding for MT-mediated immune evasion promises to lead to more effective combination therapeutic regimens. Herein, we identify the Hedgehog transcription factor, Gli2, as a key node of tumor-mediated immune evasion and immunotherapy resistance during MT.  Mechanistic studies reveal that Gli2 generates an immunotolerant tumor microenvironment through the upregulation of Wnt ligand production and increased prostaglandin synthesis. This pathway drives the recruitment, viability, and function of granulocytic myeloid-derived suppressor cells (PMN-MDSCs) while also impairing type I conventional dendritic cell, CD8+ T cell, and NK cell functionality.  Pharmacologic EP2/EP4 prostaglandin receptor inhibition and Wnt ligand inhibition each reverses a subset of these effects, while preventing primary and adaptive resistance to anti-PD-1 immunotherapy, respectively.   A transcriptional Gli2 signature correlates with resistance to anti-PD-1 immunotherapy in stage IV melanoma patients, providing a translational roadmap to direct combination immunotherapeutics in the clinic.

overall_design: BRAFV600EPTEN-/- melanoma cell lines with either an active Gli2 mutation or a non-targeted control, and Gli2 CRISPR cas9 KO or its nontargeted control

first: Brent

last: Hanks


title: Gli2 Facilitates Tumor Immune Evasion and Immunotherapeutic Resistance by Coordinating Wnt Ligand and Prostaglandin Synthesis [scRNAseq]

summary: Therapeutic resistance to immune checkpoint blockade remains a prevalent obstacle across many cancer types, and an improved mechanistic understanding of this process could lead to effective combination treatments. Herein, we identify the Hedgehog transcription factor Gli2 as a key node of tumor-mediated immune evasion during mesenchymal transition via the production of Wnt ligands and prostaglandins. This promotes an immune microenvironment rich in PMN-MDSCs, while lacking antigen-presenting cDC1s and effector cells. Gli2-active tumors resist anti-PD-1 treatment, this can be overcome with inhibition of Wnt ligand secretion and selective prostaglandin receptor blockade. The identification of a Gli2 signature in patient samples provides a translational roadmap to direct precision combination immunotherapeutics in the clinic.

overall_design: C57Bl6 mice with control (NTC) or Gli2 active (Gli2CA) BRAF V600E PTEN -/- tumors treated for ~14 days with IgG control; Gli2CA tumors treated with TPST-1495 (selective prostaglandin receptor antagonist) or ETC-159 (PORCN inhibitor)

first: Michael

last: Aksu


title: Cortex and Hypothalamus Gene Expression in bred High Responder (bHR) vs. bred Low Responder (bLR) Rats: Affymetrix Microarray Data from Generation F4

city: Ann Arbor

first: Megan

middle: Hastings

last: Hagenauer

state: Michigan

address: 109 Zina Pitcher Pl.

department: Michigan Neuroscience Institute (MNI)

institute: University of Michigan

summary: The strong pattern of comorbidity amongst psychiatric disorders is believed to be generated by a spectrum of latent liability, arising from a complex interplay of genetic risk and environmental factors, such as stress and childhood adversity. At one end of this spectrum are internalizing disorders, which are associated with neuroticism, anxiety, and depression. At the other end of the spectrum are externalizing disorders, which are associated with risk-taking and novelty-seeking, as seen in mania, substance abuse, and impulse-control disorders. We model the genetic contributions underlying both extremes of this spectrum by selectively breeding rats that react differently to a novel environment. “Bred high responder” (bHR) rats are highly exploratory with a disinhibited, novelty-seeking temperament, including hyperactivity, aggression, and drug-seeking. “Bred low responder” (bLR) rats are highly-inhibited, exhibiting reduced locomotor activity and anxious and depressive-like behavior. These behavioral propensities are robust and stable, beginning early in development similar to temperament in humans.   This Affymetrix (Rat Expression Set 230 A) microarray study examined gene expression in the hippocampus, cortex, and hypothalamus in generation F4 adult male bHR rats and bLR rats (n=6 per group). The hippocampal data was previously analyzed and uploaded as GSE140596, whereas the cortex and hypothalamus data are included in this GEO record.

overall_design: Overall Design: This microarray study examined gene expression in the hippocampus, cortex, and hypothalamus in generation F4 adult male bHR rats and bLR rats (n=6 per group). The hippocampal data was previously analyzed and uploaded as GSE140596, whereas the cortex and hypothalamus data are included in this GEO record.  Behavioral testing: Locomotor response to a novel environment was assessed between age P50–75 as part of our selective breeding paradigm (protocol: Stead et al., 2006, Behav Genet. 36: 697–712).  Sacrifice & RNA Extraction: The rats were sacrificed in adulthood (between P102-P108) via rapid decapitation followed by immediate brain extraction. The whole hippocampus, frontal cortex, and hypothalamus samples were rapidly dissected on ice, fast-frozen at –40°C, and stored at –80°C before processing. TRIzol reagent (Invitrogen, Calsbad, CA) was used to extract total RNA, followed by purification using RNeasy RNA purification columns (Qiagen, Valencia, CA). The quality and concentration of the RNA was determined using an Agilent bioanalyzer (Palo Alto, CA) and wavelength absorbance (260/280 nm ratio) by Nanodrop.  Microarray: The samples were transcriptionally profiled using Affymetrix Rat Expression Set 230 A (RAE230A) microarray according to standard manufacturer’s procedures. The samples were run in two batches: the first included the hippocampus and cortex samples and the second included the hypothalamus samples. Each brain region was run on a separate chip. Only data from the cortex and hypothalamus samples are included in this data release.  Microarray Data Preprocessing: For our current analyses, the ReadAffy() function (from R package affy, v.1.84.0; (Gautier et al., 2004)) was used to read the data from the .CEL files into R studio (v.1.0.153). An expression set was generated using the Robust Multi-Array Average method (RMA: (Irizarry et al., 2003)). An updated custom chip definition file for the RAE230A chip (.cdf (Dai et al., 2005): “rae230arnentrezgcdf_25.0.0”) was used to summarize the probe signals into probesets targeting genes annotated by Entrez Gene ID (downloaded from http://brainarray.mbni.med.umich.edu/Brainarray/Database/CustomCDF/25.0.0/entrezg.asp on Dec 9 2024, released on Jan 5, 2021). Additional gene annotation was later added using the org.Rn.eg.db package (v.3.20.0, (Carlson, 2019)). Quality control identified two hippocampal samples with low sample-sample correlation values (median R= 0.68 & 0.89, respectively), leaving a final sample size of n=5/group for the hippocampus, and n=6/group for both the cortex and hypothalamus. Separate bHR/bLR differential expression analyses were conducted for each brain region using the limma pipeline (no covariates, v.3.62.1, (Ritchie et al., 2015)), followed by an empirical Bayes correction. Nominal p-values were corrected for multiple comparisons using the Benjamini-Hochberg method (False Discovery Rate or FDR) within the multtest package (v. 2.62.0, (Pollard et al., 2005)).The full analysis code is available at: https://github.com/hagenaue/NIDA_bLRvsbHR_F2Cross_HC_RNASeq.

middle: H

first: John

middle: D

last: Stead

first: Stanley

middle: J

last: Watson

first: Huda

last: Akil


title: Gli2 Facilitates Tumor Immune Evasion and Immunotherapeutic Resistance by Coordinating Wnt Ligand and Prostaglandin Signaling [Bulk_Tumor_RNAseq]

overall_design: BRAFV600EPTEN-/- melanoma cell lines with either an active Gli2 mutation or a non-targeted control, implanted into C57Bl/6 mice


title: Landscape transcriptomics identify

city: University Park

first: Justin

last: Waraniak

state: PA

address: 421 Forestry Resources Building

department: Ecosystem Sciences and Management

institute: Penn State University

summary: Rapid heating events, such as heatwaves, are becoming more frequent and intense as a result of climate change. Importantly, such extreme weather events can be more important drivers of extirpation and selection than changes in annual or seasonal averages and they pose a particularly large threat to poikilothermic organisms. In this study, we evaluate the thermal stress response of a coldwater adapted fish species, the eastern brook trout (Salvelinus fontinalis), to two successive heatwaves during July and August 2022. We sampled brook trout at eight time points from four streams (N=116 fish) that differed in thermal habitat, sequenced mRNA from gill samples using TagSeq, and quantified expression levels of 32,670 unique transcripts. Multivariate analyses found that overall expression patterns in response to water temperature change were similar among streams. These analyses futher detected groups of genes involved in immune response and oxygen carrier activity that were upregulated and downregulated respectively at higher temperatures. We also detected 43 genes that were differentially expressed at different time points and followed the same expression pattern during the two heatwaves. Of these genes, 42 covaried with water temperature and most (27, 62.8%) exhibited responses that varied by stream. Some of the differentially expressed genes, including heat shock proteins and cold-inducible RNA binding proteins, have been widely linked to temperature responses in experimental studies, whereas other genes we identified have functions that have not been well-studied in relationship to temperature (e.g., collagen organization) or have unknown functions. This study shows the utility of landscape transcriptomic approaches to identify important biological processes governing wild organismal responses to short-term stressors. The results of this study can guide future investigations to identify phenotypic and genetic diversity that contribute to adaptive response to heatwaves and improve predictions of how populations will respond to climate change.

overall_design: Samples were taken from gill filaments of brook trout captured in four streams near State College, PA, USA during July & August 2022. Each stream was sampled on eight occasions which coincided with different points of two successive heatwave events. Streams were sampled  on three occasions 3-4 days apart starting at the onset of the heatwaves, with time points representing periods of rising temperatures, peak temperatures, and declining post-peak temperatures. A fourth sample was taken 6-7 days after the third sample during a period of non-stressful temperatures to capture post-heatwave recovery responses. Three to five fish were sampled during each sampling event. Samples were only taken from individuals >120mm total length, and all individuals were sexed based on PCR amplification of sdY primers developed by Rud and Buchatsky (2014) and Webster et al. (2021).

first: Jason

last: Keagy


title: Gli2 Facilitates Tumor Immune Evasion and Immunotherapeutic Resistance by Coordinating Wnt Ligand and Prostaglandin Signaling

summary:
Therapeutic resistance to immune checkpoint blockade has been commonly linked to the process of mesenchymal transformation (MT) and remains a prevalent obstacle across many cancer types. An improved mechanistic understanding for MT-mediated immune evasion promises to lead to more effective combination therapeutic regimens. Herein, we identify the Hedgehog transcription factor, Gli2, as a key node of tumor-mediated immune evasion and immunotherapy resistance during MT. Mechanistic studies reveal that Gli2 generates an immunotolerant tumor microenvironment through the upregulation of Wnt ligand production and increased prostaglandin synthesis. This pathway drives the recruitment, viability, and function of granulocytic myeloid-derived suppressor cells (PMN-MDSCs) while also impairing type I conventional dendritic cell, CD8+ T cell, and NK cell functionality. Pharmacologic EP2/EP4 prostaglandin receptor inhibition and Wnt ligand inhibition each reverses a subset of these effects, while preventing primary and adaptive resistance to anti-PD-1 immunotherapy, respectively. A transcriptional Gli2 signature correlates with resistance to anti-PD-1 immunotherapy in stage IV melanoma patients, providing a translational roadmap to direct combination immunotherapeutics in the clinic.
FFPE tumor samples from metastatic melanoma patients were extracted and a Nanostring Wnt Pathway panel was performed. Includes previously published samples from GSE165745 with updated clinical data

overall_design: Wnt pathway components with custom immune markers were selected to determine differences between responders and non-responders to anti-PD-1


title: Transcriptional and chromatin accessibility landscapes of hematopoiesis in a mouse model of breast cancer

city: Saint Louis

first: Changxu

last: Fan

state: Missouri

address: 4515 McKinley Avenue, Room 5213

department: Genetics

institute: Washington University in St. Louis

summary: Hematopoietic abnormalities, including myeloid-skewing, anemia, and extramedullary hematopoiesis, are often observed in cancer patients, with evidence suggesting that they promote the development of myeloid-derived suppressor cells. However, our understanding of the mechanisms underlying these hematopoietic phenotypes remains incomplete. Here, using the MMTV-PyMT mouse model of breast cancer, we characterize the transcriptional and chromatin accessibility landscapes of bone marrow and spleen hematopoietic progenitors using single-cell ATAC+RNA sequencing. We show that for the uncommitted hematopoietic stem and progenitor cells (HSPCs) in the BM, tumor growth induces a moderate upregulation of the myeloid-bias transcriptional signature and no significant chromatin accessibility changes. Compared to BM HSPCs, spleen HSPCs do not show a further upregulation of the myeloid-bias signature, and instead show the signature of Notch signaling, which has been associated with erythroid commitment. In addition, we also identify a cluster of spleen HSPCs in tumor-bearing animals with a transcriptional signature of mobilization, while our paired chromatin data suggest that AP-1 factors play a key role in HSPC mobilization. Furthermore, in contrast to the subtle tumor-induced changes in BM HSPCs, prominent transcriptional and chromatin changes are observed in dendritic cell (DC) progenitors, consistent with known cancer-associated DC defects. Our chromatin data also confirms, in an unbiased manner, that Stat3 and CEBP are both key contributors to DC defects. Overall, we provide a comprehensive dataset for understanding the hematopoietic consequences of cancer.

overall_design: single cell multiome ATAC + GEX assays were performed on bone marrow or spleen hematopoietic progenitors from control or tumor-bearing animals.

first: Fan

last: Changxu

first: Wu

last: Jun

first: Xing

last: Xiaoyun

first: Choi

last: Kyunghee

first: Wang

last: Ting


title: A single-cell atlas of circulating immune cells over the first 2 months of age in extremely premature infants

city: New Haven

first: Liza

last: Konnikova

state: CT

address: 375 Congress Ave

institute: Yale University

summary: Extremely premature infants (EPI), or those born prior to 30 weeks’ gestation, are highly susceptible to infection and inflammation. The impact of pre- and postnatal exposures on the immune response is poorly understood. We analyzed over 200, 000 circulating immune cells from 100-200L of whole blood collected from 25 donors including longitudinal samples from 10 EPI that were compared to healthy adults and to cord blood from term and preterm infants. We performed single-cell RNA-, T- and B-cell receptor sequencing, and phosphoprotein mass cytometry from matched samples and proteomics on dried blood spots from the same donors. Our data defined the trajectory of circulating T-, B-, myeloid and natural killer cells. Peripheral T cell development rapidly progressed over the first month of EPI’s life with an increase in the proportion of naïve CD4+, regulatory, and cycling T cells, accompanied by increased STAT5 signaling. Simultaneously, the transcription of IL2, which is essential for T cell activation and proliferation, increased in the lymphocytes. T cell repertoire diversity, clonality and gene usage increased rapidly over the first 60 days of life in EPI. CD38hi B cells were directly proportional to gestational age at birth. Interestingly, in utero exposure to inflammation (chorioamnionitis) altered the abundance, transcriptome and signaling of circulating T cells and monocytes specifically at 1 month of life in EPIs. Finally, we demonstrate the feasibility of a robust multi-omic longitudinal analysis in EPI from minute amounts of blood, developing a resource for studying the impact of early life exposures on immune development.

overall_design:
Circulating immune cells were isolated from blood samples obtained within the first week, one month, and at two months of age from EPI and compared to cord blood from term and preterm infants and to samples from healthy adults. Single-cell cDNA with matched TCR and BCR VDJ libraries were prepared using the 10x Chromium Next GEM 5′ reagent kit (v2) according to the manufacturer’s instructions. Sequencing was performed on the NovaSeq 6000 sequencer (Illumina) to obtain paired end reads.

PTCB is preterm cord blood samples
TCB is term cord blood samples
S1 is 1 week samples
S2 and S3 are 1-month samples
S5 is 2 months samples
adult is adult samples


title: N-acetyltransferase 10 catalyzes RNA N4-acetylcytidine affecting photosynthesis in rice

city: Changsha

first: Chongsheng

last: He

state: Hunan

address: 27 Tianma road

institute: Hunan university

summary: Using acRIP-seq, we present transcriptome-wide atlases of ac4C in wild type rice and osnat10. Analysis of ac4C distribution reveals ac4C is greatly reduced in osnat10 compared to wild type. We then performed RNA-seq to analyze differentially expressed genes between wild type and osnat10.

overall_design: This series includes samples for acRIP-seq: two biological replicates for ac4C IP and input samples of wild type rice and osnat10, samples for RNA-seq: three biological replicates from wild type rice and osnat10.

first: Linjun

last: Cai

first: Bin

last: Li

first: Qiting

last: Zhou

first: Wenxing

last: Yang

first: Lisha

last: Shen


title: Identification of heterogenous expressed transcripts that mitigate drug-induced replication stress

first: Frank

last: Riemers

overall_design: All samples consist of human RPE cell lines harboring the Tet Repressor, FUCCI4 system, fluorescently tagged 53BP1 and HRASG12V, which are described in Segeren et al 2022. Cells with WT HRAS and with doxycycline-inducible oncogenic HRASG12V were treated with replication stress inducing drugs, gemcitabine + prexasertib (referred to as GP). Single cells cells were sorted based on their degree of DNA damage as determined by the level of intracellular yH2AX staining. S-phase cells only were selected based on the level of DAPI staining. After sorting, scRNA sequencing was performed to correlate the degree of yH2AX staining to transcriptional profiles of individual cells. One 384-well plate was sorted for the Veh and Dox treated cells that were not treated with gemcitabine/prexasertib, and 3 384-well plates were sorted for the Veh and dox treated cells that were treated with gemcitabine/prexasertib.

first: Hendrika

last: Segeren

first: Kathryn

last: Wierenga

first: Bart

last: Westendorp


title: A novel conserved transcription factor MtTrh-1 regulates cellulase genes expression through controlling the Hac-1 and Cbh-1 in thermophilic fungus Myceliophthora thermophila

city: Tianjin

first: Liu

last: Qian

address: Xi 7 Dao No.32 Airport Economic Area

institute: TIB CAS

summary: In thermophilic fungi, work on the ER stress and UPR signalling is largely unknown,we here identified a new Cys2His2 transcription factor and found its knockout strain with significantly decreased cellulases production on crystalline cellulose.Here, we show that loss-of-function mutations of this regulator resulted in the reduced expression of Mthac-1 via RNA-seq under Avicel and artificial ER-stress.

sample_organism: Thermothelomyces thermophilus ATCC 42464

overall_design: mRNA profiles of Myceliophthora thermophila cells exposed to 4h Avicel.

first: Qian

last: Liu

first: Defei

first: Chaoguang

last: Tian


title: Interferon regulatory factor 4 mediates non-enzymatic IRE1 dependency in multiple myeloma cells [ChIP-Seq]

status: Public on Feb 04 2025

city: South San Francisco

first: Ioanna

last: Oikonomidi

state: California

address: 1 DNA Way

department: Research Biology

institute: Genentech Inc

type: Genome binding/occupancy profiling by high throughput sequencing

summary: Multiple Myeloma (MM) arises through oncogenic transformation of immunoglobulin-secreting plasma cells. MM often co-opts the central endoplasmic-reticulum (ER)-stress mitigator, inositol-requiring enzyme 1 (IRE1), to sustain malignant growth. While certain MMs require enzymatic IRE1-dependent activation of the transcription factor XBP1s, others display a nonenzymatic IRE1 dependency that is not yet mechanistically understood. Here we identify interferon regulatory factor 4 (IRF4), which stimulates genes that promote immune-cell proliferation, as a key conduit for IRE1’s nonenzymatic control of cell-cycle progression in MM. IRE1 silencing increased inhibitory S114/S270 phosphorylation on IRF4, disrupting IRF4’s chromatin-binding and transcriptional activity. IRF4 knockdown recapitulated, whereas IRF4 repletion reversed the anti-proliferative phenotype of IRE1 silencing. Furthermore, phospho-deficient, but not phospho-mimetic, IRF4 mutants rescued proliferation under IRE1 silencing. Functional studies revealed that IRF4 engages the E2F1 and CDC25A genes and promotes CDK2 activation to drive cell-cycle progression. Our results advance mechanistic understanding of IRE1 and IRF4 in MM.

overall_design: To identify IRF4-dependent loci, we performed ChIP-seq analysis in AMO1 cells proficient vs deficient of IRF4. Towards this, IRF4 (Antibody: #4964, CST) or RNAPII-pS2 (Antibody: ab5095, Abcam) were immunoprecipitated in AMO1 shIRF4 cells (conditionally expressing shRNAs against IRF4 upon doxycycline treatment) in the presence or absence of Doxycycline (Dox, 0.2 ug/mL) for 24 h.

first: Oikonomidi

last: Ioanna

first: Kameswaran

last: Vasumathi

first: Daniel

last: Bence

first: Modrusan

last: Zora

first: Ashkenazi

last: Avi


title: RNA-seq analysis of SPL integrin b7hi or integrin b7lo PCs, or BM PCs

city: Suita

first: Katsuyuki

last: Shiroguchi

state: Osaka

address: 6-2-3 Furuedai

department: Center for Biosystems Dynamics Research

institute: RIKEN

summary: Newly generated plasma cells in secondary lymphoid organs migrate to niches in the bone marrow, wherein they survive as long-lived plasma cells (LLPCs). Although characterization of LLPCs has been well performed, it is still unclear what is key determinant(s) for longevity of plasma cells. One postulated model is that heterogeneity of plasma cells is formed by such as BCR affinity and/or microenvironment at the induction site, and some of these variables may instruct their longevity. Here, we found that, among newly generated IgG plasma cells, integrin b7hi marks plasma cells prone to home to the bone marrow, whereas integrin b7lo cells remain in secondary lymphoid organs.

overall_design: B6 mice were transferred with splenic B cells from B1-8germ S1PR2-CreERT2 R26-LSL-tdTomato CD45.1 mice, immunized with NP-CGG in alum and then treated with tamoxifen at day 7 and 8 after immunization. At day 12 after immunization, IgG1+ CD45.1+ tdTomato+ SPL integrin b7hi or integrin b7lo PCs (CD138+ TACI+), or IgG1+ CD45.1+ tdTomato+ BM PCs were sorted by using FACSAriaII

first: Takuya

last: Koike

first: Nozomi

last: Hojo

first: Wataru

last: Ise

first: Tomohiro

last: Kurosaki


title: Interferon regulatory factor 4 mediates non-enzymatic IRE1 dependency in multiple myeloma cells [RNA-Seq]

overall_design: To follow the consequences of IRF4 silencing, we performed an RNA sequencing transcriptomic analysis of AMO1 and KMS27 human multiple myeloma cells subjected to IRF4 vs. NTC (non-targeting control) knockdown. AMO1 shIRF4 cl.1, cl. 4-3 or shNTC cells were harvested in biologic triplicates at 0 (Mock), 8, 16 and 24 h after treatment with Doxycycline (Dox, 0.2 μg/ml). KMS27 shIRF4 Cl. 20 or shNTC cells were harvested in biologic triplicates at 0 (Mock), 20 and 30 h after treatment with Doxycycline (Dox, 0.2 μg/ml).

first: Pradhan

last: Rachana


title: Control of coronary lymphangiogenesis by epicardial VEGFC/D

city: Madrid

first: Manuel

last: Gomez

state: Madrid

address: Melchor Fernández Almagro, 3

institute: CNIC

summary: The contractile ability of the mammalian heart critically relies on blood coronary circulation, essential to provide oxygen and nutrients to myocardial cells. In addition, the lymphatic vasculature is essential for the myocardial immune response, extracellular fluid homeostasis and response to injury. Recent studies identified different origins of coronary lymphatic endothelial cells, however, the cues that govern coronary lymphangiogenesis remain unknown. Here we show that the coronary lymphatic vasculature develops in intimate contact with the epicardium and with epicardial-derived cells. The epicardium expresses the lymphangiogenic cytokine VEGFC and its conditional elimination from the epicardium abrogates coronary lymphatic vasculature development. Interestingly, VEGFD is also expressed in the epicardium and cooperates with VEGFC in coronary lymphangiogenesis, but it does so only in females, uncovering an unsuspected sex-specific role for this cytokine. These results identify a role for the epicardium/subepicardium as a signalling niche required for coronary lymphangiogenesis and VEGFC/D as essential mediators of this role.

overall_design: We studied the transcriptome of dissected epicardial and subepicardial regions from wild type hearts by RNAseq comparing dorsal to ventral heart regions and males to females

first: Ester

last: De la Cruz

first: Vanessa

last: Cadenas

first: Miguel

last: Torres


title: Requirements for the neurodevelopmental disorder-associated gene ZNF292 in human cortical interneuron development and function

first: Kristen

last: Kroll

state: MO

address: 320 McDonnell Sciences/660 S. Euclid Ave.

department: Developmental Biology

institute: Washington University School of Medicine

first: Komal

last: Kaushik

first: Gareth

last: Chapman

middle: L


title: Single-cell analyses reveal increased gene expression variability in human neurodevelopmental conditions

city: Cambridge

first: Suraj

last: Upadhya

state: MA

address: 415 Main St

institute: Broad Institute

summary: Interindividual variation in phenotypic penetrance and severity is found in many neurodevelopmental conditions, including the autosomal aneuploidy Trisomy 21 (T21). However, mechanisms underlying such variation remain incompletely resolved. Here, we investigate the hypothesis that T21 can drive differential gene variance, not just differential gene expression. Leveraging multiple, independent single-cell and single-nucleus RNA sequencing datasets from human brain-relevant cell and tissue types, we identify a significant increase in transcriptional variance driven by T21 as well as CHD8 haploinsufficiency. Our analyses are consistent with a global and in part stochastic increase in transcriptional variance which is uncoupled from changes in transcript abundance. Highly variable genes tend to be lineage-restricted with modest enrichment for H3K27me3, while least variable genes are more likely to be constrained and associate with active histone marks. Our results show that neurodevelopmental conditions can drive increased transcriptional variance in brain cell types, and provide a scaffold for understanding variance in disease.

overall_design: Single cell RNA sequencing of neural progenitor cells derived from human iPSCs

first: Jenny

last: Klein

first: Anna

last: Nathanson

first: Kristina

last: Holton

first: Lindy

last: Barrett


title: ZNF292 genome-wide occupancy during cortical interneuron development

summary: Human embryonic stem cell (hESC) models of ZNF292 deficiency were generated, including a KRAB-only control and two CRISPRi knockdown models (ZNF-G1 and ZNF-G2). These hESC models were used to derived MGE-like ventral telencephalic neural progenitors (day 15; D15) and cortical interneurons (day 30; D30). CUT&Tag was utilized to define ZNF292 genome-wide occupancy at D15 and D30 during this process.

overall_design: For each time point, 4 biological replicates generated from independent differentiations of hESCs were used to assess differences in ZNF292 genome-wide occupancy between control (KRAB) and ZNF292 CRISPRi-mediated deficiency (ZNF-G1 and ZNF-G2) using CUT&Tag for ZNF292


title: Gli2 Facilitates Tumor Immune Evasion and Immunotherapeutic Resistance by Coordinating Wnt Ligand and Prostaglandin Signaling [Sorted_tumor_DC RNAseq]

first: Nicholas

last: DeVito

overall_design: BRAFV600EPTEN-/- melanoma cell lines with either an active Gli2 mutation or a non-targeted control, implanted into C57Bl/6 mice, followed by flow cytometry sorting of dendritic cells

first: Brent

last: Hanks


title: Transcriptional heterogeneity shapes stress-adaptive responses in yeast

first: Francesc

last: Posas

address: Carrar Baldiri Reixac 10

institute: Cell Signaling Group, IRB Barcelona

summary: In response to stress, cells activate signaling pathways that coordinate broad changes in gene expression to enhance cell survival. Remarkably, complex variations in gene expression occur even in isogenic populations and in response to similar signaling inputs. However, the molecular mechanisms underlying this variability and their influence on adaptive cell fate decisions are not fully understood. Here, we use scRNA-seq to longitudinally assess transcriptional dynamics during osmoadaptation in yeast. Our findings reveal highly heterogeneous expression of the osmoresponsive program, which organizes into combinatorial patterns that generate distinct cellular programs. The induction of these programs is favored by global transcriptome repression upon stress. Cells displaying basal expression of the osmoresponsive program are hyper-responsive and resistant to stress. Through a transcription-focused analysis of more than 300 RNA-barcoded deletion mutants, we identify genetic factors that shape the heterogeneity of the osmostress-induced transcriptome, define regulators of stress-related subpopulations and find a link between transcriptional heterogeneity and increased cell fitness. Our findings provide a regulatory map of the complex transcriptional phenotypes underlying osmoadaptation in yeast and highlight the importance of transcriptional heterogeneity in generating distinct adaptive strategies.

overall_design: S. cerevisiae (BY4741) were genetically barcoded either with resistance markers (WT and hog1 dataset) or through a modification of the yeast knock out collection deletion (deletion based scRNA-seq screening). For both experiments, all cells were grown individually and pooled before subjecting them or not to osmotic stress (0.4M NaCl, 15 mins). Cells were harvested by centrifugation and immediatelly fixed with ice cold methanol 80% and stored at -20ºC following 10X genomics meethanol fixation instructions. Frozen cells were equilibrated at 4ºC for 30 minutes and rehydrated following manufacturer's instructions. scRNA-seq profiling was performed using 10X genomics, each condition or time point was loaded into a lane.

first: Mariona

last: Nadal-Ribelles

first: Carme

last: Sole Serra

first: Eulalia

last: de Nadal


title: Global transcriptional profiles of intestinal stem cells, progenitor cells and Paneth cells during aging [bulk RNA-seq]

city: Kyoto

first: Takuya

last: Yamamoto

address: 53 Kawahara-cho, Shogoin, Sakyo-ku

institute: Kyoto University

summary: We report aging-induced transcriptional alterations of mouse intestinal stem cells (ISCs), progenitor cells and Paneth cells.

overall_design: ISCs, progenitor cells and stem cell niche (Paneth cells) were isolated from young (2- to 3-month-old) and aged (23- to 27-month-old) Lgr5-eGFP-IRES-CreERT2 mice. For each condition (cell type, age and sex), 5 to 7 biological replicates were analyzed. For RNA-seq libraries of Paneth cell samples, technical replicates were prepared. mRNA profiles were generated by the SC3-seq method (Nakamura et al., Nucleic Acids Res., 2015), using Illumina NextSeq500.

first: May

last: Nakajima-Koyama

first: Mio

last: Kabata


title: MAIT cells protect against sterile lung injury (BRD509)

city: Oxford

first: Shuailin

last: Li

address: Old Road Campus Research Building

country: United Kingdom

institute: University of Oxford

summary: Mucosal-associated invariant T (MAIT) cells, the most abundant unconventional T cells in the lung, have been recently linked to tissue protection and repair. Their role, especially in sterile lung injury, is unknown. Using single cell RNA sequencing (scRNA-seq), spectral analysis and adoptive transfer in a bleomycin-induced sterile lung injury, we found that bleomycin activates murine pulmonary MAIT cells and induces an accompanying tissue repair programme, associated with a protective role against bleomycin-induced lung injury. MAIT cells drive the accumulation of type 1 conventional dendritic cells (cDC1), limiting tissue damage in a DNGR-1 dependent manner. Human scRNA-seq data revealed that MAIT cells were activated, with increased cDC populations in idiopathic pulmonary fibrosis patients. Thus, MAIT cells enhance defence against sterile lung injury by fostering cDC1-driven anti-fibrotic pathways.

overall_design: For RNA-seq of MAIT cells, MAIT cells were isolated from lung of C57BL/6 mouse challenged by bleomycin or PR8 IAV with previous intranasal Salmonella Typhimurium BRD509 infection. For MAIT cells isolated after bleomycin challenge, mouse lung was collected on day 0 (with PBS challenge), day 3, day 7, day 14, day 21 and day 28 post bleomycin challenge. For MAIT cells isolated after PR8 IAV challenge, mouse lung was collected on day 0 (with PBS challenge), day 3, day 5, day 7 and day 13 post PR8 challenge. For total lung RNA-seq, total lung was collected from C57BL/6 and Mr1<tm1Gfn> mouse challenged by bleomycin day 0 (with PBS challenge), day 3, day 7, day 14 and day 21 post bleomycin challenge.

first: Xiawei

last: Zhang


title: Transcriptomic analysis uncovers an unfolded protein response in ADNP syndrome

city: Philadelphia

first: Phillip

last: Wulfridge

state: Pennsylvania

address: 3601 Spruce St

institute: The Wistar Institute

summary: Activity dependent neuroprotective protein (ADNP) is mutated in Helsmoortel–Van der Aa syndrome, an autism spectrum disorder characterized by intellectual deficiency and developmental delays. We used induced pluripotent stem cells derived from ADNP syndrome patients as a model to test the effects of syndromic ADNP mutations on gene expression and neurodifferentiation. We found that some ADNP mutations result in retention of the truncated ADNP proteins, which displayed aberrant subcellular localization. Gene expression analyses revealed widespread transcriptional deregulation in all tested mutants. Interestingly, mutants that retain ADNP fragments show ER stress as evidenced by activation of the unfolded protein response (UPR). The mutants showing the greatest UPR pathway activation associated with the most severe neurodifferentiation and survival defects. Our results reveal the potential to explore UPR activation as a new biomarker for ADNP syndrome severity.

overall_design: RNA-Seq was performed on human induced pluripotent stem cells (hiPSCs) and on hiPSCs differentiated to neural progenitor cells (NPCs).

last: Bieluszewska

first: Kuo-Chen

last: Fang

first: Yan

last: Hong

first: Tomoyo

last: Sawada

first: Jennifer

last: Erwin

first: Guo-li

last: Ming

first: Hongjun

last: Song

first: Kavitha

last: Sarma


title: RNA-seq analysis of integrin β7hi or integrin β7lo PCs, or klf2-sufficient or -deficient PCs

first: Daisuke

last: Motooka

address: 3-1, Yamadaoka

institute: NGS core facility, Research Institute for Microbial Diseases, Osaka University

summary: We found that, among newly generated IgG plasma cells, integrin b7hi marks plasma cells prone to home to the bone marrow, whereas integrin b7lo cells remain in secondary lymphoid organs. Moreover, we also found that the differences in KLF2 expression between b7hi and b7lo populations in secondary lymphoid organs is likely to explain that only integrin b7hi plasma cells can egress. Next, to look for the functional target(s) downstream of KLF2 involved in egress of newly generated plasma cells, we carried out RNA-seq analysis of KLF2-sufficient and -deficient splenic plasma cells. Wildtype control splenic plasma cells were further divided into integrin b7hi and b7lo populations. We identified S1pr1 and Itgam as functional targets downstream of KLF2.

overall_design: B6 mice were transferred with splenic B cells from B1-8hi R26-creERT2 CD45.1 Klf2 +/+ or Klf2 fl/fl mice, immunized with NP-CGG in alum and then treated with tamoxifen at day 3, 4, and 5 after immunization. At day 7 after immunization, donor-derived IgG1+ PCs (CD138+ TACI+) derived from Klf2 +/+ or Klf2 fl/fl mice were sorted by using FACSAriaII. In addition, Klf2+/+ PCs were divided into Integrin b7 hi and b7 lo cells and collected as other samples.

last: Koike

last: Okuzaki

first: Wataru

last: Ise

first: Tomohiro

last: Kurosaki


title: Whole transcriptome analysis of Mycoplasma bovis-host interactions under in vitro and in vivo conditions

city: Knoxville

first: Aga

middle: Edema

last: Gelgie

state: Tennessee

address: 2506 River Dr

department: Animal Science

institute: The University of Tennessee

summary: Mycoplasma bovis mastitis is becoming increasingly problematic for dairy cattle farming in different parts of the world. M. bovis is inherently resistant to several antimicrobial classes and there is no effective vaccine. The major constraints to developing effective control tools are limited knowledge of M. bovis virulence factors and the underlying pathogenic mechanisms. The objective of this study was to determine virulence-associated genes of M. bovis and host immune response genes expressed during the early stages of host-pathogen interactions. In this study, we conducted in vitro infection of mammary epithelial cell (MAC-T) lines and in vivo intramammary infection of dairy cows with M. bovis strain PG45 and evaluated whole transcriptome differential gene expression and pathway enrichment analysis. We found a total of 614 (304 up-regulated, 310 down-regulated) and 7161 (3935 up-regulated, 3226 down-regulated) genes of M. bovis and bovine host cells were differentially expressed, respectively. Of the up-regulated genes of M. bovis, insertion sequence (IS) genes that are involved in transposase activity such as ISMbov1, ISMbov2, ISMbov3, and ISMbov9 were significantly up-regulated, whereas protein translation-associated genes were significantly down-regulated. In MAC-T cells, genes involved in apoptosis pathways and proinflammatory cytokines were significantly up-regulated, whereas genes involved in cell cycle, ribosome biogenesis, and steroid biosynthesis were significantly down-regulated. Genes encoding formation of neutrophil extracellular traps and proinflammatory cytokines, were significantly up-regulated in the mammary gland of M. bovis challenged cows, whereas genes involved in steroid biosynthesis and metabolism were significantly down-regulated. In conclusion, there were increased expressions of IS elements which are involved in transposase activity during early stages of M. bovis infection. This observation warrants further detailed investigation to determine important specific potential targets involved in the transposition for intervention. Apoptosis might be in favor of progression of M. bovis infection but intervention that reduces apoptosis and enhances quick and effective polymorphonuclear neutrophilic recruitment with increased extracellular trap activity may improve M. bovis clearance. Our findings are key to determining important targets for immunity-based intervention. To our knowledge, this is the first whole transcriptome analysis in both M. bovis and the host under the same in vitro and in vivo conditions. Therefore, we recommend additional metatranscriptomic, metaproteomic, metabolomic and metagenomic evaluation of M. bovis and other potentially non-culturable microbes in the mammary glands during the pathogenesis of M. bovis mastitis in the bovine host to understand a complete picture of these interactions in maintaining healthy homeostatic state or mastitis.

sample_organism:
Bos taurus
Mycoplasmopsis bovis

platform_organism:
Bos taurus
Mycoplasmopsis bovis

overall_design: MAC-T (bovine mammary epithelial cells) and dairy cows were infected using Mycoplasma bovis to investigate early stage transcriptomic changes both in the pathogen and host. Mycoplasma bovis only, MAC-T only and uninfected cow controls were included in the study. All samples (5) were prepared in duplicate.

middle: E

first: Benti

last: Gelalcha

first: Trevor

last: Freeman

first: Taylor

middle: B

last: Ault-Seay

first: Jonathan

last: Beever

first: Oudessa

middle: K

last: Dego


title: Effect of escalating doses of docetaxel on chemoresistant-related gene expression of laryngeal cancer cells

city: Montreal

first: Alain

last: Pacis

state: QC

address: 1010 Sherbrooke #1800

institute: Canadian Centre for Computational Genomics (C3G)

summary: Laryngeal cancer is a sub-type of head and neck cancer, and its treatment frequently entails the use of chemotherapy drugs like docetaxel. Nevertheless, the development of docetaxel resistance mechanisms presents a substantial challenge. Analyzing the transcriptome has become a fundamental approach for understanding the underlying molecular factors contributing to resistance and for pinpointing potential treatment targets. This type of analysis provides a comprehensive overview of the changes in gene expression that occur in laryngeal cancer cells that have become resistant to docetaxel. By comparing the transcriptomes of resistant and sensitive cells, we can identify potential biomarkers and targets for therapy. We have effectively created a laryngeal cancer cell line that is resistant to docetaxel, as evidenced by the increased expression of genes associated with various processes, such as microtubules (TUBB3, EML4ALK), receptors (TRK, PDGFR, GPCR, NOTCH), epithelial adherence (CDH1), metabolism (PIK3CA, DEPTOR, GSK-3β, PLCβ, CYP24A1), hypoxia (HIF-1α, HIF-1β), apoptosis (TNFRSF11A, p21, MDM2), cytokine production (STAT2/4/6, iNOS, IL-6, IL-8, GMCSFR) and autophagy (CFLAR, GABARAP, ATG4A, RasGEF). Taken together, our data provide a framework that encompasses therapeutic targets for inhibition of upregulated gene expression of docetaxel resistance on laryngeal cancer cells.

overall_design:
To engineer a docetaxel-resistant laryngeal cancer cell line (DR-LSCC), we expose a commercially available laryngeal cancer cell line with escalating doses of docetaxel up to 4 months
We then performed gene expression profiling analysis using data obtained from RNSA-seq of 5 samples of DR-LSCC and two non-resistant cell controls (laryngeal cancer and laryngeal epithelia)
Comparative gene expression profiling analysis of RNA-seq data of DR-LSCC compared to non-resistant controls of laryngeal cancer (LSCC) and laryngeal epithelia (LSEC)

first: Christian

middle: R

last: Moya-Garcia

first: Alan

first: Nader

last: Sadeghi

first: Maryam

last: Tabrizian

first: Nicole

middle: Y

last: Li-Jessen


title: Sequential histone H1 N76/77 deamidation and K75 acetylation facilitate chromatin relaxation and genome stability by DNA repair

status: Public on Feb 03 2025

city: Shenzhen

first: Jun

last: Zhang

state: Guangdong Province

address: Xueyuan Avenue

department: School of Medicine

institute: Shenzhen University

summary: The formation of accessible chromatin around DNA double-strand breaks is essential for their efficient repair1. Although the linker histone H1 is known to facilitate higher-order chromatin compaction2,3, the mechanisms by which H1 modifications regulate chromatin relaxation in response to DNA damage are unclear. Here we show that CTP synthase 1 (CTPS1)-catalysed deamidation of H1 asparagine residues 76 and 77 triggers the sequential acetylation of lysine 75 following DNA damage, and this dual modification of H1 is associated with chromatin opening. Mechanistically, the histone acetyltransferase p300 showed a preference for deamidated H1 as a substrate, establishing H1 deamidation as a prerequisite for subsequent acetylation. Moreover, high expression of CTPS1 was associated with resistance to cancer radiotherapy, in both mouse xenograft models and clinical cohorts. These findings provide new insights into how linker histones regulate dynamic chromatin alterations in the DNA damage response.

overall_design: Examination of Linker Hinstone H1.4 modifications signal distribution upon DNA damage.

first: Yuan

last: Tian

first: Tingting

last: Feng

first: Wei-Guo

last: Zhu


title: MAIT cells protect against sterile lung injury (scRNA-Seq)

first: Shuailin

last: Li

overall_design: Lung was collected from C57BL/6 and Mr1<tm1Gfn> mouse day 0 (with PBS challenge), day 3, day 7 post bleomycin challenge. Each genotype and time point had 3 replicates. Live cells were sorted. Cells from 2 mice were mixed together for single cell sequencing after staining with TotalSeq-C Mouse Universal Cocktail (BioLegend, Cat. No. 199903) and TotalSeq-C HashTag Antibody (BioLegend, Cat. No. 155861, 155863).

first: Xiawei


title: NLRP3-mediated glutaminolysis regulates microglia in Alzheimer’s disease [ChIP-Seq]

city: La Jolla

first: Johannes

last: Schlachetzki

address: 9500 Gilman Drive

institute: UCSD

summary: Activation of the NLRP3 inflammasome has been implicated in the pathogenesis of Alzheimer’s disease (AD), via the characterised release of IL-1β and ASC specks. However, whether NLRP3 was involved in pathways beyond this remained unknown. Here we show that loss of NLRP3 influences glutamine/glutamate-related metabolism and increases expression of microglial Slc1a3, which was associated with enhanced mitochondrial and metabolic activity. The generation of α-ketoglutarate during this process impacted cellular function including more significant clearance of Aβ peptides, epigenetic and gene transcription changes. This pathway is conserved between murine and human cells. Critically, we can mimic this effect pharmacologically using NLRP3-specific inhibitors, but only with chronic NLRP3 inhibition. Together, these data demonstrates a new role for NLRP3, where it can modulate mitochondrial and metabolic function, with important downstream consequences for the progression of AD.

overall_design: H3K27ac ChIP-seq of wild type (C57BL/6N genetic background) and Cias1-knockout mice (named Nlrp3-/-; Millennium Pharmaceuticals)

first: Róisín

last: McManus

first: Christopher

last: Glass

first: Eicke

last: Latz

first: Michael

last: Heneka


title: Drosophila WDFY3/ Bchs overexpression impairs neural function

city: Leipzig

first: Diana

last: Le Duc

address: Philipp-Rosenthal-Str. 55

country: Germany

institute: Institute of Human Genetics,University of Leipzig Medical Center

summary: Pathogenic variants in WDFY3, a gene encoding for an autophagy adaptor termed ALFY, are linked to neurodevelopmental delay and altered brain size in human probands. While the role of WDFY3 loss-of-function is extensively studied in neurons, little is known about the effects of WDFY3 upregulation in different cell types of the central nervous system (CNS). We show that overexpression of the Drosophila melanogaster WDFY3 ortholog, Bchs, in either glia or neurons impaired autophagy and locomotion. Bchs glial overexpression also increased VNC size and glial nuclei number significantly, whereas neuronal Bchs overexpression affected wing and thorax morphology. We identified 79 genes that were differentially expressed and overlapped in flies that overexpress Bchs in glial and neuronal cells, respectively. Additionally, upon neuronal Bchs overexpression differentially expressed genes clustered in gene ontology categories associated with autophagy and mitochondrial function. Our data indicate that glial as well as neuronal Bchs upregulation can have detrimental outcomes on neural function.

overall_design: To understand which molecular pathways are affected by Bchs overexpression and how this impacts brain function, we performed RNA-seq on heads from adult flies (22-31 h after eclosion) overexpressing Bchs in glia (repo-Gal4/UAS-bchs::HA) or neurons (nSyb-Gal4/UAS-bchs::HA). We performed differential gene expression analysis comparing the Bchs overexpression condition to the respective Gal4- and UAS-controls.

first: Marek

last: Körner

first: Akhil

last: Velluva

first: Linneaus

last: Bundalian

first: Knut

last: Krohn

first: Kathleen

last: Schön

first: Isabell

last: Schumann

first: Jessica

last: Kromp

first: Andreas

middle: S

last: Thum

first: Antje

last: Garten

first: Julia

last: Hentschel

first: Rami

last: Jamra

first: Achmed

last: Mrestani

last: Scholz

first: Tobias

last: Langenhan


title: Characterization of rationally designed CRISPR/Cas9-based DNA methyltransferases with distinct methyltransferase and gene silencing activities in human cell lines and primary human T cells

city: Houston

first: Isaac

middle: B.

last: Hilton

state: TX

address: 6500 Main Street

institute: Rice University

type: Methylation profiling by genome tiling array

summary: Nuclease-deactivated Cas (dCas) proteins can be used to recruit epigenetic effectors, and this class of epigenetic editing technologies has revolutionized the ability to synthetically control the mammalian epigenome and transcriptome. DNA methylation is one of the most important and well-characterized epigenetic modifications in mammals, and while many different forms of dCas-based DNA methyltransferases (dCas-DNMTs) have been developed for programmable DNA methylation, these tools are frequently poorly tolerated and/or lowly-expressed in mammalian cell types. Further, the use of dCas-DNMTs has largely been restricted to cell lines, which limits mechanistic insights in karyotypically normal contexts and hampers translational utility in the longer-term. Here we extend previous insights into the rational design of the catalytic core of the mammalian DNMT3A methyltransferase and test three dCas9-DNMT3A/3L variants across different human cell lines and in primary donor-derived human T cells. We find that mutations within the catalytic core of DNMT3A stabilize the expression of dCas9-DNMT3A/3L fusion proteins in Jurkat T cells without sacrificing DNA methylation or gene-silencing performance. We also show that these rationally engineered mutations in DNMT3A alter DNA methylation profiles at loci targeted with dCas9-DNMT3A/3L in cell lines and donor-derived human T cells. Finally, we leverage the transcriptionally repressive effects of dCas9-DNMT3A/3L variants to functionally link the expression of a key immunomodulatory transcription factor to cytokine secretion in donor-derived T cells. Overall, our work expands the synthetic biology toolkit for epigenetic editing and provides a roadmap for the use of engineered dCas-based DNMTs in primary mammalian cell types.

overall_design: We aimed to evaluate the genome-scale differences in DNA methylation patterns for each dCas9-DNMT variant: WT refers to the original dCas9-DNMT3A/3L, V1 refers to the variant containing the R887E mutation in DNMT3A catalytic domain, V2 refers to the variant containing the E814G mutation in DNMT3A catalytic domain, V3 refers to the variant containing both R887E and E814G mutations, and catd refers to the variant containing the C706A and R832E mutations which inactivate catalytic activity. Non-transduced (NTC) primary human T cells are used as a control. Two replicates per condition are included in the analyses and are denoted as rep1 and rep2.


title: Targeting PRC2 enhances the antitumor cytotoxic capacity of anti-CD19 CAR-T cells against hematological malignancies

city: São Paulo

first: Alexandre

last: Defelicibus

address: Rua Tagua

country: Brazil

summary: Chimeric Antigen Receptor (CAR) T cell therapy was adopted as a clinical modality for patients with  relapsed/refractory hematological malignancies. Despite the clinical efficacy of CAR-T cell therapy,  a considerable fraction of patients still relapses during the first months following CAR-T cell infusion.  The limited CAR-T cell efficiency is thought to relate to epigenetic mechanisms involved in T cell suppression and dysfunction. Here, screening of multiple epigenetic inhibitors revealed that targeting  PRC2 consistently enhanced the cytotoxic/effector phenotype of CD8 T cells. Notably, PRC2 inhibition  promoted the differentiation of GZMB+ effector memory 19BBζ CAR-T cells and enhanced their antitumor  activity both in vitro and in vivo. Consistent with their long-lasting antitumor activity, PRC2-inhibited 19BBζ CAR-T cells did not exhibit any signs of dysfunctionality/exhaustion. Furthermore, TCR restimulation  along with PRC2 inhibition of patient-derived anti-CD19 CAR-T cells also induced the development of GZMB+ effector memory cells and elicited potent antitumor responses against CD19+ Daudi cells. In line  with this, the gene signature derived from in-house PRC2-inhibited 19BBζ CAR-T cells was enriched in tisagenlecleucel (tisa-cel) BBζ CAR-T cell therapy responders with large B-cell lymphoma. Collectively, our results demonstrated that targeting PRC2 may be a promising approach to enhance a functional effector program in CAR-T cells against hematological malignancies.

overall_design:
Leukocyte removal filters from healthy donors were used to isolate PBMCs through Ficoll-Paque PLUS density gradient medium (Cytiva), followed by T cell isolation using CD3 MicroBeads (Miltenyi Biotec). CAR-T cells were generated using the "Sleeping Beauty" Transposon System.  CD3 T cells were resuspended in 100μL of 1SM buffer, 10μg PT3-19BBz and 1μg SB100X plasmids, and then transferred into a 0.2 cm cuvette (Mirus Biotech®, Madison, WI). T cells were then electroporated (U-014 program) using the Lonza® Nucleofector® II electroporation system. After 24h resting, T cells were in vitro expanded with TransactTM CD3/CD28 T-cell activator [1:200] (Miltenyi Biotec) in media containing 50U recombinant human IL-2 (Thermo Fisher) together with  PRC2 inhibitors or their corresponding negative controls for 7 days.
Total RNA from CAR-T cells was extracted using ReliaPrep RNA Tissue Miniprep System (Promega) according to the manufacturer’s instructions.The quality of total RNA extracted from in house-produced CAR-T cells was assessed by RNA Screen Tape on TapeStation  (Agilent). cDNA libraries were constructed from 400 ng of RNA using the Stranded mRNA Prep Ligation kit (Illumina). Sequencing was performed using the Illumina NextSeq 2000 system (P2, 200 cycles kit), and at least 50 million paired-end  reads were obtained for each sample. Reads were aligned to the GHCh38/hg38 human genome (GTF v109) using STAR v2.7.10b (--twopassMode None --outSAMunmapped Within --chimSegmentMin 20 --limitIObufferSize 30000000 62500000) and quantified using htseq v2.0.3 (-m union -i gene_id -r pos -s no), with default parameters unless otherwise specified. Expression levels were calculated as log2(TPM+1).

first: Maria Leticia Rodrigues

last: Carvalho

first: Clara de Oliveira

last: Andrade

first: Mariela Pires

last: Cabral-Piccin

first: Gabriela Sarti

last: Kinker

first: Glauco Akelinghton Freite

last: Vitiello

first: Raylane Adrielle Gonçalves

last: Cambui

first: Luiza de Macedo

last: Abdo

first: Eduardo Mannarino

last: Correia

first: Bruno Dalbelo da Silva

last: Elias

first: Emmanuel Vinicius Oliveira

last: Araujo

first: Alexandre Silva

last: Chaves

first: Maria Luiza Marques

last: Pierre

first: Pedro Henrique Barbosa

last: Pereira

first: Karina Lobo

last: Hajdu

first: Rondinelli

last: Amanda

first: Arianne Fagotti

last: Gusmao

first: Igor

last: Brasil-Costa

first: Caroline Pouza

last: Zanella

first: Marta Maria Moreira

last: Lemos

first: Marjorie Vieira

last: Batista

first: Jayr

last: Schmidt Filho

first: Cheryl H

last: Arrowsmith

first: Vladmir Claudio Cordeiro

last: de Lima

first: Martin Hernan

last: Bonamino

first: Tiago da Silva

last: Medina


title: Consequences of ZNF292 deficiency for human cortical interneuron development

city: Saint Louis

first: Kristen

last: Kroll

summary: Human embryonic stem cell (hESC) models of ZNF292 deficiency were generated, including a KRAB-only control and two CRISPRi knockdown models (ZNF-G1 and ZNF-G2). These hESC models were used to derived MGE-like ventral telencephalic neural progenitors (day 15; D15) and cortical interneurons (day 30; D30). RNA-seq was conducted in these models to define transcriptional changes that result from ZNF292 deficiency during this process.

overall_design: For each time point, 4 biological replicates generated from independent differentiations of hESCs were used for comparison of transcriptomic differences between control (KRAB) and ZNF292 CRISPRi-mediated deficient (ZNF-G1 and ZNF-G2) progenitors and neurons.

first: Komal

last: Kaushik

first: Gareth

last: Chapman


title: Oncohistone H3.3K27M-driven CREB5/ID1 Axis Mediates the Malignant Cell States of Diffuse Intrinsic Pontine Glioma (DIPG) [HiC]

city: Beijing

first: Qiaoran

last: Xi

address: Haidian District

department: School of Life Sciences

institute: Tsinghua University

summary: Diffuse intrinsic pontine glioma (DIPG), an exceptionally lethal pediatric cancer with limited treatment options, is characterized by relentless proliferation and differentiation arrest, primarily influenced by H3K27M oncohistones. This study unveils that ID1 activation occurs independently of BMP signaling in H3.3K27M/ACVR1WT DIPG subtype. ID1 inhibition promotes cell differentiation and apoptosis in DIPG. Additionally, we discover the intricate regulation of ID1 tied to the lineage-specific transcription factor CREB5—an oncogenic contributor hindering differentiation in the H3.3K27M subtype of DIPG. The SWI/SNF complex is identified as a co-regulator of CREB5, further promoting tumorigenesis. Intriguingly, H3.3K27M oncohistones act as a trigger for CREB5 overexpression, facilitating the formation of super-enhancer promoter loops. Moreover, our research highlights the potent anti-tumor effect of ABBV-075 in treating DIPG. In essence, this study elucidates the oncohistone-driven H3.3K27M/CREB5/ID1 axis, providing insights into the malignant cellular state within a DIPG subtype and offering potential therapeutic avenues for this devastating pediatric cancer.

overall_design: To investigate the DNA interactions impact of Creb5, the HiC-seq experiments were conducted in mouse forebrain and hindbrain NPC cells from E13.5 embryos.

first: Wei

last: Zhou


title: Compact transcription factor cassettes generate functional, engraftable motor neurons by direct conversion [re-analysis]

first: Kate

last: Galloway

address: 25 Ames St

department: Chemical Engineering

institute: MIT

summary: Direct conversion generates patient-specific, disease-relevant cell types such as neurons that are rare, limited, or difficult to isolate from common and easily accessible cells such as skin cells. However, low rates of direct conversion and complex protocols limit scalability and thus the potential of cell-fate conversion for biomedical applications. Systems-level optimization of conversion protocols across molecular and cellular scales can support scalable cell manufacturing for diverse application. We use insights from our work on how proliferation and transcription factor levels act together to drive direct conversion to optimize the conversion protocol by examining process parameters including transcript design, delivery via AAV, retrovirus and lentivirus, cell seeding density, and the impact of media conditions. Thus, we report a compact, portable conversion process that boosts proliferation and increases direct conversion of mouse fibroblasts to induced motor neurons to achieve high conversion rates of above 1,000%, corresponding to more than 10 motor neurons yielded per cell seeded which we achieve through expansion. Our optimized, direct conversion process generates functional motor neurons at scales relevant for cell therapies (>10^7 cells) that graft with the mouse central nervous system. High-efficiency, compact, direct conversion systems will support scaling to patient-specific, neural cell therapies.

relation:
Reanalysis of: GSM3964244
Reanalysis of: GSM3964254
Reanalysis of: GSM3964255
Reanalysis of: GSM3964251
Reanalysis of: GSM3964252

overall_design:
Transcriptional profiling of cells generated by direct conversion of mouse embryonic fibroblasts to induced motor neurons (iMNs) at the terminal stage at 14 dpi. Samples include Hb9::GFP+ sorted iMNs generated with 6FDDRR (6F with p53DD, hRASG12V, RepSox) as well as MEFs and embryonic motor neurons (embMN) for reference populations. The 6F are Ascl1, Brn2, Myt1l, Ngn2, Isl1, Lhx3.

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The table below lists GEO accessions reused/reanalyzed for this study.
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first: Nathan

last: Wang


title: CDX2 downregulation regulates intrinsic WNT pathway activation, dictating metastasis in APC and CTNNB1 wildtype colorectal cancer

city: Amsterdam

first: Arezo

last: Torang

address: Meibergdreef 9

institute: Amsterdam UMC

summary: Colorectal cancer (CRC) can be divided into 4 subtypes of which consensus molecular subtype 4 (CMS4) is associated with metastasis and poor survival. Previously, we reported that the KPN mouse model resembles human CMS4. Strikingly, although tumor formation in this model is slow and limited, effective metastasis is observed. To understand this aggressive behavior, we compared two distinct in vitro KPN models, organoids and tumoroids. The organoid model only carries the original mutations, while the tumoroids are derived from in vivo grown tumors that underwent selection during development. Here, we reveal that tumoroids harbor endogenous WNT pathway activity, which can be driven by tankyrase activity and Cdx2 downregulation. Importantly, WNT pathway activation was heterogeneous in nature, subject to regulation and allowed for a mixture of WNT-driven and YAP-driven cells within tumoroids. This unique type of WNT pathway activation is not crucial for colonic tumor growth, but results in metastatic spreading. Intriguingly, these findings reflect a specific subset of aggressive human CMS4 cancers that display low CDX2 expression and lack of classical WNT pathway mutations, while having a higher tendency to metastasize. Together, these data propose a novel mechanism for WNT pathway activation that drives metastasis formation in aggressive CRC.

overall_design: To investigate mechanisms induced by selective pressure in vivo, we compared KPN mouse organoids (in vitro recombined) and KPN mouse tumoroids (derived from in vivo recombined tumors). To exclude interference of heterogeneity in the tumoroid cultures, single cell clones derived from bulk tumoroids were grown. Both bulk KPN organoids/tumoroids lines and single cell clonal lines derived KPN tumoroids were sequenced and checked for geneset enrichment and differentially expressed genes.

first: Valérie

last: Wouters

first: Roxan

middle: F

last: Helderman

middle: C

last: Cameron

first: Sander

last: van Hooff

first: Saskia

last: van den Bergh

first: Rene

last: Jackstadt

first: Sanne

last: van Neerven

first: Owen

last: Sansom

first: Jan Paul

last: Medema


title: KMT2C/KMT2D-dependent H3K4me1 mediates DNA replication timing and origin firing

city: New York

first: Tony

middle: T

last: Huang

state: NY

address: 450 East 29th St, Rm 807

department: Biochemistry and Molecular Pharmacology

institute: NYU School of Medicine

summary: Mammalian genomes are replicated in a precise, cell-type-specific order during the S phase, a process that correlates with local transcriptional activity, chromatin modifications, and chromatin architecture. However, the causal relationships between these features and DNA replication timing (RT), particularly during cell fate transitions, remain largely unknown. Here, we employed machine learning to quantify chromatin features — including epigenetic marks, histone variants, and chromatin architectural factors — that best predict local RT under steady-state conditions and RT changes during early embryonic stem (ES) cell differentiation. We found that approximately one-third of the genome exhibits RT changes during differentiation. Chromatin features collectively predicted steady-state RT and RT changes with high accuracy. Notably, histone H3 lysine 4 monomethylation (H3K4me1), catalyzed by KMT2C/D, emerged as a top predictor. Genetic deletion of Kmt2c/d (but not Kmt2c alone) or their enzymatic activities erased genome-wide RT dynamics during cell differentiation. Sites that typically gain H3K4me1 in a KMT2C/D-dependent manner during differentiation failed to transition towards earlier RT, often without affecting transcriptional activation. Further analysis at KMT2C/D binding sites revealed a local requirement for KMT2C/D in promoting DNA replication origin firing. Our findings identify KMT2C/D-dependent H3K4me1 as a functional regulator of RT and origin firing, highlighting a causal relationship between the epigenome and DNA replication that is largely independent of transcription. These insights should be relevant to various diseases associated with KMT2C/D mutations. Mammalian genomes are replicated in a precise, cell-type-specific order during the S phase, a process that correlates with local transcriptional activity, chromatin modifications, and chromatin architecture. However, the causal relationships between these features and DNA replication timing (RT), particularly during cell fate transitions, remain largely unknown. Here, we employed machine learning to quantify chromatin features — including epigenetic marks, histone variants, and chromatin architectural factors — that best predict local RT under steady-state conditions and RT changes during early embryonic stem (ES) cell differentiation. We found that approximately one-third of the genome exhibits RT changes during differentiation. Chromatin features collectively predicted steady-state RT and RT changes with high accuracy. Notably, histone H3 lysine 4 monomethylation (H3K4me1), catalyzed by KMT2C/D, emerged as a top predictor. Genetic deletion of Kmt2c/d (but not Kmt2c alone) or their enzymatic activities erased genome-wide RT dynamics during cell differentiation. Sites that typically gain H3K4me1 in a KMT2C/D-dependent manner during differentiation failed to transition towards earlier RT, often without affecting transcriptional activation. Further analysis at KMT2C/D binding sites revealed a local requirement for KMT2C/D in promoting DNA replication origin firing. Our findings identify KMT2C/D-dependent H3K4me1 as a functional regulator of RT and origin firing, highlighting a causal relationship between the epigenome and DNA replication that is largely independent of transcription. These insights should be relevant to various diseases associated with KMT2C/D mutations.

overall_design: Okazaki-fragment (Ok-seq) sequencing was performed on naïve (N) and transformed epi-blast-like (formative, F) mouse embryonic stem cells. Three experimental conditions were analyzed for each naive and formative ES cell conditions: control cell lines with loss of KMT2C (CKO), loss of both KMT2C and KMT2D (DKO), and double catalytic mutant KMT2C and KMT2D cells. Two biological replicates of Ok-seq were performed per condition.

first: Liana

last: Goehring

first: Deniz

last: Gökbuget

first: Robert

last: Blelloch


title: Transcriptomic analysis of microglia from young and aged wild-type, NLRP3-/-, APP/PS1 and APP/PS1.NLRP3-/- mice.

city: Luxembourg

first: Michael T.

last: Heneka

address: 6, Avenue du Swing,  4367 Esch-Belval

country: Luxembourg

institute: University of Luxembourg

summary: Activation of the NLRP3 inflammasome has been implicated in the pathogenesis of Alzheimer’s disease (AD), via the characterised release of IL-1β and ASC specks. However, whether NLRP3 was involved in pathways beyond this remained unknown. Here were enriched CD11b+ cells from the brains of 4, 6 an 12 month old wild-type, NLRP3-/-, APP/PS1 and APP/PS1.NLRP3-/- mice for bulk RNA sequencing. We found that amyloid deposition was associated with an increase in the expression of genes encoding inflammatory or phagocytic proteins from 6 months onwards. Interestingly, loss of NLRP3 influences glutamine/glutamate-related metabolism and increases expression of microglial Slc1a3. The increase in Slc1a3 was observed in all mice on a NLRP3-/- background and at all ages examined, demonstrating a new role for this transporter in microglia.

overall_design: Comparative gene expression analysis of RNA-seq for 4, 6 an 12 month oldwild-type, NLRP3-/-, APP/PS1 and APP/PS1.NLRP3-/- mice.

first: Róisín

middle: M

last: McManus

first: Alberto

last: Maillo

first: David

last: Gomez-Cabrero

first: Jesper

last: Tegner

first: Michael


title: Meiotic Maturation-associated RNA G-quadruplexes removal is Crucial for Translational Activation in Mouse Oocytes [LACE-seq]

city: Nanning

first: liu

last: shaoyuan

address: Guangxi province of china

institute: Guangxi university

summary: Following meiosis resumption, the transcription was silencing; and large number of maternal mRNA was actively translated and degraded during oocyte maturation. However, due to the limitation of sample number and technology, the dynamic distribution of RNA structure and its correlation with RNA dynamic change have never been revealed. RNA G-quadruplex (rG4) was a four-stranded RNA secondary structure related to translation regulation and RNA stability. In this study, we developed a low-input technique of rG4 detection (G4-lace-seq), and found that rG4s were highly abundant in fully grown oocytes and reduced during oocyte maturation. We ingeniously applied rG4 stable ligand BYBX in aim to eliminate the DNA G-quadruplex obstruction, and phenotypic analysis showed that rG4 accumulation severely impaired oocytes maturation. Above all, RBPs proteomic spectra revealed RNA structure change caused by G4 accumulation directly weakened the binding of RBPs to RNA, especially the enrichment of G4s affect the RBP binding associated with RNA metabolism, and G4s on the 5′-UTR hindered 40s ribosome movement and binding of 60s ribosome eukaryotic translation initiation factors to RNA. Combined analysis of G4-Lace-seq with Ribo-lite and RNA-seq, further proved that rG4s accumulation on UTRs disrupted translational efficiency and maternal mRNA degradation during oocyte maturation. Overexpression of DHX36, a G4s helicase, rescued the defects caused by BYBX to oocytes and restored the development rates. Jointly analyzing the function and distribution of rG4s and the metabolic signature of maternal mRNA, we hypothesized that a large amount of rG4s present before meiosis resumption is necessary to protect long-term mRNA stability from degradation in a state of poor efficiency translation. Following meiosis resumption, rG4s were cleaned up, translation efficiency increased, and the mRNA became unstable and was rapidly degraded. Collectively, our results announced for the first time that G4, as a secondary structure, had a hand in in the special dynamic rule of RNA content and translation. rG4s clearance determined the fate of cytoplasmic maturation during oocyte maturation.

overall_design: The 4-week-old female mice were injected with 5 IU of pregnant mare’s serum gonadotropin (PMSG, Ningbo Sansheng Pharmaceutical) and humanely euthanized 44h later. Oocytes were obtained in 37°C pre-warmed M2 medium (M7167; Sigma-Aldrich) at the GV stage and cultured in M16 medium (M7292; Sigma-Aldrich) covered with mineral oil (M5310; Sigma-Aldrich) at 37°C in a 5% CO2 atmosphere. BYBX (5 mM) was diluted to a gradient concentration by DMSO and added to M16 medium. Milrinone (2 μM) was applied with the culture medium to inhibit spontaneous GVBD in some experiments. G4-LACE-seq experiments were improved according to published procedure. Briefly, 50 oocytes were gathered in 1.5 ml Eppendorf LoBind microcentrifuge tubes and swiftly settled at the base using a mini centrifuge.

first: Qiongwen

last: Lu

first: Shaoyuan

last: Liu

first: Yujing

first: Qianqian

last: Sha


title: MAIT cells protect against sterile lung injury

first: Shuailin

last: Li

summary: Mucosal-associated invariant T (MAIT) cells, the most abundant unconventional T cells in the lung, have been recently linked to tissue protection and repair. Their role, especially in sterile lung injury, is unknown. Using single cell RNA sequencing (scRNA-seq), spectral analysis and adoptive transfer in a bleomycin-induced sterile lung injury, we found that bleomycin activates murine pulmonary MAIT cells and induces an accompanying tissue repair programme, associated with a protective role against bleomycin-induced lung injury. MAIT cells drive the accumulation of type 1 conventional dendritic cells (cDC1), limiting tissue damage in a DNGR-1 dependent manner. Human scRNA-seq data revealed that MAIT cells were activated, with increased cDC populations in idiopathic pulmonary fibrosis patients. Thus, MAIT cells enhance defence against sterile lung injury by fostering cDC1-driven anti-fibrotic pathways.

overall_design: MAIT cells were isolated from lung of C57BL/6 mouse challenged by bleomycin without previous intranasal BRD509 infection. Mouse Lung was collected before bleomycin challenge and 3 days and 7 days post bleomycin challenge. 27 mice were used, lung of 9 mice were collected before challenge, day 3 or day 7 post challenge, respectively. Resuspended lung cells from 3 mice were pool together to sort MAIT cells so that each time point has 3 samples of sorted MAIT cells for sequencing.

first: Xiawei

last: Zhang


title: RIPK3 promotes neuronal survival by suppressing excitatory neurotransmission during CNS viral infection

city: Piscataway

first: Brian

last: Daniels

state: NJ

address: 604 Allison Road

department: Cell Biology and Neuroscience

institute: Rutgers, The State University of New Jersey

summary: While recent work has identified roles for cytokines and inflammation in the regulation of neural activity, the capacity for cell intrinsic innate immune signaling within neurons to influence neurotransmission remains poorly understood. However, the existing evidence linking immune signaling with neuronal activity suggests that modulation of neurotransmission may serve previously undefined roles in host protection and pathogen control within the central nervous system. Here, we identify a specialized function for RIPK3, a kinase traditionally associated with necroptotic cell death, in preserving neuronal survival during neurotropic flavivirus infection through the suppression of excitatory neurotransmission. We show that RIPK3 coordinates transcriptomic changes in neurons that suppress neuronal glutamate signaling, thereby desensitizing neurons to excitotoxic cell death. These effects occur independently of the traditional functions of RIPK3 in promoting MLKL-dependent necroptosis and NFκB-mediated inflammatory transcription. Instead, RIPK3 promotes phosphorylation of the key neural regulatory kinase CAMKII, which in turn activates the transcription factor CREB to drive a neuroprotective transcriptional program and suppress deleterious glutamatergic signaling. These findings identify an unexpected function for a canonical cell death protein in promoting neuronal survival during viral infection through the modulation of neuronal activity, highlighting new mechanisms of neuroimmune crosstalk.

overall_design: We performed bulk RNA sequencing of primary cerebral cortical neurons derived from E15.5 embryos harboring a chemogenetically activatable verson of RIPK3 (RIPK3-2xFV) that can be activated with the drug B/B homodimerizer (B/B). Neurons were treated with B/B or vehicle solution in the presence of KN93 (CaMKII inhibitor), 666-15 (CREB inhibitor), or control solution for 24 hours prior to harvest.

first: Irving

last: Estevez

first: Benjamin

last: Buckley

first: Nicholas

last: Panzera

first: Marissa

last: Lindman

first: Tsui-Wen

last: Chou

first: Micheal

last: McCourt

first: Brandon

last: Vaglio

first: Colm

last: Atkins

first: Bonnie

last: Firestein

middle: P


title: Meiotic Maturation-associated RNA G-quadruplexes removal is Crucial for Translational Activation in Mouse Oocytes

first: liu

overall_design: Oocytes were collected at the indicated time after DMSO and BYBX treatment (10 oocytes per sample). Each sample was lysed using 4 μl lysis buffer (0.2% Triton X-100, RNase inhibitor, dNTPs, oligo-dT primers, and 100 pg external RNA controls consortium (ERCC) mRNA spike-in) and immediately used for cDNA synthesis based on Smart-seq2 method. Sequencing libraries were constructed from 500 pg of amplified cDNA using the TruePrep DNA Library Prep Kit V2 for Illumina (Vazyme; TD503) according to the manufacturer’s instructions. Barcoded libraries were pooled and sequenced on the Illumina HiSeq X Ten platform using 150 bp paired-end reads.


title: Subcutaneous adipose gene expression comparison between NW, OW, PT2, and T2D individuals

city: Ciudad de México

first: Ricardo

last: Orozco-Solis

state: TLALPAN

address: Periférico Sur 4809, Arenal Tepepan

country: Mexico

institute: Instituto Nacional de Medicina Genomica

summary: The prevalence of type 2 diabetes (T2D) has increased significantly over the past three decades, with an estimated 30-40% of cases remaining undiagnosed. Brown and beige adipose tissues are known for their remarkable catabolic capacity, and their ability to diminish blood glucose plasma concentration. Beige adipose tissue can be differentiated from adipose-derived stem cells or through transdifferentiation from white adipocytes. However, the impact of T2D progression on beige adipocytes' functional capacity remains unclear. Transcriptomic profiling of subcutaneous adipose tissue biopsies from healthy normal-weight, obese, prediabetic obese, and obese subjects diagnosed with T2D, reveals a progressive alteration in cellular processes associated with catabolic metabolism, circadian rhythms, thermogenesis-related signaling pathways, cellular stress, and inflammation. MAX is a potential transcription factor that links inflammation with the circadian clock and thermogenesis during the progression of T2D. This study unveils an unrecognized transcriptional circuit that increasingly disrupts subcutaneous adipose tissue oxidative capacity during the progression of T2D. These findings could open new research venues for developing chrono-pharmaceutical strategies to treat and prevent T2D.

overall_design: Subcutaneous adipose tissue from four groups of participants, divided into normal weight (NW), overweight/obesity (OW), overweight/obesity pre-type 2 diabetes (PD), and overweight/obesity type 2 diabetes (T2D) based on their clinical parameters, was studied.

first: R

first: C

last: Aguilar-Salinas


title: Oncohistone H3.3K27M-driven CREB5/ID1 Axis Mediates the Malignant Cell States of Diffuse Intrinsic Pontine Glioma (DIPG) [RNA-seq]

first: Qiaoran

last: Xi

overall_design:
To investigate the transcriptome impact of CREB5 in the regulation of DIPG malignant cell state, we performed total RNA-seq experiments in WT, CREB5 knock down (KD) DIPG17 cells.
To investigate the transcriptome impact of ABBV-075 in the regulation of DIPG malignant cell state, we performed total RNA-seq experiments in DIPG17 cells treated with DMSO and ABBV-075.

first: Wei

last: Zhou


title: Compact transcription factor cassettes generate functional, engraftable motor neurons by direct conversion

city: Cambridge

first: Kate

last: Galloway

summary: Direct conversion generates patient-specific, disease-relevant cell types such as neurons that are rare, limited, or difficult to isolate from common and easily accessible cells such as skin cells. However, low rates of direct conversion and complex protocols limit scalability and thus the potential of cell-fate conversion for biomedical applications. Systems-level optimization of conversion protocols across molecular and cellular scales can support scalable cell manufacturing for diverse application. We use insights from our work on how proliferation and transcription factor levels act together to drive direct conversion to optimize the conversion protocol by examining process parameters including transcript design, delivery via AAV, retrovirus and lentivirus, cell seeding density, and the impact of media conditions. Thus, we report a compact, portable conversion process that boosts proliferation and increases direct conversion of mouse fibroblasts to induced motor neurons to achieve high conversion rates of above 1,000%, corresponding to more than 10 motor neurons yielded per cell seeded which we achieve through expansion. Our optimized, direct conversion process generates functional motor neurons at scales relevant for cell therapies (>107 cells) that graft with the mouse central nervous system. High-efficiency, compact, direct conversion systems will support scaling to patient-specific, neural cell therapies.

overall_design: Transcriptional profiling of cells during the process of direct conversion of mouse embryonic fibroblasts to induced motor neurons (iMNs) and at the terminal stage at 14 dpi. Terminal samples at 14 dpi include Hb9::GFP+ sorted iMNs by fluorescence-activated cell sorting (FACS) generated with LNI + DDRR (LNI is mLhx3-P2A-mNgn2x3HA-T2A-Isl1; DDRR is HRAS(G12V)-IRES-p53DD) in the presence and absence of supplements (neurotrophic growth factors: BDNF, CNTF, GDNF, FGF; and RepSox). Samples at 4 dpi in conversion for LNI + DDRR with supplements for CellTrace-FarRed-low population sorted by FACS represent cells in the intermediate stages of direct conversion.

first: Nathan

last: Wang