Cell Type-specific Enhancer Research Articles

Selective Enhancer Gain of Function Deregulates MYC Expression in Multiple Myeloma

Abstract MYC deregulation occurs in the majority of multiple myeloma (MM) cases and is associated with progression and worse prognosis. Enhanced MYC expression occurs in about 70% of MM patients, but it is known to be driven by translocation or amplification events in only ~40% of myelomas. Here, we used CRISPR interference (CRISPRi) to uncover an epigenetic mechanism of MYC regulation whereby increased accessibility of a plasma cell-type specific enhancer leads to increased MYC expression. This native enhancer activity was not associated with enhancer hijacking events but led to specific binding of c-MAF, IRF4, and SPIB transcription factors that activated MYC expression in the absence of known genetic aberrations. In addition, focal amplification was another mechanism of activation of this enhancer in approximately 3.4% of MM patients. Together, these findings define an epigenetic mechanism of MYC deregulation in MM beyond known translocations or amplifications and point to the importance of non-coding regulatory elements and their associated transcription factor networks as drivers of MM progression.

Evaluating Methods for the Prediction of Cell Type-Specific Enhancers in the Mammalian Cortex.

Identifying cell type-specific enhancers in the brain is critical to building genetic tools for investigating the mammalian brain. Computational methods for functional enhancer prediction have been proposed and validated in the fruit fly and not yet the mammalian brain. We organized the 'Brain Initiative Cell Census Network (BICCN) Challenge: Predicting Functional Cell Type-Specific Enhancers from Cross-Species Multi-Omics' to assess machine learning and feature-based methods designed to nominate enhancer DNA sequences to target cell types in the mouse cortex. Methods were evaluated based on in vivo validation data from hundreds of cortical cell type-specific enhancers that were previously packaged into individual AAV vectors and retro-orbitally injected into mice. We find that open chromatin was a key predictor of functional enhancers, and sequence models improved prediction of non-functional enhancers that can be deprioritized as opposed to pursued for in vivo testing. Sequence models also identified cell type-specific transcription factor codes that can guide designs of in silico enhancers. This community challenge establishes a benchmark for enhancer prioritization algorithms and reveals computational approaches and molecular information that are crucial for the identification of functional enhancers for mammalian cortical cell types. The results of this challenge bring us closer to understanding the complex gene regulatory landscape of the mammalian brain and help us design more efficient genetic tools and potential gene therapies for human neurological diseases.

Enhancer hijacking: Innovative ways of carcinogenesis

Enhancer elements are specific DNA sequences that play a crucial role in regulating gene expression. Located upstream or downstream in the genomic context, enhancers enhance the transcription of linked genes. This is achieved by providing binding sites for transcription factors and other proteins, acting as a nucleation point that promotes the assembly of the transcriptional machinery. What makes enhancers unique is that located even thousands of base pairs away, they can exert their function. They can act over long distances, looping to interact with the promoter region of their target genes. Enhancers are often involved in cell type-specific gene expression as different cells express different sets of genes by activating and repressing cell type-specific enhancers. Cancer cells seem to co-opt this mechanism and hijack it for their own survival. In this process, an aberrant enhancer element activates the transcription of oncogenes (genes that have the potential to cause cancer) due to some alterations in genomic structure or regulatory elements that happen in cancer, contributing to tumorigenesis and cancer progression. Enhancer hijacking often occurs through complex genomic rearrangements such as chromosomal translocations and other structural rearrangements such as deletions or amplifications.8 These alterations can bring a usually distant, far-located enhancer into proximity with an oncogene, leading to its inappropriate activation. This is often seen in various cancers, including leukemias and solid tumors. Enhancer hijacking and its effects on gene regulation in cancer are frequently studied using methods such as RNA sequencing, chromatin conformation capture, and CRISPR-Cas9. Finding an enhancer hijacking event may be used to inform treatment choices, especially in precision medicine settings, and may also function as a biomarker for particular cancer types.

Genome-Wide Methylation Analysis Reveals a KCNK3-Prominent Causal Cascade on Hypertension.

Despite advances in understanding hypertension's genetic structure, how noncoding genetic variants influence it remains unclear. Studying their interaction with DNA methylation is crucial to deciphering this complex disease's genetic mechanisms. We investigated the genetic and epigenetic interplay in hypertension using whole-genome bisulfite sequencing. Methylation profiling in 918 males revealed allele-specific methylation and methylation quantitative trait loci. We engineered rs1275988T/C mutant mice using CRISPR (clustered regularly interspaced short palindromic repeats)/Cas9 (CRISPR-associated protein 9), bred them for homozygosity, and subjected them to a high-salt diet. Telemetry captured their cardiovascular metrics. Protein-DNA interactions were elucidated using DNA pull-downs, mass spectrometry, and Western blots. A wire myograph assessed vascular function, and analysis of the Kcnk3 gene methylation highlighted the mutation's role in hypertension. We discovered that DNA methylation-associated genetic effects, especially in non-cytosine-phosphate-guanine (non-CpG) island and noncoding distal regulatory regions, significantly contribute to hypertension predisposition. We identified distinct methylation quantitative trait locus patterns in the hypertensive population and observed that the onset of hypertension is influenced by the transmission of genetic effects through the demethylation process. By evidence-driven prioritization and in vivo experiments, we unearthed rs1275988 in a cell type-specific enhancer as a notable hypertension causal variant, intensifying hypertension through the modulation of local DNA methylation and consequential alterations in Kcnk3 gene expression and vascular remodeling. When exposed to a high-salt diet, mice with the rs1275988C/C genotype exhibited exacerbated hypertension and significant vascular remodeling, underscored by increased aortic wall thickness. The C allele of rs1275988 was associated with elevated DNA methylation levels, driving down the expression of the Kcnk3 gene by attenuating Nr2f2 (nuclear receptor subfamily 2 group F member 2) binding at the enhancer locus. Our research reveals new insights into the complex interplay between genetic variations and DNA methylation in hypertension. We underscore hypomethylation's potential in hypertension onset and identify rs1275988 as a causal variant in vascular remodeling. This work advances our understanding of hypertension's molecular mechanisms and encourages personalized health care strategies.

Cell type- and transcription-independent spatial proximity between enhancers and promoters.

Cell type-specific enhancers are critically important for lineage specification. The mechanisms that determine cell-type specificity of enhancer activity, however, are not fully understood. Most current models for how enhancers function invoke physical proximity between enhancer elements and their target genes. Here, we use an imaging-based approach to examine the spatial relationship of cell type-specific enhancers and their target genes with single-cell resolution. Using high-throughput microscopy, we measure the spatial distance from target promoters to their cell type-specific active and inactive enhancers in individual pancreatic cells derived from distinct lineages. We find increased proximity of all promoter-enhancer pairs relative to non-enhancer pairs separated by similar genomic distances. Strikingly, spatial proximity between enhancers and target genes was unrelated to tissue-specific enhancer activity. Furthermore, promoter-enhancer proximity did not correlate with the expression status of target genes. Our results suggest that promoter-enhancer pairs exist in a distinctive chromatin environment but that genome folding is not a universal driver of cell-type specificity in enhancer function.

The chromatin accessibility dynamics during cell fate specifications in zebrafish early embryogenesis.

Chromatin accessibility plays a critical role in the regulation of cell fate decisions. Although gene expression changes have been extensively profiled at the single-cell level during early embryogenesis, the dynamics of chromatin accessibility at cis-regulatory elements remain poorly studied. Here, we used a plate-based single-cell ATAC-seq method to profile the chromatin accessibility dynamics of over 10000 nuclei from zebrafish embryos. We investigated several important time points immediately after zygotic genome activation (ZGA), covering key developmental stages up to dome. The results revealed key chromatin signatures in the first cell fate specifications when cells start to differentiate into enveloping layer (EVL) and yolk syncytial layer (YSL) cells. Finally, we uncovered many potential cell-type specific enhancers and transcription factor motifs that are important for the cell fate specifications.

Integrative genomic analyses reveal putative cell type-specific targets of the Drosophila ets transcription factor Pointed

The Ets domain transcription factors direct diverse biological processes throughout all metazoans and are implicated in development as well as in tumor initiation, progression and metastasis. The Drosophila Ets transcription factor Pointed (Pnt) is the downstream effector of the Epidermal growth factor receptor (Egfr) pathway and is required for cell cycle progression, specification, and differentiation of most cell types in the larval eye disc. Despite its critical role in development, very few targets of Pnt have been reported previously. Here, we employed an integrated approach by combining genome-wide single cell and bulk data to identify putative cell type-specific Pnt targets. First, we used chromatin immunoprecipitation with high-throughput sequencing (ChIP-seq) to determine the genome-wide occupancy of Pnt in late larval eye discs. We identified enriched regions that mapped to an average of 6,941 genes, the vast majority of which are novel putative Pnt targets. Next, we integrated ChIP-seq data with two other larval eye single cell genomics datasets (scRNA-seq and snATAC-seq) to reveal 157 putative cell type-specific Pnt targets that may help mediate unique cell type responses upon Egfr-induced differentiation. Finally, our integrated data also predicts cell type-specific functional enhancers that were not reported previously. Together, our study provides a greatly expanded list of putative cell type-specific Pnt targets in the eye and is a resource for future studies that will allow mechanistic insights into complex developmental processes regulated by Egfr signaling.

Genome-wide binding analysis unveils critical implication of B-Myb-mediated transactivation in cancers.

B-Myb, also known as MYB proto-oncogene like 2 (MYBL2), is an important transcription factor implicated in transcription regulation, cell cycle and tumorigenesis. However, the molecular mechanism underlying B-Myb-controlled transactivation in different cell contexts as well as its functional implication in cancers remains elusive. In this study, we have conducted a comprehensive genome-wide analysis of B-Myb binding sites in multiple immortalized or cancer cell lines and identified its critical target genes. The results revealed that B-Myb regulates a common set of core cell cycle genes and cell type-specific genes through collaboration with other important transcription factors (e.g. NFY and MuvB complex) and binding to cell type-invariant promoters and cell type-specific enhancers and super-enhancers. KIF2C, UBE2C and MYC were further validated as B-Myb target genes. Loss-of-function analysis demonstrated that KIF2C knockdown inhibited tumor cell growth both in vitro and in vivo, suppressed cell motility and cell cycle progression, accompanied with defects in microtubule organization and mitosis, strongly suggesting that KIF2C is a critical regulator of cancer cell growth and mitosis, and maintains high cancer cell motility ability and microtubule dynamics. Pan-cancer transcriptomic analysis revealed that the overexpression of both B-Myb and KIF2C presents as independent prognostic markers in various types of cancer. Notably, B-Myb associates with NFYB, binds to target gene promoters, enhancers and super-enhancers, and provokes a cascade of oncogenic gene expression profiles in cancers. Overall, our results highly suggest the critical implication of B-Myb-mediated gene regulation in cancers, and the promising therapeutic and prognostic potentials of B-Myb and KIF2C for cancer diagnosis and treatment.

An intronic LINE-1 regulates IFNAR1 expression in human immune cells

BackgroundDespite their origins as selfish parasitic sequences, some transposons in the human genome have been co-opted to serve as regulatory elements, contributing to the evolution of transcriptional networks. Most well-characterized examples of transposon-derived regulatory elements derive from endogenous retroviruses (ERVs), due to the intrinsic regulatory activity of proviral long terminal repeat regions. However, one subclass of transposable elements, the Long Interspersed Nuclear Elements (LINEs), have been largely overlooked in the search for functional regulatory transposons, and considered to be broadly epigenetically repressed.ResultsWe examined the chromatin state of LINEs by analyzing epigenomic data from human immune cells. Many LINEs are marked by the repressive H3K9me3 modification, but a subset exhibits evidence of enhancer activity in human immune cells despite also showing evidence of epigenetic repression. We hypothesized that these competing forces of repressive and activating epigenetic marks might lead to inducible enhancer activity. We investigated a specific L1M2a element located within the first intron of Interferon Alpha/Beta Receptor 1 (IFNAR1). This element shows epigenetic signatures of B cell-specific enhancer activity, despite being repressed by the Human Silencing Hub (HUSH) complex. CRISPR deletion of the element in B lymphoblastoid cells revealed that the element acts as an enhancer that regulates both steady state and interferon-inducible expression of IFNAR1.ConclusionsOur study experimentally demonstrates that an L1M2a element was co-opted to function as an interferon-inducible enhancer of IFNAR1, creating a feedback loop wherein IFNAR1 is transcriptionally upregulated by interferon signaling. This finding suggests that other LINEs may exhibit cryptic cell type-specific or context-dependent enhancer activity. LINEs have received less attention than ERVs in the effort to understand the contribution of transposons to the regulatory landscape of cellular genomes, but these are likely important, lineage-specific players in the rapid evolution of immune system regulatory networks and deserve further study.

A marmoset brain cell census reveals regional specialization of cellular identities.

The mammalian brain is composed of many brain structures, each with its own ontogenetic and developmental history. We used single-nucleus RNA sequencing to sample over 2.4 million brain cells across 18 locations in the common marmoset, a New World monkey primed for genetic engineering, and examined gene expression patterns of cell types within and across brain structures. The adult transcriptomic identity of most neuronal types is shaped more by developmental origin than by neurotransmitter signaling repertoire. Quantitative mapping of GABAergic types with single-molecule FISH (smFISH) reveals that interneurons in the striatum and neocortex follow distinct spatial principles, and that lateral prefrontal and other higher-order cortical association areas are distinguished by high proportions of VIP+ neurons. We use cell type-specific enhancers to drive AAV-GFP and reconstruct the morphologies of molecularly resolved interneuron types in neocortex and striatum. Our analyses highlight how lineage, local context, and functional class contribute to the transcriptional identity and biodistribution of primate brain cell types.

Clustered and diverse transcription factor binding underlies cell type specificity of enhancers for housekeeping genes.

Housekeeping genes are considered to be regulated by common enhancers across different tissues. Here we report that most of the commonly expressed mouse or human genes across different cell types, including more than half of the previously identified housekeeping genes, are associated with cell type-specific enhancers. Furthermore, the binding of most transcription factors (TFs) is cell type-specific. We reason that these cell type specificities are causally related to the collective TF recruitment at regulatory sites, as TFs tend to bind to regions associated with many other TFs and each cell type has a unique repertoire of expressed TFs. Based on binding profiles of hundreds of TFs from HepG2, K562, and GM12878 cells, we show that 80% of all TF peaks overlapping H3K27ac signals are in the top 20,000-23,000 most TF-enriched H3K27ac peak regions, and approximately 12,000-15,000 of these peaks are enhancers (nonpromoters). Those enhancers are mainly cell type-specific and include those linked to the majority of commonly expressed genes. Moreover, we show that the top 15,000 most TF-enriched regulatory sites in HepG2 cells, associated with about 200 TFs, can be predicted largely from the binding profile of as few as 30 TFs. Through motif analysis, we show that major enhancers harbor diverse and clustered motifs from a combination of available TFs uniquely present in each cell type. We propose a mechanism that explains how the highly focused TF binding at regulatory sites results in cell type specificity of enhancers for housekeeping and commonly expressed genes.

Chromatin accessibility differences between alpha, beta, and delta cells identifies common and cell type-specific enhancers

BackgroundHigh throughput sequencing has enabled the interrogation of the transcriptomic landscape of glucagon-secreting alpha cells, insulin-secreting beta cells, and somatostatin-secreting delta cells. These approaches have furthered our understanding of expression patterns that define healthy or diseased islet cell types and helped explicate some of the intricacies between major islet cell crosstalk and glucose regulation. All three endocrine cell types derive from a common pancreatic progenitor, yet alpha and beta cells have partially opposing functions, and delta cells modulate and control insulin and glucagon release. While gene expression signatures that define and maintain cellular identity have been widely explored, the underlying epigenetic components are incompletely characterized and understood. However, chromatin accessibility and remodeling is a dynamic attribute that plays a critical role to determine and maintain cellular identity.ResultsHere, we compare and contrast the chromatin landscape between mouse alpha, beta, and delta cells using ATAC-Seq to evaluate the significant differences in chromatin accessibility. The similarities and differences in chromatin accessibility between these related islet endocrine cells help define their fate in support of their distinct functional roles. We identify patterns that suggest that both alpha and delta cells are poised, but repressed, from becoming beta-like. We also identify patterns in differentially enriched chromatin that have transcription factor motifs preferentially associated with different regions of the genome. Finally, we not only confirm and visualize previously discovered common endocrine- and cell specific- enhancer regions across differentially enriched chromatin, but identify novel regions as well. We compiled our chromatin accessibility data in a freely accessible database of common endocrine- and cell specific-enhancer regions that can be navigated with minimal bioinformatics expertise.ConclusionsBoth alpha and delta cells appear poised, but repressed, from becoming beta cells in murine pancreatic islets. These data broadly support earlier findings on the plasticity in identity of non-beta cells under certain circumstances. Furthermore, differential chromatin accessibility shows preferentially enriched distal-intergenic regions in beta cells, when compared to either alpha or delta cells.

Super-enhancer-associated transcription factors collaboratively regulate trophoblast-active gene expression programs in human trophoblast stem cells.

The placenta is an essential organ that supports the growth and development of the fetus during pregnancy. However, cell type-specific enhancers and transcription factors (TFs), and the mechanisms underlying the maintenance and differentiation of trophoblast stem cell (TSC) populations in the human placenta remain elusive. Here, using human TSCs as a model system, we identify 31,362 enhancers that are enriched with the motifs of previously reported TSC-pivotal TFs, including TEAD4, GATA2/3 and TFAP2C. Subsequently, we identify 580 super-enhancers (SEs) and 549 SE-associated genes. These genes are robustly expressed in the human placenta and include numerous TFs, implying that SE-associated TFs (SE-TFs) may play crucial roles in placental development. Additionally, we identify the global binding sites of five TSC-pivotal SE-TFs (FOS, GATA2, MAFK, TEAD4 and TFAP2C), revealing that they preferentially co-occupy enhancers, regulate each otherand form a trophoblast-active gene regulatory network. Loss-of-function studies unveil that the five TFs promote self-renewal of TSCs by activating proliferation-associated genes while repressing developmental genes. We further reveal that the five TFs exert conserved and unique functions on placental development between humans and mice. Our study provides important insights into the roles of human TSC-pivotal TFs in regulating placenta-specific gene expression programs.

Learning single-cell chromatin accessibility profiles using meta-analytic marker genes.

Single-cell assay for transposase accessible chromatin using sequencing (scATAC-seq) is a valuable resource to learn cis-regulatory elements such as cell-type specific enhancers and transcription factor binding sites. However, cell-type identification of scATAC-seq data is known to be challenging due to the heterogeneity derived from different protocols and the high dropout rate. In this study, we perform a systematic comparison of seven scATAC-seq datasets of mouse brain to benchmark the efficacy of neuronal cell-type annotation from gene sets. We find that redundant marker genes give a dramatic improvement for a sparse scATAC-seq annotation across the data collected from different studies. Interestingly, simple aggregation of such marker genes achieves performance comparable or higher than that of machine-learning classifiers, suggesting its potential for downstream applications. Based on our results, we reannotated all scATAC-seq data for detailed cell types using robust marker genes. Their meta scATAC-seq profiles are publicly available at https://gillisweb.cshl.edu/Meta_scATAC. Furthermore, we trained a deep neural network to predict chromatin accessibility from only DNA sequence and identified key motifs enriched for each neuronal subtype. Those predicted profiles are visualized together in our database as a valuable resource to explore cell-type specific epigenetic regulation in a sequence-dependent and -independent manner.

Abstract IA014: SWI/SNF complexes in development and disease

Abstract The Hargreaves laboratory is focused on understanding the molecular mechanisms of SWI/SNF complex function in immune cell differentiation and response relating to cancer and inflammation. SWI/SNF complexes are broadly required for embryonic development and cellular differentiation. Mutations in SWI/SNF complex genes are observed in ~20% of human cancers and in a number of rare intellectual disability disorders. However, it remains unclear how SWI/SNF complexes mechanistically regulate cellular differentiation through complex targeting, engagement with transcription factors, and function in transcription. We approach these questions in immune cells by employing molecular and proteomic methods in conjunction with next-generation sequencing approaches with the ultimate goal of connecting the functions of specific SWI/SNF subunits to the regulation of specific gene networks important for inflammatory responses and immune cell differentiation. This approach has allowed us to reveal the role of the canonical BAF complex (cBAF) in the establishment and maintenance of epigenetic transitions that occur during cellular differentiation, including activation of cell type-specific enhancers and engagement of transcription factors. Further, we and others have identified a new variant, referred to as the noncanonical BAF (ncBAF) complex, which exhibits distinct modes of targeting, unique regulatory interactions, and specific function in inflammatory gene transcription (Ahmed, Gatchalian et al PNAS 2022). Following up on the identification of the ncBAF subunit Brd9 in a CRISPR genome wide screen for regulators of Foxp3 performed in collaboration with the Zheng lab at the Salk Institute, we performed sgRNA knockdown to identify the role of each of the three SWI/SNF complex variants (BAF, ncBAF, and PBAF) (Loo, Gatchalian et al Immunity 2020). We found that ncBAF positively regulates the expression of Foxp3, while PBAF complexes negatively regulate Foxp3. Using epigenomic and transcriptomic analysis in Tregs following knockdown or chemical degradation of BRD9, we found that BRD9 positively regulates Foxp3 expression and its target genes by stabilizing Foxp3 binding at BRD9/Foxp3 co-bound sites, including at the Foxp3 locus. In an MC38 colorectal cancer model, transfer of Brd9 deficient Tregs resulted in significantly slower tumor growth than control Tregs, with increased infiltration of IFN-γ producing CD8 and CD4 T cells, suggesting that Brd9 deletion in Tregs reduces Treg-mediated suppression of anti-tumor responses. Thus, these results highlight BRD9 and the ncBAF complex as novel targets that could be exploited to manipulate Treg function in cancer. Citation Format: Jovylyn Gatchalian, Nasiha Ahmed, Chin-San Loo, Ye Zheng, Diana C. Hargreaves. SWI/SNF complexes in development and disease. [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 IA014.

Diff-ATAC-STARR-Seq: A Method for Genome-Wide Functional Screening of Enhancer Activity in Vivo.

Transcriptional regulatory elements, including promoters and enhancers, play a key role in the cell-type specific regulation of the transcriptome. Application of rapidly evolving genetic tools, such as optogenetic/chemogenetic actuators and fluorescent reporters to elucidate the function of cell subtypes in vivo necessitates cell-type specific promoters or enhancers. In this context, methods for genome-wide functional screening of cis-regulatory elements, including enhancers, are of utmost importance. In this study, we describe a novel method for genome-wide functional screening of enhancer activity in vivo with minimal handling. Application of the method to cells from different brain structures and subsequent differential analysis allow identification of active enhancers in the target tissue or brain structures. To demonstrate proof of concept, we applied this method to samples from the dorsal raphe nucleus (DRN) and the medial prefrontal cortex of the mouse brain and successfully identified six enhancers with highly biased activity towards the dorsal raphe nucleus. Considering that these two structures consist of largely similar cell types whereas serotonin and dopamine neurons exist only in the DRN, our results confirm the validity of this method in identifying cell-type specific and brain-structure specific enhancers. Overall, this method will be helpful in identifying cis-regulatory elements suitable for cell-type specific manipulations.

Mitochondrial stress induces AREG expression and epigenomic remodeling through c-JUN and YAP-mediated enhancer activation.

Nucleus–mitochondria crosstalk is essential for cellular and organismal homeostasis. Although anterograde (nucleus-to-mitochondria) pathways have been well characterized, retrograde (mitochondria-to-nucleus) pathways remain to be clarified. Here, we found that mitochondrial dysfunction triggered a retrograde signaling via unique transcriptional and chromatin factors in hepatic cells. Our transcriptomic analysis revealed that the loss of mitochondrial transcription factor A led to mitochondrial dysfunction and dramatically induced expression of amphiregulin (AREG) and other secretory protein genes. AREG expression was also induced by various mitochondria stressors and was upregulated in murine liver injury models, suggesting that AREG expression is a hallmark of mitochondrial damage. Using epigenomic and informatic approaches, we identified that mitochondrial dysfunction-responsive enhancers of AREG gene were activated by c-JUN/YAP1/TEAD axis and were repressed by chromatin remodeler BRG1. Furthermore, while mitochondrial dysfunction-activated enhancers were enriched with JUN and TEAD binding motifs, the repressed enhancers possessed the binding motifs for hepatocyte nuclear factor 4α, suggesting that both stress responsible and cell type-specific enhancers were reprogrammed. Our study revealed that c-JUN and YAP1-mediated enhancer activation shapes the mitochondrial stress-responsive phenotype, which may shift from metabolism to stress adaptation including protein secretion under such stressed conditions.

A sequence-based global map of regulatory activity for deciphering human genetics

Epigenomic profiling has enabled large-scale identification of regulatory elements, yet we still lack a systematic mapping from any sequence or variant to regulatory activities. We address this challenge with Sei, a framework for integrating human genetics data with sequence information to discover the regulatory basis of traits and diseases. Sei learns a vocabulary of regulatory activities, called sequence classes, using a deep learning model that predicts 21,907 chromatin profiles across >1,300 cell lines and tissues. Sequence classes provide a global classification and quantification of sequence and variant effects based on diverse regulatory activities, such as cell type-specific enhancer functions. These predictions are supported by tissue-specific expression, expression quantitative trait loci and evolutionary constraint data. Furthermore, sequence classes enable characterization of the tissue-specific, regulatory architecture of complex traits and generate mechanistic hypotheses for individual regulatory pathogenic mutations. We provide Sei as a resource to elucidate the regulatory basis of human health and disease.

Heterogeneity of enhancers embodies shared and representative functional groups underlying developmental and cell type-specific gene regulation

While enhancers in a particular tissue coordinately fulfill regulatory functions, these functions are heterogeneous in nature and comprise of multiple enhancer subclasses and the associated regulatory mechanisms. In this work, we used multiple cell lines to identify enhancer subclasses linked to development, differentiation, and cellular identity. We found that enhancer functional heterogeneity during development encompasses subclasses of ubiquitous functions (11%), development specific regulatory activity (62%), and chromatin interactions (12%). In differentiated cell lines, ubiquitous enhancers (10%) stay active across multiple cell lines.They are accompanied by a large enhancer subclass (ranging from 33% to 63%) with functions specific to the corresponding lineage. The remaining enhancers (27–40%) establish regulatory chromatin structure and facilitate interactions of cell type-specific enhancers with their target promoters. In addition to specialized functions of cell type-specific enhancers, we show that proper accounting of enhancer heterogeneity leads to a 10% increase in accuracy of enhancer classification, which significantly improves the modeling of enhancers and identification of underlying regulatory mechanisms. In summary, our observations suggest that although cell type-specific enhancers are heterogeneous and coordinate different regulatory programs, enhancers from different cell lines maintain common categories of functional groups across developmental and differentiation stages, indicating a higher order rule followed by enhancer-gene regulation.

Enhancers regulate 3′ end processing activity to control expression of alternative 3′UTR isoforms

Multi-UTR genes are widely transcribed and express their alternative 3′UTR isoforms in a cell type-specific manner. As transcriptional enhancers regulate mRNA expression, we investigated if they also regulate 3′UTR isoform expression. Endogenous enhancer deletion of the multi-UTR gene PTEN did not impair transcript production but prevented 3′UTR isoform switching which was recapitulated by silencing of an enhancer-bound transcription factor. In reporter assays, enhancers increase transcript production when paired with single-UTR gene promoters. However, when combined with multi-UTR gene promoters, they change 3′UTR isoform expression by increasing 3′ end processing activity of polyadenylation sites. Processing activity of polyadenylation sites is affected by transcription factors, including NF-κB and MYC, transcription elongation factors, chromatin remodelers, and histone acetyltransferases. As endogenous cell type-specific enhancers are associated with genes that increase their short 3′UTRs in a cell type-specific manner, our data suggest that transcriptional enhancers integrate cellular signals to regulate cell type-and condition-specific 3′UTR isoform expression.