title: TurboCas: A method for locus-specific labeling of genomic regions and isolating their associated protein interactome [RNAseq]

status: Public on Dec 21 2024

city: Chicago

first: Ali

last: Shilatifard

state: IL

address: 320 E Superior St

department: Department of Biochemistry and Molecular Genetics

institute: Northwestern University Feinberg School of Medicine

summary: Regulation of gene expression during development and stress response requires the concerted action of transcription factors and chromatin-binding proteins. Because this process is cell-type specific and varies with cellular conditions, mapping of chromatin factors at individual regulatory loci is crucial for understanding cis-regulatory control. Previous methods only characterize static protein binding. We present “TurboCas,” a method combining a proximity-labeling (PL) enzyme, miniTurbo, with CRISPR-dCas9 that allows for efficient and site-specific labeling of chromatin factors in mammalian cells. Validating TurboCas at the FOS promoter, we identify proteins recruited upon heat shock, cross-validated via RNA polymerase II and P-TEFb immunoprecipitation. These methodologies reveal canonical and uncharacterized factors that function to activate expression of heat-shock-responsive genes. Applying TurboCas to the MYC promoter, we identify two P-TEFb coactivators, the super elongation complex (SEC) and BRD4, as MYC co-regulators. TurboCas provides a genome-specific targeting PL, with the potential to deepen our molecular understanding of transcriptional regulatory pathways in development and stress response.

overall_design:
The miniTurbo proximity labeling enzyme was fused to the N terminus of a catalytically dead Cas9 and cloned into a lentiviral expression vector with an in-frame blasticidin selection cassette, separated by a 2A autocatalytic peptide. HEK293T human embryonic kidney cells and HCT116 human colon carcinoma cells were used to stably express TurboCas or MiniTurbo. To target TurboCas to the FOS locus, sgRNA were designed against regions ~600bp and ~300bp upstream of the transcription start site (TSS) and the first FOS exon (hereafter referred to as FOS-600, FOS-300, FOX-EX1). TurboCas or MiniTurbo cell lines were then transduced with LentiGuide-Puro virus containing the sgRNA and selected with Puromycin to generate stable sgRNA-expressing cell lines. To confirm that TurboCas was specifically binding at the intended sgRNA target sites, we assessed TurboCas chromatin binding by performing ChIP-seq with a commercial polyclonal dCas9 antibody. In addition to the three sgRNA cell lines (TurboCas-Fos600, TurboCas-Fos300, TurboCas-FosEX1), the experiments were also performed in the MiniTurbo and TurboCas-empty controls. To functionally validate hits identified through TurboCas mass spectrometry, we selected the top hits as targets  and performed shRNA knockdown of these genes in HCT116 cells. Cells were selected with puromycin for 10 days and then heat shocked for 3 hours, followed by RNA extraction and RNA-seq both to evaluate shRNA efficiency and to assess gene expression patterns.
For dCas9 ChIP, 5 x 107 cells were used. For Pol2 ChIP, 1 x 107 cells were used. In brief, HCT116 or HEK293T cells were fixed in 1% formaldehyde for 15 minutes, followed by quenching, cell lysis and chromatin shearing with an E220 focused ultrasonicator (Covaris). Sheared chromatin was subjected to immunoprecipitation with a Cas9 antibody (Diagenode, #C15310258-100) or a Pol2 antibody (Cell Signaling Technologies, 14958). Immunoprecipitated DNA was then washed, eluted, revers-crosslinked and purified using a ChIP DNA Clean&Concentrator kit (Zymo Research, D5205) prior to library preparation using the KAPA HTP library preparation kit (KAPA Biosystems). RNA from HCT116 cells was isolated using the RNeasy mini kit (Qiagen, #74106) as per manufacturer’s instructions. RNA-seq libraries were prepared using the NEBNext® Ultra™ II Directional RNA Library Prep Kit for Illumina® (New England Biolabs, # E7760L).
ChIP-seq and RNA-seq libraries were paired-end read sequenced on the NovaSeq 6000 (Illumina). Raw BCL output files were processed using bcl2fastq (Illumina, version 2.20) prior to quality trimming using CutAdapt1.14. Trimmed ChIP-seq reads were aligned to the human genome (University of California at Santa Cruz [UCSC] hg38 build) using Bowtie v2.2.6. Only uniquely mapped reads satisfying the two-mismatch maximum threshold within the entire length of the gene were considered for ensuing analyses. Mapped ChIP-seq reads with MAPQ quality score >=30 were extended to 150bp to represent sequenced fragments. Raw read counts from both ChIP-seq were normalized to total reads per million (RPM). Output BAM files were converted into bigwig coverage plots to generate UCSC genome browser tracks. Peak calling was performed using MACS2 with the  default (narrow) option. Called peaks were annotated with HOMER program annotatePeaks.pl. Heatmaps and metaplots from bigwig files were generated using deepTools v3.1.1. For region-based categorization of dCas9 signal, bed files were split by chromosome and coordinate (in the case of chromosome 14), these split files were then used as inputs for profile plotting with deepTools. RNA-seq reads were aligned to the hg38 human genome using STAR aligner v2.5.2 and outputted bam files were sorted using samtools v1.6. For RNA-seq, exonic reads were assigned to specific genes from Ensembl release 72 and genome assembly GRCh38.p13 using the python package HTSeq-0.6.1.

first: Bercin

last: Cenik

first: Yuki

last: Aoi

first: Marta

last: Iwanaszko


title: Phase separation of RXRγ drives tumor chemoresistance and represents a therapeutic target for small cell lung cancer [ChIP-Seq]

status: Public on Dec 31 2024

city: Guangzhou

first: Hong

last: Wang

address: 132, Waihuandonglu, Daxue Cheng

department: School Of Pharmaceutical Sciences

country: China

institute: Sun Yat-sen University

type: Genome binding/occupancy profiling by high throughput sequencing

summary: Small cell lung cancer (SCLC) is the most lethal type of lung cancer, characterized by limited treatment options and rapid evolution from chemosensitivity to chemoresistance. However, the mechanisms underlying this evolution remain poorly understood. Identifying the druggable drivers and developing pharmacological strategies to overcome chemoresistance are imperative. Here, we show that Retinoid X receptor γ (RXRγ) is uniquely overexpressed in chemo-resistant SCLC tumors, and that RXRγ serves as an essential factor driving chemoresistance in SCLC. RXRγ forms phase-separated droplets with LSD1 in the nucleus, which enhances RXRγ-mediated gene transcription activity and reprograms gene expression, promoting tumor stemness and metastasis, and eventually driving SCLC chemoresistance. In turn, RXRγ antagonist disrupts RXRγ-LSD1 interaction, reducing their binding to the target gene locus, markedly suppressing the expression of the RXRγ target gene network. Finally, RXRγ antagonists strongly suppress tumor growth and metastasis and restore SCLC vulnerability to chemotherapy in multiple preclinical SCLC models, without overt toxicity, in mice. Thus, these results establish RXRγ as a key player in SCLC by phase separation and as a potential therapeutic target for this deadly disease.

overall_design: ChIP-Seq

first: Jie

last: Huang

first: Zhenhua

last: Zhang

first: Yana

last: An


title: Lactylated histone promotes rheumatoid arthritis progression by increasing NFATc2 expression and the production of anti-lactylated histone autoantibodies

status: Public on Dec 27 2024

city: Wenzhou

first: Wu

last: Gan

address: Chashan Street, Ouhai District

institute: Wenzhou Medical University

type: Expression profiling by high throughput sequencing

summary: The elevated lactate in the joint microenvironment of patients with rheumatoid arthritis (RA) is crucial for disease progression, although the underlying mechanism remains to be elucidated. In this study, we observed significantly increased global lactylation levels within fibroblast-like synoviocytes (FLS) of RA patients compared to HC (healthy controls), and lactylated proteins were enriched in histones. Furthermore, we found that anti-lactated histone antibodies were detected in almost RA patients and positively correlated with Disease Activity Score 28 (DAS28). Then we identified NFATc2 as a key target gene regulated by histone H3 lysine 9 lactylation (H3K9la) using CUT&Tag combined with RNA-seq. Functional studies revealed that NFATc2 promotes migration of RA-FLSs. Additionally, using server combined immune-deficiency (SCID) and collagen-induced arthritis (CIA) mouse models, we demonstrated that NFATc2 exacerbates RA disease progression through enhanced FLS cartilage invasion function. Collectively, our findings suggest that upregulated target gene NFATc2 by lactate-dependent histone lactylation, can be used as a potential therapeutic target for intervention, anti-lactated histone autoantibodies is promising as a diagnostic marker for RA.

overall_design: RNA-seq profiling of fibroblast-like synoviocytes (FLS) from RA patients and healthy controls

first: Gan

last: Wu


title: TurboCas: A method for locus-specific labeling of genomic regions and isolating their associated protein interactome [ChIPseq]

country: USA

middle: K


title: Genome-wide CRISPR knockout screening with viral replicons for identification of host factors involved in viral replication

status: Public on Dec 20 2024

city: San Francisco

first: Amy

middle: L

last: Kistler

state: CA

address: 499 Illinois St, 4th floor

institute: Chan Zuckerberg Biohub

summary: Pooled CRISPR knockout (KO) screens using live viruses are a proven and valuable approach for identifying essential host factors acting across viral life cycles. Here we describe the development of a pooled genome-wide CRISPR KO screening approach using stable viral replicon cell lines to specifically identify host factors essential for viral replication. Virus replicons are non-infectious, therefore enabling the study of highly virulent viruses under standard biosafety level 2 containment. We developed a stable fluorescent dengue virus type 2 (DENV-2) replicon cell line to perform a pooled genome-wide FACS-based CRISPR KO screen. This benchmark DENV-2 replicon screen successfully identified host genes previously known to be required for viral DENV-2 replication (e.g., endoplasmic reticulum membrane complex and oligosaccharyltransferase complex components) and confirmed two additional genes (DOHH and ZFP36L2) involved in replication that have not been recovered in prior live virus screens. We applied this replicon screening approach to two highly divergent viruses: chikungunya virus (CHIKV), a positive-sense RNA virus that replicates at the plasma membrane, and Ebola virus (EBOV), a negative-sense RNA virus that replicates in cytoplasmic inclusion bodies. The CHIKV replicon screen identified two genes known to be required for replication (G3BP1 and G3BP2) and several additional, novel genes (CLEC4G, CSDE1, GOLGA7, HNF1A, and PCBD1). We verified two of them (CSDE1 and GOLGA7) in live CHIKV replication assays. A distinct set of genes (EHMT1, EHMT2, and USP7) were recovered in the EBOV replicon screen and were further confirmed using independent transient transfection assays. Thus, viral replicon-based screens provide a useful approach that can be extended to viruses of diverse taxa to identify host pathways essential for viral replication and to uncover potential novel targets for host-directed medical countermeasures.

overall_design: Comparative sequence analysis of sgRNAs integrated in genomic DNA collected from 2 populations of Huh7.5.1-Cas9 cells harboring different stable fluorescent viral replicons (DENV2, CHIKV, and ZEBOV). Each replicon cell line was transduced with a pool of lentiviruses harboring the Brunello sgRNA library. An unsorted cell populuation was harvested at day 0 post-transduction, and FACS-sorted populations with different types of fluorescent replicon signal (GFP-low and/or GFP-high) were harvested 19 days post-transduction. For each viral replicon cell line, gDNA was extracted from the sorted cells and unsorted cells, sequenced, and compared to compute enrichment scores of targeted genes recovered in the sorted vs unsorted cell populations.

first: Karen

last: Cheng

first: Madhura

last: Bhave

first: Andrew

last: Markhard

first: Duo

last: Peng

first: Karan

middle: D

last: Bhatt

first: Katherine

last: Travisano

first: Josette

middle: V

last: Medicielo

first: Leila

last: Njoya

first: Sanae

last: Lembirik

first: Manu

last: Anantpadma

first: Andreas

middle: S

last: Puschnik

first: Jens

middle: H

last: Kuhn


title: Characterization of the cellular components of mouse collecting lymphatic vessels reveals that lymphatic muscle cells are the innate pacemaker cells regulating lymphatic contractions

city: Columbia

first: Sathesh

last: Sivasankaran

state: MO

address: Bond Life Sciences Center Suite 106, 1201 Rollins Street

institute: University of Missouri

summary: Collecting lymphatic vessels (cLVs) exhibit spontaneous contractions with a pressure-dependent frequency, but the identity of the lymphatic pacemaker cell is still debated. By analogy to pacemakers in the GI and lower urinary tracts, proposed cLV pacemaker cells include interstitial cells of Cajal like cells (ICLC) or the lymphatic muscle (LMCs) cells themselves. Here we examined the cellular constituents of the mouse cLV wall and assessed whether any cell type exhibited morphological and functional processes characteristic of pacemaker cells: a continuous if not contiguous network; spontaneous Ca2+ transients;  and depolarization-induced propagated contractions. We employed inducible Cre (iCre) mouse models routinely used to target these specific cell populations including: c-kitCreERT2 to target ICLC; PdgfrβCreERT2 to target pericyte like cells; PdgfrαCreERTM to target CD34+ adventitial cells and ICLC; and Myh11CreERT2 to target LMCs directly. These specific inducible Cre lines were crossed to the fluorescent reporter ROSA26mT/mG, the genetically encoded Ca2+ sensor GCaMP6f, and the light-activated cation channel rhodopsin2 (ChR2). c-KitCreERT2 labeled both a sparse population of LECs and round adventitial cells that responded to the mast cell activator compound 48-80. PdgfrβCreERT2 drove recombination in both adventitial cells and LMCs, limiting its power to discriminate a pericyte specific population. PdgfrαCreERTM labeled a large population of interconnected, oak leaf-shaped cells primarily along the adventitial surface of the vessel. Of these cells, only LMCs consistently, but heterogeneously, displayed spontaneous Ca2+ events during the diastolic period of the contraction cycle, and whose frequency was modulated in a pressure-dependent manner. Optogenetic depolarization through the expression of ChR2 under control of Myh11CreERT2, but not PdgfrαCreERTM or c-KitCreERT2, resulted in a propagated contraction upon photo-stimulation. Membrane potential recording in LMCs demonstrated that the diastolic depolarization was significantly correlated with contraction frequency. These findings support the conclusion that LMCs, or a subset of LMCs, are responsible for mouse cLV pacemaking.

overall_design: IALVs we used a total of 10 Rosa26mTmGmice, without Cre and no tamoxifen, with equivalent representation of sex (5 males and 5 females). Full length IALVs from both the left and right side of each mouse ROSA26mTmG were isolated and cleaned of excessive matrix and adipose tissue. Isolated vessels were digested into single cell suspensions as described above and the cells were kept on ice following single cell suspension until all the tissues had been processed. Cells from all vessels were combined and FACs purified by tdTomato expression to remove debris and concentrate the cells for downstream single cell 3’ RNA-Seq libraries creation with 10x Genomics Chromium Chip and Chromium Next GEM Single Cell 3’ RNA-Seq reagents. Samples were sequenced with the NovaSeq 6000 S4-PE100 flow cell

first: S

last: Zawieja

first: G

middle: A

last: Pea

middle: E

last: Broyhill

first: A

last: Patro

first: K

last: Bromert

first: C

last: Norton

first: H

middle: J

last: Kim

first: M

last: Li

first: J

last: Castorena-Gonzalez

first: B

middle: T

last: Drumm

last: Davis


title: Plasma multi-omics identify early pathogenesis and biomarkers of SCD post-AMI

city: Harbin

first: Maomao

last: Zhang

state: Heilongjiang Province

address: Baojian Road Sub district, Nangang District

institute: The Second Affiliated Hospital of Harbin Medical University

summary: Early identification and treatment of Sudden Cardiac Death (SCD) post-myocardial infarction (MI) are currently limited by unclear warning indicators and elusive pathogenesis. Here, we constructed a comprehensive multi-omics blood atlas in the acute phase of AMI from 55 pairs of SCD patients and matched survivors, confirmed immune dysregulation and metabolic disorders as the biological hubs leading to post-MI SCD. Targeted proteome quantification identified a TOP4 protein-based biomarker panel, which were validated by internal and external cohorts, and confirmed a superior early prediction power of SCD to traditional clinical risk models. Further interventions targeting complement factor D (CFD) suggested a potential therapeutic strategy for high-risk MI. Our study provided valuable knowledge about the molecular changes and pathogenesis of SCD, shed light on accurate risk stratification and potential therapeutic targets in high SCD-risk MI.

overall_design: After informed consent was obtained, fasting blood samples (5 mL) were collected within 24 hours after emergent admission using EDTA tubes.We collected  110 samples (55 SCD and their matched survivor).Total RNA from peripheral blood mononuclear cells (PBMCs) which were separated within 20 min after blood collection was isolated using TRIzol™ reagent (Invitrogen, Carlsbad, CA, USA), followed by 15 min incubation at room temperature and subsequent freezing at -80°C before total RNA extraction.


title: Effect of osteocytes mitochondria on chondrocytes

city: Shanghai

first: Junjie

last: Gao

address: 600 Yishan Road

institute: Shanghai Sixth People’s Hospital Affiliated to Shanghai Jiao Tong University School of Medicine

summary:
To investigate the regulatory role of MLO-Y4 mitochondria on chondrocytes, we isolated mitochondria from MLO-Y4 and transplanted to chondrocytes.
We then performed gene expression profiling analysis using data obtained from RNA-seq of chondrocytes from vehicle group and mitochondria transplanted group.

overall_design: Comparative gene expression profiling analysis of RNA-seq data for normar chondrocytes and chondrocytes tranplanted with mitochondria.

first: Yiyang

last: Ma

first: Changqing


title: Giardiavirus rewires host translation and glycolytic metabolism to support its replication in Giardia duodenalis

city: changchun

first: lu

last: li

institute: Jilin university

summary: Giardia duodenalis is a prevalent intestinal pathogens known to cause giardiasis, a condition characterized by diarrhea and frequently linked to malnutrition and growth impairments in children. The presence of Giardiavirus (GLV) in Giardia strains has been associated with heightened immune cytokine responses in the host compared to the GLV-free strains. However, the transmission mode and biological significance of GLV remain unclear. In this study, by using G. duodenalis DH (GLV-containing) and WBC6 (GLV-free) strains, we demonstrated that the DH strain produced extracellular vesicles (EVs), which originated from unique peripheral vesicle and bead-like structures in the ventrolateral flange. Nanoparticle tracking analysis revealed that GLV infected-G. duodenalis DH strain secreted fewer EVs than the GLV-free WBC6 strain. Biochemical and electron microscopy demonstrated that GLV virions can exploit the Giardia EVs pathway to facilitate their spread among parasites. Giardia uptake of GLV-containing EVs occured through clathrin-mediated endocytosis, leading to rapid infection of trophozoites by GLV. Furthermore, GLV infection enhanced messenger RNA translation efficiency, influencing protein abundance in Giardia trophozoites. The presence of GLV also upregulated the glycolytic pathway, with Giardia enolase closely associated with GLV replication. Importantly, Giardia infected with GLV alleviated the pathogenicity of parasite compared to the GLV-freeGiardia strain. These findings highlight the pivotal role of GLV in regulating Giardia biology, suggesting its potential for informing the development of novel intervention strategies against Giardia infections.

overall_design: Comparative genes translation efficiency on the three species by Monosomes RNA-seq and polysomes RNA-seq analyses

first: Lu


title: RNA sequencing of TA skeletal muscle of mice with conditional ablation of dystrophin within the myofiber

status: Public on Dec 25 2024

city: Birmingham

first: Matthew

last: Alexander

state: AL

address: 1900 University Blvd. THT 929 Box# 96

department: Pediatrics

institute: University of Alabama at Birmingham

summary: We generated and analyzed a conditional Dystrophin flox52/Y: human alpha-skeletal actin muscle knockout (Dmd flox52/Y: HSA mKO) model by targeting exon 52 deletion in the Dystrophin gene to study the consequences of dystrophin loss in skeletal muscle lineages. The generated DMD mouse model ablates dystrophin protein expression after mating with a Cre recombinase transgenic mouse under the control of HSA promoter element that is restricted to the skeletal muscle. The resulting Dmd mKO mice have been assessed using histopathological, phenotypical, functional and biochemical assays based on TRET-NMD standard operating protocols (SOPs) for mdx mouse models. Phenotypic analysis of these conditional Dmd mKO mice revealed a significant decline in locomotor activity and reduced muscle force, motor and muscular function. The histochemical analysis revealed an increase in centralized myonuclei and fibrotic area similar to mdx mice. Immunoassays including western blot and immunohistochemistry confirmed low expression levels of dystrophin in skeletal muscles of Dmd mKO mice. Bulk RNA sequencing analysis revealed that dystrophin loss in myofiber significantly disrupted the expression of cytokines and extracellular matrix genes.

overall_design:
To investigate the molecular changes of myofiber dystophin loss in Dmd flox52/Y: HSA mKO mice
Five mice per group were used for RNA-sequencing and gene expression analysis. Total RNA was extracted from tibialis anterior (TA) muscle using Trizol reagent. Quality of RNA was assessed by Nanodrop and Bioanalyzer. Purified RNA was used to generate cDNA. Duplicates of cDNA samples were amplified on the QuantStudio 3 Real-Time PCR system using PowerUp SYBR Green Master Mix. RNA sequencing library construction and next-generation sequencing were performed by Novogene. Purified RNA from TA muscles of Dmd flox52/Y: HSA mKO and Dmd flox52/Y mice was measured by Agilent Bioanalyzer. High quality total RNA with RNA integrity value higher than 7 was used for library construction. After mRNA isolation, RNA was fragmented in First Strand synthesis buffer. Then, first-strand CDNA synthesis using reverse transcriptase and random hexamer primer was performed. Synthesis of cDNA using second-strand master mix, end-repair and dA-tailing were performed. The library was amplified and purified using AMPure beads. The library size and mass were evaluated and quantititative PCR was performed to validate the libraries. Barcoded samples were pooled equimolarly for simultanoeus sequencing. The quality of RNA-sequencing data was assessed for downstream analysis. Low-quality sequences and reads were removed. The expected read counts and FPKM were used for further analysis.

first: Muthukumar

last: Karuppasamy


title: Breast cancer brain metastasis

city: sadel crick road

first: Mohd

middle: wasim

last: Nasser

state: Omaha

address: 42 and Emile

department: BMB

institute: UNMC

summary: Investigating the differential expression of gene during breast cancer brain metastasis

overall_design: We then performed gene expression profiling analysis using data obtained from RNA-seq of cells. Comparative gene expression profiling analysis of RNA-seq data of cells (Breast cancer and breast cancer brain metastatic cells).

first: Surinder

last: Batra

middle: W

first: Jawed

last: Siddiqui


title: liver GR epigenomic changes with rs6190 SNP genocopy

status: Public on Dec 19 2024

city: Cincinnati

first: Mattia

last: Quattrocelli

state: OH

address: 240 Sabin Way

institute: Cincinnati Children's Hospital Medical Center

summary: this dataset encompasses the liver GR epigenomic changes elicited in 4-month-old mice by the rs6190 genocopy allele

overall_design: We compared 4 months-old mice (C57BL/6J background) homozygous for the rs6190 SNP genocopy allele (R24K-homo) vs homozygous for the reference allele (R24K-WT). Livers were collected at ~ZT4 and immediately flash frozen for further processing.

first: Hima

middle: B

last: Durumutla


title: Differential Gene Expression in oxaliplatin-resistant versus unteated PANC1 cells

city: Boston

first: Jonathan

middle: P

last: Celli

state: MA

address: 100 Morrissey Blvd

institute: University of Massachusetts Boston

summary: We sought to examine changes in gene expression of PANC1 cells with acquired resistance to oxaliplatin chemotherapy. Cultures of PANC1 cells, an established pancreatic cancer cell line, were exposed to oxaliplatin chemotherapy over multiple passages and acquired a stable drug-resistant phenotype labeled as PANC1OR. Previously reported immunofluorescence and western blots indicate that PANC1OR exhibit increased EMT (increased cytoskeletal vimentin, decreased E-cadherin and fibroblast-like morphology). Here we sought to examine more broadly the differential expression of genes associated with acquired drug resistance in these cells.

overall_design: As reported in Cramer et al Mol Cancer Res (2017) PANC1 cells were grown with increasing concentrations of oxaliplatin (Tocris) in regular complete media over successive passages until a stable proliferative phenotype (PANC1OR) without chemotherapy was observed and maintained following cryopreservation and thawing. Drug resistance was confirmed by comparative dose response and measurement of a statistically significant increase in IC50. RNA was extracted from triplicate cell cultures of PANC1 and PANC1OR cells using the TRIzol reagent (Life Technologies Corporation, Carlsbad, CA). RNAseq was performed by the Center for Personalized Therapy Genomics Core at University of Massachusetts Boston. The quality of the raw fastq files were assessed using FastQC (v.0.11.5). Adaptor sequences, “AGATCGGAAGAGCACACGTCTGAACTCCAGTCA”, and “AGATCGGAAGAGCGTCGTGTAGGGAAAGAGTGT" were trimmed from the 3’ end of the reads using Cutadapt commandline tool form the Trime Galore package (v.0.4.2). The trimmed reads were mapped against the human reference genome (Ensemble, GRCh38) using STAR/2.5 using default parameters25. The average alignment rate was 96%. The sorted BAM files generated by STAR were used to estimate the transcript abundance per sample using featureCount from the Subread package (v.1.6.2)26. Gene expression analysis was performed using the edgR Bioconductor R package (v.3.24.3)27. The edgeR TMM method (trimmed mean of M values) was applied to the filtered genes utilizing the DGElist(), calcNormFactors(), estimateGLMCommonDisp(), estimateGLMTrendedDisp(), estimateGLMTagwiseDisp() functions. The glmFit and glmLRT functions from edgeR were used to fit a negative binomial generalized log-linear model to the read counts. The expression of the genes was ranked by logFoldChange (logFC) and false discovery rate (FDR). Differentially expressed genes (DEGs) were determined using abs(logFC) > 2 and FDR < 0.01 cutoffs, resulting in 1342 protein coding DEGs.

first: Eric

last: Struth

first: Maryam

last: Labaf

first: Vida

last: Karimnia

first: Yiran

last: Liu

first: Gwendolyn

middle: M

last: Cramer

first: Joanna

last: Dahl

first: Frank

last: Slack

first: Kourosh

last: Zarringhalam


title: Gene expression data of NMDAR-E Associated with Ovarian Teratoma

first: Hexia

last: Xia

state: --- Select One ---

address: No.419 Fangxie Rd. Shanghai

institute: Obstetrics and Gynecology Hospital of Fudan University

summary: Purpose: To identify significantly differentially expressed genes and to investigate the intricate molecular regulatory network underlying anti-NMDA receptor encephalitis associated with ovarian teratoma. Methods: This retrospective study analyzed ovarian teratoma samples from patients with and without NMDAR-E. We employed RNA sequencing  for gene expression profiling. qPCR and Western blotting were used for gene and protein expression validation. Results: We identified 2524 significantly differentially expressed genes. The changes were notable in mRNA  levels in ovarian teratomas associated with NMDAR-E. Functional enrichment analysis highlighted extracellular matrix and immune activation pathways. Key genes and proteins involved in ferroptosis and immune activation, including SLC40A1, GGH, FKBP11, CCDC80, and ANK3, showed significant expression differences.Conclusions: Our findings offer deeper insights into the pathophysiology of NMDAR-encephalitis associated with ovarian teratomas. The identified biomarkers, particularly in ferroptosis and immune activation pathways, provide potential targets for diagnosis and treatment.

overall_design: To identify significantly differentially expressed genes and to investigate the intricate molecular regulatory network underlying anti-NMDA receptor encephalitis associated with ovarian teratoma, four ovarian teratoma samples from patients with NMDAR-E and three samples without NMDAR-E were collected. We then performed gene expresssion analysis of RNA-seq data of these samples.

first: Lingwei

last: Ma

first: Xinmen

last: Sun

first: Wei

last: Zhang


title: liver transcriptional changes with rs6190 SNP genotype (R24K in GR protein)

summary: this dataset encompasses the transcriptomic changes elicited in liver tissues of 4-months-old mice with either the artificially-knock-in rs6190 genotype, or the control non-mutant (WT) genotype.

overall_design: Mice were obtained and interbred from the CCHMC Transgenic Core through CRISPR-mediated genomic engineering. Analyses were performed at ~16 weeks of age, comparing livers from littermate female mice homozygous for the SNP or homozygous for the reference allele. Tissues were harvested at ~ZT4.


title: Noncanonical role of ALAS1 as a heme-independent inhibitor of small RNA–mediated silencing [hepatocyte_sRNA-seq]

city: New York

first: Seungjae

last: Lee

state: NY

address: 430 East 67th St.

department: Department of Developmental Biology

institute: Sloan-Kettering Institute

type: Non-coding RNA profiling by high throughput sequencing

summary: RNA interference (RNAi) is a fundamental strategy for gene silencing via small interfering RNAs (siRNAs) that program Argonaute (Ago) effectors, and is related to endogenous silencing via microRNAs (miRNAs). Although most efforts focus on factors that are required for miRNA/RNAi function, biological systems are typically subject to repression. Using molecular genetic approaches, we uncover ALAS1, the first enzyme in the heme biosynthesis pathway, as a repressor of miRNA accumulation in cells and in mice. This was non-intuitive, since heme is known to play a positive role as a cofactor for the nuclear miRNA processing machinery. Instead, we show that ALAS1 (but not other core heme biosynthesis factors) limits RISC assembly and activity. Notably, ALAS1 is the target of one the few current FDA-approved siRNA drugs (Givosiran). Our data have implications for side effects of Givosiran, but also for RNAi therapies in general. We provide evidence that depletion of ALAS1 enhances siRNA-mediated knockdown, suggesting that Givosiran is a viable RNAi adjuvant.

overall_design: To investigate the role of ALAS in small RNA regulation, we established ALAS1/2 mutant hepatocyte and validated small RNA expressions

first: Sangmi

first: Robert

last: Desnick

first: Makiko

last: Yasuda

middle: C

last: Lai


title: Noncanonical role of ALAS1 as a heme-independent inhibitor of small RNA–mediated silencing [hepatocyte RNA-Seq]

overall_design: To investigate the cooperative function of Alas in mouse hepatocytes, we engineered a conditional mouse allele that permits Cre-inducible deletion of Alas1 exons 4-5 and further depleted Alas2 by injecting GalNAc-conjugated siRNAs.


title: Longitudinal gene expression profiling in Nodal-induced and control zebrafish embryonic explants

status: Public on Dec 16 2024

city: Houston

first: Amrit

last: Koirala

state: TX

address: 1 Baylor Plaza

institute: Baylor College of Medicine

summary: We report RNA-sequencing from zebrafish explants generated from embryos injected with mRNA encoding the Nodal ligand ndr2 (N), constitutively active Nodal receptor CA-acvr1b* (T), and uninjected (U) controls. Explants of all 3 conditions were compared at each of 7 developmental stages: sphere (SR), 30% epiboly (30), 50% epiboly (50), shield (SH), 75% epiboly (75), 90% epiboly (90), and 2-somite (2S) (corresponding to 4, 4.7, 5.3, 6, 8, 9, and 11 hours post fertilization, respectively).

overall_design: Sequencing of polyadenylated mRNAs from ndr2 (N), CA-acvr1b*(T), and uninjected (U) explants with 3 biological replicates (A-C) per condition per stage (SR - 2S).

first: Margot

last: Kossmann Williams

first: Cristian

last: Coarfa


title: Noncanonical role of ALAS1 as a heme-independent inhibitor of small RNA–mediated silencing [HEK293T_sRNA-seq]

overall_design: To investigate the role of ALAS1 in small RNA regulation, we established ALAS1 knock-out HEK293T cells and validated small RNA expressions

first: Eric


title: CTCF-mediated 3D chromatin predetermines the gene expression program in the male germline (ChIP-seq)

status: Public on Dec 13 2024

city: Davis

first: Satoshi

last: Namekawa

address: One Shields Avenue

department: Department of Molecular and Cellular Biology

institute: University of California, Davis

summary: Spermatogenesis is a unidirectional differentiation process to produce haploid sperm. However, it remains largely unknown how the gene expression program is determined to direct a unidirectional differentiation. Here we unveil the high-resolution 3D chromatin architecture of male germ cells and show that CTCF-mediated 3D chromatin predetermines the gene expression program required for spermatogenesis. At the mitosis-to-meiosis transition, the germline-specific Polycomb protein SCML2 resolves chromatin loops specific to mitotic spermatogonia. On autosomes, CTCF-mediated 3D chromatin manifests the structural feature of meiosis-specific super-enhancer (meiotic SE) loci already in undifferentiated spermatogonia. In meiotic spermatocytes, the master transcription factor A-MYB is recruited to these meiotic SE loci to strengthen their 3D contacts to instruct the burst of meiotic gene expression. Further, SCML2 and A-MYB establish unique 3D chromatin of the sex chromosomes in meiotic sex chromosome inactivation. We propose that CTCF-mediated 3D chromatin underlines epigenetic priming to direct unidirectional differentiation, thereby determining the cellular identity of the male germline.

overall_design: Chromatin immunoprecipitation DNA-sequencing (ChIP-seq) for CTCF in THY1+ spermatogonia and pachytene spermatocytes.

first: Yuka

last: Kitamura

first: Kazuki

last: Takahashi

first: So

last: Maezawa

first: Yasuhisa

last: Munakata

first: Akihiko

last: Sakashita

first: Noam

last: Kaplan


title: CTCF-mediated 3D chromatin predetermines the gene expression program in the male germline (Hi-C)

overall_design: Determination of 3D chromatin structures of THY1+ and KIT+ spermatogonia, pachytene spermatocytes and round spermatids by Hi-C analyses.


title: CTCF-mediated 3D chromatin predetermines the gene expression program in the male germline (RNA-Seq)

summary: Spermatogenesis is a unidirectional differentiation process that generates haploid sperm, but how the gene expression program that directs this process is established is largely unknown. Here we determine the high-resolution 3D chromatin architecture of male germ cells during spermatogenesis and show that CTCF-mediated 3D chromatin predetermines the gene expression program required for spermatogenesis. In undifferentiated spermatogonia, CTCF-mediated chromatin interactions between meiosis-specific super-enhancers (SE) loci and target genes precede activation of meiosis-specific SEs on autosomes. These meiotic SE recruit the master transcription factor A-MYB in meiotic spermatocytes, which strengthens their 3D contacts and instructs a burst of meiotic gene expression. We also find that at the mitosis-to-meiosis transition, the germline-specific Polycomb protein SCML2 resolves chromatin loops that are specific to mitotic spermatogonia. Moreover, SCML2 and A-MYB establish the unique 3D chromatin organization of sex chromosomes during meiotic sex chromosome inactivation. We propose that CTCF-mediated 3D chromatin organization enforces epigenetic priming that directs unidirectional differentiation, thereby determining the cellular identity of the male germline.

overall_design: Testis were collected from P17 or 18 A-myb+/- and A-myb-/- mice and pachytene spermatocytes were isolated using fluorescence-activated cell sorting (FACS) with Vybrant DyeCycle Violet Stain (DCV)


title: Senescent cells cluster CTCF on nuclear speckles to sustain their splicing program

status: Public on Dec 12 2024

city: Göttingen

first: Argyris

last: Papantonis

address: Robert-Koch-Str. 40

department: Institute of Pathology

country: Germany

institute: University Medical Center Göttingen

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

overall_design: Refer to individual Series


title: Nuclear expansion drives chromatin structure remodeling in aging neurons

status: Public on Dec 09 2024

city: Moscow

first: Ekaterina

last: Khrameeva

address: Bolshoy Boulevard 30, bld. 1

department: Center of Life Sciences

country: Russia

institute: Skolkovo institute of science and technology

summary: Aging, particularly in the brain, involves impairments in multiple cellular and molecular functions, many of which are regulated at the nucleus. Chromatin structure plays a critical role in the regulation of gene expression and the maintenance of genomic stability. During differentiation, chromosomes acquire their unique topology depending on the cell type that should be kept for a lifetime, but this may deteriorate as we age. However, the effects of aging on the chromatin 3D structure of neurons remain largely unknown and much has been inferred from senescent cells. By combining chromosome conformation capture and microscopy techniques, we investigated cortical neurons of young and aged mice and discovered neuronal nuclear expansion during neuronal aging, leading to increased distances between chromosomes. This expansion alters the topology of compartments, topologically associating domains (TADs) and chromatin loops. While larger TADs tend to dissociate, smaller TADs and loops exhibit strengthened interactions to maintain the cohesiveness of chromatin in aged neurons. These topological changes impact the borders of TADs, resulting in their overall weakening. Interestingly, we attribute these alterations to changes in the physical forces of an expanding nucleus, filling a growing nuclear area, affecting downstream gene expression and chromatin topology, further contributing to the functional declines observed during aging.

overall_design: We performed Hi-C in neurons isolated using FACS from the cortex tissue of the aging, adult, and control mice.

first: Deborah

last: Toiber

first: Dmitrii

last: Kriukov


title: Development of Fit Epidermal Resident Memory T Cells Requires Antigen Encounter in the Skin.

status: Public on Dec 10 2024

first: Liang

last: Yang

address: 77 Avenue Louis Pasteur

department: Pathology

institute: Harvard Medical School

summary: CD8+ tissue resident memory T cells (TRM) develop from effectors that are recruited into peripheral tissues by infection or inflammation where they persist and provide defense against subsequent challenges.  Long-term persistence of epidermal TRM requires autocrine TGFβ that is transactivated by integrins expressed on keratinocytes.  In response to inflammation, the availability of TGFβ transactivation is limited.  Antigen-experienced TRM that encountered antigen in the epidermis during their development are more fit and outcompete bystander TRM for TGFβ transactivation resulting in their enrichment in the epidermis.  Transcriptomic analysis of endogenous epidermal TRM revealed that antigen-experienced TRM were enriched for TRM signature genes while bystander TRM retained the transcriptome of an earlier developmental state.  Following antigen recall, antigen-experienced TRM displayed accelerated proliferation kinetics compared with bystanders that was determined by the TCR signal-strength during their differentiation the skin.  Finally, antigen experienced TRM expressed TGFβRIII, which increases avidity of the TGFβRI/II receptor complex.  Selective ablation of Tgfbr3 from antigen experienced TRM reduced their capacity to persist when TGFβ activation was limited thus rendering them phenotypically like bystander TRM.  In sum, antigen-driven TCR signaling in the epidermis during TRM differentiation represents a required final step in ontogeny resulting in a lowered requirement on TGFβ transactivation for persistence and increased proliferation during recall responses that together result in the enhanced fitness of antigen-experience epidermal TRM.

overall_design:
Cohorts of wildtype C57BL/6 mice were infected on the left flank with VV by skin scarification.  DNFB was applied to the right flank 5 days post infection to recruit into the skin circulating CD8 effectors expanded by infection.  Mice were then rested for 50+ days to allow for the formation of TRM. Cells were pooled from 10 mice, enriched by percoll gradient centrifugation and purified by double FACSorting based on expression of CD90 and CD8.
On August 15, 2025, the Series was reorganized in order to include tissue-specific samples.

last: Weiss

first: Toshiro

last: Hirai

first: Haiyue

last: Li

first: Andrew

last: Lui

first: Shannon

last: Baker

first: Ian

last: Magill

first: Jingyu

last: Fan

first: David

last: Masopust

first: Niroshana

last: Anandasabapathy

first: Harinder

last: Singh

last: Zemmour

first: Laura

last: MacKay

first: Daniel


title: A biologic- and chemical-induced rat model of glomerular and tubular injury

status: Public on Dec 06 2024

city: Groton

first: Thomas

last: Lanz

state: CT

address: Eastern Point Rd

department: Multi-Omics & Biomarkers

institute: Pfizer

summary: Nonsurgical rodent chronic kidney disease (CKD) models for both glomerular and tubular injuries are currently limited. This study aimed to develop a rat model of CKD by combining anti-Fx1A with N(ω)-nitrophenyl-L-arginine methyl ester (L-NAME) administration. The rats were assigned to groups receiving L-NAME, anti-Fx1A, anti-Fx1A + L-NAME, or vehicle. Renal function, stiffness, injury biomarkers, histopathology and genome-wide transcriptomic changes were evaluated. Protein and renal injury biomarker levels in the urine were elevated in the anti-Fx1A alone and combination groups. Shear wave elastography revealed increased stiffness of the kidneys in all treatment groups. Histopathological evaluation revealed glomerular injury, characterized by enlarged glomeruli with increased hyaline materials in both anti-Fx1A groups and tubular degeneration/regeneration in the renal cortex of all treated groups, with the highest incidence and severity in the combination group. These tubular changes were sometimes accompanied by interstitial mononuclear cell infiltrates and interstitial fibrosis. Proteinuria and mild changes in blood, urine renal injury biomarkers and imaging endpoints were noted in association with these histopathologic changes. The concurrence and higher incidence and/or severity of glomerular and tubular injuries in the combination group indicate that this would be a useful and relevant CKD model suitable for mechanistic, pharmacologic and toxicological investigations.

overall_design: Kidneys were processed by RNAseq from rats following 7 weeks of treatment with L-NAME, anti-Fx1A, or a combination of the two drugs.

first: Seo-Kyoung

last: Hwang

first: Leah

last: Newman

first: Jessie

last: Qian

first: Thomas A

first: Chang-Ning

last: Liu


title: Sample multiplexed scRNA-seq using Nanocoding method for sample barcode labeling

city: Urbana

first: Yujun

last: Feng

address: 1304 W Green St.

department: Materials Science and Engineering

institute: University of Illinois, Urbana-Champaign

summary: Sample multiplexed scRNA-seq is a promising strategy to overcome current barriers in high cost and potential technical variations by multiple scRNA-seq tests. In this study, we developed a highly efficienct novel sample barcode labeling method using DNA-encoded Lipid Nanoparticles ('Nanocoding') that could label cells with minimal dependence on their type or sample conditions. This method provids a roubust and general protocol for sample barcoding and multiplexing in scRNA-seq. We demonstrated the performance of Nanocoding through three scRNA-seq studies, which include:  1. mouse spleen cells mix (one dataset including 6 mouse spleen tissues samples); 2. HeLa-mouse Stromal Vascular Fraction(SVF) cells mix (one dataset containing mixed HeLa cell and SVF cell); 3. Aged-Young SVF cells mix (one dataset containing two SVF samples) tests. These studies showcased the biomodal distribution of barcode counts in different models with high signal-to-background ratio, as well as pan-cell labeling activity for efficient and accurate sample-multiplexing. By using Nanocoding, we profiled obsity and age related change in lipid metabolism associated genes or inflammatory related features, in various cell types from spleen or adipose tissues.

overall_design: Male C57BL/6J Diet-Induced-Obese (DIO, N=3) and age-matched C57BL/6J control lean mice (8 month old, N=3) were used for speen collection and following multiplexed scRNA-seq. Each sample is labeled with a unique sample barcode using Nanocoding before mixing. Male C57BL/6J DIO mice that are 8 month old are used for visceral adipose tissue (VAT) collections and SVF cell collection in HeLa-SVF cell mixing test. Male C57BL/6J DIO mice that are 11 month old (N=3) or 4 month old (N=3) are used for visceral adipose tissue (VAT) collections and SVF cell collation in Young-Aged SVF cell mix test.

first: Fangxiu

last: Xu

first: Chris

last: Wright

last: Smith


title: PD-1 Mediated Regulation of Unique Activated CD8+ T Cells by NK Cells in the Submandibular Gland

status: Public on Dec 11 2024

first: Liang

last: Yang

summary: The increasing use of anti-PD-1 immune checkpoint blockade has led to the emergence of immune-related adverse events (irAEs), including dysfunction of the submandibular gland (SMG). In this study, we investigated the immunoregulatory mechanism contributing to the susceptibility of the SMG to irAEs. Here, we found that the SMG of PD-1 deficient mice and anti-PD-L1 treated mice harbor an expanded population of CD8+ T cells. We demonstrate that NK cells expressing PD-L1 tightly regulate CD8+ T cells in the SMG. When this immunoregulation is disrupted, CD8+ T cells clonally expand and acquire a unique transcriptional profile consistent with TCR activation. These clonally expanded cells phenotypically overlapped with cytotoxic GzmK+ CD8+ T autoimmune cells identified in primary Sjögren’s syndrome patients. Understanding how NK cells modulate CD8+ T cell activity in the SMG opens new avenues for preventing irAEs in patients undergoing checkpoint blockade therapies.

overall_design:
T cells were extracted from the submandibular glands of WT and PD-1KO mice, and the spleen of WT mice for transcriptomic, proteomic, and TCR repertoire analysis
On August 15, 2025, the Series was reorganized in order to include tissue-specific samples.

first: Sam

last: Borys

first: Shanelle

last: Reilly

first: Laurent

last: Brossay


title: MicroRNA-directed control of complex mRNA stability patterns across cell types

city: Seattle

first: Lukas

last: Oesinghaus

state: WA

address: 3946 W Stevens Wy NE

institute: University of Washington

summary: Limiting expression to target cell types or states is a longstanding goal in gene therapy, which could be met by sensing endogenous microRNA. However, an unclear association between microRNA expression and activity currently hampers such an approach. Here, we probe this relationship by measuring the stability of synthetic microRNA-responsive 3'UTRs across 10 cell lines in a library format. By systematically addressing biases in microRNA expression data and confounding factors such as microRNA crosstalk, we demonstrate that a straightforward model can quantitatively predict the stability of reporters containing full microRNA target sites purely from expression data. We use this model to design constructs with previously unattainable response patterns across our cell lines. We then generalize this approach to primary tissues and cells and show that mRNA can be engineered to be differentially active between rested and exhausted T cell states. The rules we derive for microRNA expression data selection and processing should apply to microRNA-responsive devices for any environment with available expression data.

sample_organism:
Homo sapiens
synthetic construct

platform_organism:
Homo sapiens
synthetic construct

overall_design: Libraries were prepared using four different protocols. All protocols start with plasmid libraries containing reporters with microRNA target sequences in their 3'UTR or 5' UTR, which were also sequenced. 1) 3' or 5' UTR libraries were transiently transfected into cell lines for n=2 replicates. After two days, library RNA was extracted, reverse transcribed, and sequenced. 2) Plasmid libraries were transfected with helper plasmids for murine stem cell virus (MSCV) into HEK293T. MSCV particles were purified and their RNA sequenced. MSCV particles were used to transduce either P2C2 cells or primary mouse CD8 T cells. T cells were expanded according to various protocols, then their library RNA and/or library constructs integrated into gDNA were extracted and sequenced. 3) Plasmid library was transiently transfected into K562 cells using electroporation. 48 hours later, Actinomycin D was added. Samples were extracted at 0 min, 30 min, 1h, 2h, 4h, 8h, 12h, and 24h after the addition of ActD. Sample library RNA was sequenced. 4) Plasmid library was transiently transfected into HEK293T or HeLa cells. 24 hours later, samples were lysed in the presence of cycloheximide and underwent a polysome profiling workflow. Individual polysome fractions were sequenced as separate libraries.

first: Sebastian

last: Castillo-Hair

first: Morgan

last: Bean

first: Nicole

last: Ludwig

first: Shusruto

last: Rishik

first: Hao Yuan

last: Kueh

first: Andreas

last: Keller

first: Georg

last: Seelig


title: Noncanonical role of ALAS1 as a heme-independent inhibitor of small RNA–mediated silencing [Ago2-CLEAR-CLIP]

first: Seungjae

last: Lee

overall_design: To investigate functional effects on miRNA-mediated targeting upon loss of ALAS1, we performed Ago2 CLEAR-CLIP

first: Sangmi

first: Robert

last: Desnick

first: Makiko

last: Yasuda

first: Eric

last: Lai


title: Amphiregulin Upregulated in Esophageal Squamous Cell Carcinoma Cells upon Direct Contact with Cancer-Associated Fibroblasts

status: Public on Dec 04 2024

city: Kamakura

first: Satoshi

last: Kondo

state: Kanagawa

address: Tebiro 6-10-1

department: New Projects Development Division

country: Japan

institute: Toray Industries,Inc.

summary:
Cancer-associated fibroblasts (CAFs) are a pivotal component of the tumor microenvironment, significantly contributing to the progression of esophageal squamous cell carcinoma (ESCC). Direct co-culture of human bone marrow-derived mesenchymal stem cells (MSCs), one of the origins of CAFs, with ESCC cell line, led to increased amphiregulin (AREG) expression and secretion in the ESCC cells. Consequently, the expression and phosphorylation of the AREG receptor EGFR were upregulated in co-cultured ESCC cells. Moreover, AREG treatment enhanced ESCC cell survival and migration through the EGFR-Erk/p38 signaling pathway.
Immunohistochemical analysis of AREG using human ESCC tissues showed a positive correlation between the intensity of AREG expression in tumor invasive front and the expression level of the CAF marker FAP. Furthermore, bioinformatics database analysis confirmed a significant upregulation of AREG expression in ESCC tissues compared to normal tissues. These findings suggest that AREG is involved in CAF-mediated ESCC progression and could be a novel therapeutic target for ESCC.

overall_design: MSCs and ESCC cells were seeded in the same dish and co-cultured for 4 days. Monocultures of both cell types served as controls. Following co-culture or monoculture, cells were washed with phosphate-buffered saline (FUJIFILM Wako Pure Chemical Corporation) and detached themselves from the dish using trypsin (FUJIFILM Wako Pure Chemical Corporation) treatment. The harvested cells were then mixed with anti-CD326 (epithelial cell adhesion molecule; EpCAM) microbeads (130-061-101; Miltenyi Biotec) and subjected to magnetic separation using the autoMACS Pro Separator (Miltenyi Biotec) to isolate high-purity tumor cells. cDNA microarray analysis was performed on monocultured and co-cultured TE-9 cells.

first: T

last: Nakanishi

first: YI

last: Koma


title: CTCF-mediated 3D chromatin predetermines the gene expression program in the male germline

last: Namekawa


title: Early response to cyclic stretch in human intestinal smooth muscle activates transition to a synthetic, proinflammatory phenotype hypothesized to affect gene expression in nearby cells in the bowel wall

city: Philadelphia

first: Katherine

last: Beigel

state: PA

address: 2716 South Street

department: Department of Biomedical and Health Informatics

institute: Children's Hospital of Philadelphia

summary: Background and Aims: Bowel smooth muscle experience mechanical stress constantly during normal function, and unusual mechanical stressors in various disease settings. Here, we test the hypothesis that pathologic mechanical stress could alter transcription to induce smooth muscle phenotypic class switching. Methods: Primary human intestinal smooth muscle cells (HISMCs), seeded on electrospun aligned poly-ε-caprolactone nano-fibrous scaffolds, were subjected to uniaxial 3% cyclic stretch at 1 Hz (loaded) or kept unloaded in culture for 6 hours. Bulk total RNA sequencing, RT-qPCR, and quantitative immunohistochemistry defined loading-induced changes in gene expression. NicheNet predicted how differentially expressed genes might impact HISMCs and other bowel cells. Results: Loading induced differential expression of 2500 genes in HISMCs. Loaded HISMCs had a less contractile phenotype, with increased expression of synthetic SMC genes, proinflammatory cytokines, and altered expression of axon guidance molecules, growth factors and morphogens. Many differentially expressed genes encode secreted ligands that could act cell-autonomously on smooth muscle, but also on other cells in the bowel wall. Discussion: HISMCs demonstrate remarkably rapid phenotypic plasticity in response to mechanical stress that may convert contractile HISMC into proliferative fibroblast-like cells or proinflammatory cells. These mechanical stress-induced changes in HISMC gene expression may be relevant for human bowel disease.

overall_design: To test the hypothesis that mechanical stress could rapidly alter gene expression in visceral SMCs and to gain insight into early changes in SMC phenotype events, we decided to evaluate gene expression in cultured human intestinal smooth muscle cells (HISMCs) after only 6 hours with or without cyclic stretching. Compared to most prior studies, we used a very mild mechanical stress (3%, 1 Hz), which rapidly altered the expression of 2500 genes (genes with log2 fold change > 0.48 or < -0.48, out of 4537 genes with adjusted p-value < 0.05)mRNAin loaded cultured human intestinal smooth muscle compared to unloaded. Compared to unstretched HISMCs, stretched cells had increased expression of genes typically produced in synthetic phenotype SMCs, increased production of many cytokines, chemokines, cytokine receptors, axon guidance molecules, junctional proteins, and altered levels of many signaling molecules predicted to act on nearby cells in the bowel. Cyclic stretching at this frequency also enhanced expression of many genes known to be preferentially expressed in vascular compared to visceral SMCs. Collectively, these data suggest that bowel SMC phenotype, in part, depends on the unique physical forces these cells experience as nutrients move through the bowel, as waste is eliminated, and in response to various types of bowel injury or disease.

first: Sharon

last: Wolfson

first: Sierra

last: Anderson

first: Molly

last: Weiner

first: Deanne

last: Taylor

first: Su-Jin

last: Heo

middle: O

last: Heuckeroth

first: Sohaib

last: Hashmi


title: Global analysis of H3K36 methylation in the MM genome

city: Shimotsuke

first: Sae

last: Matsuoka

state: Tochigi

department: Center for Molecular Medicine

institute: Jichi  Medical  University

summary: We analyzed histone H3K36 methylation in multiple myeloma using ChIP-seq.

overall_design: We isolated fixed chromatin from KMS34 cells and performed immunoprecipitation with anti-histone H3K36me2 antibody, followed by deep sequencing.

first: Sae Matsuoka

last: MD

first: Jiro Kikuchi

last: PhD


title: Striga hermonthica kibos ecotype microRNA annotations

city: University Park

first: Michael

last: Axtell

address: 280 Mueller Lab

department: Biology

institute: Pennsylvania State University

summary: The purpose of this study was to annotate microRNAs in the parasitic plant Striga hermonthica and investigate any haustorial specific expression of novel microRNAs.

overall_design: Haustorial and leaf tissue were collected from Striga hermonthica kibos ecotype grown on roots of Oryza sativa japonica cultivar KitaakeX. Small RNA libraries were prepared from total RNA and sequenced using Illumina HiSeq 2000.

first: Allison

last: Vanek

first: Saima

last: Shahid


title: 3D Chromatin Structures Foreshadow the Drosophila Fate Map

city: Princeton

first: Yuri

last: Pritykin

state: NJ

address: 245 Carl Icahn Lab

department: QCB

institute: Princeton

summary: 3D structure of chromatin is thought to be critical for the regulation of gene expression during development. Here we employ the Micro-C assay to achieve 100 bp resolution for the genome organization of Drosophila melanogaster throughout the first half of embryogenesis. The resulting contact maps enable the identification of fine-scaled structures such as individual loops and boundaries delineating TADs. We observe that 3D structures form prior to zygotic genome activation in precellular embryos, and many of these organizational features persist during successive mitotic cycles. 3D structures are classified through the use of 149 ChIP-seq datasets. We present evidence for specialized elements associated with housekeeping genes that are enriched for BEAF-32 but not CTCF. We also characterize a distinct class of elements associated with developmental gene regulation that are enriched with GAF and Zld binding. This binding is maintained during mitotic cycles and exhibits evolutionary conservation of both sequence and 3D structures. In sum, this work provides a comprehensive genome-wide characterization at unprecedented resolution of the role of 3D chromatin organization in gene regulation during development. We propose that the 3D organization of the pre-cellular embryo facilitates deployment of the developmental control genes defining the Drosophila fate map.

sample_organism:
Drosophila melanogaster
Drosophila virilis

overall_design: Micro-C in Drosophila embryos at stages nc1-8, nc12 (mitotic), nc14, and s10-12 with two biological replicates and two technical replicates.

first: Xinyang

last: Bing

first: Evan

last: Cofer

first: Gabriel

last: Dolsten

first: Benjamin

last: Brack

first: Marcus

last: Curlin

first: Chandra

last: Theesfeld

first: Olga

last: Troyanskaya

last: S Levine


title: Hi-C Calibration by Chemically Induced Chromosomal Interactions

city: State College

first: Yi

last: Li

address: 406 SOUTH FREAR, UNIVERSITY PARK

institute: Penn State University

summary: The genome-wide chromosome conformation capture method, Hi-C, has greatly advanced our understanding of genome organization. However, its quantitative properties, including sensitivity, bias, and linearity, remain challenging to assess. Measuring these properties in vivo is difficult due to the heterogenous and dynamic nature of chromosomal interactions. Here, using Chemically Induced Chromosomal Interaction (CICI) method, we create stable intra- and inter-chromosomal interactions in G1-phase budding yeast across a broad range of contact frequencies. Hi-C analysis of these engineered cell populations demonstrates that static intra-chromosomal loops do not generate Topologically Associated Domains (TADs) and only promote 3D proximity within ~50kb flanking regions. At moderate sequencing depth, Hi-C is sensitive enough to detect interactions occurring in a few percent of cells. It also shows no inherent bias toward intra- versus inter-chromosomal interactions. Furthermore, we observe a linear relationship between Hi-C signal intensity and contact frequency. These findings illuminate the intrinsic properties of the Hi-C assay and provide a robust framework for its calibration.

overall_design: Hi-C experiments are performed on three yeast strains with intra-chromosomal CICI pairs and two yeast strains with inter-chromosomal CICI pairs. For each strain, 5 populations with different absolute contact frequencies between CICI loci are generated and their Hi-C contacts are measured.

first: Christoph

last: Fischer

first: Fan

last: Zou

first: Manyu

last: Du

first: Lu

last: Bai


title: Transcriptomic and Epigenomic Signatures Distinguish High- and Low-Risk Endotypes for Liver Tumor Development [ChIP-Seq]

first: Amrit

last: Koirala

summary: The epigenome is a target for environmental exposures, and a potential determinant of inter-individual differences in response.  In genetically identical C57Bl/6 mice exposed from gestation to weaning to the endocrine disrupting chemical (EDC) tributyltin (TBT), tumor development later in life varied across multiple cohorts over time and depending on sex and diet.  In one cohort where approximately half of TBT-exposed male mice developed tumors at 10 months (Katz, 2020), transcriptomic (RNA-seq) and epigenomic (ChIP-seq) profiling was performed on blood and liver tissue from mice that developed tumors (i.e. “high-risk”) and equivalently exposed mice did not (i.e “low-risk”).  Blood transcriptomic signatures separated TBT-exposed from vehicle controls but did not discriminate between animals that developed tumors vs those that did not.  However, uninvolved liver tissue of mice with tumors exhibited transcriptomic and epigenomic signatures distinct from liver tissue of mice without tumors and had many features in common with tumors.  These high-risk transcriptomic and epigenomic features were also found in 10/26 TBT-exposed mice at 5 months, indicating that this risk signature preceded tumor development.  Thus, while early life exposure to TBT exhibits variable penetrance for tumor development, indicating TBT-exposure is not sufficient for tumorigenesis, increased risk for tumor development is linked to epigenomic and transcriptomic reprogramming of the liver induced by this EDC.

overall_design: This dataset consists of ChIP-Seq data generated from the livers of5-month or 10-month-old WT C57Bl/6 mice in response to maternal exposure to tributyltin (TBT) through weaning

first: Cheryl

last: Walker

first: Cristian

last: Coarfa

first: Rahul

middle: K

last: Jangid

first: Sandra

last: Grimm

first: Tia

last: Talley


title: Nonuple atg8 mutant provides genetic evidence for functional specialization of ATG8 isoforms in Arabidopsis thaliana

city: Vienna

first: Yasin

last: Dagdas

address: Dr. Bohr-Gasse 3

country: Austria

institute: Gregor Mendel Institute

summary: Autophagy is a vital cellular process that maintains organismal health by recycling damaged or superfluous cellular components within autophagosomes. This process is mediated by conserved ATG proteins, which coordinate autophagosome biogenesis and selective cargo degradation. Among these, the ubiquitin-like ATG8 protein plays a central role by linking cargo to the growing autophagosomes through interactions with selective autophagy receptors. Unlike most ATG proteins, which are represented by single or few isoforms, the ATG8 gene family is expanded in vascular plants, yet the extent of functional specialization within ATG8 isoforms remains largely unknown. Using transcriptional and translational reporters in Arabidopsis thaliana, we revealed that ATG8 isoforms are differentially expressed across tissues and form distinct autophagosomes within the same cell. To explore ATG8 specialization, we generated the nonuple Δatg8mutant lacking all nine ATG8 isoforms. The mutant displayed hypersensitivity to carbon and nitrogen starvation, coupled with defects in bulk and selective autophagy as shown by biochemical and ultrastructural analyses. Complementation experiments demonstrated that ATG8A could rescue both carbon and nitrogen starvation phenotypes, whereas ATG8H could only complement carbon starvation. Proximity labeling proteomics further identified isoform-specific interactors under nitrogen starvation, underscoring their functional divergence. These findings provide genetic evidence for the specialization of ATG8 isoforms in plants and lay a foundation for investigating their roles in diverse cell types and stress conditions.

overall_design: For standard plant growth, Arabidopsis seeds were gas sterilized with sodium hypochlorite + HCl (10:1 v/v), sown on water-saturated soil and grown in 16h light/8h dark photoperiod with 165 μmol m^-2 s^-1 light intensity. For in vitrogrowth, Arabidopsis seeds were surface sterilized in 70% ethanol for 10 minutes twice, then rinsed in absolute ethanol and dried on sterile paper. Seeds were sown in ½ MS liquid medium (Murashige and Skoog salt + Gamborg B5 vitamin mixture [Duchefa] supplemented with 0.5 g/liter MES and 1% sucrose, pH 5.7), vernalized at 4 °C in the dark for 2 days, and then grown under LEDs with 85μM/m²/s with a 14 h light/10 h dark photoperiod.High-quality DNA was extracted using the cetyltrimethylammonium bromide (CTAB) method and used to construct Illumina-compatible libraries with the Nextera XT DNA Library Preparation Kit, following the manufacturer’s instructions. Sequencing was performed on an Illumina NextSeq instrument in paired-end 150 bp mode. Raw FASTQ files obtained from sequencing were quality-checked and adapter-trimmed using TrimGalore (https://github.com/FelixKrueger/TrimGalore) with default settings. The trimmed FASTQ files were aligned to the TAIR10 genome using Bowtie2 (Langmead & Salzberg, 2012) with the parameters -D 15 -R 2 -N 0 -L 22 -i S,1,1.15. The resulting aligned BAM files were sorted and indexed using SAMtools (Danecek et al., 2021) and manually inspected for insertions or deletions using the Integrative Genomics Viewer (IGV).


title: DNA hypomethylation promotes UHRF1- and SUV39H1/H2-dependent crosstalk between H3K18ub and H3K9me3 to reinforce heterochromatin states [RNA-Seq]

status: Public on Dec 03 2024

city: Grand Rapids

first: Scott

last: Rothbart

state: MI

address: 333 Bostwick Avenue NE

institute: Van Andel Research Institute

summary: Mono-ubiquitination of lysine 18 on histone H3 (H3K18ub), catalyzed by UHRF1, is a DNMT1 docking site that facilitates replication-coupled DNA methylation maintenance. Its functions beyond this are unknown. Here, we genomically map simultaneous increases in UHRF1-dependent H3K18ub and SUV39H1/H2-dependent H3K9me3 following DNMT1 inhibition. Mechanistically, transient accumulation of hemi-methylated DNA at CpG islands facilitates UHRF1 recruitment and E3 ligase activity towards H3K18. Notably, H3K18ub enhances SUV39H1/H2 methyltransferase activity and, in colon cancer cells, nucleates new H3K9me3 domains at CpG island promoters of DNA methylation-silenced tumor suppressor genes. Disrupting UHRF1 enzyme activity prevents H3K9me3 accumulation while promoting PRC2-dependent H3K27me3 as a tertiary layer of gene repression in these regions. In contrast, disrupting H3K18ub-dependent SUV39H1/H2 activity enhances the transcriptional activating and antiproliferative effects of DNMT1 inhibition. Collectively, these findings reveal roles for UHRF1 and H3K18ub in regulating a hierarchy of repressive histone methylation signaling and rationalize a combination strategy for epigenetic cancer therapy.

overall_design: RNA-seq in RKO cells with SUV39H2 KO and dox-inducible SUV39H1 shRNA KD. There are four treatment groups: cells were treated with 1) DMSO control, 2) 20ng/mL doxycycline (SUV39H1 KD), 3) 1uM GSK3484862(DNMT1 inhibition), and 4) doxycycline+GSK3484862 (SUV39H1 KD + DNMT1 inhibition) for 5 days before RNA collection.

first: Yanqing

last: Liu

first: Rochelle

middle: L

last: Tiedemann


title: Sensory input, sex, and function shape hypothalamic cell type development

city: Cambridge

first: Harris

last: Kaplan

address: 16 Divinity Ave., Room 4019

department: MCB

institute: Harvard University

summary: Mammalian behavior and physiology undergo dramatic changes in early life. Young animals rely on conspecifics to meet their needs and start displaying nutritional independence and sex-specific social interactions at weaning and puberty, respectively. How neuronal populations regulating homeostatic functions and social behaviors develop during these transitions remains unclear. We used paired transcriptomic and chromatin accessibility profiling to examine the developmental trajectories of neuronal populations in the hypothalamic preoptic region, where cell types with key roles in physiological and behavioral control have been identified1–6. These data reveal a remarkable diversity of developmental trajectories shaped by the sex of the animal, and the location and behavioral or physiological function of the corresponding cell types. We identify key stages of preoptic development, including early diversification, perinatal emergence of sex differences, postnatal maturation and refinement of signaling networks, and nonlinear transcriptional changes accelerating at the time of weaning and puberty. We assessed preoptic development in various sensory mutants and find a major role for vomeronasal sensing in the timing of preoptic cell type maturation. These results provide novel insights into the development of neurons controlling homeostatic functions and social behaviors and lay ground for examining the dynamics of these functions in early life.

overall_design:
To build a transcriptional atlas of POA development, we performed droplet-based paired snRNA-seq and snATAC-seq at nine ages from E14 to P65, using the 10x Multiome kit, separately in males and females. We performed POA multiome sequencing on mutant and littermate control strains as well, focusing on mutants that affect specific sensory modalities, as well as dark-reared animals.
All raw data are available here within each GSM sample page, via SRA.
Processed data are available here on GEO:
- individual RDS files for snRNA-seq - the field "my.cell.type" contains cell type IDs used in the publication.
- fragment files for snATAC-seq - within each GSM page for each sample ending with "_scATAC", or all together in the GSE280964_RAW.tar file.

first: Catherine

last: Dulac


title: DNA hypomethylation promotes UHRF1- and SUV39H1/H2-dependent crosstalk between H3K18ub and H3K9me3 to reinforce heterochromatin states [EPIC]

overall_design: EPIC array analysis in WI-38 cells following DMSO control treatment or DNMT1 inhibition to profile DNA methylation changes following indicated treatment.


title: DNA hypomethylation promotes UHRF1- and SUV39H1/H2-dependent crosstalk between H3K18ub and H3K9me3 to reinforce heterochromatin states

type: Methylation profiling by genome tiling array

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

overall_design: Refer to individual Series


title: Transcriptomic and Epigenomic Signatures Distinguish High- and Low-Risk Endotypes for Liver Tumor Development

institute: Baylor College of Medicine


title: Proteogenomics Study of Lung Cancer

status: Public on Dec 02 2024

first: Sung Wook

last: Kang

address: one plaza

department: Surgery

institute: Baylor college of medicine

summary: Lung cancer remains the leading cause of cancer-related mortality worldwide, with limited treatment options for advanced stages. This proteogenomics study aims to integrate multi-omics approaches, including proteomics, genomics, and transcriptomics, to elucidate the molecular mechanisms underlying lung cancer progression and treatment resistance. By leveraging cutting-edge technologies, this study seeks to identify novel biomarkers and therapeutic targets, enabling personalized medicine strategies to improve patient outcomes. The integration of proteogenomic data will provide a comprehensive understanding of tumor biology, revealing critical pathways and interactions that drive tumorigenesis and immune evasion.

first: Hyun-Sung

last: Lee


summary: Mono-ubiquitination of lysine 18 on histone H3 (H3K18ub), catalyzed by UHRF1, is a DNMT1 docking site that facilitates replication-coupled DNA methylation maintenance. Its functions beyond this are unknown. Here, we genomically map simultaneous increases in UHRF1-dependent H3K18ub and SUV39H1/H2-dependent H3K9me3 following DNMT1 inhibition. Mechanistically, transient accumulation of hemi-methylated DNA at CpG islands facilitates UHRF1 recruitment and E3 ligase activity towards H3K18. Notably, H3K18ub enhances SUV39H1/H2 methyltransferase activity and, in colon cancer cells, nucleates new H3K9me3 domains at CpG island promoters of DNA methylation-silenced tumor suppressor genes. Disrupting UHRF1 enzyme activity prevents H3K9me3 accumulation while promoting PRC2-dependent H3K27me3 as a tertiary layer of gene repression in these regions. In contrast, disrupting H3K18ub- dependent SUV39H1/H2 activity enhances the transcriptional activating and antiproliferative effects of DNMT1 inhibition. Collectively, these findings reveal roles for UHRF1 and H3K18ub in regulating a hierarchy of repressive histone methylation signaling and rationalize a combination strategy for epigenetic cancer therapy.

overall_design: Chromatin immunoprecipitation sequencing (ChIP-seq) for H3K9me3, UHRF1, H3K18ub, and H3K27me3 in RKO cells following DMSO control treatment, UHRF1 knockdown, DNMT1 inhibition, and UHRF1 KD+DNMT1 inhibition.

first: Bradley

last: Dickson


overall_design: RNA-seq for RKO cells following DMSO control treatment, UHRF1 knockdown, DNMT1 inhibition, and UHRF1 KD+DNMT1 inhibition to define transcriptome changes following indicated treatments.


title: The roles of CLOCK and CYCLE in the reproductive system

status: Public on Nov 22 2024

city: Beijing

first: Pengfei

last: Lv

address: No. 2, Old Summer Palace West Road

institute: China agricultural university

summary: To investigate whether the regulatory roles of the circadian genes CLOCK and CYCLE as transcription factors in the fruit fly testes and ovaries are similar to those in the head tissue, we conducted ChIP-seq analyses on these three tissues, respectively.

overall_design: Chromatin immunoprecipitation DNA sequencing (ChIP-seq) for CLOCK and CYCLE in the head, testis, and ovary.


title: Age is an intrinsic driver of inflammatory responses to malaria

city: Brisbane

first: Zuleima

last: Pava Imitola

state: Queensland

address: 300 Herston road

country: Australia

institute: QIMR Berghofer

summary: Age is a critical factor influencing the host immune response and disease pathogenesis. In malaria, the risk of severe disease increases with age in non-immune individuals. Malaria disease is in part driven by inflammation, but the specific cells and mechanisms contributing to age-dependent disease risk are incompletely understood. Here, we assessed inflammatory cytokines in malaria in non-immune children and adults, and the phenotypic, functional and transcriptional differences of innate immune cell responders to malaria parasites in malaria-naïve children and adults. During naturally acquired malaria, age was associated with increased plasma levels of inflammatory chemokines CCL2, CCL3, CXCL8, CXLC9, along with CRP, and IDO, which were associated with clinical symptoms. In malaria naïve individuals, classical monocyte and Vδ2+ δ T cell responses from adults were characterized by increased inflammatory cytokine production, and higher transcriptional activation following stimulation with malaria parasites. Classical monocyte responses in adults were dominated by CCL2 production, while in children the response had increased IL-10 production and enrichment in IL-10 signaling pathways upon parasite stimulation. This heightened inflammatory response in adults was not mitigated by regulatory T cells (Tregs). Taken together, these findings identify cellular mechanisms of age-dependent host responses that play crucial roles in driving inflammatory responses in malaria.

overall_design: PBMCs were co-cultured at 37 °C, 5% CO2 in a 96-well U-bottom plate for 4 hours at a 1:1 cell ratio with 1x106 mature trophozoite stage pRBCs. After incubation classical monocytes and Vδ2 T cells subsets were FACS sorted using the BD FACSAria™ III Cell Sorter. RNA was extracted from isolated cell population lysates using the QIAGEN PicoPureTM RNA isolation kit (Applied Biosystems™, KIT0204), and RNA quality confirmed with the 2200 TapeStation system (G2964AA) by High Sensitivity RNA ScreenTape (5067- 5579). RNA sequencing libraries were constructed using the NEBNext® Single Cell/Low Input RNA Library Prep Kit for Illumina® (E6420S) and NEBNext® Multiplex Oligos for Illumina® (96 Unique Dual Index Primer Pairs) (E6440S). The libraries were sequenced using a NextSeq 550 system

first: Jessica

middle: R

last: Loughland

first: Nicholas

last: Dooley

last: Pava

first: Arya

last: SheelaNair

first: Dean

last: Andrew

first: Peta

last: Tipping

first: Peter

last: Bourke

first: Christian

last: Engwerda

first: Alejandro

last: Lopez

first: Kim

last: Piera

first: Tonia

last: Woodberry

first: Timothy

last: William

first: Bridget

last: Barber

first: Matthew

last: Grigg

last: Anstey

first: Gabriela

last: Minigo

first: Michelle

last: Boyle


first: Scott

last: Rothbart

overall_design: EPIC array analysis in RKO cells following DMSO control treatment, UHRF1 knockdown, DNMT1 inhibition, and UHRF1 KD+DNMT1 inhibition to profile DNA methylation changes following indicated treatment.

first: Yanqing

last: Liu

first: Rochelle

last: Tiedemann


title: High affinity chimeric antigen receptor signaling induces an inflammatory program in human regulatory T cells

status: Public on Nov 30 2024

city: Charleston

first: Stefano

last: Berto

state: SC

address: 68 President Street, BE208

institute: MUSC

summary: Regulatory T cells (Tregs) are promising cellular therapies to induce immune tolerance in organ transplantation and autoimmune disease. The success of chimeric antigen receptor (CAR) T-cell therapy for cancer has sparked interest in using CARs to generate antigen-specific Tregs. Here, we compared CAR with endogenous T cell receptor (TCR)/CD28 activation in human Tregs. Strikingly, CAR Tregs displayed increased cytotoxicity and diminished suppression of antigen-presenting cells and effector T (Teff) cells compared with TCR/CD28 activated Tregs. RNA sequencing revealed that CAR Tregs activate Teff cell gene programs. Indeed, CAR Tregs secreted high levels of inflammatory cytokines, with a subset of FOXP3+ CAR Tregs uniquely acquiring CD40L surface expression and producing IFNγ. Interestingly, decreasing CAR antigen affinity reduced Teff cell gene expression and inflammatory cytokine production by CAR Tregs. Our findings showcase the impact of engineered receptor activation on Treg biology and support tailoring CAR constructs to Tregs for maximal therapeutic efficacy.

overall_design: CAR Tregs and CAR Teff cells were co-cultured with irradiated K562 (No Activation), CD19-K562 (CAR Activation) or CD64-CD80-K562 loaded with anti-CD3 antibody (TCR/CD28 Activation) at a 1:1 ratio in RPMI10 medium. CAR Treg co-cultures were supplemented with 1,000 IU/ml IL-2. After 24h, CD4+ cells were isolated using the EasySep Human CD4 Positive Selection Kit (STEMCELL Technologies), following the manufacturer’s instructions. At least two independent blood donors per condition were analyzed. With this experimental design, we kept CAR lentivirus transduction and sorting for CAR+ cells constant across all samples and kept the number of irradiated K562 target cells constant across the three modes of activation to rigorously compare the impact of activation via endogenous CD3 and CD28 with activation via CAR intracellular domain CD3 and CD28.

first: Bryan

last: Granger

first: Russell W.

last: Cochrane

first: Leonardo M.R.

last: Ferreira


title: Golgi-ER Axis Regulates Anti-Tumor T Cell Immunotherapeutic Response

status: Public on Nov 20 2024

summary: The cumulative role of cellular organelle’s in shaping the life and function of a cell has been long acknowledged. While each organelle plays a specific role in the growth and development of T cells, numerous studies have thus far focused on targeting mitochondria, endoplasmic reticulum, or lysosome related pathways to improve anti-tumor T cell immune response. Here we highlight the tumor microenvironment (TME) mediated disruption of Golgi architecture and function, termed Golgi stress, contributes to altered redox signaling and affects cell survival. Importantly, the decreased expression if GM130, a marker of Golgi stress and indicating the disruption of Golgi architecture, was reverted upon treatment with hydrogen sulphide (H2S) donor GYY4137, or over expressing cystathionine β-synthase (Cbs), an enzyme involved in biosynthesis of endogenous H2S. Activation and expansion of tumor reactive TCR or chimeric antigen receptor bearing T cells expanded with exogenous H2S promoted stemness, antioxidant capacity and exhibited an increase effector cytokine secretion that correlated to increased protein translation. In in vivo models of melanoma and lymphoma, anti-tumor T cells conditioned ex vivo with exogenous H2S or overexpressing Cbs demonstrated superior tumor control upon ACT. Further, sorting anti- tumor T cells based on Golgi structure and abundance resulted in identification of a Golgihi subset of T cells with a unique metabolic signature, superior fitness, and enhanced anti-tumor capacity. These data suggest that H2S can be used an immunomodulatory agent to enhance the efficacy of ACT, and that the structure and function of the Golgi apparatus is an important feature of T cells that determines their anti-tumor capacity.

overall_design: Splenocytes isolated from the spleens of Pmel-1 mice were activated with gp100 peptide in the presence or absence of H2S (GYY4137 0.5mM) treatment for 3 days and expanded for 3 more days. Control and H2S-treated T cells were then isolated for mRNA isolation.

first: Nathaniel

last: Oberholtzer

first: Shikhar

last: Mehrotra


title: DUX4 induced by HSV-1 infection promotes viral replication

status: Public on Nov 24 2024

city: Wuhan

first: Junjie

last: Zhang

state: Hubei

address: Donghu Road, No. 115

department: Medical Research Institute

institute: Wuhan University

summary: RNAseq analysis of THP-1 cells mock-infected or infected with HSV-1 without or with DUX4 knockdown

overall_design: THP-1 cells (2 × 106, three replicates per sample) were mock-infected or infected with HSV-1 (MOI=1) for 12 h. RNA was extracted and RNA-seq was performed. Three replicates are included for each samples.

first: Ming

last: Gao


title: Sequencing data of a novel piperine derivative HJ-4 against colorectal cancer

status: Public on Nov 28 2024

city: lianyungang

first: Luyao

address: tongguan

institute: Jiangsu Ocean University

summary: In order to explore the mechanism of HJ-4, we used RNA-seq technology to extract and analyze the total RNA of cells treated with DMSO and HJ-4, and analyzed the RNA data.

overall_design: After reaching 80% density, HCT116 cells were divided into two groups, with no less than 5×10^6 cells in each group. One group was treated with 32µM HJ-4 drug, and the other group was treated with DMSO as a control. Both groups of cells were incubated at 37°C. One day later, the original culture medium was removed and washed with 2 mL PBS using an RNaseA-free pipette tip. 1 mL of total RNA extraction reagent (Biyuntian, China) was added to the culture dish until the cells were completely covered. The culture dish was gently shaken and allowed to stand for 2-3 minutes. Mixed by blowing with an RNaseA-free pipette tip and aspirated into an RNaseA-free cryovial. Quickly frozen in liquid nitrogen and stored in a -80°C freezer. Subsequent RNA sequencing analysis was performed by Sangyo Bioengineering (Shanghai).

first: Bin


title: Sp140L Is a Herpesvirus Restriction Factor [Day5_LPKO_vs_SP100_SP140L_KO_BulkRNAseq]

city: Durham

first: Jana

last: Cable

state: NC

address: 213 Research Dr.

department: Molecular Genetics and Microbiology

institute: Duke University

summary: Herpesviruses, including the oncogenic Epstein-Barr Virus (EBV), must bypass host DNA sensing mechanisms to drive infection and pathogenesis. The first viral latency protein expressed, EBNA-LP, is essential for the transformation of naïve B cells, yet its role in evading host defenses remains unclear. Using single-cell RNA sequencing of EBNA-LP-Knockout (LPKO)-infected B cells, we reveal an antiviral response landscape implicating the ‘speckled proteins’ as key restriction factors countered by EBNA-LP. Specifically, loss of SP100 or the primate-specific SP140L reverses the restriction of LPKO, suppresses a subset of canonically interferon-stimulated genes, and restores viral gene transcription and cellular proliferation. Notably, we also identify Sp140L as a restriction target of the herpesvirus saimiri ORF3 protein, implying a role in immunity to other DNA viruses. This study reveals Sp140L as a restriction factor that we propose links sensing and transcriptional suppression of viral DNA to an IFN-independent innate immune response, likely relevant to all nuclear DNA viruses.

overall_design: Bulk RNAseq of B cells 5 days post-infection with LPKO virus and CD46 alone or CD46 and Sp100 or Sp140L knocked out, or WT with CD46 knocked out.

last: M Cable

first: Wiyada

last: Wongwiwat

first: Jenna

last: C Grabowski

first: Robert

last: E White

first: Micah

last: A Luftig


title: Total RNA-seq from mice of NAc and mPFC

status: Public on Nov 27 2024

summary: Total RNA-seq approach identified mRNAs  and non-coding RNAs in the mouse NAc after Cocaine CPP conditioning and in the mPFC after social defeat stress.

overall_design: Adult male C57Bl6J mice were subjected to cocaine (7.5 mg/kg, i.p.) conditioned place preference conditioning procedure, experienced social defeat stress by aggressive male CD1 mice, or stayed in home cage as no stimulation control. The NAc and mPFC tissue were dissected out at 15 min after emotional experiences. The RNAs were then subjected to total RNA-seqencing processes with BGI Genomics.

first: Setefano

first: Makoto

last: Taniguchi

first: Christopher

last: Cowan

first: Rose

last: Akiki


title: Phosphorylation of HP1/Swi6 relieves competition with Suv39/Clr4 on nucleosomes and enables H3K9 trimethyl spreading.

city: San Francisco

first: Bassem

last: Al-Sady

state: CA

address: 513 Parnassus Ave HSW 1501

institute: UCSF

summary: Heterochromatin formation in Schizosaccharomyces pombe requires the spreading of histone 3 (H3) Lysine 9 (K9) methylation (me) from nucleation centers by the H3K9 methylase, Suv39/Clr4, and the reader protein, HP1/Swi6. To accomplish this, Suv39/Clr4 and HP1/Swi6 have to associate with nucleosomes both nonspecifically, binding DNA and octamer surfaces and specifically, via recognition of methylated H3K9 by their respective chromodomains. However, how both proteins avoid competition for the same nucleosomes in this process is unclear. Here, we show that phosphorylation tunes the nucleosome affinity of HP1/Swi6 such that it preferentially partitions onto Suv39/Clr4’s trimethyl product rather than its unmethylated substrates. Preferential partitioning enables efficient conversion from di-to trimethylation on nucleosomes in vitro and H3K9me3 spreading in vivo. Together, our data suggests that phosphorylation of HP1/Swi6 creates a regime that relieves competition with the “read-write” mechanism of Suv39/Clr4 for productive heterochromatin spreading.

overall_design: Chromatin immunoprecipitation followed by sequencing (ChIP-seq) probing H3K9me2 and H3K9me3 levels in the context of delta swi6, wildtype swi6, or S18A/S24A swi6. Strain background contained our heterochromatin spreading sensor at the MAT locus.

first: Dana

last: Kennedy

first: Eric

last: Simental


title: DNA hypomethylation promotes UHRF1- and SUV39H1/H2-dependent crosstalk between H3K18ub and H3K9me3 to reinforce heterochromatin states [ChIP-Seq]

first: Scott

last: Rothbart

overall_design: Chromatin immunoprecipitation sequencing (ChIP-seq) for H3K9me3 in WI-38 cells following DMSO control treatment, UHRF1 knockdown, DNMT1 inhibition, and UHRF1 KD+DNMT1 inhibition.

first: Yanqing

last: Liu

first: Rochelle

last: Tiedemann

first: Bradley

last: Dickson


title: Sp140L Is a Herpesvirus Restriction Factor [scRNA-seq]

overall_design: Single cell RNAseq data from B cells in a timecourse of LPKO or WT EBV infection. Samples including uninfected (Day 0), and 2, 5, and 8 days post-infection.


title: Temporal Dynamics of Gene and Protein Expression Follow Volumetric Muscle Loss

status: Public on Nov 26 2024

city: Palo Alto

first: Ishita

last: Jain

address: 3801 Miranda Avenue

institute: Stanford University

summary: Here, we performed time-series analysis of transcriptomic and proteomic changes associated with VML in a mouse model, focusing on the dynamics of gene and protein expression patterns at up to three weeks after muscle injury. We identified signaling pathways associated with temporal expression patterns that fail to restore within 3 weeks after VML, including those with sustained upregulation or downregulation.  Using temporal expression analysis, we identified Sp1 as a novel molecular mediator of dysregulated muscle recovery after VML as well as elucidated pro-inflammatory and extracellular matrix (ECM) remodeling pathways mediating the remodeling process.  These insights pave the way for the development of new targets that promote muscle regeneration and functional recovery of traumatically injured muscle.

overall_design: We utilized a well-established mouse partial-thickness VML model, in which 40% of the tibialis anterior muscle in C57BL/6 (8-week-old male) mice was unilaterally excised, based on our previous work .  At time points of 3, 7, 14, and 21 days after the induction of VML, the tibialis anterior muscles were explanted for RNA extraction (n=5-6 samples per time point).

first: Beu

last: Oropeza

first: Caroline

last: Hu

first: Gladys

last: Chiang

first: Ngan

last: Huang


title: Cathepsin L as a dual-target to mitigate muscle wasting while enhancing anti-tumor efficacy of anti-PD-L1

status: Public on Nov 15 2024

city: Busan

first: Haeseung

last: Lee

address: 63 Beon-gil 2, Busandaehag-ro, Geumjeong-gu

department: College of Pharmacy

country: South Korea

institute: Pusan National University

summary: Immune checkpoint inhibitors (ICIs) have revolutionized cancer therapy; however, their use is frequently associated with immune-related adverse events (irAEs). In this study, anti-PD-L1 therapy exacerbates muscle wasting in tumor-bearing male mice despite its anti-tumor efficacy, accompanied by an accumulation of CD8+ T cells in muscle. Single-cell RNA sequencing identifies these cells as tissue-resident memory-like CD49a+ CD8+ T cells. While CD8+ T cell depletion prevents muscle wasting, it compromises the anti-tumor efficacy of anti-PD-L1. To resolve this paradox, we identify cathepsin L (CTSL) as a dual-target capable of suppressing both tumor progression and CD8+ T cell-mediated muscle wasting, through integrative transcriptomic analysis. Pharmacological inhibition of CTSL not only mitigates anti-PD-L1-induced muscle wasting but also further suppresses tumor growth, potentially via downregulation of BNIP3. Here, we show that CTSL is a dual-action target to uncouple anti-tumor efficacy from muscle-specific irAEs, offering a strategy to improve clinical outcomes of ICIs.

overall_design: To identify effective therapeutic targets for both cachexia and muscle-related immune-related adverse events (irAEs), we generated RNA-seq data from muscles in a tumor bearing mouse model treated with IgG, anti-PD-L1, and anti-CD8a

first: Nayoung

last: Song


title: Human introns contain conserved tissue-specific cryptic poison exons

city: Moscow

first: Dmitri

middle: D

last: Pervouchine

address: Bolshoy Bul'var, 30, str 1

department: Skoltech Center for Molecular and Cellular Biology

institute: Skolkovo Institute of Science and Technology

summary: Eukaryotic cells express a large number of transcripts from a single gene due to alternative splicing. Despite hundreds of thousands of splice isoforms being annotated in databases, it has been reported that the current exon catalogs remain incomplete. At the same time, introns of human protein-coding genes contain a large number of evolutionarily conserved elements with unknown function. Here, we explore the possibility that some of them represent cryptic exons that are expressed in rare conditions. We identified a group of cryptic exons that are similar to the annotated exons in terms of evolutionary conservation and RNA-seq read coverage in the GTEx dataset. Most of them were poison, i.e. generated an NMD isoform upon inclusion, and many showed signs of tissue-specific and cancer-specific expression and regulation. We performed RNA-seq in A549 cell line treated with cycloheximide to inactivate NMD, and confirmed using qPCR that seven of eight exons tested are, indeed, expressed. This study shows that introns of human protein-coding genes contain cryptic poison exons, which reside in conserved intronic regions and remain not fully annotated due to insufficient representation in RNA-seq libraries.

overall_design: To investigate whether novel poison exons are indeed targeted by NMD, we performed RNA-seq in A549 cell line treated with cycloheximide to inactivate NMD. We then compared the inclusion level of novel and annotated cassette exons in cycloheximide-treated and untreated cells.

first: Sergey

last: Margasyuk

first: Antonina

last: Kuznetsova

first: Lev

last: Zavileyskiy

first: Maria

last: Vlasenok

first: Dmitry

last: Skvortsov


title: A microbial-inspired antibody-drug conjugate for cancer immunotherapy

status: Public on Dec 01 2024

city: Houston

first: Benjamin

last: Schrank

address: 1400 Pressler St

institute: MD Anderson Cancer Center

summary: Generating antitumor immune responses requires the phagocytosis of tumor cells and subsequent cross-presentation of tumor-derived antigens by antigen-presenting cells. However, these processes are impeded by phagocytosis checkpoints and inefficient cytosolic transport of antigenic peptides from phagolysosomes. Here, using a microbial-inspired strategy, we engineered an antibody-drug conjugate (ADC) that targets the “don’t eat me” signal CD47 linked to the bacterial toxin listeriolysin O from the intracellular bacterium Listeria monocytogenes via a cleavable linker (CD47-LLO). CD47-LLO promotes the phagocytosis of cancer cells followed by the release and activation of LLO to form pores on phagolysosomal membranes that allow cytosolic entry of tumor-derived contents, leading to enhanced antigen cross-presentation of tumor-derived peptides and activation of cytosolic immune sensors. CD47-LLO treatment in vivo significantly inhibited the growth of both localized and metastatic tumors and improved animal survival as monotherapy or in combination with checkpoint blockade. Together, these results demonstrate that designing ADCs to promote immune recognition of tumor cells represents a promising therapeutic strategy for treating multiple cancers.

overall_design: To define the identities of innate and adaptive immune cells that accumulate in tumors upon CD47-LLO treatment, we performed single cell RNA (scRNA-seq) on CD45+ cells isolated from EO771 tumors. CD45+ cells were rare in IgG or anti-CD47 treated animals (Fig. 4a,b). In contrast, we observed a 30-fold increase in CD45+ cells upon CD47-LLO treatment. After rigorous quality control, we used our previously published methodsto annotate cells and interpret changes in their abundance and gene expression (n= 13,891 cells total).

first: Wen

last: Jiang

first: Kyuson

last: Yum

first: Nhat

last: Tran


title: Effect of feeding on gene expression in HEK-293E cells either treated with Torin1 or not.

city: Higashihiroshima

first: Takafumi

last: Ogawa

state: Hiroshima

address: 1-3-1 Kagamiyama

institute: Hiroshima University

summary: We examined how mTORC1 influences mRNA expression and alternative splicing during a growth response in human embryonic kidney (HEK) 293E cells, in which mTORC1 is not hyperactive.  We released a starvation-induced growth arrest by refeeding, then analyzed mRNA levels and alternative splicing events one and four hours later under either control or Torin1 treatment conditions.

overall_design: To investigate the nutrient response on gene expression, we treated HEK-293E cells with either 1 or 4 hours of feeding, with or without Torin1 treatment.

first: Ziyun

last: Wu

first: Meltem

last: Isik

first: T

last: Blackwell