Distal Regulatory Elements Research Articles

An Atlas of Promoter Chromatin Modifications and HiChIP Regulatory Interactions in Human Subcutaneous Adipose-Derived Stem Cells.

The genome of human adipose-derived stem cells (ADSCs) from abdominal and gluteofemoral adipose tissue depots are maintained in depot-specific stable epigenetic conformations that influence cell-autonomous gene expression patterns and drive unique depot-specific functions. The traditional approach to explore tissue-specific transcriptional regulation has been to correlate differential gene expression to the nearest-neighbor linear-distance regulatory region defined by associated chromatin features including open chromatin status, histone modifications, and DNA methylation. This has provided important information; nonetheless, the approach is limited because of the known organization of eukaryotic chromatin into a topologically constrained three-dimensional network. This network positions distal regulatory elements in spatial proximity with gene promoters which are not predictable based on linear genomic distance. In this work, we capture long-range chromatin interactions using HiChIP to identify remote genomic regions that influence the differential regulation of depot-specific genes in ADSCs isolated from different adipose depots. By integrating these data with RNA-seq results and histone modifications identified by ChIP-seq, we uncovered distal regulatory elements that influence depot-specific gene expression in ADSCs. Interestingly, a subset of the HiChIP-defined chromatin loops also provide previously unknown connections between waist-to-hip ratio GWAS variants with genes that are known to significantly influence ADSC differentiation and adipocyte function.

Dynamics of chromatin accessibility and genome wide control of desiccation tolerance in the resurrection plant Haberlea rhodopensis

BackgroundDrought is one of the main consequences of global climate change and this problem is expected to intensify in the future. Resurrection plants evolved the ability to withstand the negative impact of long periods of almost complete desiccation and to recover at rewatering. In this respect, many physiological, transcriptomic, proteomic and genomic investigations have been performed in recent years, however, few epigenetic control studies have been performed on these valuable desiccation-tolerant plants so far.ResultsIn the present study, for the first time for resurrection plants we provide evidences about the differential chromatin accessibility of Haberlea rhodopensis during desiccation stress by ATAC-seq (Assay for Transposase Accessible Chromatin with high-throughput sequencing). Based on gene similarity between species, we used the available genome of the closely related resurrection plant Dorcoceras hygrometricum to identify approximately nine hundred transposase hypersensitive sites (THSs) in H. rhodopensis. The majority of them corresponds to proximal and distal regulatory elements of different genes involved in photosynthesis, carbon metabolism, synthesis of secondary metabolites, cell signalling and transcriptional regulation, cell growth, cell wall, stomata conditioning, chaperons, oxidative stress, autophagy and others. Various types of binding motifs recognized by several families of transcription factors have been enriched from the THSs found in different stages of drought. Further, we used the previously published RNA-seq data from H. rhodopensis to evaluate the expression of transcription factors putatively interacting with the enriched motifs, and the potential correlation between the identified THS and the expression of their corresponding genes.ConclusionsThese results provide a blueprint for investigating the epigenetic regulation of desiccation tolerance in resurrection plant H. rhodopensis and comparative genomics between resurrection and non-resurrection species with available genome information.

Targeting Super-Enhancers Driving EVI1 Expression in Leukemia By Inhibition of p300/CBP

Introduction One of the most aggressive types of acute myeloid leukemia (AML) is caused by aberrant activation of EVI1 as a result of the hijacking of enhancers repositioned by structural rearrangements. One of these enhancers, the MYC-blood enhancer cluster ( MYC-BENC) (Bahr et al. 2018) activates EVI1 in AML patients with t(3;8). MYC-BENC consists of several enhancer modules, of which only one is essential for activating EVI1: deletion of this element abrogates MYC-BENC to EVI1 promoter interaction as well as transcription of EVI1 (Ottema et al. 2021). Approach Given the central role of acetyltransferases p300 and CBP in enhancer activation, in particular super-enhancers such as MYC-BENC, we wondered whether we could specifically inhibit MYC-BENC driven EVI1 transcription using p300/CBP inhibitors. We studied this in a previously reported t(3;8) K562 model (Ottema et al. 2021), in which we introduced GFP 3' of EVI1 as a readout for MYC-BENC-driven EVI1 expression. Results ChIP-seq in t(3;8) cells showed that p300 binds to the promoter of EVI1 and to MYC-BENC, whereas CBP only binds to MYC-BENC (Fig. A). Following rapid p300/CBP degradation (3 hours) with the heterobifunctional p300/CBP degrader dCBP-1 (Vannam et al. 2021), we found a severe reduction of nascent EVI1 (and MYC) transcripts (SLAM-seq), as well as of EVI1 protein levels, i.e. by EVI1-eGFP, intracellular anti-EVI1-PE staining (Fig. B) and western blot.. Importantly, p300/CBP degradation was accompanied by loss of H3K27ac at MYC-BENC, but not at the promoter of EVI1 (Fig. A). Thus, the activation of EVI1 in t(3;8) AML appears strongly dependent on the presence of p300/CBP at MYC-BENC super-enhancer. Importantly, in healthy HSPCs, we observed that EVI1 expression was not strongly downregulated upon p300/CBP degradation (Fig. B). These data suggest that MYC-BENC driven EVI1 transcription in AML can be targeted, while leaving EVI1 transcription in normal HSPCs unaffected. We next studied whether loss of enhancer activity was dependent on functional domains within p300/CBP using specific inhibitors of either the acetyltransferase domain (A485) or bromodomain (GNE781). Treatment of t(3;8) EVI1-GFP cells with single inhibitors had a mild effect on EVI1 expression, but simultaneous A485/GNE781 treatment showed strong synergy in reducing EVI1 levels (Fig. B). Dual inhibition also led to a profound loss of H3K27ac signal at the MYC-BENC locus, but not at the EVI1 promoter (Fig. A). Thus, both domains in CBP/p300 are required to activate EVI1 expression via the hijacked MYC-BENC enhancer. As with dCBP-1, we found that EVI1 expression levels in healthy HSPCs were not affected by A485 and GNE781 treatment (Fig. B). We hypothesized that A485/GNE781 treatment would only cause loss of p300/CBP activity, but p300 ChIP-sequencing showed that A485/GNE781 treatment caused strong loss of p300 binding at the enhancer while the total protein levels of p300 and CBP were not affected. p300 binding at the EVI1 promoter was not decreased by A485/GNE781 treatment. In line with this, we observed that genome-wide p300 binding at enhancers was reduced at 15939 out of 41160 (39%) putative enhancers upon A485/GNE781 exposure, whereas dual inhibitor treatment only affected p300 binding at 429 out of 12452 promoters (3.5%) (Fig. A), suggesting a high selectivity of p300 inhibitors for long distant regulatory elements. Conclusion In summary, EVI1 is sensitive to pharmacological p300/CBP inhibition when activated by the hijacked MYC-BENC enhancer, but not in normal HSPCs. Globally, the sensitivity of genomic regions to bromo- or acetyltransferase inhibition is highly locus-dependent and is mainly restricted to enhancers. Our findings demonstrate that aberrantly controlled expression of oncogenes by hijacked enhancers in leukemia can be pharmacologically targeted, pointing to a potential therapeutic avenue for patients who are refractory to current treatment regimens.

Differential usage of DNA modifications in neurons, astrocytes, and microglia

BackgroundCellular identity is determined partly by cell type-specific epigenomic profiles that regulate gene expression. In neuroscience, there is a pressing need to isolate and characterize the epigenomes of specific CNS cell types in health and disease. In this study, we developed an in vivo tagging mouse model (Camk2a-NuTRAP) for paired isolation of neuronal DNA and RNA without cell sorting and then used this model to assess epigenomic regulation, DNA modifications in particular, of gene expression between neurons and glia.ResultsAfter validating the cell-specificity of the Camk2a-NuTRAP model, we performed TRAP-RNA-Seq and INTACT-whole genome oxidative bisulfite sequencing (WGoxBS) to assess the neuronal translatome and epigenome in the hippocampus of young mice (4 months old). WGoxBS findings were validated with enzymatic methyl-Seq (EM-Seq) and nanopore sequencing. Comparing neuronal data to microglial and astrocytic data from NuTRAP models, microglia had the highest global mCG levels followed by astrocytes and then neurons, with the opposite pattern observed for hmCG and mCH. Differentially modified regions between cell types were predominantly found within gene bodies and distal intergenic regions, rather than proximal promoters. Across cell types there was a negative correlation between DNA modifications (mCG, mCH, hmCG) and gene expression at proximal promoters. In contrast, a negative correlation of gene body mCG and a positive relationship between distal promoter and gene body hmCG with gene expression was observed. Furthermore, we identified a neuron-specific inverse relationship between mCH and gene expression across promoter and gene body regions.ConclusionsNeurons, astrocytes, and microglia demonstrate different genome-wide levels of mCG, hmCG, and mCH that are reproducible across analytical methods. However, modification-gene expression relationships are conserved across cell types. Enrichment of differential modifications across cell types in gene bodies and distal regulatory elements, but not proximal promoters, highlights epigenomic patterning in these regions as potentially greater determinants of cell identity. These findings also demonstrate the importance of differentiating between mC and hmC in neuroepigenomic analyses, as up to 30% of what is conventionally interpreted as mCG can be hmCG, which often has a different relationship to gene expression than mCG.

Identification of conserved skeletal enhancers associated with craniosynostosis risk genes.

Craniosynostosis, defined by premature fusion of one or multiple cranial sutures, is a common congenital defect affecting more than 1/2000 infants and results in restricted brain expansion. Single gene mutations account for 15%-20% of cases, largely as part of a syndrome, but the majority are nonsyndromic with complex underlying genetics. We hypothesized that the two noncoding genomic regions identified by a GWAS for craniosynostosis contain distal regulatory elements for the risk genes BMPER and BMP2. To identify such regulatory elements, we surveyed conserved noncoding sequences from both risk loci for enhancer activity in transgenic Danio rerio. We identified enhancers from both regions that direct expression to skeletal tissues, consistent with the endogenous expression of bmper and bmp2. For each locus, we also found a skeletal enhancer that also contains a sequence variant associated with craniosynostosis risk. We examined the activity of each enhancer during craniofacial development and found that the BMPER-associated enhancer is active in the restricted region of cartilage closely associated with frontal bone initiation. The same enhancer is active in mouse skeletal tissues, demonstrating evolutionarily conserved activity. Using enhanced yeast one-hybrid assays, we identified transcription factors that bind each enhancer and observed differential binding between alleles, implicating multiple signaling pathways. Our findings help unveil the genetic mechanism of the two craniosynostosis risk loci. More broadly, our combined in vivo approach is applicable to many complex genetic diseases to build a link between association studies and specific genetic mechanisms.

Mutations on a conserved distal enhancer in the porcine C-reactive protein gene impair its expression in liver.

C-reactive protein (CRP) is an evolutionary highly conserved protein. Like humans, CRP acts as a major acute phase protein in pigs. While CRP regulatory mechanisms have been extensively studied in humans, little is known about the molecular mechanisms that control pig CRP gene expression. The main goal of the present work was to study the regulatory mechanisms and identify functional genetic variants regulating CRP gene expression and CRP blood levels in pigs. The characterization of the porcine CRP proximal promoter region revealed a high level of conservation with both cow and human promoters, sharing binding sites for transcription factors required for CRP expression. Through genome-wide association studies and fine mapping, the most associated variants with both mRNA and protein CRP levels were localized in a genomic region 39.3 kb upstream of CRP. Further study of the region revealed a highly conserved putative enhancer that contains binding sites for several transcriptional regulators such as STAT3, NF-kB or C/EBP-β. Luciferase reporter assays showed the necessity of this enhancer-promoter interaction for the acute phase induction of CRP expression in liver, where differences in the enhancer sequences significantly modified CRP activity. The associated polymorphisms disrupted the putative binding sites for HNF4α and FOXA2 transcription factors. The high correlation between HNF4α and CRP expression levels suggest the participation of HNF4α in the regulatory mechanism of porcine CRP expression through the modification of its binding site in liver. Our findings determine, for the first time, the relevance of a distal regulatory element essential for the acute phase induction of porcine CRP in liver and identify functional polymorphisms that can be included in pig breeding programs to improve immunocompetence.

High-sensitive nascent transcript sequencing reveals BRD4-specific control of widespread enhancer and target gene transcription

Gene transcription by RNA polymerase II (Pol II) is under control of promoters and distal regulatory elements known as enhancers. Enhancers are themselves transcribed by Pol II correlating with their activity. How enhancer transcription is regulated and coordinated with transcription at target genes has remained unclear. Here, we developed a high-sensitive native elongating transcript sequencing approach, called HiS-NET-seq, to provide an extended high-resolution view on transcription, especially at lowly transcribed regions such as enhancers. HiS-NET-seq uncovers new transcribed enhancers in human cells. A multi-omics analysis shows that genome-wide enhancer transcription depends on the BET family protein BRD4. Specifically, BRD4 co-localizes to enhancer and promoter-proximal gene regions, and is required for elongation activation at enhancers and their genes. BRD4 keeps a set of enhancers and genes in proximity through long-range contacts. From these studies BRD4 emerges as a general regulator of enhancer transcription that may link transcription at enhancers and genes.

N -acetyltransferase 2 haplotype modifies risks for both dyslipidemia and urinary bladder cancer.

A novel haplotype in N -acetyltransferase 2 ( NAT2 ) composed of seven non-coding variants (rs1495741, rs4921913, rs4921914, rs4921915, rs146812806, rs35246381, and rs35570672) has been linked to dyslipidemia by multiple, independent genome-wide association studies. The haplotype is located approximately 14 kb downstream of NAT2-coding region (ch8:18,272,377-18,272,881; GRCh38/hg38) and represents a non-coding, intergenic haplotype. Interestingly, the same dyslipidemia NAT2 haplotype is also linked to urinary bladder cancer risk. Dyslipidemia risk alleles are associated with rapid acetylator phenotype, whereas bladder cancer risk alleles are associated with slow acetylator, suggesting that the level of systemic NAT2 activity modifies the risk of these pathologies. We speculate that rs1495741 (and its associated haplotype) belongs to a distal regulatory element of human NAT2 gene (e.g., enhancer or silencer), and the genetic variation at the newly discovered haplotype results in a differential level of NAT2 gene expression. Understanding how this NAT2 haplotype contributes to not only urinary bladder cancer but also to dyslipidemia will ultimately help devise strategies to identify and protect susceptible individuals.

An Integrated Workflow to Study the Promoter-Centric Spatio-Temporal Genome Architecture in Scarce Cell Populations.

Spatiotemporal gene transcription is tightly regulated by distal regulatory elements, such as enhancers and silencers, which rely on physical proximity with their target gene promoters to control transcription. Although these regulatory elements are easy to identify, their target genes are difficult to predict, since most of them are cell-type specific and may be separated by hundreds of kilobases in the linear genome sequence, skipping over other non-target genes. For several years, Promoter Capture Hi-C (PCHi-C) has been the gold standard for the association of distal regulatory elements to their target genes. However, PCHi-C relies on the availability of millions of cells, prohibiting the study of rare cell populations such as those commonly obtained from primary tissues. To overcome this limitation, low input Capture Hi-C (liCHi-C), a cost-effective and customizable method to identify the repertoire of distal regulatory elements controlling each gene of the genome, has been developed. liCHi-C relies on a similar experimental and computational framework as PCHi-C, but by employing minimal tube changes, modifyingthe reagent concentration and volumes, and swapping or eliminating steps, it accountsfor minimal material loss during library construction. Collectively, liCHi-C enables the study of gene regulation and spatiotemporal genome organization in the context of developmental biology and cellular function.

Connectome and regulatory hubs of CAGE highly active enhancers

Evidence indicates that enhancers are transcriptionally active. Herein, we investigated transcriptionally active enhancers by using cap analysis of gene expression (CAGE) combined with epigenetic marks and chromatin interactions. We identified CAGE-tag highly active (CHA) enhancers as distant regulatory elements with CAGE-tag ≥ 90th percentile and overlapping with H3K27ac peaks (4.5% of enhancers). CHA enhancers were conserved between mouse and man and were independent from super-enhancers in predicting cell identity with lower P-values. CHA enhancers had increased open chromatin and a higher recruitment of cell-specific transcription factors as well as molecules involved in 3D genome interactions. HiChIP analysis of enhancer-promoter looping indicated that CHA enhancers had a higher density of anchor loops when compared to regular enhancers. A subset of CHA enhancers and promoters characterized by a high density of chromatin loops and forming hub regulatory units were connected to the promoter of immediate early response genes, genes involved in cancer and encoding for transcription factors. Promoter of genes within hub CHA regulatory units were less likely to be paused. CHA enhancers were enriched in gene variants associated with autoimmune disorders and had looping with causal candidate genes as revealed by Mendelian randomization. Hence, CHA enhancers form a dense hierarchical network of chromatin interactions between regulatory elements and genes involved in cell identity and disorders.

Abstract 4742: Familial adenomatous polyposis epigenetic landscape as a precancer model of colorectal cancer

Abstract Aberrant shifts in DNA methylation have long been regarded as an early biomarker for cancer onset and progression. However, it is unclear when methylation aberrance starts and how it interacts with other epigenomic modifications. To address how epigenomic changes occur and interact during the transformation from normal healthy colon tissue to malignant colorectal cancer (CRC), we collected 51 samples from 15 familial adenomatous polyposis (FAP) and non-FAP colorectal cancer patients. We generated 30-70x of whole-genome enzymatic methylation sequencing (WGEM-seq) data via the novel Ultima Genomics ultra high-throughput sequencing platform. We observed hypermethylation and hypomethylation emerge early in the malignant transformation process in gene promoters and distal regulatory elements. We performed multifaceted analysis on methylation alterations with whole-genome sequencing (WGS), transposase accessibility (ATAC-seq), high-resolution chromatin accessibility (Tri-C), and gene expression (RNA-seq) data. Our multidimensional analysis demonstrates how collectively epigenomic alterations have affected gene expression throughout normal colon mucosa, benign and dysplasia polyps to adenocarcinoma. Epigenomic changes start as early as benign polyps, followed by other epigenomic shifts, including bivalent domains. Various epigenomic aberrances are associated with concomitant gene expression level changes. Our integrative analysis of multi-epigenomics data implicates collective and cumulative epigenomic instability in the early onset of colon carcinogenesis. Citation Format: Hayan Lee, Gat Krieger, Tyson Clark, Yizhou Zhu, Aziz Khan, Casey R. Hanson, Aaron Horning, Edward D. Esplin, Mohan Badu, Kristina Paul, Roxanne Chiu, Bahareh Bahmani, Stephanie Nevins, Annika K. Weimer, Ariel Jaimovich, Christina Curtis, William Greenleaf, James M. Ford, Doron Lipson, Zohar Shipony, Michael P. Snyder. Familial adenomatous polyposis epigenetic landscape as a precancer model of colorectal cancer. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4742.

Deletion mapping of regulatory elements for GATA3 in T cells reveals a distal enhancer involved in allergic diseases

GATA3 is essential for Tcell differentiation and is surrounded by genome-wide association study (GWAS) hits for immune traits. Interpretation of these GWAS hits is challenging because gene expression quantitative trait locus (eQTL) studies lack power to detect variants with small effects on gene expression in specific cell types and the genome region containing GATA3 contains dozens of potential regulatory sequences. To map regulatory sequences for GATA3, we performed a high-throughput tiling deletion screen of a 2 Mb genome region in Jurkat Tcells. This revealed 23 candidate regulatory sequences, all but one of which is within the same topological-associating domain (TAD) as GATA3. We then performed a lower-throughput deletion screen to precisely map regulatory sequences in primary T helper 2 (Th2) cells. We tested 25 sequences with ∼100bp deletions and validated five of the strongest hits with independent deletion experiments. Additionally, we fine-mapped GWAS hits for allergic diseases in a distal regulatory element, 1 Mb downstream of GATA3, and identified 14 candidate causal variants. Small deletions spanning the candidate variant rs725861 decreased GATA3 levels in Th2 cells, and luciferase reporter assays showed regulatory differences between its two alleles, suggesting a causal mechanism for this variant in allergic diseases. Our study demonstrates the power of integrating GWAS signals with deletion mapping and identifies critical regulatory sequences for GATA3.

DNA methylation trajectories during innate and adaptive immune responses of human B lymphocytes.

B lymphocytes can engage in either a rapid T cell-independent pathway (TI) or a delayed long-lasting T cell-independent (TD) response through highly ordered transcriptional programs, yet the detailed underlying mechanisms are unclear. Since DNA methylation plays a key role in controlling gene expression and lineage specification, we explored the dynamics of whole-genome cytosine modifications during the ex vivo response of human B cells isolated from normal individuals by negative selection. We found that B cell differentiation following TI and TD signalling is accompanied by extensive remodelling of the epigenome, including global gain and loss of DNA methylation. The epigenetic changes map to different regions of the B cell genome, including non- C-phosphate-G CpG islands, indicating that modifications of distal regulatory elements likely regulate specific gene transcription in B cells. Non-CpG methylation also occurs in differentiating human B cells, suggesting that this DNA modification is involved in transcriptional regulation of B cell genes with promoters exhibiting a low-density methylation, possibly by changing the chromatin shape that could have an impact on gene expression. Most strikingly, compared to TD activation, stimulation of B cells through an innate pathway induced higher levels of DNA methylation modifications at CpG, CHG and CGG contexts, supporting the view that DNA methylation modifications are used in distinct trajectories to specify the TI and TD B lymphocyte responses.

Pioneer factor ASCL1 cooperates with the mSWI/SNF complex at distal regulatory elements to regulate human neural differentiation.

Pioneer transcription factors are thought to play pivotal roles in developmental processes by binding nucleosomal DNA to activate gene expression, though mechanisms through which pioneer transcription factors remodel chromatin remain unclear. Here, using single-cell transcriptomics, we show that endogenous expression of neurogenic transcription factor ASCL1, considered a classical pioneer factor, defines a transient population of progenitors in human neural differentiation. Testing ASCL1's pioneer function using a knockout model to define the unbound state, we found that endogenous expression of ASCL1 drives progenitor differentiation by cis-regulation both as a classical pioneer factor and as a nonpioneer remodeler, where ASCL1 binds permissive chromatin to induce chromatin conformation changes. ASCL1 interacts with BAF SWI/SNF chromatin remodeling complexes, primarily at targets where it acts as a nonpioneer factor, and we provide evidence for codependent DNA binding and remodeling at a subset of ASCL1 and SWI/SNF cotargets. Our findings provide new insights into ASCL1 function regulating activation of long-range regulatory elements in human neurogenesis and uncover a novel mechanism of its chromatin remodeling function codependent on partner ATPase activity.

Comprehensive in silico and functional studies for classification of EPAS1/HIF2A genetic variants identified in patients with erythrocytosis.

Gain-of-function mutations in the EPAS1/HIF2A gene have been identified in patients with hereditary erythrocytosis that can be associated with the development of paraganglioma, pheochromocytoma and somatostatinoma. In the present study, we describe a unique European collection of 41 patients and 28 relatives diagnosed with an erythrocytosis associated with a germline genetic variant in EPAS1. In addition we identified two infants with severe erythrocytosis associated with a mosaic mutation present in less than 2% of the blood, one of whom later developed a paraganglioma. The aim of this study was to determine the causal role of these genetic variants, to establish pathogenicity, and to identify potential candidates eligible for the new hypoxia-inducible factor-2 α (HIF-2α) inhibitor treatment. Pathogenicity was predicted with in silico tools and the impact of 13 HIF-2b variants has been studied by using canonical and real-time reporter luciferase assays. These functional assays consisted of a novel edited vector containing an expanded region of the erythropoietin promoter combined with distal regulatory elements which substantially enhanced the HIF-2α-dependent induction. Altogether, our studies allowed the classification of 11 mutations as pathogenic in 17 patients and 23 relatives. We described four new mutations (D525G, L526F, G527K, A530S) close to the key proline P531, which broadens the spectrum of mutations involved in erythrocytosis. Notably, we identified patients with only erythrocytosis associated with germline mutations A530S and Y532C previously identified at somatic state in tumors, thereby raising the complexity of the genotype/phenotype correlations. Altogether, this study allows accurate clinical follow-up of patients and opens the possibility of benefiting from HIF-2α inhibitor treatment, so far the only targeted treatment in hypoxia-related erythrocytosis disease.

β-actin mediated H3K27ac changes demonstrate the link between compartment switching and enhancer-dependent transcriptional regulation

BackgroundRecent work has demonstrated that three-dimensional genome organization is directly affected by changes in the levels of nuclear cytoskeletal proteins such as β-actin. The mechanisms which translate changes in 3D genome structure into changes in transcription, however, are not fully understood. Here, we use a comprehensive genomic analysis of cells lacking nuclear β-actin to investigate the mechanistic links between compartment organization, enhancer activity, and gene expression.ResultsUsing HiC-Seq, ATAC-Seq, and RNA-Seq, we first demonstrate that transcriptional and chromatin accessibility changes observed upon β-actin loss are highly enriched in compartment-switching regions. Accessibility changes within compartment switching genes, however, are mainly observed in non-promoter regions which potentially represent distal regulatory elements. Our results also show that β-actin loss induces widespread accumulation of the enhancer-specific epigenetic mark H3K27ac. Using the ABC model of enhancer annotation, we then establish that these epigenetic changes have a direct impact on enhancer activity and underlie transcriptional changes observed upon compartment switching. A complementary analysis of fibroblasts undergoing reprogramming into pluripotent stem cells further confirms that this relationship between compartment switching and enhancer-dependent transcriptional change is not specific to β-actin knockout cells but represents a general mechanism linking compartment-level genome organization to gene expression.ConclusionsWe demonstrate that enhancer-dependent transcriptional regulation plays a crucial role in driving gene expression changes observed upon compartment-switching. Our results also reveal a novel function of nuclear β-actin in regulating enhancer function by influencing H3K27 acetylation levels.

Low input capture Hi-C (liCHi-C) identifies promoter-enhancer interactions at high-resolution

Long-range interactions between regulatory elements and promoters are key in gene transcriptional control; however, their study requires large amounts of starting material, which is not compatible with clinical scenarios nor the study of rare cell populations. Here we introduce low input capture Hi-C (liCHi-C) as a cost-effective, flexible method to map and robustly compare promoter interactomes at high resolution. As proof of its broad applicability, we implement liCHi-C to study normal and malignant human hematopoietic hierarchy in clinical samples. We demonstrate that the dynamic promoter architecture identifies developmental trajectories and orchestrates transcriptional transitions during cell-state commitment. Moreover, liCHi-C enables the identification of disease-relevant cell types, genes and pathways potentially deregulated by non-coding alterations at distal regulatory elements. Finally, we show that liCHi-C can be harnessed to uncover genome-wide structural variants, resolve their breakpoints and infer their pathogenic effects. Collectively, our optimized liCHi-C method expands the study of 3D chromatin organization to unique, low-abundance cell populations, and offers an opportunity to uncover factors and regulatory networks involved in disease pathogenesis.

Dr.Nod: computational framework for discovery of regulatory non-coding drivers in tissue-matched distal regulatory elements.

The discovery of cancer driver mutations is a fundamental goal in cancer research. While many cancer driver mutations have been discovered in the protein-coding genome, research into potential cancer drivers in the non-coding regions showed limited success so far. Here, we present a novel comprehensive framework Dr.Nod for detection of non-coding cis-regulatory candidate driver mutations that are associated with dysregulated gene expression using tissue-matched enhancer-gene annotations. Applying the framework to data from over 1500 tumours across eight tissues revealed a 4.4-fold enrichment of candidate driver mutations in regulatory regions of known cancer driver genes. An overarching conclusion that emerges is that the non-coding driver mutations contribute to cancer by significantly altering transcription factor binding sites, leading to upregulation of tissue-matched oncogenes and down-regulation of tumour-suppressor genes. Interestingly, more than half of the detected cancer-promoting non-coding regulatory driver mutations are over 20 kb distant from the cancer-associated genes they regulate. Our results show the importance of tissue-matched enhancer-gene maps, functional impact of mutations, and complex background mutagenesis model for the prediction of non-coding regulatory drivers. In conclusion, our study demonstrates that non-coding mutations in enhancers play a previously underappreciated role in cancer and dysregulation of clinically relevant target genes.

Structural variants drive context-dependent oncogene activation in cancer.

Higher-order chromatin structure is important for the regulation of genes by distal regulatory sequences1,2. Structural variants (SVs) that alter three-dimensional (3D) genome organization can lead to enhancer-promoter rewiring and human disease, particularly in the context of cancer3. However, only a small minority of SVs are associated with altered gene expression4,5, and it remains unclear why certain SVs lead to changes in distal gene expression and others do not. To address these questions, we used a combination of genomic profiling and genome engineering to identify sites of recurrent changes in 3D genome structure in cancer and determine the effects of specific rearrangements on oncogene activation. By analysing Hi-C data from 92 cancer cell lines and patient samples, we identified loci affected by recurrent alterations to 3D genome structure, including oncogenes such as MYC, TERT and CCND1. By using CRISPR-Cas9 genome engineering to generate de novo SVs, we show that oncogene activity can be predicted by using 'activity-by-contact' models that consider partner region chromatin contacts and enhancer activity. However, activity-by-contact models are only predictive of specific subsets of genes in the genome, suggesting that different classes of genes engage in distinct modes of regulation by distal regulatory elements. These results indicate that SVs that alter 3D genome organization are widespread in cancer genomes and begin to illustrate predictive rules for the consequences of SVs on oncogene activation.

Identification of cis‐regulatory elements associated with MAPT expression in human induced pluripotent stem cell derived neural cells.

AbstractBackgroundTau is encoded by MAPT and abnormal aggregates of tau are a hallmark of many neurodegenerative diseases. MAPT is highly expressed in neurons but much more lowly expressed in other cell types, including neural precursor cells (NPCs), suggesting that its expression may be controlled by neuron‐specific transcription factors and cis‐regulatory elements. Tau reduction, even of endogenous tau, is a current therapeutic strategy in both model systems and humans; therefore, understanding MAPT regulation may point to new strategies to achieve tau reduction. Additionally, genome sequencing studies continue to identify disease‐associated variants in non‐coding regions of the genome. These variants are likely located in regulatory regions and affect the expression of target genes whose function contributes to neurodegeneration. Therefore, an additional benefit of this study is that high confidence regulatory regions for MAPT may provide a way to identify important non‐coding variants associated with disease.MethodsThree‐dimensional chromatin conformation capture (Capture‐C), single‐cell multiomics (RNAseq + ATACseq), and chromatin marks including H3K27ac, H3K4me1, and DNase hypersensitivity were used to identify candidate enhancers of MAPT in human NPCs, NPCs differentiated to neurons, and pure inhibitory or excitatory neuron cultures (all lines were H1/H1). Approaches included nomination of elements with signals specific to neurons or neuronal subtypes. The nominated regions were then assessed using luciferase assays to test sufficiency for regulatory activity as well as CRISPR‐dCas9‐KRAB to assess the effect of nominated elements on MAPT expression.ResultsWe identified both proximal and distal regulatory elements for MAPT with function confirmed by up to four orthogonal approaches (chromatin capture, single cell multiomics, a reporter assay, and element inhibition using dCas9‐KRAB). Several of these elements were specific to neurons. One particularly important region was centromeric to MAPT beyond the H2 inversion breakpoint.ConclusionsOverall, these results indicate that MAPT regulation is likely controlled by neuron‐specific cis‐regulatory elements, including both proximal and distal elements. Next steps include study of the effect of trans‐regulatory elements (i.e., transcription factors) that may control recruitment of these cis‐regulatory elements for expression of MAPT and assessment of the effects of the disease‐associated genetic variation on cis‐regulatory element function and expression of MAPT.