ABSTRACT
Three-dimensional (3D) genome folding has a fundamental role in the regulation of developmental genes by facilitating or constraining chromatin interactions between cis-regulatory elements (CREs). Polycomb response elements (PREs) are a specific kind of CRE involved in the memory of transcriptional states in Drosophila melanogaster. PREs act as nucleation sites for Polycomb group (PcG) proteins, which deposit the repressive histone mark H3K27me3, leading to the formation of a class of topologically associating domain (TAD) called a Polycomb domain. PREs can establish looping contacts that stabilize the gene repression of key developmental genes during development. However, the mechanism by which PRE loops fine-tune gene expression is unknown. Using clustered regularly interspaced short palindromic repeats and Cas9 genome engineering, we specifically perturbed PRE contacts or enhancer function and used complementary approaches including 4C-seq, Hi-C and Hi-M to analyze how chromatin architecture perturbation affects gene expression. Our results suggest that the PRE loop at the dac gene locus acts as a constitutive 3D chromatin scaffold during Drosophila development that forms independently of gene expression states and has a versatile function; it restricts enhancer-promoter communication and contributes to enhancer specificity.
ABSTRACT
Cancer initiation and progression are typically associated with the accumulation of driver mutations and genomic instability. However, recent studies demonstrated that cancer can also be driven purely by epigenetic alterations, without driver mutations. Specifically, a 24-h transient downregulation of polyhomeotic (ph-KD), a core component of the Polycomb complex PRC1, is sufficient to induce epigenetically initiated cancers (EICs) in Drosophila, which are proficient in DNA repair and characterized by a stable genome. Whether genomic instability eventually occurs when PRC1 downregulation is performed for extended periods of time remains unclear. Here, we show that prolonged depletion of PH, which mimics cancer initiating events, results in broad dysregulation of DNA replication and repair genes, along with the accumulation of DNA breaks, defective repair, and widespread genomic instability in the cancer tissue. A broad misregulation of H2AK118 ubiquitylation and to a lesser extent of H3K27 trimethylation also occurs and might contribute to these phenotypes. Together, this study supports a model where DNA repair and replication defects accumulate during the tumorigenic transformation epigenetically induced by PRC1 loss, resulting in genomic instability and cancer progression.
Subject(s)
DNA Repair , Epigenesis, Genetic , Genomic Instability , Animals , Drosophila Proteins/metabolism , Drosophila Proteins/genetics , Polycomb Repressive Complex 1/metabolism , Polycomb Repressive Complex 1/genetics , Neoplasms/metabolism , Neoplasms/genetics , Neoplasms/pathology , Drosophila/metabolism , Drosophila melanogaster/metabolism , Polycomb-Group Proteins/metabolism , Polycomb-Group Proteins/geneticsABSTRACT
Polycomb (Pc) group proteins are transcriptional regulators with key roles in development, cell identity, and differentiation. Pc-bound chromatin regions form repressive domains that interact in 3D to assemble repressive nuclear compartments. Here, we use multiplexed chromatin imaging to investigate whether Pc compartments involve the clustering of multiple Pc domains during Drosophila development. Notably, 3D proximity between Pc targets is rare and involves predominantly pairwise interactions. These 3D proximities are particularly enhanced in segments where Pc genes are co-repressed. In addition, segment-specific expression of Hox Pc targets leads to their spatial segregation from Pc-repressed genes. Finally, non-Hox Pc targets are more proximal in regions where they are co-expressed. These results indicate that long-range Pc interactions are temporally and spatially regulated during differentiation and development but do not induce frequent clustering of multiple distant Pc genes.
Subject(s)
Chromatin , Drosophila Proteins , Polycomb-Group Proteins , Animals , Chromatin/metabolism , Polycomb-Group Proteins/metabolism , Polycomb-Group Proteins/genetics , Drosophila Proteins/metabolism , Drosophila Proteins/genetics , Drosophila melanogaster/genetics , Drosophila melanogaster/metabolism , Gene Expression Regulation, DevelopmentalABSTRACT
Despite significant progress in understanding epigenetic reprogramming of cells, the mechanistic basis of "organ reprogramming" by (epi-)gene-environment interactions remained largely obscure. Here, we use the ether-induced haltere-to-wing transformations in Drosophila as a model for epigenetic "reprogramming" at the whole organism level. Our findings support a mechanistic chain of events explaining why and how brief embryonic exposure to ether leads to haltere-to-wing transformations manifested at the larval stage and on. We show that ether interferes with protein integrity in the egg, leading to altered deployment of Hsp90 and widespread repression of Trithorax-mediated establishment of active H3K4me3 chromatin marks throughout the genome. Despite this global reduction, Ubx targets and wing development genes preferentially retain higher levels of H3K4me3 that predispose these genes for later up-regulation in the larval haltere disc, hence the wing-like outcome. Consistent with compromised protein integrity during the exposure, the penetrance of bithorax transformations increases by genetic or chemical reduction of Hsp90 function. Moreover, joint reduction in Hsp90 and trx gene dosage can cause bithorax transformations without exposure to ether, supporting an underlying epistasis between Hsp90 and trx loss-of-functions. These findings implicate environmental disruption of protein integrity at the onset of histone methylation with altered epigenetic regulation of developmental patterning genes. The emerging picture provides a unique example wherein the alleviation of the Hsp90 "capacitor function" by the environment drives a morphogenetic shift towards an ancestral-like body plan. The morphogenetic impact of chaperone response during a major setup of epigenetic patterns may be a general scheme for organ transformation by environmental cues.
Subject(s)
Drosophila Proteins , Drosophila melanogaster , Epigenesis, Genetic , HSP90 Heat-Shock Proteins , Histones , Wings, Animal , Animals , Drosophila Proteins/metabolism , Drosophila Proteins/genetics , Histones/metabolism , HSP90 Heat-Shock Proteins/metabolism , HSP90 Heat-Shock Proteins/genetics , Wings, Animal/metabolism , Wings, Animal/growth & development , Drosophila melanogaster/genetics , Drosophila melanogaster/metabolism , Larva/metabolism , Larva/genetics , Larva/growth & development , Gene Expression Regulation, Developmental , Gene-Environment Interaction , Chromosomal Proteins, Non-Histone/metabolism , Chromosomal Proteins, Non-Histone/genetics , Chromatin/metabolism , Homeodomain Proteins/metabolism , Homeodomain Proteins/genetics , Epigenetic Memory , Transcription FactorsABSTRACT
Polycomb-associated chromatin and pericentromeric heterochromatin form genomic domains important for the epigenetic regulation of gene expression. Both Polycomb complexes and heterochromatin factors rely on 'read and write' mechanisms, which, on their own, are not sufficient to explain the formation and the maintenance of these epigenetic domains. Microscopy has revealed that they form specific nuclear compartments separated from the rest of the genome. Recently, some subunits of these molecular machineries have been shown to undergo phase separation, both in vitro and in vivo, suggesting that phase separation might play important roles in the formation and the function of these two kinds of repressive chromatin. In this review, we will present the recent advances in the field of facultative and constitutive heterochromatin formation and maintenance through phase separation.
Subject(s)
Chromatin , Epigenesis, Genetic , Heterochromatin , Polycomb-Group Proteins , Heterochromatin/genetics , Heterochromatin/metabolism , Polycomb-Group Proteins/genetics , Polycomb-Group Proteins/metabolism , Chromatin/genetics , Chromatin/metabolism , Animals , Humans , Histones/genetics , Histones/metabolism , Chromatin Assembly and Disassembly/genetics , Phase SeparationABSTRACT
Enzymes of the ten-eleven translocation (TET) family play a key role in the regulation of gene expression by oxidizing 5-methylcytosine (5mC), a prominent epigenetic mark in many species. Yet, TET proteins also have less characterized noncanonical modes of action, notably in Drosophila, whose genome is devoid of 5mC. Here, we show that Drosophila TET activates the expression of genes required for larval central nervous system (CNS) development mainly in a catalytic-independent manner. Genome-wide profiling shows that TET is recruited to enhancer and promoter regions bound by Polycomb group complex (PcG) proteins. We found that TET interacts and colocalizes on chromatin preferentially with Polycomb repressor complex 1 (PRC1) rather than PRC2. Furthermore, PRC1 but not PRC2 is required for the activation of TET target genes. Last, our results suggest that TET and PRC1 binding to activated genes is interdependent. These data highlight the importance of TET noncatalytic function and the role of PRC1 for gene activation in the Drosophila larval CNS.
Subject(s)
Drosophila Proteins , Polycomb Repressive Complex 1 , Animals , Central Nervous System/metabolism , Chromatin/metabolism , Chromatin/genetics , Drosophila/metabolism , Drosophila/genetics , Drosophila melanogaster/genetics , Drosophila melanogaster/metabolism , Drosophila Proteins/metabolism , Drosophila Proteins/genetics , Gene Expression Regulation , Gene Expression Regulation, Developmental , Larva/metabolism , Larva/genetics , Polycomb Repressive Complex 1/metabolism , Polycomb Repressive Complex 1/genetics , Promoter Regions, Genetic , Protein BindingABSTRACT
Cancer initiation and progression are typically associated with the accumulation of driver mutations and genomic instability. However, recent studies demonstrated that cancers can also be purely initiated by epigenetic alterations, without driver mutations. Specifically, a 24-hours transient down-regulation of polyhomeotic (ph-KD), a core component of the Polycomb complex PRC1, is sufficient to drive epigenetically initiated cancers (EICs) in Drosophila, which are proficient in DNA repair and are characterized by a stable genome. Whether genomic instability eventually occurs when PRC1 down-regulation is performed for extended periods of time remains unclear. Here we show that prolonged depletion of a PRC1 component, which mimics cancer initiating events, results in broad dysregulation of DNA replication and repair genes, along with the accumulation of DNA breaks, defective repair, and widespread genomic instability in the cancer tissue. A broad mis-regulation of H2AK118 ubiquitylation and to a lesser extent of H3K27 trimethylation also occurs, and might contribute to these phenotypes. Together, this study supports a model where DNA repair and replication defects amplify the tumorigenic transformation epigenetically induced by PRC1 loss, resulting in genomic instability and cancer progression.
ABSTRACT
ABSTRACT: Plasma cells (PCs) are highly specialized cells representing the end stage of B-cell differentiation. We have shown that PC differentiation can be reproduced in vitro using elaborate culture systems. The molecular changes occurring during PC differentiation are recapitulated in this in vitro differentiation model. However, a major challenge exists to decipher the spatiotemporal epigenetic and transcriptional programs that drive the early stages of PC differentiation. We combined single cell (sc) RNA sequencing (RNA-seq) and assay for transposase-accessible chromatin with high throughput sequencing (scATAC-seq) to decipher the trajectories involved in PC differentiation. ScRNA-seq experiments revealed a strong heterogeneity of the preplasmablastic and plasmablastic stages. Among genes that were commonly identified using scATAC-seq and scRNA-seq, we identified several transcription factors with significant stage specific potential importance in PC differentiation. Interestingly, differentially accessible peaks characterizing the preplasmablastic stage were enriched in motifs of BATF3, FOS and BATF, belonging to activating protein 1 (AP-1) transcription factor family that may represent key transcriptional nodes involved in PC differentiation. Integration of transcriptomic and epigenetic data at the single cell level revealed that a population of preplasmablasts had already undergone epigenetic remodeling related to PC profile together with unfolded protein response activation and are committed to differentiate in PC. These results and the supporting data generated with our in vitro PC differentiation model provide a unique resource for the identification of molecular circuits that are crucial for early and mature PC maturation and biological functions. These data thus provide critical insights into epigenetic- and transcription-mediated reprogramming events that sustain PC differentiation.
Subject(s)
Cell Differentiation , Chromatin , Gene Expression Profiling , Plasma Cells , Single-Cell Analysis , Humans , Cell Differentiation/genetics , Plasma Cells/metabolism , Plasma Cells/cytology , Single-Cell Analysis/methods , Chromatin/metabolism , Chromatin/genetics , Transcriptome , Epigenesis, Genetic , Cells, CulturedABSTRACT
Evolutionary analyses have estimated that â¼60% of nucleotides in intergenic regions of the Drosophila melanogaster genome are functionally relevant, suggesting that regulatory information may be encoded more densely in intergenic regions than has been revealed by most functional dissections of regulatory DNA. Here, we approached this issue through a functional dissection of the regulatory region of the gene shavenbaby (svb). Most of the â¼90â kb of this large regulatory region is highly conserved in the genus Drosophila, though characterized enhancers occupy a small fraction of this region. By analyzing the regulation of svb in different contexts of Drosophila development, we found that the regulatory information that drives svb expression in the abdominal pupal epidermis is organized in a different way than the elements that drive svb expression in the embryonic epidermis. While in the embryonic epidermis svb is activated by compact enhancers separated by large inactive DNA regions, svb expression in the pupal epidermis is driven by regulatory information distributed over broader regions of svb cis-regulatory DNA. In the same vein, we observed that other developmental genes also display a dense distribution of putative regulatory elements in their regulatory regions. Furthermore, we found that a large percentage of conserved noncoding DNA of the Drosophila genome is contained within regions of open chromatin. These results suggest that part of the evolutionary constraint on noncoding DNA of Drosophila is explained by the density of regulatory information, which may be greater than previously appreciated.
Subject(s)
Drosophila Proteins , Drosophila , Animals , Drosophila/metabolism , Transcription Factors/metabolism , Drosophila melanogaster/genetics , Drosophila melanogaster/metabolism , DNA-Binding Proteins/genetics , Drosophila Proteins/genetics , Drosophila Proteins/metabolism , Gene Expression Regulation, Developmental , DNA , DNA, Intergenic/genetics , DNA, Intergenic/metabolism , Enhancer Elements, GeneticABSTRACT
Since the Modern Synthesis, our ideas of evolution have mostly centered on the information encoded in the DNA molecule and their mechanisms of heredity. Increasing evidence, however, suggests that epigenetic mechanisms have the potential to perpetuate gene activity states in the context of the same DNA sequence. Here, we discuss recent compelling evidence showing that epigenetic signals triggered by environmental stress can persist over very long timeframes, contributing to phenotypic changes in relevant traits upon which selection could act. We argue that epigenetic inheritance plays an important role in fast phenotypic adaptation to fluctuating environments, ensuring the survival of the organisms of a population under environmental stress in the short term while maintaining a "bet-hedging" strategy of reverting to the original state if the environment returns to standard conditions. These examples call for a reevaluation of the role of nongenetic information in adaptive evolution, raising questions about its broader relevance in nature.
Subject(s)
Biological Evolution , Heredity , Humans , Phenotype , Adaptation, Physiological/genetics , Epigenesis, GeneticABSTRACT
Drosophila insulators were the first DNA elements found to regulate gene expression by delimiting chromatin contacts. We still do not know how many of them exist and what impact they have on the Drosophila genome folding. Contrary to vertebrates, there is no evidence that fly insulators block cohesin-mediated chromatin loop extrusion. Therefore, their mechanism of action remains uncertain. To bridge these gaps, we mapped chromatin contacts in Drosophila cells lacking the key insulator proteins CTCF and Cp190. With this approach, we found hundreds of insulator elements. Their study indicates that Drosophila insulators play a minor role in the overall genome folding but affect chromatin contacts locally at many loci. Our observations argue that Cp190 promotes cobinding of other insulator proteins and that the model, where Drosophila insulators block chromatin contacts by forming loops, needs revision. Our insulator catalog provides an important resource to study mechanisms of genome folding.
Subject(s)
Drosophila Proteins , Drosophila , Animals , Drosophila/metabolism , Chromatin/genetics , Chromatin/metabolism , DNA-Binding Proteins/metabolism , Drosophila Proteins/genetics , Drosophila Proteins/metabolism , Insulator Elements/genetics , Nuclear Proteins/metabolism , Microtubule-Associated Proteins/metabolism , CCCTC-Binding Factor/genetics , CCCTC-Binding Factor/metabolismABSTRACT
The structural organization of the genome is emerging as a crucial regulator of the cell state, affecting gene transcription, DNA replication, and repair. Over the last twenty years, increasing evidence prompted the development of new experimental techniques to study genome structure. In parallel with the complexity of the novel techniques, computational approaches have become an essential tool in any structural genomics laboratory to analyze and model the data. For biologists to be able to apply the most appropriate modeling approach, it is fundamental to understand the conceptual bases of distinct methods and the insights they can provide. Here, we will discuss recent advances that were possible thanks to 3D genome modeling, discuss their limitations and highlight future perspectives.
Subject(s)
Genome , Genomics , Genome/genetics , Genomics/methods , DNA Replication , Chromatin/geneticsABSTRACT
Regulatory landscapes drive complex developmental gene expression, but it remains unclear how their integrity is maintained when incorporating novel genes and functions during evolution. Here, we investigated how a placental mammal-specific gene, Zfp42, emerged in an ancient vertebrate topologically associated domain (TAD) without adopting or disrupting the conserved expression of its gene, Fat1. In ESCs, physical TAD partitioning separates Zfp42 and Fat1 with distinct local enhancers that drive their independent expression. This separation is driven by chromatin activity and not CTCF/cohesin. In contrast, in embryonic limbs, inactive Zfp42 shares Fat1's intact TAD without responding to active Fat1 enhancers. However, neither Fat1 enhancer-incompatibility nor nuclear envelope-attachment account for Zfp42's unresponsiveness. Rather, Zfp42's promoter is rendered inert to enhancers by context-dependent DNA methylation. Thus, diverse mechanisms enabled the integration of independent Zfp42 regulation in the Fat1 locus. Critically, such regulatory complexity appears common in evolution as, genome wide, most TADs contain multiple independently expressed genes.
Subject(s)
Chromatin , Placenta , Animals , CCCTC-Binding Factor/metabolism , Chromatin Assembly and Disassembly , Enhancer Elements, Genetic , Evolution, Molecular , Female , Genome , Mammals/metabolism , Placenta/metabolism , Pregnancy , Promoter Regions, Genetic , Transcription Factors/genetics , Transcription Factors/metabolismABSTRACT
Gene duplications and transcriptional enhancer emergence/modifications are thought having greatly contributed to phenotypic innovations during animal evolution. Nevertheless, little is known about how enhancers evolve after gene duplication and how regulatory information is rewired between duplicated genes. The Drosophila melanogaster bric-a-brac (bab) complex, comprising the tandem paralogous genes bab1 and bab2, provides a paradigm to address these issues. We previously characterized an intergenic enhancer (named LAE) regulating bab2 expression in the developing legs. We show here that bab2 regulators binding directly the LAE also govern bab1 expression in tarsal cells. LAE excision by CRISPR/Cas9-mediated genome editing reveals that this enhancer appears involved but not strictly required for bab1 and bab2 co-expression in leg tissues. Instead, the LAE enhancer is critical for paralog-specific bab2 expression along the proximo-distal leg axis. Chromatin features and phenotypic rescue experiments indicate that LAE functions partly redundantly with leg-specific regulatory information overlapping the bab1 transcription unit. Phylogenomics analyses indicate that (i) the bab complex originates from duplication of an ancestral singleton gene early on within the Cyclorrhapha dipteran sublineage, and (ii) LAE sequences have been evolutionarily-fixed early on within the Brachycera suborder thus predating the gene duplication event. This work provides new insights on enhancers, particularly about their emergence, maintenance and functional diversification during evolution.
Subject(s)
Drosophila Proteins , Drosophila , Animals , DNA-Binding Proteins/genetics , DNA-Binding Proteins/metabolism , Drosophila/genetics , Drosophila Proteins/genetics , Drosophila Proteins/metabolism , Drosophila melanogaster/genetics , Drosophila melanogaster/metabolism , Enhancer Elements, Genetic , Transcription Factors/genetics , Transcription Factors/metabolismABSTRACT
Background: Human multiple myeloma (MM) cell lines (HMCLs) have been widely used to understand the molecular processes that drive MM biology. Epigenetic modifications are involved in MM development, progression, and drug resistance. A comprehensive characterization of the epigenetic landscape of MM would advance our understanding of MM pathophysiology and may attempt to identify new therapeutic targets. Methods: We performed chromatin immunoprecipitation sequencing to analyze histone mark changes (H3K4me1, H3K4me3, H3K9me3, H3K27ac, H3K27me3 and H3K36me3) on 16 HMCLs. Results: Differential analysis of histone modification profiles highlighted links between histone modifications and cytogenetic abnormalities or recurrent mutations. Using histone modifications associated to enhancer regions, we identified super-enhancers (SE) associated with genes involved in MM biology. We also identified promoters of genes enriched in H3K9me3 and H3K27me3 repressive marks associated to potential tumor suppressor functions. The prognostic value of genes associated with repressive domains and SE was used to build two distinct scores identifying high-risk MM patients in two independent cohorts (CoMMpass cohort; n = 674 and Montpellier cohort; n = 69). Finally, we explored H3K4me3 marks comparing drug-resistant and -sensitive HMCLs to identify regions involved in drug resistance. From these data, we developed epigenetic biomarkers based on the H3K4me3 modification predicting MM cell response to lenalidomide and histone deacetylase inhibitors (HDACi). Conclusions: The epigenetic landscape of MM cells represents a unique resource for future biological studies. Furthermore, risk-scores based on SE and repressive regions together with epigenetic biomarkers of drug response could represent new tools for precision medicine in MM.
Subject(s)
Histones , Multiple Myeloma , Epigenesis, Genetic/genetics , Epigenomics , Histone Code , Histones/genetics , Histones/metabolism , Humans , Multiple Myeloma/drug therapy , Multiple Myeloma/geneticsABSTRACT
BACKGROUND: With the development of HiC technology, more and more HiC sequencing data have been produced. Although there are dozens of packages that can turn sequencing data into contact maps, there is no appropriate tool to query contact maps in order to extract biological information from HiC datasets. RESULTS: We present HiCmapTools, a tool for biologists to efficiently calculate and analyze HiC maps. The complete program provides multi-query modes and analysis tools. We have validated its utility on two real biological questions: TAD loop and TAD intra-density. CONCLUSIONS: HiCmapTools supports seven access options so that biologists can quantify contact frequency of the interest sites. The tool has been implemented in C++ and R and is freely available at https://github.com/changlabtw/hicmaptools and documented at https://hicmaptools.readthedocs.io/ .
Subject(s)
Chromatin , GenomicsABSTRACT
Cancer arises from a multitude of disorders resulting in loss of differentiation and a stem cell-like phenotype characterized by uncontrolled growth. Polycomb Group (PcG) proteins are members of multiprotein complexes that are highly conserved throughout evolution. Historically, they have been described as essential for maintaining epigenetic cellular memory by locking homeotic genes in a transcriptionally repressed state. What was initially thought to be a function restricted to a few target genes, subsequently turned out to be of much broader relevance, since the main role of PcG complexes is to ensure a dynamically choregraphed spatio-temporal regulation of their numerous target genes during development. Their ability to modify chromatin landscapes and refine the expression of master genes controlling major switches in cellular decisions under physiological conditions is often misregulated in tumors. Surprisingly, their functional implication in the initiation and progression of cancer may be either dependent on Polycomb complexes, or specific for a subunit that acts independently of other PcG members. In this review, we describe how misregulated Polycomb proteins play a pleiotropic role in cancer by altering a broad spectrum of biological processes such as the proliferation-differentiation balance, metabolism and the immune response, all of which are crucial in tumor progression. We also illustrate how interfering with PcG functions can provide a powerful strategy to counter tumor progression.
Subject(s)
Drosophila Proteins , Neoplasms , Chromatin , Drosophila Proteins/genetics , Genes, Homeobox , Humans , Neoplasms/genetics , Polycomb Repressive Complex 1 , Polycomb-Group Proteins/genetics , Polycomb-Group Proteins/metabolismABSTRACT
Increasing evidence indicates that non-DNA sequence-based epigenetic information can be inherited across several generations in organisms ranging from yeast to plants to humans. This raises the possibility of heritable 'epimutations' contributing to heritable phenotypic variation and thus to evolution. Recent work has shed light on both the signals that underpin these epimutations, including DNA methylation, histone modifications and non-coding RNAs, and the mechanisms by which they are transmitted across generations at the molecular level. These mechanisms can vary greatly among species and have a more limited effect in mammals than in plants and other animal species. Nevertheless, common principles are emerging, with transmission occurring either via direct replicative mechanisms or indirect reconstruction of the signal in subsequent generations. As these processes become clearer we continue to improve our understanding of the distinctive features and relative contribution of DNA sequence and epigenetic variation to heritable differences in phenotype.