Transcription Factor Occupancy Research Articles

Abstract A031: Deciphering the transcriptomic landscape of AKT isoforms and its prognostic impact in patients with lung adenocarcinoma

Abstract AKT kinase is a regulator of several crucial biological functions including metabolism, cell cycle progression and proliferation. Its three isoforms, namely AKT1, AKT2 and AKT3, although they display significant similarities in their sequences, their functions largely differ and often antagonize each other. In the context of human cancer, several studies have previously demonstrated that each three of the AKT isoforms often act as oncogenes or tumor suppressors in the same type of cancer, and especially in lung adenocarcinoma (LUAD). These differences could be attributed to the plethora of different targets and pathways that they directly regulate through phosphorylation or indirectly through different intermediate effectors. One of these targets and pathways can be the regulation of transcription. Based on recent reports, several transcription factors can be directly phosphorylated by AKT kinase in human cancer, globally changing their chromatin occupancy and transcriptional landscape, thus altering transcription regulation. Nevertheless, a comprehensive analysis of the impact of each AKT isoform on transcription regulation and transcription factor enrichment in lung adenocarcinoma was yet to be performed. Here, we were willing to elucidate the genome-wide transcriptional landscape, driven by each AKT isoform, and its prognostic effect in patients with lung adenocarcinoma. To this extent, we utilized Next-Generation Sequencing data from The Cancer Genome Atlas (TCGA) to identify the transcriptomic changes related with the expression of each isoform in LUAD patients. Gene Set Enrichment Analysis revealed enrichment of unique pathways between the three isoforms. In parallel analysis, Transcription Factor enrichment and ATAC-Seq analysis from the same LUAD patients revealed a unique set of transcription factors that are differentially enriched between each AKT isoform group. More importantly, clinical and survival analysis revealed that the top 50 Differentially Expressed Genes (DEG), ranked by q-value, for each isoform correlated with poor overall survival, prognosis free survival, acting as potential prognostic factor in patients with LUAD. In addition, by analyzing publicly available LUAD datasets, through Gene Expression Omnibus (GEO), we demonstrate that the same gene signature is also enriched in relapsing patients. Collectively, our atlas of AKT isoform-mediated transcriptomic landscapes establishes a role of the AKT isoforms in the regulation of transcription by governing transcription factor occupancy in target genes. Given the lack of isoform specific AKT inhibitors, the deep knowledge of the transcriptional landscape driven by each isoform in human lung adenocarcinoma, can open new avenues on the design of precision-medicine markers and therapeutics that can preserve the normal AKT functions, while targeting the oncogenic pathways that these isoforms regulate. Citation Format: George Laliotis, Pauline Lin. Deciphering the transcriptomic landscape of AKT isoforms and its prognostic impact in patients with lung adenocarcinoma. [abstract]. In: Proceedings of the AACR Special Conference: Cancer Epigenomics; 2022 Oct 6-8; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2022;82(23 Suppl_2):Abstract nr A031.

Integrating transcription factor occupancy with transcriptome-wide association analysis identifies susceptibility genes in human cancers

Transcriptome-wide association studies (TWAS) have successfully discovered many putative disease susceptibility genes. However, TWAS may suffer from inaccuracy of gene expression predictions due to inclusion of non-regulatory variants. By integrating prior knowledge of susceptible transcription factor occupied elements, we develop sTF-TWAS and demonstrate that it outperforms existing TWAS approaches in both simulation and real data analyses. Under the sTF-TWAS framework, we build genetic models to predict alternative splicing and gene expression in normal breast, prostate and lung tissues from the Genotype-Tissue Expression project and apply these models to data from large genome-wide association studies (GWAS) conducted among European-ancestry populations. At Bonferroni-corrected P < 0.05, we identify 354 putative susceptibility genes for these cancers, including 189 previously unreported in GWAS loci and 45 in loci unreported by GWAS. These findings provide additional insight into the genetic susceptibility of human cancers. Additionally, we show the generalizability of the sTF-TWAS on non-cancer diseases.

A Multi-Omics Approach Reveals Features That Permit Robust and Widespread Regulation of IFN-Inducible Antiviral Effectors.

The antiviral state, an initial line of defense against viral infection, is established by a set of IFN-stimulated genes (ISGs) encoding antiviral effector proteins. The effector ISGs are transcriptionally regulated by type I IFNs mainly via activation of IFN-stimulated gene factor 3 (ISGF3). In this study, the regulatory elements of effector ISGs were characterized to determine the (epi)genetic features that enable their robust induction by type I IFNs in multiple cell types. We determined the location of regulatory elements, the DNA motifs, the occupancy of ISGF3 subunits (IRF9, STAT1, and STAT2) and other transcription factors, and the chromatin accessibility of 37 effector ISGs in murine dendritic cells. The IFN-stimulated response element (ISRE) and its tripartite version occurred most frequently in the regulatory elements of effector ISGs than in any other tested ISG subsets. Chromatin accessibility at their promoter regions was similar to most other ISGs but higher than at the promoters of inflammation-related cytokines, which were used as a reference gene set. Most effector ISGs (81.1%) had at least one ISGF3 binding region proximal to the transcription start site (TSS), and only a subset of effector ISGs (24.3%) was associated with three or more ISGF3 binding regions. The IRF9 signals were typically higher, and ISRE motifs were "stronger" (more similar to the canonical sequence) in TSS-proximal versus TSS-distal regulatory regions. Moreover, most TSS-proximal regulatory regions were accessible before stimulation in multiple cell types. Our results indicate that "strong" ISRE motifs and universally accessible promoter regions that permit robust, widespread induction are characteristic features of effector ISGs.

Therapeutic Targeting of EP300/CBP By Bromodomain Inhibition in Acute Myeloid Leukemia

CCS1477 (also known as inobrodib) is a potent and selective inhibitor of the bromodomains of EP300 and CBP, two homologous histone acetyltransferases with critical roles in cellular growth and differentiation. In pre-clinical analyses we found that CCS1477 induced dose-dependent growth inhibition of a wide range of acute myeloid leukemia (AML) cell lines and primary patient samples of diverse molecular subtypes at low nanomolar concentrations. Growth inhibition was predominantly due to cell cycle arrest and differentiation; there was only modest apoptosis. In vivo, in contrast to control mice which all developed short latency AML, all animals with experimentally initiated MLL-AF9 and MLL-AF10 driven AMLs (in a retroviral transduction and transplantation syngeneic model) treated with 30mg/kg daily PO CCS1477 survived to termination of the experiment at 220 days. In subcutaneous xenograft models, there was dose-dependent growth inhibition of MOLM16 AML cell tumours, with 20mg/kg daily for 21 days completely suppressing tumour growth and no tumour regrowth until 14 days following the end of treatment. Mechanistically, we found that 100nM CCS1477 induced rapid and selective depletion of EP300 in particular from MYB binding sites in THP1 AML cells. ChIPseq demonstrated that in steady state the strongest recruitment of EP300 to chromatin in THP1 AML cells was at sites of MYB and CEBPA transcription factor occupancy, although EP300 was also found genome-wide at sites occupied by other factors including RUNX1 and ETS. Following CCS1477 treatment there was, within six hours, selective loss of EP300 at MYB occupied sites, co-localized reduced chromatin accessibility and histone H3K27 acetylation, and stalling of RNA polymerase at subordinate genes with reduced transcription. This was associated with a dose-dependent redistribution of EP300 to alternate sites and up regulation of differentiation genes. In our ongoing phase I/IIa clinical trial investigating the safety and efficacy of CCS1477 in advanced haematological malignancies (NCT04068597), we have observed therapeutically beneficial granulocytic differentiation responses in patients with high-risk myelodysplasia or relapsed AML. For example, a patient with myelodysplasia with excess blasts 2 (MDS-EB2) evolving to AML who had received three cycles of azacitidine without response was treated with CCS1477 50mg PO (once daily, three days/week). Pre-CCS1477 treatment there was profound neutropenia and multiple admissions for sepsis. After CCS1477 treatment the neutrophil count rapidly normalized and, despite a brief admission for splenic infarction in the first week, the patient remained well for 5 months with no infective episodes. Treatment was stopped due to a rising leukocyte count. Similar responses were seen in other patients. In one of these, single cell RNA sequencing of serial blood and bone marrow samples confirmed appearance of mature myeloid cells on CCS1477 treatment and, within the blast population, up regulation of a multilineage differentiation programme and down regulation of a stem cell programme. Reflecting our findings in cell line models, there was also up regulation of genes induced by MYB knockdown and concordant up and down regulation of genes regulated by CEBPA in THP1 AML cells. Finally, we evaluated the activity of CCS1477 in xenograft models in combination with existing standard of care agents. Azacitidine and CCS1477 in particular demonstrated synergistic growth inhibitory activity in the MOLM16 model, as did venetoclax in MOLM16 and MV(4;11) models. These encouraging pre-clinical and early phase clinical findings set the stage for the clinical trial evaluation of CCS1477 in combination with azacitidine and venetoclax.

Chromatin accessibility-based characterisation of brain gene regulatory networks in three distinct honey bee polyphenisms.

The honey bee genome has the capacity to produce three phenotypically distinct organisms (two diploid female castes: queen and worker, and a haploid male drone). Previous studies have implicated metabolic flux acting via epigenetic regulation in directing nutrition-driven phenotypic plasticity in the honey bee. However, the cis-acting DNA regulatory elements that establish tissue and polyphenism -specific epigenomes and gene expression programmes, remain unclear. Using a high resolution multiomic approach including assay for transposase-accessible chromatin by sequencing (ATAC-seq), RNA-seq and ChIP-seq, we produce the first genome-wide maps of the regulatory landscape across all three adult honey bee phenotypes identifying >5000 regulatory regions in queen, 7500 in worker and 6500 in drone, with the vast majority of these sites located within intronic regions. These regions are defined by positive enrichment of H3K27ac and depletion of H3K4me3 and show a positive correlation with gene expression. Using ATAC-seq footprinting we determine queen, worker and drone -specific transcription factor occupancy and uncover novel phenotype-specific regulatory networks identifying two key nuclear receptors that have previously been implicated in caste-determination and adult behavioural maturation in honey bees; ecdysone receptor and ultraspiracle. Collectively, this study provides novel insights into key gene regulatory networks that are associated with these distinct polyphenisms in the honey bee.

Prenatal immune stress blunts microglia reactivity, impairing neurocircuitry.

Recent studies suggested that microglia, the primary brain immune cells, can affect circuit connectivity and neuronal function1,2. Microglia infiltrate the neuroepithelium early in embryonic development and are maintained in the brain throughout adulthood3,4. Several maternal environmental factors-such as an aberrant microbiome, immune activation and poor nutrition-can influence prenatal brain development5,6. Nevertheless, it is unknown how changes in the prenatal environment instruct the developmental trajectory of infiltrating microglia, which in turn affect brain development and function. Here we show that, after maternal immune activation (MIA) in mice, microglia from the offspring have a long-lived decrease in immune reactivity (blunting) across the developmental trajectory. The blunted immune response was accompanied by changes in chromatin accessibility and reduced transcription factor occupancy of the open chromatin. Single-cell RNA-sequencing analysis revealed that MIA does not induce a distinct subpopulation but, rather, decreases the contribution to inflammatory microglia states. Prenatal replacement of microglia from MIA offspring with physiological infiltration of naive microglia ameliorated the immune blunting and restored a decrease in presynaptic vesicle release probability onto dopamine receptor type-two medium spiny neurons, indicating that aberrantly formed microglia due to an adverse prenatal environment affect the long-term microglia reactivity and proper striatal circuit development.

M6A RNA Methylation-Based Epitranscriptomic Modifications in Plasticity-Related Genes via miR-124-C/EBPα-FTO-Transcriptional Axis in the Hippocampus of Learned Helplessness Rats.

Impaired synaptic plasticity has been linked to dynamic gene regulatory network changes. Recently, gene regulation has been introduced with the emerging concept of unique N6-methyladenosine (m6A)-based reversible transcript methylation. In this study, we tested whether m6A RNA methylation may potentially serve as a link between the stressful insults and altered expression of plasticity-related genes. Expression of plasticity genes Nr3c1, Creb1, Ntrk2; m6A-modifying enzymes Fto, methyltransferase like (Mettl)-3 and 14; DNA methylation enzymes Dnmt1, Dnmt3a; transcription factor C/ebp-α; and miRNA-124-3p were determined by quantitative polymerase chain reaction (qPCR) in the hippocampus of rats that showed susceptibility to develop stress-induced depression (learned helplessness). M6A methylation of plasticity-related genes was determined following m6A mRNA immunoprecipitation. Chromatin immunoprecipitation was used to examine the endogenous binding of C/EBP-α to the Fto promoter. MiR-124-mediated post-transcriptional inhibition of Fto via C/EBPα was determined using an in vitro model. Hippocampus of learned helplessness rats showed downregulation of Nr3c1, Creb1, and Ntrk2 along with enrichment in their m6A methylation. A downregulation in demethylating enzyme Fto and upregulation in methylating enzyme Mettl3 were also noted. The Fto promoter was hypomethylated due to the lower expression of Dnmt1 and Dnmt3a. At the same time, there was a lower occupancy of transcription factor C/EBPα on the Fto promoter. Conversely, C/ebp-α transcript was downregulated via induced miR-124-3p expression. Our study mechanistically linked defective C/EBP-α-FTO-axis, epigenetically influenced by induced expression of miR-124-3p, in modifying m6A enrichment in plasticity-related genes. This could potentially be linked with abnormal neuronal plasticity in depression.

Subtype and cell type specific expression of lncRNAs provide insight into breast cancer

Long non-coding RNAs (lncRNAs) are involved in breast cancer pathogenesis through chromatin remodeling, transcriptional and post-transcriptional gene regulation. We report robust associations between lncRNA expression and breast cancer clinicopathological features in two population-based cohorts: SCAN-B and TCGA. Using co-expression analysis of lncRNAs with protein coding genes, we discovered three distinct clusters of lncRNAs. In silico cell type deconvolution coupled with single-cell RNA-seq analyses revealed that these three clusters were driven by cell type specific expression of lncRNAs. In one cluster lncRNAs were expressed by cancer cells and were mostly associated with the estrogen signaling pathways. In the two other clusters, lncRNAs were expressed either by immune cells or fibroblasts of the tumor microenvironment. To further investigate the cis-regulatory regions driving lncRNA expression in breast cancer, we identified subtype-specific transcription factor (TF) occupancy at lncRNA promoters. We also integrated lncRNA expression with DNA methylation data to identify long-range regulatory regions for lncRNA which were validated using ChiA-Pet-Pol2 loops. lncRNAs play an important role in shaping the gene regulatory landscape in breast cancer. We provide a detailed subtype and cell type-specific expression of lncRNA, which improves the understanding of underlying transcriptional regulation in breast cancer.

IKKε Inhibitor Amlexanox Promotes Olaparib Sensitivity through the C/EBP-β-Mediated Transcription of Rad51 in Castrate-Resistant Prostate Cancer.

Simple SummaryMost men with advanced hormone-sensitive prostate cancer (HSPC) treated with androgen deprivation therapy will develop castrate resistant prostate cancer (CRPC), a lethal form of prostate cancer (PC). Our group has previously shown that IKKε expression is stronger in CRPC tumors and correlates with aggressive PC. Moreover, we have shown that IKKε depletion or inhibition (BX795, Amlexanox) decrease CRPC cell proliferation and tumor volume in an in vivo mouse model. We also demonstrate that IKKε inhibitors specifically target CRPC to induce a senescent phenotype as well as DNA damage and genomic instability. In this study, we demonstrated that IKKε depletion or inhibition block C/EBP-β recruitment on Rad51 promoter to decrease promoter activity. We have also shown that Amlexanox treatment sensitizes CRPC cells to Olaparib in vitro and in mouse models. Taken together, targeting IKKε with Amlexanox combined with Olaparib may lead to additional effective therapeutic strategies in the management of patients with CRPC.The progression of prostate cancer (PC) is often characterized by the development of castrate-resistant PC (CRPC). Patients with CRPC are treated with a variety of agents including new generation hormonal therapies or chemotherapy. However, as the cancer develops more resistance mechanisms, these drugs eventually become less effective and finding new therapeutic approaches is critical to improving patient outcomes. Previously, we have shown that IKKε depletion and IKKε inhibitors, BX795 and Amlexanox, decrease CRPC cell proliferation in vitro and in vivo and that IKKε inhibitors induce a senescence phenotype accompanied by increased DNA damage and genomic instability in CRPC cells. Here, we describe a new role for IKKε in DNA damage repair involving Rad51 and examine the therapeutic potential of Amlexanox combined with the PARP inhibitor Olaparib in CRPC cell lines. Combining Amlexanox with Olaparib decreased CRPC cell proliferation and enhanced DNA damage through the inhibition of Olaparib-induced Rad51 recruitment and expression in CRPC cells or IKKε-depleted PC-3 cells. We demonstrated that Rad51 promoter activity, measured by luciferase assay, was decreased with Amlexanox treatment or IKKε depletion and that Amlexanox treatment decreased the occupancy of transcription factor C/EBP-β on the Rad51 promoter. Our mouse model also showed that Amlexanox combined with Olaparib inhibited tumor growth of CRPC xenografts. Our study highlights a new role for IKKε in DNA damage repair through the regulation of Rad51 transcription and provides a rationale for the combination of Amlexanox and Olaparib in the treatment of patients with CRPC.

ATAC-STARR-seq reveals transcription factor-bound activators and silencers within chromatin-accessible regions of the human genome.

Massively parallel reporter assays (MPRAs) test the capacity of putative gene regulatory elements to drive transcription on a genome-wide scale. Most gene regulatory activity occurs within accessible chromatin, and recently described methods have combined assays that capture these regions—such as assay for transposase-accessible chromatin using sequencing (ATAC-seq)—with self-transcribing active regulatory region sequencing (STARR-seq) to selectively assay the regulatory potential of accessible DNA (ATAC-STARR-seq). Here, we report an integrated approach that quantifies activating and silencing regulatory activity, chromatin accessibility, and transcription factor (TF) occupancy with one assay using ATAC-STARR-seq. Our strategy, including important updates to the ATAC-STARR-seq assay and workflow, enabled high-resolution testing of ∼50 million unique DNA fragments tiling ∼101,000 accessible chromatin regions in human lymphoblastoid cells. We discovered that 30% of all accessible regions contain an activator, a silencer, or both. Although few MPRA studies have explored silencing activity, we demonstrate that silencers occur at similar frequencies to activators, and they represent a distinct functional group enriched for unique TF motifs and repressive histone modifications. We further show that Tn5 cut-site frequencies are retained in the ATAC-STARR plasmid library compared to standard ATAC-seq, enabling TF occupancy to be ascertained from ATAC-STARR data. With this approach, we found that activators and silencers cluster by distinct TF footprint combinations, and these groups of activity represent different gene regulatory networks of immune cell function. Altogether, these data highlight the multilayered capabilities of ATAC-STARR-seq to comprehensively investigate the regulatory landscape of the human genome all from a single DNA fragment source.

ScPCOR-seq enables co-profiling of chromatin occupancy and RNAs in single cells

Cell-to-cell variation in gene expression is a widespread phenomenon, which may play important roles in cellular differentiation, function, and disease development1–9. Chromatin is implicated in contributing to the cellular heterogeneity in gene expression10–16. Fully understanding the mechanisms of cellular heterogeneity requires simultaneous measurement of RNA and occupancy of histone modifications and transcription factors on chromatin due to their critical roles in transcriptional regulation17,18. We generally term the occupancy of histone modifications and transcription factors as Chromatin occupancy. Here, we report a technique, termed scPCOR-seq (single-cell Profiling of Chromatin Occupancy and RNAs Sequencing), for simultaneously profiling genome-wide chromatin protein binding or histone modification marks and RNA expression in the same cell. We demonstrated that scPCOR-seq can profile either H3K4me3 or RNAPII and RNAs in a mixture of human H1, GM12878 and 293 T cells at a single-cell resolution and either H3K4me3, RNAPII, or RNA profile can correctly separate the cells. Application of scPCOR-seq to the in vitro differentiation of the erythrocyte precursor CD36 cells from human CD34 stem or progenitor cells revealed that H3K4me3 and RNA exhibit distinct properties in clustering cells during differentiation. Overall, our work provides a promising approach to understand the relationships among different omics layers.

Toward a base-resolution panorama of the in vivo impact of cytosine methylation on transcription factor binding

BackgroundWhile methylation of CpG dinucleotides is traditionally considered antagonistic to the DNA-binding activity of most transcription factors (TFs), recent in vitro studies have revealed a more complex picture, suggesting that over a third of TFs may preferentially bind to methylated sequences. Expanding these in vitro observations to in vivo TF binding preferences is challenging since the effect of methylation of individual CpG sites cannot be easily isolated from the confounding effects of DNA accessibility and regional DNA methylation. Thus, in vivo methylation preferences of most TFs remain uncharacterized.ResultsWe introduce joint accessibility-methylation-sequence (JAMS) models, which connect the strength of the binding signal observed in ChIP-seq to the DNA accessibility of the binding site, regional methylation level, DNA sequence, and base-resolution cytosine methylation. We show that JAMS models quantitatively explain TF occupancy, recapitulate cell type-specific TF binding, and have high positive predictive value for identification of TFs affected by intra-motif methylation. Analysis of 2209 ChIP-seq experiments results in high-confidence JAMS models for 260 TFs, revealing a negative association between in vivo TF occupancy and intra-motif methylation for 45% of studied TFs, as well as 16 TFs that are predicted to bind to methylated sites, including 11 novel methyl-binding TFs mostly from the multi-zinc finger family.ConclusionsOur study substantially expands the repertoire of in vivo methyl-binding TFs, but also suggests that most TFs that prefer methylated CpGs in vitro present themselves as methylation agnostic in vivo, potentially due to the balancing effect of competition with other methyl-binding proteins.

Abstract 5900: Integrating prior knowledge of transcription factor occupied elements with transcriptome-wide association analysis identifies 153 breast cancer susceptibility genes

Abstract Transcriptome-wide association studies (TWAS) have been successful in uncovering large numbers of putative disease susceptibility genes. TWAS approaches such as S-PrediXcan, FUSION and TIGAR, show comparable overall performance for susceptibility gene discovery; however, integrating prior disease-specific regulatory information is lacking. To improve accuracy of gene expression prediction by integrating information on susceptible transcription factor (sTF) occupancy in target cells and tissues, we developed an analytical framework, called sTF-TWAS. We evaluated six sets of putative regulatory variants selected from sTF-occupied sites, and performed generalized Berk-Jones (GBJ) analysis to combine sets and quantify overall associations. We demonstrated that sTF-TWAS outperforms S-PrediXcan in both simulation and real data analyses. Using summary statistics data from the Breast Cancer Association Consortium (BCAC), we also incorporated alternative splicing (sp) data in TF-TWAS and identified 76 and 88 significant genes for breast cancer risk at a Bonferroni-corrected P &amp;lt;0.05 from sTF-TWAS and sp-sTF-TWAS, respectively. Of 153 putative susceptibility genes from both sTF-TWAS and sp-sTF-TWAS, 14 genes in 12 loci had novel associations with breast cancer risk and 90 genes were previously unreported by breast cancer GWAS. We also provide strong evidence for 15 previously unreported genes, including 11 essential for breast cell proliferation and five suggested cancer predisposition or driver genes: RAD50, IGHMBP2, ATXN3, KANSL1, and IL6ST. Overall, we demonstrated that our approach has significant advantages over regular TWAS and our findings provide additional insights into the genetic susceptibility of breast cancer. Citation Format: Xingyi Guo, Jingni He, Alicia Beeghly-Fadiel, Zhishan Chen, Chen Cao, Xiao-ou Shu, Wei Zheng, Quan Long. Integrating prior knowledge of transcription factor occupied elements with transcriptome-wide association analysis identifies 153 breast cancer susceptibility genes [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5900.

SpyChIP identifies cell type-specific transcription factor occupancy from complex tissues

Chromatin immunoprecipitation (ChIP) is an important technique for characterizing protein-DNA binding invivo. One drawback of ChIP-based techniques is the lack of cell type-specificity when profiling complex tissues. To overcome this limitation, we developed SpyChIP to identify cell type-specific transcription factor (TF) binding sites in native physiological contexts without tissue dissociation or nuclei sorting. SpyChIP takes advantage of a specific covalent isopeptide bond that rapidly forms between the 15-amino acid SpyTag and the 17-kDa protein SpyCatcher. In SpyChIP, the target TF is fused with SpyTag by genome engineering, and an epitope tagged SpyCatcher is expressed in cell populations of interest, where it covalently binds to SpyTag-TF. Cell type-specific ChIP is obtained by immunoprecipitating chromatin prepared from whole tissues using antibodies directed against the epitope-tagged SpyCatcher. Using SpyChIP, we identified the genome-wide binding profiles of the Hox protein Ultrabithorax (Ubx) in two distinct cell types of the Drosophila haltere imaginal disc. Our results revealed extensive region-specific Ubx-DNA binding events, highlighting the significance of cell type-specific ChIP and the limitations of whole-tissue ChIP approaches. Analysis of Ubx::SpyChIP results provided insights into the relationship between chromatin accessibility and Ubx-DNA binding, as well as different mechanisms Ubx employs to regulate its downstream cis-regulatory modules. In addition to SpyChIP, we suggest that SpyTag-SpyCatcher technology, as well as other protein pairs that form covalent isopeptide bonds, will facilitate many additional invivo applications that were previously impractical.

Profiling the quantitative occupancy of myriad transcription factors across conditions by modeling chromatin accessibility data.

Over a thousand different transcription factors (TFs) bind with varying occupancy across the human genome. Chromatin immunoprecipitation (ChIP) can assay occupancy genome-wide, but only one TF at a time, limiting our ability to comprehensively observe the TF occupancy landscape, let alone quantify how it changes across conditions. We developed TF occupancy profiler (TOP), a Bayesian hierarchical regression framework, to profile genome-wide quantitative occupancy of numerous TFs using data from a single chromatin accessibility experiment (DNase- or ATAC-seq). TOP is supervised, and its hierarchical structure allows it to predict the occupancy of any sequence-specific TF, even those never assayed with ChIP. We used TOP to profile the quantitative occupancy of hundreds of sequence-specific TFs at sites throughout the genome and examined how their occupancies changed in multiple contexts: in approximately 200 human cell types, through 12 h of exposure to different hormones, and across the genetic backgrounds of 70 individuals. TOP enables cost-effective exploration of quantitative changes in the landscape of TF binding.

Abstract 532: Dioxin Modifies The Transcriptomic And Epigenetic Landscape Of Smooth Muscle Cells Leading To Adverse Remodeling Of Atherosclerosis

Background: Environmental exposure to dioxin, an active component of cigarette smoke and air pollution has been linked to increased myocardial infarction. Smooth muscle cells (SMC) in the coronary vasculature play a critical role in atherosclerotic plaque remodeling due to their phenotypic plasticity. We have previously found strong dioxin response element activity in modulated SMC of atherosclerotic lesion. In this study, we aimed to understand the effect of dioxin on vascular SMC and atherosclerosis phenotype. Methods: Primary human coronary artery SMC (HCASMC) treated in culture with 2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) or vehicle control were used to perform RNA-Seq and ATAC-Seq. ChIP-Seq was performed with antibodies against Aryl-hydrocarbon receptor (AHR) and TCF21. Tamoxifen inducible SMC-lineage tracing reporter mice were used to assess the in-vivo effect of TCDD on aortic SMC. After 8 weeks of high fat diet (HFD), followed by 8 weeks of TCDD injection and HFD, the aortic sinus was collected for histology and single-cell sequencing (10X Genomics). Results: Analysis of the RNA-Seq from TCDD treated HCASMC showed differential enrichment of pathways related to cell migration and vascular development. Further, ATAC-Seq data showed a significant enrichment for pathways regulating vascular development, cell migration, and apoptosis. There was an overall increase in chromatin accessibility suggesting transcriptional activation with TCDD. We observed enrichment for AHR ChIP-Seq peaks in the open chromatin regions along with a significant reciprocal reduction in TCF21 occupancy. The scRNA-Seq of mice treated with TCDD exposure showed differential gene expression of SMC-lineage cells enriching for extracellular matrix (ECM) organization, inflammation, unfolded protein response, and apoptotic process, and histological analysis of the aortic sinus showed increased lesion area with increased presence of SMC-lineage cells and Cd68 + cells compared to control. Conclusion: Dioxin adversely remodels atherosclerotic plaque affecting pathways of ECM organization, inflammation and unfolded protein response in SMC, partly by modifying chromatin accessibility and occupancy of key transcription factors of phenotypic modulation.

H1.0 C Terminal Domain Is Integral for Altering Transcription Factor Binding within Nucleosomes.

The linker histone H1 is a highly prevalent protein that compacts chromatin and regulates DNA accessibility and transcription. However, the mechanisms behind H1 regulation of transcription factor (TF) binding within nucleosomes are not well understood. Using in vitro fluorescence assays, we positioned fluorophores throughout human H1 and the nucleosome, then monitored the distance changes between H1 and the histone octamer, H1 and nucleosomal DNA, or nucleosomal DNA and the histone octamer to monitor the H1 movement during TF binding. We found that H1 remains bound to the nucleosome dyad, while the C terminal domain (CTD) releases the linker DNA during nucleosome partial unwrapping and TF binding. In addition, mutational studies revealed that a small 16 amino acid region at the beginning of the H1 CTD is largely responsible for altering nucleosome wrapping and regulating TF binding within nucleosomes. We then investigated physiologically relevant post-translational modifications (PTMs) in human H1 by preparing fully synthetic H1 using convergent hybrid phase native chemical ligation. Both individual PTMs and combinations of phosphorylation and citrullination of H1 had no detectable influence on nucleosome binding and nucleosome wrapping, and had only a minor impact on H1 regulation of TF occupancy within nucleosomes. This suggests that these H1 PTMs function by other mechanisms. Our results highlight the importance of the H1 CTD, in particular, the first 16 amino acids, in regulating nucleosome linker DNA dynamics and TF binding within the nucleosome.

Altered BAF occupancy and transcription factor dynamics in PBAF-deficient melanoma.

SUMMARYARID2 is the most recurrently mutated SWI/SNF complex member in melanoma; however, its tumor-suppressive mechanisms in the context of the chromatin landscape remain to be elucidated. Here, we model ARID2 deficiency in melanoma cells, which results in defective PBAF complex assembly with a concomitant genomic redistribution of the BAF complex. Upon ARID2 depletion, a subset of PBAF and shared BAF-PBAF-occupied regions displays diminished chromatin accessibility and associated gene expression, while BAF-occupied enhancers gain chromatin accessibility and expression of genes linked to the process of invasion. As a function of altered accessibility, the genomic occupancy of melanoma-relevant transcription factors is affected and significantly correlates with the observed transcriptional changes. We further demonstrate that ARID2-deficient cells acquire the ability to colonize distal organs in multiple animal models. Taken together, our results reveal a role for ARID2 in mediating BAF and PBAF subcomplex chromatin dynamics with consequences for melanoma metastasis.

Global and context-specific transcriptional consequences of oncogenic Fbw7 mutations.

The Fbw7 ubiquitin ligase targets many proteins for proteasomal degradation, which include oncogenic transcription factors (TFs) (e.g., c-Myc, c-Jun, and Notch). Fbw7 is a tumor suppressor and tumors often contain mutations in FBXW7, the gene that encodes Fbw7. The complexity of its substrate network has obscured the mechanisms of Fbw7-associated tumorigenesis, yet this understanding is needed for developing therapies. We used an integrated approach employing RNA-Seq and high-resolution mapping (cleavage under target and release using nuclease) of histone modifications and TF occupancy (c-Jun and c-Myc) to examine the combinatorial effects of misregulated Fbw7 substrates in colorectal cancer (CRC) cells with engineered tumor-associated FBXW7 null or missense mutations. Both Fbw7 mutations caused widespread transcriptional changes associated with active chromatin and altered TF occupancy: some were common to both Fbw7 mutant cell lines, whereas others were mutation specific. We identified loci where both Jun and Myc were coregulated by Fbw7, suggesting that substrates may have synergistic effects. One coregulated gene was CIITA, the master regulator of MHC Class II gene expression. Fbw7 loss increased MHC Class II expression and Fbw7 mutations were correlated with increased CIITA expression in TCGA colorectal tumors and cell lines, which may have immunotherapeutic implications for Fbw7-associated cancers. Analogous studies in neural stem cells in which FBXW7 had been acutely deleted closely mirrored the results in CRC cells. Gene set enrichment analyses revealed Fbw7-associated pathways that were conserved across both cell types that may reflect fundamental Fbw7 functions. These analyses provide a framework for understanding normal and neoplastic context-specific Fbw7 functions.

Antibody-free profiling of transcription factor occupancy during early embryogenesis by FitCUT&RUN.

Key transcription factors (TFs) play critical roles in zygotic genome activation (ZGA) during early embryogenesis, whereas genome-wide occupancies of only a few factors have been profiled during ZGA due to the limitation of cell numbers or the lack of high-quality antibodies. Here, we present FitCUT&RUN, a modified CUT&RUN method, in which an Fc fragment of immunoglobulin G is used for tagging, to profile TF occupancy in an antibody-free manner and demonstrate its reliability and robustness using as few as 5000 K562 cells. We applied FitCUT&RUN to zebrafish undergoing embryogenesis to generate reliable occupancy profiles of three known activators of zebrafish ZGA: Nanog, Pou5f3, and Sox19b. By profiling the time-series occupancy of Nanog during zebrafish ZGA, we observed a clear trend toward a gradual increase in Nanog occupancy and found that Nanog occupancy prior to the major phase of ZGA is important for the activation of some early transcribed genes.