Transcription Factor Motifs Research Articles

Single-housing-induced islet epigenomic changes are related to polymorphisms in diabetic KK mice.

A lack of social relationships is increasingly recognized as a type 2 diabetes (T2D) risk. To investigate the underlying mechanism, we used male KK mice, an inbred strain with spontaneous diabetes. Given the association between living alone and T2D risk in humans, we divided the non-diabetic mice into singly housed (KK-SH) and group-housed control mice. Around the onset of diabetes in KK-SH mice, we compared H3K27ac ChIP-Seq with RNA-Seq using pancreatic islets derived from each experimental group, revealing a positive correlation between single-housing-induced changes in H3K27ac and gene expression levels. In particular, single-housing-induced H3K27ac decreases revealed a significant association with islet cell functions and GWAS loci for T2D and related diseases, with significant enrichment of binding motifs for transcription factors representative of human diabetes. Although these H3K27ac regions were preferentially localized to a polymorphic genomic background, SNVs and indels did not cause sequence disruption of enriched transcription factor motifs in most of these elements. These results suggest alternative roles of genetic variants in environment-dependent epigenomic changes and provide insights into the complex mode of disease inheritance.

Real-Time Measurement of a Weak Interaction of a Transcription Factor Motif with a Protein Hub at Single-Molecule Precision.

Transcription factors often interact with other protein cofactors, regulating gene expression. Direct detection of these brief events using existing technologies remains challenging due to their transient nature. In addition, intrinsically disordered domains, intranuclear location, and lack of cofactor-dependent active sites of transcription factors further complicate the quantitative analysis of these critical processes. Here, we create a genetically encoded label-free sensor to identify the interaction between a motif of the MYC transcription factor, a primary cancer driver, and WDR5, a chromatin-associated protein hub. Using an engineered nanopore equipped with this motif, WDR5 is probed through reversible captures and releases in a one-by-one and time-resolved fashion. Our single-molecule kinetic measurements indicate a weak-affinity interaction arising from a relatively slow complex association and a fast dissociation of WDR5 from the tethered motif. Further, we validate this subtle interaction by determinations in an ensemble using single nanodisc-wrapped nanopores immobilized on a biolayer interferometry sensor. This study also provides the proof-of-concept for a sensor that reveals unique recognition signatures of different protein binding sites. Our foundational work may be further developed to produce sensing elements for analytical proteomics and cancer nanomedicine.

Terminal deoxynucleotidyl transferase and CD84 identify human multi-potent lymphoid progenitors

Lymphoid specification in human hematopoietic progenitors is not fully understood. To better associate lymphoid identity with protein-level cell features, we conduct a highly multiplexed single-cell proteomic screen on human bone marrow progenitors. This screen identifies terminal deoxynucleotidyl transferase (TdT), a specialized DNA polymerase intrinsic to VDJ recombination, broadly expressed within CD34+ progenitors prior to B/T cell emergence. While these TdT+ cells coincide with granulocyte-monocyte progenitor (GMP) immunophenotype, their accessible chromatin regions show enrichment for lymphoid-associated transcription factor (TF) motifs. TdT expression on GMPs is inversely related to the SLAM family member CD84. Prospective isolation of CD84lo GMPs demonstrates robust lymphoid potentials ex vivo, while still retaining significant myeloid differentiation capacity, akin to LMPPs. This multi-omic study identifies human bone marrow lymphoid-primed progenitors, further defining the lympho-myeloid axis in human hematopoiesis.

Cellular zinc status alters chromatin accessibility and binding of p53 to DNA.

Zn2+ is an essential metal required by approximately 850 human transcription factors. How these proteins acquire their essential Zn2+ cofactor and whether they are sensitive to changes in the labile Zn2+ pool in cells remain open questions. Using ATAC-seq to profile regions of accessible chromatin coupled with transcription factor enrichment analysis, we examined how increases and decreases in the labile zinc pool affect chromatin accessibility and transcription factor enrichment. We found 685 transcription factor motifs were differentially enriched, corresponding to 507 unique transcription factors. The pattern of perturbation and the types of transcription factors were notably different at promoters versus intergenic regions, with zinc-finger transcription factors strongly enriched in intergenic regions in elevated Zn2+ To test whether ATAC-seq and transcription factor enrichment analysis predictions correlate with changes in transcription factor binding, we used ChIP-qPCR to profile six p53 binding sites. We found that for five of the six targets, p53 binding correlates with the local accessibility determined by ATAC-seq. These results demonstrate that changes in labile zinc alter chromatin accessibility and transcription factor binding to DNA.

A spatially resolved multiomic single-cell atlas of soybean development.

Cis-regulatory elements (CREs) precisely control spatiotemporal gene expression in cells. Using a spatially resolved single-cell atlas of gene expression with chromatin accessibility across ten soybean tissues, we identified 103 distinct cell types and 303,199 accessible chromatin regions (ACRs). Nearly 40% of the ACRs showed cell-type-specific patterns and were enriched for transcription factor (TF) motifs defining diverse cell identities. We identified de novo enriched TF motifs and explored conservation of gene regulatory networks underpinning legume symbiotic nitrogen fixation. With comprehensive developmental trajectories for endosperm and embryo, we uncovered the functional transition of the three sub-cell types of endosperm, identified 13 sucrose transporters sharing the DOF11 motif that were co-up-regulated in late peripheral endosperm and identified key embryo cell-type specification regulators during embryogenesis, including a homeobox TF that promotes cotyledon parenchyma identity. This resource provides a valuable foundation for analyzing gene regulatory programs in soybean cell types across tissues and life stages.

Genomic background sequences systematically outperform synthetic ones in de novo motif discovery for ChIP-seq data.

Efficient de novo motif discovery from the results of wide-genome mapping of transcription factor binding sites (ChIP-seq) is dependent on the choice of background nucleotide sequences. The foreground sequences (ChIP-seq peaks) represent not only specific motifs of target transcription factors, but also the motifs overrepresented throughout the genome, such as simple sequence repeats. We performed a massive comparison of the 'synthetic' and 'genomic' approaches to generate background sequences for de novo motif discovery. The 'synthetic' approach shuffled nucleotides in peaks, while in the 'genomic' approach selected sequences from the reference genome randomly or only from gene promoters according to the fraction of A/T nucleotides in each sequence. We compiled the benchmark collections of ChIP-seq datasets for mouse, human and Arabidopsis, and performed de novo motif discovery. We showed that the genomic approach has both more robust detection of the known motifs of target transcription factors and more stringent exclusion of the simple sequence repeats as possible non-specific motifs. The advantage of the genomic approach over the synthetic approach was greater in plants compared to mammals. We developed the AntiNoise web service (https://denovosea.icgbio.ru/antinoise/) that implements a genomic approach to extract genomic background sequences for twelve eukaryotic genomes.

Dynamics of DNA methylation during osteogenic differentiation of porcine synovial membrane mesenchymal stem cells from two metabolically distinct breeds

ABSTRACT Mesenchymal stem cells (MSCs), with the ability to differentiate into osteoblasts, adipocytes, or chondrocytes, show evidence that the donor cell’s metabolic type influences the osteogenic process. Limited knowledge exists on DNA methylation changes during osteogenic differentiation and the impact of diverse donor genetic backgrounds on MSC differentiation. In this study, synovial membrane mesenchymal stem cells (SMSCs) from two pig breeds (Angeln Saddleback, AS; German Landrace, DL) with distinct metabolic phenotypes were isolated, and the methylation pattern of SMSCs during osteogenic induction was investigated. Results showed that most differentially methylated regions (DMRs) were hypomethylated in osteogenic-induced SMSC group. These DMRs were enriched with genes of different osteogenic signalling pathways at different time points including Wnt, ECM, TGFB and BMP signalling pathways. AS pigs consistently exhibited a higher number of hypermethylated DMRs than DL pigs, particularly during the peak of osteogenesis (day 21). Predicting transcription factor motifs in regions of DMRs linked to osteogenic processes and donor breeds revealed influential motifs, including KLF1, NFATC3, ZNF148, ASCL1, FOXI1, and KLF5. These findings contribute to understanding the pattern of methylation changes promoting osteogenic differentiation, emphasizing the substantial role of donor the metabolic type and epigenetic memory of different donors on SMSC differentiation.

REUNION: transcription factor binding prediction and regulatory association inference from single-cell multi-omics data.

Profiling of gene expression and chromatin accessibility by single-cell multi-omics approaches can help to systematically decipher how transcription factors (TFs) regulate target gene expression via cis-region interactions. However, integrating information from different modalities to discover regulatory associations is challenging, in part because motif scanning approaches miss many likely TF binding sites. We develop REUNION, a framework for predicting genome-wide TF binding and cis-region-TF-gene "triplet" regulatory associations using single-cell multi-omics data. The first component of REUNION, Unify, utilizes information theory-inspired complementary score functions that incorporate TF expression, chromatin accessibility, and target gene expression to identify regulatory associations. The second component, Rediscover, takes Unify estimates as input for pseudo semi-supervised learning to predict TF binding in accessible genomic regions that may or may not include detected TF motifs. Rediscover leverages latent chromatin accessibility and sequence feature spaces of the genomic regions, without requiring chromatin immunoprecipitation data for model training. Applied to peripheral blood mononuclear cell data, REUNION outperforms alternative methods in TF binding prediction on average performance. In particular, it recovers missing region-TF associations from regions lacking detected motifs, which circumvents the reliance on motif scanning and facilitates discovery of novel associations involving potential co-binding transcriptional regulators. Newly identified region-TF associations, even in regions lacking a detected motif, improve the prediction of target gene expression in regulatory triplets, and are thus likely to genuinely participate in the regulation. All source code is available at https://github.com/yangymargaret/REUNION.

DNA Transposons Favor De Novo Transcript Emergence Through Enrichment of Transcription Factor Binding Motifs.

De novo genes emerge from noncoding regions of genomes via succession of mutations. Among others, such mutations activate transcription and create a new open reading frame (ORF). Although the mechanisms underlying ORF emergence are well documented, relatively little is known about the mechanisms enabling new transcription events. Yet, in many species a continuum between absent and very prominent transcription has been reported for essentially all regions of the genome. In this study, we searched for de novo transcripts by using newly assembled genomes and transcriptomes of seven inbred lines of Drosophila melanogaster, originating from six European and one African population. This setup allowed us to detect sample specific de novo transcripts, and compare them to their homologous nontranscribed regions in other samples, as well as genic and intergenic control sequences. We studied the association with transposable elements (TEs) and the enrichment of transcription factor motifs upstream of de novo emerged transcripts and compared them with regulatory elements. We found that de novo transcripts overlap with TEs more often than expected by chance. The emergence of new transcripts correlates with regions of high guanine-cytosine content and TE expression. Moreover, upstream regions of de novo transcripts are highly enriched with regulatory motifs. Such motifs are more enriched in new transcripts overlapping with TEs, particularly DNA TEs, and are more conserved upstream de novo transcripts than upstream their 'nontranscribed homologs'. Overall, our study demonstrates that TE insertion is important for transcript emergence, partly by introducing new regulatory motifs from DNA TE families.

Unfolding the mysteries of heterogeneity from a high-resolution perspective: integration analysis of single-cell multi-omics and spatial omics revealed functionally heterogeneous cancer cells in ccRCC.

The genomic landscape of clear cell renal cell carcinoma (ccRCC) has a considerable intra-tumor heterogeneity, which is a significant obstacle in the field of precision oncology and plays a pivotal role in metastasis, recurrence, and therapeutic resistance of cancer. The mechanisms of intra-tumor heterogeneity in ccRCC have yet to be fully established. We integrated single-cell RNA sequencing (scRNA-seq) and transposase-accessible chromatin sequencing (scATAC-seq) data from a single-cell multi-omics perspective. Based on consensus non-negative matrix factorization (cNMF) algorithm, functionally heterogeneous cancer cells were classified into metabolism, inflammatory, and EMT meta programs, with spatial transcriptomics sequencing (stRNA-seq) providing spatial information of such disparate meta programs of cancer cells. The bulk RNA sequencing (RNA-seq) data revealed high clinical prognostic values of functionally heterogeneous cancer cells of three meta programs, with transcription factor regulatory network and motif activities revealing the key transcription factors that regulate functionally heterogeneous ccRCC cells. The interactions between varying meta programs and other cell subpopulations in the microenvironment were investigated. Finally, we assessed the sensitivity of cancer cells of disparate meta programs to different anti-cancer agents. Our findings inform on the intra-tumor heterogeneity of ccRCC and its regulatory networks and offers new perspectives to facilitate the designs of rational therapeutic strategies.

Bioinformatics Analysis in Predicting Transcription Factors of Robo3 Gene in Drosophila melanogaster

In bilaterian animals, axon guidance decisions are regulated by many transmembrane receptor proteins called Roundabout (Robo) family members. During the developmental stages of fruit flies (Drosophila melanogaster), three Robo family members play unique roles in the central nervous system. Robo3 is revolutionarily conserved among taxa and studies show that Robo3 regulates mediolateral axonal navigation. Recent studies suggest that Robo3 guides longitudinal axons in a manner independent of its ligand (slit). The expression patterns of Robo3 are controlled by transcription factors (TFs) that play a significant role in gene regulation, and it is not a fully understood mechanism. Knowing the transcription factor binding sites (TFBS) of Robo3 would help to predict TFs that regulate Robo3. In this study, bioinformatics tools MEME Suite, TOMTOM, and MAST were utilized to analyze the Robo3 DNA sequence to identify putative TFs that assist as docking regions for TFs involved in the regulation of Robo3 gene expression. We found seven putative TFs: Btd, Opa, Mad, Odd, Twi, CF2, and h. Mapping these TF motifs against the Robo3 sequence showed that these motifs are located in many regions of the Robo3 gene. Understanding the roles of these TFs in Robo3 gene regulation would help to implement novel strategies to control and overcome disorders related to the Robo3 gene. This study aims to identify the unknown TFs that may play a critical role in Robo3 gene expression.

DIPG-24. COMBINED EPIGENETIC THERAPY WITH FACT AND BET INHIBITORS REMODELS CHROMATIN AND DISRUPTS ONCOGENIC TRANSCRIPTION IN DIFFUSE MIDLINE GLIOMA

Abstract BACKGROUND Aberrant epigenetic regulation is a hallmark of Diffuse Midline Glioma (DMG), an incurable pediatric brain tumor. The H3K27M driver histone mutation leads to transcriptional dysregulation, indicating that targeting the epigenome and transcription may be a key therapeutic strategy against this aggressive cancer. One such target is the Facilitates Chromatin Transcription (FACT) histone chaperone. FACT is targeted by the curaxin compound CBL0137 which is currently in Phase I/II Children’s Oncology Group clinical trial as a single agent for pediatric cancer patients (NCT04870944). METHODS We applied a multi-omics approach by integrating epigenetic profiling with transcriptomics (CUT&RUN, ATAC-seq and RNA-seq) to interrogate the role of FACT in DMG. We then employed in vitro cytotoxicity assays and in vivo orthotopic patient-derived xenograft (PDX) pre-clinical models to test a novel, mechanistic-anchored epigenetic combination therapy. RESULTS We found FACT to be enriched at developmental gene promoters, coinciding with regions of open chromatin and binding motifs of core DMG regulatory transcription factors (OLIG2, TCF12, and SNAI1). FACT interacted and co-localized with the Bromodomain and Extra-Terminal Domain (BET) protein BRD4 at active promoters and enhancers, suggesting functional cooperation between FACT and BRD4 in DMG. Investigating the combination of CBL0137 with two BET inhibitors (JQ1 and trotabresib) revealed potent cytotoxicity in vitro, particularly against H3K27M-mutant cells. Furthermore, this approach was recapitulated in vivo, significantly extending the survival of three independent orthotopic PDX models of DMG. Mechanistically, CBL0137 decreased chromatin accessibility, synergising with BET inhibition to disrupt transcription, leading to transcriptional silencing of several key oncogenes (MYC, PDGFRA and MDM4) as well as causing alterations to the splicing landscape. CONCLUSIONS This study highlights the therapeutic promise of simultaneously targeting FACT and BRD4 in DMG, and provides the rationale for a combination clinical trial, currently in development.

FLI1 is associated with regulation of DNA methylation and megakaryocytic differentiation in FPDMM caused by a RUNX1 transactivation domain mutation

Familial platelet disorder with associated myeloid malignancies (FPDMM) is an autosomal dominant disease caused by heterozygous germline mutations in RUNX1. It is characterized by thrombocytopenia, platelet dysfunction, and a predisposition to hematological malignancies. Although FPDMM is a precursor for diseases involving abnormal DNA methylation, the DNA methylation status in FPDMM remains unknown, largely due to a lack of animal models and challenges in obtaining patient-derived samples. Here, using genome editing techniques, we established two lines of human induced pluripotent stem cells (iPSCs) with different FPDMM-mimicking heterozygous RUNX1 mutations. These iPSCs showed defective differentiation of hematopoietic progenitor cells (HPCs) and megakaryocytes (Mks), consistent with FPDMM. The FPDMM-mimicking HPCs showed DNA methylation patterns distinct from those of wild-type HPCs, with hypermethylated regions showing the enrichment of ETS transcription factor (TF) motifs. We found that the expression of FLI1, an ETS family member, was significantly downregulated in FPDMM-mimicking HPCs with a RUNX1 transactivation domain (TAD) mutation. We demonstrated that FLI1 promoted binding-site-directed DNA demethylation, and that overexpression of FLI1 restored their megakaryocytic differentiation efficiency and hypermethylation status. These findings suggest that FLI1 plays a crucial role in regulating DNA methylation and correcting defective megakaryocytic differentiation in FPDMM-mimicking HPCs with a RUNX1 TAD mutation.

Acute depletion of BRG1 reveals its primary function as an activator of transcription

The mammalian SWI/SNF-like BAF complexes play critical roles during animal development and pathological conditions. Previous gene deletion studies and characterization of human gene mutations implicate that the complexes both repress and activate a large number of genes. However, the direct function of the complexes in cells remains largely unclear due to the relatively long-term nature of gene deletion or natural mutation. Here we generate a mouse line by knocking in the auxin-inducible degron tag (AID) to the Smarca4 gene, which encodes BRG1, the essential ATPase subunit of the BAF complexes. We show that the tagged BRG1 can be efficiently depleted by osTIR1 expression and auxin treatment for 6 to 10 h in CD4 + T cells, hepatocytes, and fibroblasts isolated from the knock-in mice. The acute depletion of BRG1 leads to decreases in nascent RNAs and RNA polymerase II binding at a large number of genes, which are positively correlated with the loss of BRG1. Further, these changes are correlated with diminished accessibility at DNase I Hypersensitive Sites (DHSs) and p300 binding. The acute BRG1 depletion results in three major patterns of nucleosome shifts leading to narrower nucleosome spacing surrounding transcription factor motifs and at enhancers and transcription start sites (TSSs), which are correlated with loss of BRG1, decreased chromatin accessibility and decreased nascent RNAs. Acute depletion of BRG1 severely compromises the Trichostatin A (TSA) -induced histone acetylation, suggesting a substantial interplay between the chromatin remodeling activity of BRG1 and histone acetylation. Our data suggest BRG1 mainly plays a direct positive role in chromatin accessibility, RNAPII binding, and nascent RNA production by regulating nucleosome positioning and facilitating transcription factor binding to their target sites.

IL-4–STAT6 axis amplifies histamine-induced vascular endothelial dysfunction and hypovolemic shock

BackgroundMast cell (MC)-derived mediators induce vasodilatation and fluid extravasation, leading to cardiovascular failure in severe anaphylaxis. We have previously revealed a synergistic interaction between the cytokine IL-4 and the MC-derived mediator histamine in modulating vascular endothelial (VE) dysfunction and severe anaphylaxis. The mechanism by which IL-4 exacerbates histamine-induced VE dysfunction and severe anaphylaxis are unknown. ObjectiveTo identify the IL-4-induced molecular processes regulating the amplification of histamine-induced VE barrier dysfunction and the severity of IgE-mediated anaphylaxic reactions. MethodsRNAseq, Western blot, Ca2+ imaging and barrier functional analyses on the vascular endothelial cell line (EA.hy926). Pharmacologic degraders (selective PROTAC (proteolysis-targeting chimera) and genetic (lentiviral shRNA) inhibitors were used to determine the roles of STAT3 and STAT6 in conjunction with in vivo model systems of histamine-induced hypovolemic shock. ResultsIL-4 enhancement of histamine-induced VE barrier dysfunction was associated with increased VE-Cadherin degradation, intracellular calcium flux, phospho-Src levels and required transcription and de novo protein synthesis. RNAseq analyses of IL-4 stimulated VE cells identified dysregulation of genes involved in cell proliferation, cell development, and cell growth and transcription factor motif analyses revealed a significant enrichment of differential expressed genes (DEGs) with putative STAT3 and STAT6 motif. IL-4 stimulation in EA.hy926 cells induced both STAT3Y705 and STAT3S727 phosphorylation. Genetic and pharmacologic ablation of VE STAT3 activity revealed a role for STAT3 in basal VE barrier function, however IL-4 enhancement and histamine-induced VE barrier dysfunction was predominantly STAT3-independent. In contrast, IL-4 enhancement and histamine-induced VE barrier dysfunction was STAT6-dependent. Consistent with this finding, pharmacologic knockdown of STAT6 abrogated IL-4-mediated amplification of histamine-induced hypovolemia. ConclusionsThese studies unveil a novel role of the IL-4/ STAT6 signaling axis in the priming of VE cells predisposing to exacerbation of histamine-induced anaphylaxis.

Cumin Seed Oil Induces Oxidative Stress-Based Antifungal Activities on Fusarium graminearum.

In this study, the antifungal activity of cumin seed oil (CSO) was tested on Fusarium graminearum. (i) Minimum inhibitory concentrations (MICs) and related concentrations (IC75, IC50, and IC25) were detected; (ii) toxicity was evaluated by a water-soluble tetrazolium salt-1 (WST-1) assay; (iii) genomic/epigenomic alterations were evaluated by the coupled restriction enzyme digestion-random amplification (CRED-RA) method; (iv) oxidative stress was investigated by CAT expression, catalase activity, and DCF-DA staining; (v) deoxynivalenol biosynthesis was evaluated by tri6 expression; (vi) and potential effects of CSO on wheat were tested by a water loss rate (WLR) assay. MIC, IC75, IC50 and IC25 values were detected at 0.5, 0.375, 0.25, and 0.125 mg mL-1. In WST-1 assays, significant decreases (p < 0.001) were detected. Genomic template stability (GTS) related to methylation differences ranged from 94.60% to 96.30%. Percentage polymorphism for HapII/MspI values were as 9.1%/15.8%. CAT (oxidative stress-related catalase) and tri6 (zinc finger motif transcription factor) gene expressions were recorded between 5.29 ± 0.74 and 0.46 ± 0.10 (p < 0.05). Increased catalase activity was detected (p < 0.05) by spectrophotometric assays. DCF-DA-stained (oxidative stressed) cells were increased in response to increased concentrations, and there were no significant changes in WLR values. It was concluded that CSO showed strong antifungal activity on F. graminearum via different physiological levels.

Abstract 2122: A Multiomic Timecourse Atlas Of Murine Vascular Disease Smooth Muscle Cell Transition Phenotypes And Their Perturbation With Tcf21 Knockout

Background: Vascular smooth muscle cells (SMC) contribute to heritable CAD risk and undergo cell changes to fibroblast-like (FMC) and osteochondrogenic (CMC) phenotypes. We perform molecular characterization of this transition process over the course of atherosclerosis development to better understand how causal SMC CAD genes such as TCF21 drive the phenotypic transition process to alter CAD risk. Methods/Results: Deep multiomic profiling of aortic roots from control atherosclerosis model ( Myh11Cre ERT2 , ROSA tdT/+ , ApoE -/- ) mice with SMC specific lineage tracing on high fat diet were harvested for 7 scRNA and 6 scATAC time points across 16 weeks. Similarly, aortic roots from Tcf21 knockout ( Myh11Cre ERT2 , Tcf21 ΔSMC/ΔSMC , ROSA tdT/+ , ApoE -/- ) were collected at 3 timepoints for paired scRNA/scATAC. Waddington-Optimal Transport (WOT) mapped transcriptional trajectories across SMC lineage clusters which terminate in FMC and CMC phenotype clusters. PANDO/CellOracle frameworks were used to create gene regulatory networks with the ability to model SMC transition perturbations in silico . These analyses generated a multiomic timecourse atlas demonstrating the lineage traced phenotypic progression of SMC to FMC and CMC. Upon knockout of Tcf21 , we discern notable shifts in the SMC to FMC transition characterized by differential scRNA transcriptional pathways and scATAC shifts in transcription factor (TF) motif accessibility which also overlap with WOT predicted TF enrichment for each cell state. These integrative analyses revealed Tcf21 regulation of Cebpb and the Tead1 -Hippo pathways as novel mechanisms for mediating its role in modulation of disease related processes such as inflammation, response to TGF- β signaling and cell proliferation. Further mechanistic validation is performed through a combination of TF binding site analysis with TEAD1 and CEBPB ChIP-Seq in human coronary artery SMCs, dual luciferase reporter assays and proximity ligation assays. Conclusion: We construct a single cell atherosclerosis timecourse to model mouse SMC cell states and perturb CAD gene Tcf21 to elucidate the interplay between epigenetic and transcriptional mechanisms which affect key SMC transition elements such as CEBPB and the TEAD1 -Hippo axis.

Abstract 1099: Single-cell Transcriptomic And Epigenic Profiling Identifies Sox6 As A Key Regulator Of Arterial Endothelial Cell Phenotype In Systemic Sclerosis-associated Pulmonary Hypertension

Background: Pulmonary hypertension (PH) frequently arises as a complication of systemic sclerosis (SSc), and is a major cause of death among patients with this condition. Pulmonary vasculopathy in SSc is characterized by endothelial dysfunction with subsequent remodeling of the pulmonary vasculature, especially arterioles and small arteries. Characterization of transcriptional regulators driving the pathological changes in arterial endothelial cells (ECs) is critical to understanding pathogenic mechanisms underlying SSc-associated vasculopathy. Methods and Results: We performed single-cell multiome sequencing to simultaneously profile the transcriptome and epigenome of explanted lung tissue from SSc-PH patients (n = 9) and healthy donors (n = 6). We identified co-regulated genes and candidate enhancer regions associated with transcription factors (TFs) across different EC cell states using SCENIC+. Gene regulatory network analysis revealed SOX6 as a key transcription factor for the arterial EC phenotype associated with SSc-PH. 71 genes and 153 regions were predicted to be regulated by SOX6. Notably, SOX6 was the only SOX family member significantly upregulated in SSc-PH arterial ECs (fold change = 40.0, adjusted p-value = 6.1*10 -21 ). Putative bound TF motif instances accounting for chromatin accessibility in the candidate enhancer regions were identified using the base-resolution deep learning model, ChromBPNet. Consistent with SCENIC+ predictions, cell-state specific ChromBPNet models revealed higher putative TF occupancy at SOX motif instances across the genome. In addition, we identified SOX/ETS composite motif instances showing higher putative occupancy in SSc-PH arterial ECs, which may support cooperative binding of SOX proteins with ETS family members. Conclusions: These data provide novel insight into transcriptional regulatory programs of arterial ECs in SSc-PH and highlight SOX6 as a key contributor.

How microRNAs go awry in cancer: Exploring the underlying factors of miRNA dysregulation

MicroRNAs (miRNA) are short, non-coding molecules (~23nt) that fine-tune mRNA expression at the post-transcriptional level by repressing or cleaving their targets. A single miRNA targets several mRNAs; dysfunctions disrupt biological systems. Unravelling the interactome of regulators and targets is vital for biological networks. Cancer research is one of the hottest fields, due to its high prevalence and financial burden. Despite improvements in prevention and therapy, challenges from disease complexity and precision medicine limit the effciency of targeted approaches. Accurate characterization of all interactions and mechanisms is essential to fight it. In this study, we matched whole genome, RNA and small RNA sequencing data acquired from the International Cancer Genome Consortium (ICGC) for Liver cancer patients, resulting in a dataset of 40 samples with the aim to explain the dysregulation of miRNA expression. DNA variants were readily extracted using the “Broad variant call pipeline” of ICGC. Utilization of DNase-seq data indicated an average promoter region activity of 1500bp upstream and 500bp downstream of the transcription start site. Variations were then intersected to promoter regions. Analysis of transcription factors (TF), which regulate the expression of DNA bound genes, as well as miRNAs, was done using FIMO prediction algorithm (25bp proximal window centered on identified variants, p-value &lt; 0.000001) combined with JASPAR transcription factor motifs to identify mutated transcription factor binding sites. Matching RNA-seq expression was then combined with small RNA-seq expression and correlation analysis was performed so as to identify dysregulated TF::miRNA interacting pairs with mutations on their regulatory elements, on the basis that their correlated expression is potentially disrupted due to overlapping variation events. A total of 172691 variants events were identified in liver promoter regions. Analysis of mutational load using chi-square test in various gene biotypes indicated differences with protein-coding and small RNA (excluding miRNA) categories exhibiting the higher rates. Variants in TF genes generally showed low frequency (3.64% of all TF genes analyzed), necessitating a deeper analysis to discover if the underling mechanisms of TFBS are affected by variation events. Of all TFs utilized in the FIMO analysis (n=39), ZNF263, which has been associated to a variety of tumours, was found to have the highest percentage of mutated binding motif (31%) followed by ZNF384 (28%). Pearson correlation analysis between the expression of miRNAs and TFs with and without mutations overlapping TF binding sites in miRNA promoters identified HNF4A (Hepatocyte Nuclear Factor 4) and hsa-mir-122 having significant difference in their correlated expression (Transcripts per million normalized values) between wildtype samples (n=20, r=0.8, p=0.00002) and mutated individuals (n=20, r=0.25, p=026), with both molecules being associated with liver localized functions in the literature. These results demonstrate the importance of further enriching the interactome towards a more complete network in order to achieve a more effective and accurate precision medicine. Hellenic Foundation for Research and Innovation (H.F.R.I.) under the ‘1st Call for H.F.R.I. Research Projects to support Faculty Members &amp; Researchers and the procurement of high-cost research equipment grant’ [2563]. Funding for open access charge. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

A Bayesian Change Point Model for Dynamic Alternative Transcription Start Site Usage During Cellular Differentiation.

ABSTRACT An alternative transcription start site (ATSS) is a major driving force for increasing the complexity of transcripts in human tissues. As a transcriptional regulatory mechanism, ATSS has biological significance. Many studies have confirmed that ATSS plays an important role in diseases and cell development and differentiation. However, exploration of its dynamic mechanisms remains insufficient. Identifying ATSS change points during cell differentiation is critical for elucidating potential dynamic mechanisms. For relative ATSS usage as percentage data, the existing methods lack sensitivity to detect the change point for ATSS longitudinal data. In addition, some methods have strict requirements for data distribution and cannot be applied to deal with this problem. In this study, the Bayesian change point detection model was first constructed using reparameterization techniques for two parameters of a beta distribution for the percentage data type, and the posterior distributions of parameters and change points were obtained using Markov Chain Monte Carlo (MCMC) sampling. With comprehensive simulation studies, the performance of the Bayesian change point detection model is found to be consistently powerful and robust across most scenarios with different sample sizes and beta distributions. Second, differential ATSS events in the real data, whose change points were identified using our method, were clustered according to their change points. Last, for each change point, pathway and transcription factor motif analyses were performed on its differential ATSS events. The results of our analyses demonstrated the effectiveness of the Bayesian change point detection model and provided biological insights into cell differentiation.