A cis-regulatory atlas in maize at single-cell resolution.

cis-regulatory elements (CREs) encode the genomic blueprints of spatiotemporal gene expression programs enabling highly specialized cell functions. Using single-cell genomics in six maize organs, we determined the cis- and trans-regulatory factors defining diverse cell identities and coordinating chromatin organization by profiling transcription factor (TF) combinatorics, identifying TFs with non-cell-autonomous activity, and uncovering TFs underlying higher-order chromatin interactions. Cell-type-specific CREs were enriched for enhancer activity and within unmethylated long terminal repeat retrotransposons. Moreover, we found cell-type-specific CREs are hotspots for phenotype-associated genetic variants and were targeted by selection during modern maize breeding, highlighting the biological implications of this CRE atlas. Through comparison of maize and Arabidopsis thaliana developmental trajectories, we identified TFs and CREs with conserved and divergent chromatin dynamics, showcasing extensive evolution of gene regulatory networks. In addition to this rich dataset, we developed single-cell analysis software, Socrates, which can be used to understand cis-regulatory variation in any species.

The cis-Regulatory Atlas of the Mouse Immune System.

A complete chart of cis-regulatory elements and their dynamic activity is necessary to understand the transcriptional basis of differentiation and function of an organ system. We generated matched epigenome and transcriptome measurements in 86 primary cell types that span the mouse immune system and its differentiation cascades. This breadth of data enable variance components analysis that suggests that genes fall into two distinct classes, controlled by either enhancer- or promoter-driven logic, and multiple regression that connects genes to the enhancers that regulate them. Relating transcription factor (TF) expression to the genome-wide accessibility of their binding motifs classifies them as predominantly openers or closers of local chromatin accessibility, pinpointing specific cis-regulatory elements where binding of given TFs is likely functionally relevant, validated by chromatin immunoprecipitation sequencing (ChIP-seq). Overall, this cis-regulatory atlas provides a trove of information on transcriptional regulation through immune differentiation and a foundational scaffold to define key regulatory events throughout the immunological genome.

Recurrent repeat expansions in human cancer genomes.

Expansion of a single repetitive DNA sequence, termed a tandem repeat (TR), is known to cause more than 50 diseases1,2. However, repeat expansions are often not explored beyond neurological and neurodegenerative disorders. In some cancers, mutations accumulate in short tracts of TRs, a phenomenon termed microsatellite instability; however, larger repeat expansions have not been systematically analysed in cancer3-8. Here we identified TR expansions in 2,622 cancer genomes spanning 29 cancer types. In seven cancer types, we found 160 recurrent repeat expansions (rREs), most of which (155/160) were subtype specific. We found that rREs were non-uniformly distributed in the genome with enrichment near candidate cis-regulatory elements, suggesting a potential role in gene regulation. One rRE, a GAAA-repeat expansion, located near a regulatory element in the first intron of UGT2B7 was detected in 34% of renal cell carcinoma samples and was validated by long-read DNA sequencing. Moreover, in preliminary experiments, treating cells that harbour this rRE with a GAAA-targeting molecule led to a dose-dependent decrease in cell proliferation. Overall, our results suggest that rREs may be an important but unexplored source of genetic variation in human cancer, and we provide a comprehensive catalogue for further study.

Understanding transcription across scales: From base pairs to chromosomes.

The influence of genome organization on transcription is central to our understanding of cell type specification. Higher-order genome organization is established through short- and long-range DNA interactions. Coordination of these interactions, from single atoms to entire chromosomes, plays a fundamental role in transcriptional control of gene expression. Loss of this coupling can result in disease. Analysis of transcriptional regulation typically involves disparate experimental approaches, from structural studies that define angstrom-level interactions to cell-biological and genomic approaches that assess mesoscale relationships. Thus, to fully understand the mechanisms that regulate gene expression, it is critical to integrate the findings gained across these distinct size scales. In this review, I illustrate fundamental ways in which cells regulate transcription in the context of genome organization.

Pioneering the developmental frontier.

Coordinated changes in gene expression allow a single fertilized oocyte to develop into a complex multi-cellular organism. These changes in expression are controlled by transcription factors that gain access to discrete cis-regulatory elements in the genome, allowing them to activate gene expression. Although nucleosomes present barriers to transcription factor occupancy, pioneer transcription factors have unique properties that allow them to bind DNA in the context of nucleosomes, define cis-regulatory elements, and facilitate the subsequent binding of additional factors that determine gene expression. In this capacity, pioneer factors act at the top of gene-regulatory networks to control developmental transitions. Developmental context also influences pioneer factor binding and activity. Here we discuss the interplay between pioneer factors and development, their role in driving developmental transitions, and the influence of the cellular environment on pioneer factor binding and activity.

Enhancer RNA m6A methylation facilitates transcriptional condensate formation and gene activation.

The mechanistic understanding of nascent RNAs in transcriptional control remains limited. Here, by a high sensitivity method methylation-inscribed nascent transcripts sequencing (MINT-seq), we characterized the landscapes of N6-methyladenosine (m6A) on nascent RNAs. We uncover heavy but selective m6A deposition on nascent RNAs produced by transcription regulatory elements, including promoter upstream antisense RNAs and enhancer RNAs (eRNAs), which positively correlates with their length, inclusion of m6A motif, and RNA abundances. m6A-eRNAs mark highly active enhancers, where they recruit nuclear m6A reader YTHDC1 to phase separate into liquid-like condensates, in a manner dependent on its C terminus intrinsically disordered region and arginine residues. The m6A-eRNA/YTHDC1 condensate co-mixes with and facilitates the formation of BRD4 coactivator condensate. Consequently, YTHDC1 depletion diminished BRD4 condensate and its recruitment to enhancers, resulting in inhibited enhancer and gene activation. We propose that chemical modifications of eRNAs together with reader proteins play broad roles in enhancer activation and gene transcriptional control.

Identification and characterization of conserved noncoding cis-regulatory elements that impact Mecp2 expression and neurological functions.

While changes in MeCP2 dosage cause Rett syndrome (RTT) and MECP2 duplication syndrome (MDS), its transcriptional regulation is poorly understood. Here, we identified six putative noncoding regulatory elements of Mecp2, two of which are conserved in humans. Upon deletion in mice and human iPSC-derived neurons, these elements altered RNA and protein levels in opposite directions and resulted in a subset of RTT- and MDS-like behavioral deficits in mice. Our discovery provides insight into transcriptional regulation of Mecp2/MECP2 and highlights genomic sites that could serve as diagnostic and therapeutic targets in RTT or MDS.

Advances in computational and experimental approaches for deciphering transcriptional regulatory networks: Understanding the roles of cis-regulatory elements is essential, and recent research utilizing MPRAs, STARR-seq, CRISPR-Cas9, and machine learning has yielded valuable insights.

Understanding the influence of cis-regulatory elements on gene regulation poses numerous challenges given complexities stemming from variations in transcription factor (TF) binding, chromatin accessibility, structural constraints, and cell-type differences. This review discusses the role of gene regulatory networks in enhancing understanding of transcriptional regulation and covers construction methods ranging from expression-based approaches to supervised machine learning. Additionally, key experimental methods, including MPRAs and CRISPR-Cas9-based screening, which have significantly contributed to understanding TF binding preferences and cis-regulatory element functions, are explored. Lastly, the potential of machine learning and artificial intelligence to unravel cis-regulatory logic is analyzed. These computational advances have far-reaching implications for precision medicine, therapeutic target discovery, and the study of genetic variations in health and disease.

ChIP-Atlas 3.0: a data-mining suite to explore chromosome architecture together with large-scale regulome data.

ChIP-Atlas (https://chip-atlas.org/) presents a suite of data-mining tools for analyzing epigenomic landscapes, powered by the comprehensive integration of over 376 000 public ChIP-seq, ATAC-seq, DNase-seq and Bisulfite-seq experiments from six representative model organisms. To unravel the intricacies of chromatin architecture that mediates the regulome-initiated generation of transcriptional and phenotypic diversity within cells, we report ChIP-Atlas 3.0 that enhances clarity by incorporating additional tracks for genomic and epigenomic features within a newly consolidated 'annotation track' section. The tracks include chromosomal conformation (Hi-C and eQTL datasets), transcriptional regulatory elements (ChromHMM and FANTOM5 enhancers), and genomic variants associated with diseases and phenotypes (GWAS SNPs and ClinVar variants). These annotation tracks are easily accessible alongside other experimental tracks, facilitating better elucidation of chromatin architecture underlying the diversification of transcriptional and phenotypic traits. Furthermore, 'Diff Analysis,' a new online tool, compares the query epigenome data to identify differentially bound, accessible, and methylated regions using ChIP-seq, ATAC-seq and DNase-seq, and Bisulfite-seq datasets, respectively. The integration of annotation tracks and the Diff Analysis tool, coupled with continuous data expansion, renders ChIP-Atlas 3.0 a robust resource for mining the landscape of transcriptional regulatory mechanisms, thereby offering valuable perspectives, particularly for genetic disease research and drug discovery.

Enhancer sequence variants and transcription-factor deregulation synergize to construct pathogenic regulatory circuits in B-cell lymphoma.

Most B-cell lymphomas arise in the germinal center (GC), where humoral immune responses evolve from potentially oncogenic cycles of mutation, proliferation, and clonal selection. Although lymphoma gene expression diverges significantly from GC B cells, underlying mechanisms that alter the activities of corresponding regulatory elements (REs) remain elusive. Here we define the complete pathogenic circuitry of human follicular lymphoma (FL), which activates or decommissions REs from normal GC B cells and commandeers enhancers from other lineages. Moreover, independent sets of transcription factors, whose expression was deregulated in FL, targeted commandeered versus decommissioned REs. Our approach revealed two distinct subtypes of low-grade FL, whose pathogenic circuitries resembled GC B or activated B cells. FL-altered enhancers also were enriched for sequence variants, including somatic mutations, which disrupt transcription-factor binding and expression of circuit-linked genes. Thus, the pathogenic regulatory circuitry of FL reveals distinct genetic and epigenetic etiologies for GC B-cell transformation.

Fruitless decommissions regulatory elements to implement cell-type-specific neuronal masculinization.

In the fruit fly Drosophila melanogaster, male-specific splicing and translation of the Fruitless transcription factor (FruM) alters the presence, anatomy, and/or connectivity of >60 types of central brain neurons that interconnect to generate male-typical behaviors. While the indispensable function of FruM in sex-specific behavior has been understood for decades, the molecular mechanisms underlying its activity remain unknown. Here, we take a genome-wide, brain-wide approach to identifying regulatory elements whose activity depends on the presence of FruM. We identify 436 high-confidence genomic regions differentially accessible in male fruitless neurons, validate candidate regions as bona fide, differentially regulated enhancers, and describe the particular cell types in which these enhancers are active. We find that individual enhancers are not activated universally but are dedicated to specific fru+ cell types. Aside from fru itself, genes are not dedicated to or common across the fru circuit; rather, FruM appears to masculinize each cell type differently, by tweaking expression of the same effector genes used in other circuits. Finally, we find FruM motifs enriched among regulatory elements that are open in the female but closed in the male. Together, these results suggest that FruM acts cell-type-specifically to decommission regulatory elements in male fruitless neurons.

A widespread sequence-specific mRNA decay pathway mediated by hnRNPs A1 and A2/B1.

3'-untranslated regions (UTRs) specify post-transcriptional fates of mammalian messenger RNAs (mRNAs), yet knowledge of the underlying sequences and mechanisms is largely incomplete. Here, we identify two related novel 3' UTR motifs in mammals that specify transcript degradation. These motifs are interchangeable and active only within 3' UTRs, where they are often preferentially conserved; furthermore, they are found in hundreds of transcripts, many encoding regulatory proteins. We found that degradation occurs via mRNA deadenylation, mediated by the CCR4-NOT complex. We purified trans factors that recognize the motifs and identified heterogeneous nuclear ribonucleoproteins (hnRNPs) A1 and A2/B1, which are required for transcript degradation, acting in a previously unknown manner. We used RNA sequencing (RNA-seq) to confirm hnRNP A1 and A2/B1 motif-dependent roles genome-wide, profiling cells depleted of these factors singly and in combination. Interestingly, the motifs are most active within the distal portion of 3' UTRs, suggesting that their role in gene regulation can be modulated by alternative processing, resulting in shorter 3' UTRs.

An ensemble of regulatory elements controls Runx3 spatiotemporal expression in subsets of dorsal root ganglia proprioceptive neurons.

The Runx3 transcription factor is essential for development and diversification of the dorsal root ganglia (DRGs) TrkC sensory neurons. In Runx3-deficient mice, developing TrkC neurons fail to extend central and peripheral afferents, leading to cell death and disruption of the stretch reflex circuit, resulting in severe limb ataxia. Despite its central role, the mechanisms underlying the spatiotemporal expression specificities of Runx3 in TrkC neurons were largely unknown. Here we first defined the genomic transcription unit encompassing regulatory elements (REs) that mediate the tissue-specific expression of Runx3. Using transgenic mice expressing BAC reporters spanning the Runx3 locus, we discovered three REs-dubbed R1, R2, and R3-that cross-talk with promoter-2 (P2) to drive TrkC neuron-specific Runx3 transcription. Deletion of single or multiple elements either in the BAC transgenics or by CRISPR/Cas9-mediated endogenous ablation established the REs' ability to promote and/or repress Runx3 expression in developing sensory neurons. Our analysis reveals that an intricate combinatorial interplay among the three REs governs Runx3 expression in distinct subtypes of TrkC neurons while concomitantly extinguishing its expression in non-TrkC neurons. These findings provide insights into the mechanism regulating cell type-specific expression and subtype diversification of TrkC neurons in developing DRGs.

Cell type- and stage-specific expression of Otx2 is regulated by multiple transcription factors and cis-regulatory modules in the retina.

Transcription factors (TFs) are often used repeatedly during development and homeostasis to control distinct processes in the same and/or different cellular contexts. Considering the limited number of TFs in the genome and the tremendous number of events that need to be regulated, re-use of TFs is necessary. We analyzed how the expression of the homeobox TF, orthodenticle homeobox 2 (Otx2), is regulated in a cell type- and stage-specific manner during development in the mouse retina. We identified seven Otx2 cis-regulatory modules (CRMs), among which the O5, O7 and O9 CRMs mark three distinct cellular contexts of Otx2 expression. We discovered that Otx2, Crx and Sox2, which are well-known TFs regulating retinal development, bind to and activate the O5, O7 or O9 CRMs, respectively. The chromatin status of these three CRMs was found to be distinct in vivo in different retinal cell types and at different stages. We conclude that retinal cells use a cohort of TFs with different expression patterns and multiple CRMs with different chromatin configurations to regulate the expression of Otx2 precisely.

Nuclear adaptor Ldb1 regulates a transcriptional program essential for the maintenance of hematopoietic stem cells.

The nuclear adaptor Ldb1 functions as a core component of multiprotein transcription complexes that regulate differentiation in diverse cell types. In the hematopoietic lineage, Ldb1 forms a complex with the non-DNA-binding adaptor Lmo2 and the transcription factors E2A, Scl and GATA-1 (or GATA-2). Here we demonstrate a critical and continuous requirement for Ldb1 in the maintenance of both fetal and adult mouse hematopoietic stem cells (HSCs). Deletion of Ldb1 in hematopoietic progenitors resulted in the downregulation of many transcripts required for HSC maintenance. Genome-wide profiling by chromatin immunoprecipitation followed by sequencing (ChIP-Seq) identified Ldb1 complex-binding sites at highly conserved regions in the promoters of genes involved in HSC maintenance. Our results identify a central role for Ldb1 in regulating the transcriptional program responsible for the maintenance of HSCs.

HRT Atlas v1.0 database: redefining human and mouse housekeeping genes and candidate reference transcripts by mining massive RNA-seq datasets.

Housekeeping (HK) genes are constitutively expressed genes that are required for the maintenance of basic cellular functions. Despite their importance in the calibration of gene expression, as well as the understanding of many genomic and evolutionary features, important discrepancies have been observed in studies that previously identified these genes. Here, we present Housekeeping and Reference Transcript Atlas (HRT Atlas v1.0, www.housekeeping.unicamp.br) a web-based database which addresses some of the previously observed limitations in the identification of these genes, and offers a more accurate database of human and mouse HK genes and transcripts. The database was generated by mining massive human and mouse RNA-seq data sets, including 11 281 and 507 high-quality RNA-seq samples from 52 human non-disease tissues/cells and 14 healthy tissues/cells of C57BL/6 wild type mouse, respectively. User can visualize the expression and download lists of 2158 human HK transcripts from 2176 HK genes and 3024 mouse HK transcripts from 3277 mouse HK genes. HRT Atlas also offers the most stable and suitable tissue selective candidate reference transcripts for normalization of qPCR experiments. Specific primers and predicted modifiers of gene expression for some of these HK transcripts are also proposed. HRT Atlas has also been integrated with a regulatory elements resource from Epiregio server.

TENET 2.0: Identification of key transcriptional regulators and enhancers in lung adenocarcinoma.

Lung cancer is the leading cause of cancer-related death and lung adenocarcinoma is its most common subtype. Although genetic alterations have been identified as drivers in subsets of lung adenocarcinoma, they do not fully explain tumor development. Epigenetic alterations have been implicated in the pathogenesis of tumors. To identify epigenetic alterations driving lung adenocarcinoma, we used an improved version of the Tracing Enhancer Networks using Epigenetic Traits method (TENET 2.0) in primary normal lung and lung adenocarcinoma cells. We found over 32,000 enhancers that appear differentially activated between normal lung and lung adenocarcinoma. Among the identified transcriptional regulators inactivated in lung adenocarcinoma vs. normal lung, NKX2-1 was linked to a large number of silenced enhancers. Among the activated transcriptional regulators identified, CENPA, FOXM1, and MYBL2 were linked to numerous cancer-specific enhancers. High expression of CENPA, FOXM1, and MYBL2 is particularly observed in a subgroup of lung adenocarcinomas and is associated with poor patient survival. Notably, CENPA, FOXM1, and MYBL2 are also key regulators of cancer-specific enhancers in breast adenocarcinoma of the basal subtype, but they are associated with distinct sets of activated enhancers. We identified individual lung adenocarcinoma enhancers linked to CENPA, FOXM1, or MYBL2 that were associated with poor patient survival. Knockdown experiments of FOXM1 and MYBL2 suggest that these factors regulate genes involved in controlling cell cycle progression and cell division. For example, we found that expression of TK1, a potential target gene of a MYBL2-linked enhancer, is associated with poor patient survival. Identification and characterization of key transcriptional regulators and associated enhancers in lung adenocarcinoma provides important insights into the deregulation of lung adenocarcinoma epigenomes, highlighting novel potential targets for clinical intervention.

Enhancer evolutionary co-option through shared chromatin accessibility input.

The diversity of forms in multicellular organisms originates largely from the spatial redeployment of developmental genes [S. B. Carroll, Cell 134, 25-36 (2008)]. Several scenarios can explain the emergence of cis-regulatory elements that govern novel aspects of a gene expression pattern [M. Rebeiz, M. Tsiantis, Curr. Opin. Genet. Dev. 45, 115-123 (2017)]. One scenario, enhancer co-option, holds that a DNA sequence producing an ancestral regulatory activity also becomes the template for a new regulatory activity, sharing regulatory information. While enhancer co-option might fuel morphological diversification, it has rarely been documented [W. J. Glassford et al., Dev. Cell 34, 520-531 (2015)]. Moreover, if two regulatory activities are borne from the same sequence, their modularity, considered a defining feature of enhancers [J. Banerji, L. Olson, W. Schaffner, Cell 33, 729-740 (1983)], might be affected by pleiotropy. Sequence overlap may thereby play a determinant role in enhancer function and evolution. Here, we investigated this problem with two regulatory activities of the Drosophila gene yellow, the novel spot enhancer and the ancestral wing blade enhancer. We used precise and comprehensive quantification of each activity in Drosophila wings to systematically map their sequences along the locus. We show that the spot enhancer has co-opted the sequences of the wing blade enhancer. We also identified a pleiotropic site necessary for DNA accessibility of a shared regulatory region. While the evolutionary steps leading to the derived activity are still unknown, such pleiotropy suggests that enhancer accessibility could be one of the molecular mechanisms seeding evolutionary co-option.

Chromatin signatures of cancer.

Changes in the pattern of gene expression play an important role in allowing cancer cells to acquire their hallmark characteristics, while genomic instability enables cells to acquire genetic alterations that promote oncogenesis. Chromatin plays central roles in both transcriptional regulation and the maintenance of genomic stability. Studies by cancer genome consortiums have identified frequent mutations in genes encoding chromatin regulatory factors and histone proteins in human cancer, implicating them as major mediators in the pathogenesis of both hematological malignancies and solid tumors. Here, we review recent advances in our understanding of the role of chromatin in cancer, focusing on transcriptional regulatory complexes, enhancer-associated factors, histone point mutations, and alterations in heterochromatin-interacting factors.

A map of cis-regulatory modules and constituent transcription factor binding sites in 80% of the mouse genome.

BACKGROUND: Mouse is probably the most important model organism to study mammal biology and human diseases. A better understanding of the mouse genome will help understand the human genome, biology and diseases. However, despite the recent progress, the characterization of the regulatory sequences in the mouse genome is still far from complete, limiting its use to understand the regulatory sequences in the human genome. RESULTS: Here, by integrating binding peaks in ~ 9,000 transcription factor (TF) ChIP-seq datasets that cover 79.9% of the mouse mappable genome using an efficient pipeline, we were able to partition these binding peak-covered genome regions into a cis-regulatory module (CRM) candidate (CRMC) set and a non-CRMC set. The CRMCs contain 912,197 putative CRMs and 38,554,729 TF binding sites (TFBSs) islands, covering 55.5% and 24.4% of the mappable genome, respectively. The CRMCs tend to be under strong evolutionary constraints, indicating that they are likely cis-regulatory; while the non-CRMCs are largely selectively neutral, indicating that they are unlikely cis-regulatory. Based on evolutionary profiles of the genome positions, we further estimated that 63.8% and 27.4% of the mouse genome might code for CRMs and TFBSs, respectively. CONCLUSIONS: Validation using experimental data suggests that at least most of the CRMCs are authentic. Thus, this unprecedentedly comprehensive map of CRMs and TFBSs can be a good resource to guide experimental studies of regulatory genomes in mice and humans.