Transcription Factor Occupancy Research Articles

A fine kinetic balance of interactions directs transcription factor hubs to genes.

Eukaryotic gene regulation relies on the binding of sequence-specific transcription factors (TFs). TFs bind chromatin transiently yet occupy their target sites by forming high-local concentration microenvironments (hubs and condensates) that increase the frequency of binding. Despite their ubiquity, such microenvironments are difficult to study in endogenous contexts due to technical limitations. Here, we use live embryo light-sheet imaging, single-molecule tracking, and genomics to overcome these limitations and investigate how hubs are localized to target genes to drive TF occupancy and transcription. By examining mutants of a hub-forming TF, Zelda, in Drosophila embryos, we find that hub formation propensity, spatial distributions, and temporal stabilities are differentially regulated by DNA binding and disordered protein domains. We show that hub localization to genomic targets is driven by a finely-tuned kinetic balance of interactions between proteins and chromatin, and hubs can be redirected to new genomic sites when this balance is perturbed.

Chromatin endogenous cleavage provides a global view of yeast RNA polymerase II transcription kinetics.

Chromatin immunoprecipitation (ChIP-seq) is the most common approach to observe global binding of proteins to DNA in vivo. The occupancy of transcription factors (TFs) from ChIP-seq agrees well with an alternative method, chromatin endogenous cleavage (ChEC-seq2). However, ChIP-seq and ChEC-seq2 reveal strikingly different patterns of enrichment of yeast RNA polymerase II. We hypothesized that this reflects distinct populations of RNAPII, some of which are captured by ChIP-seq and some of which are captured by ChEC-seq2. RNAPII association with enhancers and promoters - predicted from biochemical studies - is detected well by ChEC-seq2 but not by ChIP-seq. Enhancer/promoter bound RNAPII correlates with transcription levels and matches predicted occupancy based on published rates of enhancer recruitment, preinitiation assembly, initiation, elongation and termination. The occupancy from ChEC-seq2 allowed us to develop a stochastic model for global kinetics of RNAPII transcription which captured both the ChEC-seq2 data and changes upon chemical-genetic perturbations to transcription. Finally, RNAPII ChEC-seq2 and kinetic modeling suggests that a mutation in the Gcn4 transcription factor that blocks interaction with the NPC destabilizes promoter-associated RNAPII without altering its recruitment to the enhancer.

ScNanoSeq-CUT&Tag: a single-cell long-read CUT&Tag sequencing method for efficient chromatin modification profiling within individual cells.

Chromatin modifications are fundamental epigenetic marks that determine genome functions, but it remains challenging to profile those of repetitive elements and complex genomic regions. Here, we develop scNanoSeq-CUT&Tag, a streamlined method, by adapting modified cleavage under targets and tagmentation (CUT&Tag) to the nanopore sequencing platform for genome-wide chromatin modification profiling within individual cells. We show that scNanoSeq-CUT&Tag can accurately profile histone marks and transcription factor occupancy patterns at single-cell resolution as well as distinguish different cell types. scNanoSeq-CUT&Tag efficiently maps the allele-specific chromatin modifications and allows analysis of their neighboring region co-occupancy patterns within individual cells. Moreover, scNanoSeq-CUT&Tag can accurately detect chromatin modifications for individual copies of repetitive elements in both human and mouse genomes. Overall, we prove that scNanoSeq-CUT&Tag is a valuable single-cell tool for efficiently profiling histone marks and transcription factor occupancies, especially for previously poorly studied complex genomic regions and blacklist genomic regions.

Allele-specific transcription factor binding across human brain regions offers mechanistic insight into eQTLs.

Transcription factors (TFs) regulate gene expression by facilitating or disrupting the formation of transcription initiation machinery at particular genomic loci. Because TF occupancy is driven in part by recognition of DNA sequence, genetic variation can influence TF-DNA associations and gene regulation. To identify variants that impact TF binding in human brain tissues, we assessed allele-specific binding (ASB) at heterozygous variants for 94 TFs in nine brain regions from two donors. Leveraging graph genomes constructed from phased genomic sequence data, we compared ChIP-seq signals between alleles at heterozygous variants within each brain region and identified thousands of variants exhibiting ASB for at least one TF. ASB reproducibility was measured by comparisons between independent experiments both within and between donors. We found that rare alleles in the general population more frequently led to reduced TF binding, whereas common alleles had an equal likelihood of increasing or decreasing binding. Further, for ASB variants in predicted binding motifs, the favored allele tended to be the one with the stronger expected motif match, but this concordance was not observed within highly occupied sites. We also found that neuron-specific cis-regulatory elements (cCREs), in contrast with oligodendrocyte-specific cCREs, showed depletion of ASB variants. We identified 2670 ASB variants associated with evidence for allele-specific gene expression in the brain from GTEx data and observed increasing eQTL effect direction concordance as ASB significance increases. These results provide a valuable and unique resource for mechanistic analysis of cis-regulatory variation in human brain tissue.

An Atlas of Accessible Chromatin in Advanced Prostate Cancer Reveals the Epigenetic Evolution during Tumor Progression.

Metastatic castration-resistant prostate cancer (mCRPC) is a lethal disease that resists therapy targeting androgen signaling, the primary driver of prostate cancer. mCRPC resists androgen receptor (AR) inhibitors by amplifying AR signaling or by evolving into therapy-resistant subtypes that do not depend on AR. Elucidation of the epigenetic underpinnings of these subtypes could provide important insights into the drivers of therapy resistance. In this study, we produced chromatin accessibility maps linked to the binding of lineage-specific transcription factors (TF) by performing assay for transposase-accessible chromatin sequencing on 70 mCRPC tissue biopsies integrated with transcriptome and whole-genome sequencing. mCRPC had a distinct global chromatin accessibility profile linked to AR function. Analysis of TF occupancy across accessible chromatin revealed 203 TFs associated with mCRPC subtypes. Notably, ZNF263 was identified as a putative prostate cancer TF with a significant impact on gene activity in the double-negative subtype (AR- neuroendocrine-), potentially activating MYC targets. Overall, this analysis of chromatin accessibility in mCRPC provides valuable insights into epigenetic changes that occur during progression to mCRPC. Significance: Integration of a large cohort of transcriptome, whole-genome, and ATAC sequencing characterizes the chromatin accessibility changes in advanced prostate cancer and identifies therapy-resistant prostate cancer subtype-specific transcription factors that modulate oncogenic programs.

DFF-ChIP: a method to detect and quantify complex interactions between RNA polymerase II, transcription factors, and chromatin.

Recently, we introduced a chromatin immunoprecipitation (ChIP) technique utilizing the human DNA Fragmentation Factor (DFF) to digest the DNA prior to immunoprecipitation (DFF-ChIP) that provides the precise location of transcription complexes and their interactions with neighboring nucleosomes. Here we expand the technique to new targets and provide useful information concerning purification of DFF, digestion conditions, and the impact of crosslinking. DFF-ChIP analysis was performed individually for subunits of Mediator, DSIF, and NELF that that do not interact with DNA directly, but rather interact with RNA polymerase II (Pol II). We found that Mediator was associated almost exclusively with preinitiation complexes (PICs). DSIF and NELF were associated with engaged Pol II and, in addition, potential intermediates between PICs and early initiation complexes. DFF-ChIP was then used to analyze the occupancy of a tight binding transcription factor, CTCF, and a much weaker binding factor, glucocorticoid receptor (GR), with and without crosslinking. These results were compared to those from standard ChIP-Seq that employs sonication and to CUT&RUN which utilizes MNase to fragment the genomic DNA. Our findings indicate that DFF-ChIP reveals details of occupancy that are not available using other methods including information revealing pertinent protein:protein interactions.

Single-nucleus multi-omics identifies shared and distinct pathways in Pick's and Alzheimer's disease.

The study of neurodegenerative diseases, particularly tauopathies like Pick's disease (PiD) and Alzheimer's disease (AD), offers insights into the underlying regulatory mechanisms. By investigating epigenomic variations in these conditions, we identified critical regulatory changes driving disease progression, revealing potential therapeutic targets. Our comparative analyses uncovered disease-enriched non-coding regions and genome-wide transcription factor (TF) binding differences, linking them to target genes. Notably, we identified a distal human-gained enhancer (HGE) associated with E3 ubiquitin ligase (UBE3A), highlighting disease-specific regulatory alterations. Additionally, fine-mapping of AD risk genes uncovered loci enriched in microglial enhancers and accessible in other cell types. Shared and distinct TF binding patterns were observed in neurons and glial cells across PiD and AD. We validated our findings using CRISPR to excise a predicted enhancer region in UBE3A and developed an interactive database (http://swaruplab.bio.uci.edu/scROAD) to visualize predicted single-cell TF occupancy and regulatory networks.

Temporally discordant chromatin accessibility and DNA demethylation define short and long-term enhancer regulation during cell fate specification.

Epigenetic mechanisms govern the transcriptional activity of lineage-specifying enhancers; but recent work challenges the dogma that joint chromatin accessibility and DNA demethylation are prerequisites for transcription. To understand this paradox, we established a highly-resolved timeline of DNA demethylation, chromatin accessibility, and transcription factor occupancy during neural progenitor cell differentiation. We show thousands of enhancers undergo rapid, transient accessibility changes associated with distinct periods of transcription factor expression. However, most DNA methylation changes are unidirectional and delayed relative to chromatin dynamics, creating transiently discordant epigenetic states. Genome-wide detection of 5-hydroxymethylcytosine further revealed active demethylation begins ahead of chromatin and transcription factor activity, while enhancer hypomethylation persists long after these activities have dissipated. We demonstrate that these timepoint specific methylation states predict past, present and future chromatin accessibility using machine learning models. Thus, chromatin and DNA methylation collaborate on different timescales to mediate short and long-term enhancer regulation during cell fate specification.

An updated compendium and reevaluation of the evidence for nuclear transcription factor occupancy over the mitochondrial genome.

In most eukaryotes, mitochondrial organelles contain their own genome, usually circular, which is the remnant of the genome of the ancestral bacterial endosymbiont that gave rise to modern mitochondria. Mitochondrial genomes are dramatically reduced in their gene content due to the process of endosymbiotic gene transfer to the nucleus; as a result most mitochondrial proteins are encoded in the nucleus and imported into mitochondria. This includes the components of the dedicated mitochondrial transcription and replication systems and regulatory factors, which are entirely distinct from the information processing systems in the nucleus. However, since the 1990s several nuclear transcription factors have been reported to act in mitochondria, and previously we identified 8 human and 3 mouse transcription factors (TFs) with strong localized enrichment over the mitochondrial genome using ChIP-seq (Chromatin Immunoprecipitation) datasets from the second phase of the ENCODE (Encyclopedia of DNA Elements) Project Consortium. Here, we analyze the greatly expanded in the intervening decade ENCODE compendium of TF ChIP-seq datasets (a total of 6,153 ChIP experiments for 942 proteins, of which 763 are sequence-specific TFs) combined with interpretative deep learning models of TF occupancy to create a comprehensive compendium of nuclear TFs that show evidence of association with the mitochondrial genome. We find some evidence for chrM occupancy for 50 nuclear TFs and two other proteins, with bZIP TFs emerging as most likely to be playing a role in mitochondria. However, we also observe that in cases where the same TF has been assayed with multiple antibodies and ChIP protocols, evidence for its chrM occupancy is not always reproducible. In the light of these findings, we discuss the evidential criteria for establishing chrM occupancy and reevaluate the overall compendium of putative mitochondrial-acting nuclear TFs.

Multiomic profiling of transcription factor binding and function in human brain.

Transcription factors (TFs) orchestrate gene expression programs crucial for brain function, but we lack detailed information about TF binding in human brain tissue. We generated a multiomic resource (ChIP-seq, ATAC-seq, RNA-seq, DNA methylation) on bulk tissues and sorted nuclei from several postmortem brain regions, including binding maps for more than 100 TFs. We demonstrate improved measurements of TF activity, including motif recognition and gene expression modeling, upon identification and removal of high TF occupancy regions. Further, predictive TF binding models demonstrate a bias for these high-occupancy sites. Neuronal TFs SATB2 and TBR1 bind unique regions depleted for such sites and promote neuronal gene expression. Binding sites for TFs, including TBR1 and PKNOX1, are enriched for risk variants associated with neuropsychiatric disorders, predominantly in neurons. This work, titled BrainTF, is a powerful resource for future studies seeking to understand the roles of specific TFs in regulating gene expression in the human brain.

Genome-wide nucleosome and transcription factor responses to genetic perturbations reveal chromatin-mediated mechanisms of transcriptional regulation.

Epigenetic mechanisms contribute to gene regulation by altering chromatin accessibility through changes in transcription factor (TF) and nucleosome occupancy throughout the genome. Despite numerous studies focusing on changes in gene expression, the intricate chromatin-mediated regulatory code remains largely unexplored on a comprehensive scale. We address this by employing a factor-agnostic, reverse-genetics approach that uses MNase-seq to capture genome-wide TF and nucleosome occupancies in response to the individual deletion of 201 transcriptional regulators in Saccharomyces cerevisiae, thereby assaying nearly one million mutant-gene interactions. We develop a principled approach to identify and quantify chromatin changes genome-wide, observing differences in TF and nucleosome occupancy that recapitulate well-established pathways identified by gene expression data. We also discover distinct chromatin signatures associated with the up- and downregulation of genes, and use these signatures to reveal regulatory mechanisms previously unexplored in expression-based studies. Finally, we demonstrate that chromatin features are predictive of transcriptional activity and leverage these features to reconstruct chromatin-based transcriptional regulatory networks. Overall, these results illustrate the power of an approach combining genetic perturbation with high-resolution epigenomic profiling; the latter enables a close examination of the interplay between TFs and nucleosomes genome-wide, providing a deeper, more mechanistic understanding of the complex relationship between chromatin organization and transcription.

Effect of genomic and cellular environments on gene expression noise

BackgroundIndividual cells from isogenic populations often display large cell-to-cell differences in gene expression. This “noise” in expression derives from several sources, including the genomic and cellular environment in which a gene resides. Large-scale maps of genomic environments have revealed the effects of epigenetic modifications and transcription factor occupancy on mean expression levels, but leveraging such maps to explain expression noise will require new methods to assay how expression noise changes at locations across the genome.ResultsTo address this gap, we present Single-cell Analysis of Reporter Gene Expression Noise and Transcriptome (SARGENT), a method that simultaneously measures the noisiness of reporter genes integrated throughout the genome and the global mRNA profiles of individual reporter-gene-containing cells. Using SARGENT, we perform the first comprehensive genome-wide survey of how genomic locations impact gene expression noise. We find that the mean and noise of expression correlate with different histone modifications. We quantify the intrinsic and extrinsic components of reporter gene noise and, using the associated mRNA profiles, assign the extrinsic component to differences between the CD24+ “stem-like” substate and the more “differentiated” substate. SARGENT also reveals the effects of transgene integrations on endogenous gene expression, which will help guide the search for “safe-harbor” loci.ConclusionsTaken together, we show that SARGENT is a powerful tool to measure both the mean and noise of gene expression at locations across the genome and that the data generatd by SARGENT reveals important insights into the regulation of gene expression noise genome-wide.

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.

Probabilistic association of differentially expressed genes with cis-regulatory elements.

Differential gene expression in response to perturbations is mediated at least in part by changes in binding of transcription factors (TFs) and other proteins at specific genomic regions. Association of these cis-regulatory elements (CREs) with their target genes is a challenging task that is essential to address many biological and mechanistic questions. Many current approaches rely on chromatin conformation capture techniques or single-cell correlational methods to establish CRE-to-gene associations. These methods can be effective but have limitations, including resolution, gaps in detectable association distances, and cost. As an alternative, we have developed DegCre, a nonparametric method that evaluates correlations between measurements of perturbation-induced differential gene expression and differential regulatory signal at CREs to score possible CRE-to-gene associations. It has several unique features, including the ability to use any type of CRE activity measurement, yield probabilistic scores for CRE-to-gene pairs, and assess CRE-to-gene pairings across a wide range of sequence distances. We apply DegCre to six data sets, each using different perturbations and containing a variety of regulatory signal measurements, including chromatin openness, histone modifications, and TF occupancy. To test their efficacy, we compare DegCre associations to Hi-C loop calls and CRISPR-validated CRE-to-gene associations, establishing good performance by DegCre that is comparable or superior to competing methods. DegCre is a novel approach to the association of CREs to genes from a perturbation-differential perspective, with strengths that are complementary to existing approaches and allow for new insights into gene regulation.

Abstract LB196: An atlas of accessible chromatin in advanced prostate cancer

Abstract Although androgen deprivation therapy (ADT) and second-generation Androgen Receptor (AR) signaling inhibitors (ARSIs) extend the survival of patients with metastatic castration-resistant prostate cancer (mCRPC), these tumors almost inevitably progress on these therapies. Prolonged usage of ADT and/or ARSI is associated with the development of treatment-associated transcriptional subtypes distinguished by alterations that affect the activity of the AR and neuroendocrine (NE) genes. Genomic and transcriptomic distinctions between the mCRPC subtypes are emerging. However, relatively little is known about the epigenome of the mCRPC transcriptional subtypes. We hypothesized that epigenetic mechanisms drive lineage plasticity in mCRPC by changing chromatin accessibility at regulatory loci for transcription factors (TFs) that affect downstream gene activity. In this study, we produced high-resolution chromatin accessibility maps linked to lineage-specific TF binding by performing Assay for Transposase-Accessible Chromatin using sequencing (ATAC-seq) on 70 mCRPC tissue biopsy samples from the SU2C/PCF West Coast Prostate Cancer Dream Team cohort. Here, we demonstrate that the global chromatin accessibility profile of mCRPC is unique and primarily correlated with the functional activity of AR. We comprehensively examined genome-wide TF occupancy signals in accessible chromatin regions and identified 203 TFs associated with mCRPC subtypes. Our findings indicated that different mCRPC subtypes exhibit a specific preference for certain TFs. The AR+NE- subtype showed a greater dependence on TFs, such as FOXA1, AR, and HOXB13, while the AR-NE+ subtype was more addicted to ASCL1 and NEUROD1. We found that a group of TFs including SP1, KLF5, and ZNF263 that bind to GC-rich sequences, are likely to play a major role in the regulation of the AR-NE- subtype. Most notably, we identified a potentially novel TF in prostate cancer, ZNF263, which is estimated to have a significant impact on gene activity in the AR-NE- subtype and may play a role in activating the targets of the MYC signaling pathway. Overall, our analysis of chromatin accessibility in mCRPC provides valuable insights into epigenetic changes that occur during mCRPC progression. Citation Format: Raunak Shrestha, Lisa N. Chesner, Meng Zhang, Stanley Zhou, Adam Foye, Arian Lundberg, Alana S. Weinstein, Martin Sjöström, Xiaolin Zhu, Thaidy Moreno-Rodriguez, Haolong Li, Joshi J. Alumkal, Rahul Aggarwal, Eric J. Small, Mathieu Lupien, David A. Quigley, Felix Y. Feng. An atlas of accessible chromatin in advanced prostate cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr LB196.

Targeting the chromatin binding of exportin-1 disrupts NFAT and T cell activation.

Exportin-1 (XPO1/CRM1) plays a central role in the nuclear-to-cytoplasmic transport of hundreds of proteins and contributes to other cellular processes, such as centrosome duplication. Small molecules targeting XPO1 induce cytotoxicity, and selinexor was approved by the Food and Drug Administration in 2019 as a cancer chemotherapy for relapsed multiple myeloma. Here, we describe a cell-type-dependent chromatin-binding function for XPO1 that is essential for the chromatin occupancy of NFAT transcription factors and thus the appropriate activation of T cells. Additionally, we establish a class of XPO1-targeting small molecules capable of disrupting the chromatin binding of XPO1 without perturbing nuclear export or inducing cytotoxicity. This work defines a broad transcription regulatory role for XPO1 that is essential for T cell activation as well as a new class of XPO1 modulators to enable therapeutic targeting of XPO1 beyond oncology including in T cell-driven autoimmune disorders.

The Binding Occupancy of Pro-Hypertrophic Transcription Factors among Cardiac Developmental Enhancers

Objective: To check which of the eight pro-hypertrophic transcription factors bind in abundance to a set of 140 functionally confirmed cardiac enhancers aiming to identify potential therapeutic targets for delaying the onset of cardiac hypertrophy. Study Design: Bioinformatics, in-Silico Study.Place and Duration of Study: The study was conducted at the Department of Biological Sciences, National University of Medical Sciences (NUMS), Rawalpindi, Pakistan, from January 2022 to December 2022.Methods: For experimentally verified human and mouse noncoding regions with gene enhancer activity measured in transgenic mice, VISTA Enhancer Browser was used to obtain 140 cardiac-specific functionally confirmed enhancers. Among these enhancers, transcription factor binding sites were computationally mapped to assess transcription factor occupancy. The binding sites for all eight transcription factors were screened in the collective set of 140 cardiac enhancers via an in-house script. Results: In this study, among a small set of eight pro-hypertrophic transcription factors, occupancy of transcription factor HAND (Heart- And Neural Crest Derivatives-Expressed Protein) was found to be maximum in the collected pool of functionally confirmed cardiac enhancers. Conclusion: The transcription factor HAND was found to have maximum binding occupancy among a set of experimentally confirmed cardiac enhancers.How to cite this: Raza RZ, Hussain I, Akhtar F, Mumtaz S. The Binding Occupancy of Pro-Hypertrophic Transcription Factors among Cardiac Developmental Enhancers. Life and Science. 2024; 5(1): 22-26. doi: http://doi.org/10.37185/LnS.1.1.464

ChEC-seq2: an improved chromatin endogenous cleavage sequencing method and bioinformatic analysis pipeline for mapping in vivo protein-DNA interactions.

Defining the in vivo DNA binding specificity of transcription factors (TFs) has relied nearly exclusively on chromatin immunoprecipitation (ChIP). While ChIP reveals TF binding patterns, its resolution is low. Higher resolution methods employing nucleases such as ChIP-exo, chromatin endogenous cleavage (ChEC-seq) and CUT&RUN resolve both TF occupancy and binding site protection. ChEC-seq, in which an endogenous TF is fused to micrococcal nuclease, requires neither fixation nor antibodies. However, the specificity of DNA cleavage during ChEC has been suggested to be lower than the specificity of the peaks identified by ChIP or ChIP-exo, perhaps reflecting non-specific binding of transcription factors to DNA. We have simplified the ChEC-seq protocol to minimize nuclease digestion while increasing the yield of cleaved DNA. ChEC-seq2 cleavage patterns were highly reproducible between replicates and with published ChEC-seq data. Combined with DoubleChEC, a new bioinformatic pipeline that removes non-specific cleavage sites, ChEC-seq2 identified high-confidence cleavage sites for three different yeast TFs that are strongly enriched for their known binding sites and adjacent to known target genes.

Single-cell joint profiling of multiple epigenetic proteins and gene transcription.

Sculpting the epigenome with a combination of histone modifications and transcription factor occupancy determines gene transcription and cell fate specification. Here, we first develop uCoTarget, utilizing a split-pool barcoding strategy for realizing ultrahigh-throughput single-cell joint profiling of multiple epigenetic proteins. Through extensive optimization for sensitivity and multimodality resolution, we demonstrate that uCoTarget enables simultaneous detection of five histone modifications (H3K27ac, H3K4me3, H3K4me1, H3K36me3, and H3K27me3) in 19,860 single cells. We applied uCoTarget to the in vitro generation of hematopoietic stem/progenitor cells (HSPCs) from human embryonic stem cells, presenting multimodal epigenomic profiles in 26,418 single cells. uCoTarget reveals establishment of pairing of HSPC enhancers (H3K27ac) and promoters (H3K4me3) and RUNX1 engagement priming for H3K27ac activation along the HSPC path. We then develop uCoTargetX, an expansion of uCoTarget to simultaneously measure transcriptome and multiple epigenome targets. Together, our methods enable generalizable, versatile multimodal profiles for reconstructing comprehensive epigenome and transcriptome landscapes and analyzing the regulatory interplay at single-cell level.

MED16 Negatively Regulates Erythropoiesis and Myelopoiesis through Modulation of Chromatin Accessibility

Tissue-specific transcription factors work in conjunction with numerous coregulators to modulate transcription networks during hematopoiesis; disruptions in this process may lead to hematopoietic disorders, including myelodysplastic syndromes (MDS). While the Mediator complex is known to regulate various aspects of hematopoiesis, the mechanisms underlying subunit-specific functions have not been fully understood. Here, we uncovered that MED16, a core component of Mediator tail module, serves as a crucial negative regulator of erythropoiesis and myelopoiesis by controlling chromatin accessibility at specific target genes. To investigate the function of MED16 in vivo, we transplanted control (shSCR) or MED16-deficient (shMED16) human cord blood CD34+ hematopoietic stem and progenitor cells (HSPCs) into immunodeficient mice. Mice receiving shMED16 HSPCs showed a higher proportion of human erythroid and myeloid cells in the bone marrow (BM), while B cell and T cell reconstitution remained unaffected. MED16 deficiency led to an increased number of myeloid colonies and a more pronounced increase in erythroid colonies in the colony-forming assay. During erythroid differentiation, MED16 deficiency resulted in an elevated ratio of CD235a+ cells and increased enucleation. Furthermore, hematopoietic-specific Med16 knockout ( Mx1-Cre × Med16 -/- Med16 KO)mice showed normal BM and spleen cellularity, with an increased frequency of common myeloid progenitors (CMPs). Med16 KO mice exhibited a higher frequency of CD11b+ myeloid cells and Ter119+ erythroid cells in the BM and spleen. A higher percentage of enucleated erythrocytes were observed in the BM of Med16 KO mice. Additionally, in the model of phenylhydrazine-induced hemolytic anemia, Med16 KO mice showed accelerated red blood cell recovery and pronounced splenomegaly, highlighting the crucial role of Med16 in stress erythropoiesis. Our findings demonstrate that MED16 plays a repressive role in hematopoietic development. To elucidate how MED16 regulates gene expression, we conducted single-cell RNA-seq and ATAC-seq of cultured CD34+ cells after MED16 knockdown in primary human CD34+ HSPCs. MED16 deficiency resulted in upregulation of erythroid and innate immunity genes in erythroid and myeloid cells, respectively. Interestingly, MED16 deficiency led to significantly increased chromatin accessibility around the transcription start sites of upregulated genes. Considering the Mediator complex often interacts with transcription factors to convey information, we conducted MED16 chromatin IP sequencing (ChIP-seq). The majority of MED16 occupancy peaks were located at promoters (59.68%). Colocalization analysis of MED16 with chromatin occupancy of hematopoietic-related transcription factors revealed significant overlap with RUNX1. To unveil the mechanism of MED16-mediated transcriptional repression, we conducted immunoprecipitation coupled with mass spectrometry on cultured CD34+ cells to identify MED16-interacting nuclear proteins. We identified the FAcilitates Chromatin Transcription (FACT) complex as a potential candidate. ChIP-seq of the FACT complex subunit SPT16 showed weakened chromatin occupancy signals upon MED16 deficiency. Similar results were observed in mouse BM Ter119+ erythroblasts. These findings indicate that MED16 plays a crucial role in modulating chromatin accessibility and transcriptional repression, potentially through cooperation with RUNX1 and the FACT complex. Furthermore, we found that MED16 overexpression impeded erythroid cell enucleation, similar to MDS erythroid dysplasia. We observed higher MED16 expression in BM-derived CD34+ cells from 183 MDS patients across different subtypes compared to healthy individuals. Interestingly, knocking down MED16 in bone marrow mononuclear cells from MDS patients restored normal erythropoiesis. Transcriptome analysis revealed that MED16 deficiency upregulated innate immunity and erythroid genes. Together, these findings shed light on the previously unknown role of MED16 in hematopoiesis and its association with MDS. Our study provides mechanistic insights into the lineage commitment and developmental progression of hematopoiesis governed by Mediator and may have implications for the treatment of hematological disorders.