RESUMO
The association of nuclear DNA with histones to form chromatin is essential for temporal and spatial control of eukaryotic genomes. In this study, we examined the physical state of condensed chromatin in vitro and in vivo. Our in vitro studies demonstrate that self-association of nucleosomal arrays under a wide range of solution conditions produces supramolecular condensates in which the chromatin is physically constrained and solid-like. By measuring DNA mobility in living cells, we show that condensed chromatin also exhibits solid-like behavior in vivo. Representative heterochromatin proteins, however, display liquid-like behavior and coalesce around the solid chromatin scaffold. Importantly, euchromatin and heterochromatin show solid-like behavior even under conditions that produce limited interactions between chromatin fibers. Our results reveal that condensed chromatin exists in a solid-like state whose properties resist external forces and create an elastic gel and provides a scaffold that supports liquid-liquid phase separation of chromatin binding proteins.
Assuntos
Cromatina/metabolismo , Acetilação/efeitos dos fármacos , Animais , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Cromatina/efeitos dos fármacos , Dano ao DNA , Eucromatina/metabolismo , Fluorescência , Heterocromatina/metabolismo , Inibidores de Histona Desacetilases/farmacologia , Lasers , Camundongos , Modelos Biológicos , Concentração Osmolar , FotodegradaçãoRESUMO
To discover regulatory elements driving the specificity of gene expression in different cell types and regions of the developing human brain, we generated an atlas of open chromatin from nine dissected regions of the mid-gestation human telencephalon, as well as microdissected upper and deep layers of the prefrontal cortex. We identified a subset of open chromatin regions (OCRs), termed predicted regulatory elements (pREs), that are likely to function as developmental brain enhancers. pREs showed temporal, regional, and laminar differences in chromatin accessibility and were correlated with gene expression differences across regions and gestational ages. We identified two functional de novo variants in a pRE for autism risk gene SLC6A1, and using CRISPRa, demonstrated that this pRE regulates SCL6A1. Additionally, mouse transgenic experiments validated enhancer activity for pREs proximal to FEZF2 and BCL11A. Thus, this atlas serves as a resource for decoding neurodevelopmental gene regulation in health and disease.
Assuntos
Cromatina/genética , Cromatina/metabolismo , Elementos Facilitadores Genéticos , Regulação da Expressão Gênica no Desenvolvimento/genética , Córtex Pré-Frontal/embriologia , Telencéfalo/embriologia , Animais , Transtorno Autístico/genética , Linhagem Celular , Sequenciamento de Cromatina por Imunoprecipitação , Eucromatina/genética , Proteínas da Membrana Plasmática de Transporte de GABA/genética , Ontologia Genética , Predisposição Genética para Doença , Idade Gestacional , Humanos , Camundongos , Camundongos Transgênicos , Motivos de Nucleotídeos , Mutação Puntual , Córtex Pré-Frontal/metabolismo , Proteínas Repressoras/genética , Proteínas Repressoras/metabolismo , Análise Espaço-Temporal , Telencéfalo/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismoRESUMO
In order to provide a comprehensive resource for human structural variants (SVs), we generated long-read sequence data and analyzed SVs for fifteen human genomes. We sequence resolved 99,604 insertions, deletions, and inversions including 2,238 (1.6 Mbp) that are shared among all discovery genomes with an additional 13,053 (6.9 Mbp) present in the majority, indicating minor alleles or errors in the reference. Genotyping in 440 additional genomes confirms the most common SVs in unique euchromatin are now sequence resolved. We report a ninefold SV bias toward the last 5 Mbp of human chromosomes with nearly 55% of all VNTRs (variable number of tandem repeats) mapping to this portion of the genome. We identify SVs affecting coding and noncoding regulatory loci improving annotation and interpretation of functional variation. These data provide the framework to construct a canonical human reference and a resource for developing advanced representations capable of capturing allelic diversity.
Assuntos
Frequência do Gene/genética , Genoma Humano/genética , Variação Estrutural do Genoma/genética , Alelos , Eucromatina/genética , Genômica/métodos , Humanos , Repetições Minissatélites/genética , Análise de Sequência de DNA/métodosRESUMO
Whether and how histone post-translational modifications and the proteins that bind them drive 3D genome organization remains unanswered. Here, we evaluate the contribution of H3K9-methylated constitutive heterochromatin to 3D genome organization in Drosophila tissues. We find that the predominant organizational feature of wild-type tissues is the segregation of euchromatic chromosome arms from heterochromatic pericentromeres. Reciprocal perturbation of HP1aâ H3K9me binding, using a point mutation in the HP1a chromodomain or replacement of the replication-dependent histone H3 with H3K9R mutant histones, revealed that HP1a binding to methylated H3K9 in constitutive heterochromatin is required to limit contact frequency between pericentromeres and chromosome arms and regulate the distance between arm and pericentromeric regions. Surprisingly, the self-association of pericentromeric regions is largely preserved despite the loss of H3K9 methylation and HP1a occupancy. Thus, the HP1aâ H3K9 interaction contributes to but does not solely drive the segregation of euchromatin and heterochromatin inside the nucleus.
Assuntos
Homólogo 5 da Proteína Cromobox , Proteínas Cromossômicas não Histona , Proteínas de Drosophila , Drosophila melanogaster , Heterocromatina , Histonas , Heterocromatina/metabolismo , Heterocromatina/genética , Animais , Histonas/metabolismo , Histonas/genética , Proteínas Cromossômicas não Histona/metabolismo , Proteínas Cromossômicas não Histona/genética , Proteínas de Drosophila/metabolismo , Proteínas de Drosophila/genética , Drosophila melanogaster/genética , Drosophila melanogaster/metabolismo , Metilação , Eucromatina/metabolismo , Eucromatina/genética , Centrômero/metabolismo , Centrômero/genética , Ligação Proteica , Genoma de Inseto , Segregação de Cromossomos , Processamento de Proteína Pós-TraducionalRESUMO
RNA has been proposed to be a component of an underlying nuclear matrix. Hall et al. show that noncoding, repetitive RNAs, some derived from LINE1 elements, stably associate with interphase chromosomes and copurify with nuclear scaffold, indicating that RNAs might impact interphase chromosome architecture.
Assuntos
Cromossomos de Mamíferos/química , Eucromatina/química , Interfase , RNA não Traduzido/análise , Animais , HumanosRESUMO
Recent studies recognize a vast diversity of noncoding RNAs with largely unknown functions, but few have examined interspersed repeat sequences, which constitute almost half our genome. RNA hybridization in situ using C0T-1 (highly repeated) DNA probes detects surprisingly abundant euchromatin-associated RNA comprised predominantly of repeat sequences (C0T-1 RNA), including LINE-1. C0T-1-hybridizing RNA strictly localizes to the interphase chromosome territory in cis and remains stably associated with the chromosome territory following prolonged transcriptional inhibition. The C0T-1 RNA territory resists mechanical disruption and fractionates with the nonchromatin scaffold but can be experimentally released. Loss of repeat-rich, stable nuclear RNAs from euchromatin corresponds to aberrant chromatin distribution and condensation. C0T-1 RNA has several properties similar to XIST chromosomal RNA but is excluded from chromatin condensed by XIST. These findings impact two "black boxes" of genome science: the poorly understood diversity of noncoding RNA and the unexplained abundance of repetitive elements.
Assuntos
Cromossomos de Mamíferos/química , Eucromatina/química , Interfase , RNA não Traduzido/análise , Animais , Núcleo Celular/química , Humanos , Células Híbridas , Elementos Nucleotídeos Longos e Dispersos , Camundongos , RNA não Traduzido/genética , Sequências Repetitivas de Ácido Nucleico , Transcrição GênicaRESUMO
The prevalence of highly repetitive sequences within the human Y chromosome has prevented its complete assembly to date1 and led to its systematic omission from genomic analyses. Here we present de novo assemblies of 43 Y chromosomes spanning 182,900 years of human evolution and report considerable diversity in size and structure. Half of the male-specific euchromatic region is subject to large inversions with a greater than twofold higher recurrence rate compared with all other chromosomes2. Ampliconic sequences associated with these inversions show differing mutation rates that are sequence context dependent, and some ampliconic genes exhibit evidence for concerted evolution with the acquisition and purging of lineage-specific pseudogenes. The largest heterochromatic region in the human genome, Yq12, is composed of alternating repeat arrays that show extensive variation in the number, size and distribution, but retain a 1:1 copy-number ratio. Finally, our data suggest that the boundary between the recombining pseudoautosomal region 1 and the non-recombining portions of the X and Y chromosomes lies 500 kb away from the currently established1 boundary. The availability of fully sequence-resolved Y chromosomes from multiple individuals provides a unique opportunity for identifying new associations of traits with specific Y-chromosomal variants and garnering insights into the evolution and function of complex regions of the human genome.
Assuntos
Cromossomos Humanos Y , Evolução Molecular , Humanos , Masculino , Cromossomos Humanos Y/genética , Genoma Humano/genética , Genômica , Taxa de Mutação , Fenótipo , Eucromatina/genética , Pseudogenes , Variação Genética/genética , Cromossomos Humanos X/genética , Regiões Pseudoautossômicas/genéticaRESUMO
Human genomics is witnessing an ongoing paradigm shift from a single reference sequence to a pangenome form, but populations of Asian ancestry are underrepresented. Here we present data from the first phase of the Chinese Pangenome Consortium, including a collection of 116 high-quality and haplotype-phased de novo assemblies based on 58 core samples representing 36 minority Chinese ethnic groups. With an average 30.65× high-fidelity long-read sequence coverage, an average contiguity N50 of more than 35.63 megabases and an average total size of 3.01 gigabases, the CPC core assemblies add 189 million base pairs of euchromatic polymorphic sequences and 1,367 protein-coding gene duplications to GRCh38. We identified 15.9 million small variants and 78,072 structural variants, of which 5.9 million small variants and 34,223 structural variants were not reported in a recently released pangenome reference1. The Chinese Pangenome Consortium data demonstrate a remarkable increase in the discovery of novel and missing sequences when individuals are included from underrepresented minority ethnic groups. The missing reference sequences were enriched with archaic-derived alleles and genes that confer essential functions related to keratinization, response to ultraviolet radiation, DNA repair, immunological responses and lifespan, implying great potential for shedding new light on human evolution and recovering missing heritability in complex disease mapping.
Assuntos
População do Leste Asiático , Etnicidade , Variação Genética , Genoma Humano , Genética Humana , Grupos Minoritários , Humanos , População do Leste Asiático/classificação , População do Leste Asiático/genética , Etnicidade/genética , Genoma Humano/genética , Análise de Sequência de DNA , Raios Ultravioleta , Genética Humana/normas , Minorias Étnicas e Raciais , Padrões de Referência , Haplótipos/genética , Eucromatina/genética , Alelos , Reparo do DNA/genética , Queratinas/genética , Queratinas/metabolismo , Longevidade/genética , Imunidade/genéticaRESUMO
DNA double-strand break (DSB) repair is mediated by multiple pathways. It is thought that the local chromatin context affects the pathway choice, but the underlying principles are poorly understood. Using a multiplexed reporter assay in combination with Cas9 cutting, we systematically measure the relative activities of three DSB repair pathways as a function of chromatin context in >1,000 genomic locations. This reveals that non-homologous end-joining (NHEJ) is broadly biased toward euchromatin, while the contribution of microhomology-mediated end-joining (MMEJ) is higher in specific heterochromatin contexts. In H3K27me3-marked heterochromatin, inhibition of the H3K27 methyltransferase EZH2 reverts the balance toward NHEJ. Single-stranded template repair (SSTR), often used for precise CRISPR editing, competes with MMEJ and is moderately linked to chromatin context. These results provide insight into the impact of chromatin on DSB repair pathway balance and guidance for the design of Cas9-mediated genome editing experiments.
Assuntos
Proteína 9 Associada à CRISPR/metabolismo , Cromatina/metabolismo , Quebras de DNA de Cadeia Dupla , Reparo do DNA , Sequência de Bases , Reparo do DNA por Junção de Extremidades , Eucromatina/metabolismo , Rearranjo Gênico , Genoma Humano , Heterocromatina/metabolismo , Humanos , Mutação INDEL/genética , Células K562 , Cinética , Ligação Proteica , Reprodutibilidade dos TestesRESUMO
Nuclear chromosomes transcribe far more RNA than required to encode protein. Here we investigate whether non-coding RNA broadly contributes to cytological-scale chromosome territory architecture. We develop a procedure that depletes soluble proteins, chromatin, and most nuclear RNA from the nucleus but does not delocalize XIST, a known architectural RNA, from an insoluble chromosome "scaffold." RNA-seq analysis reveals that most RNA in the nuclear scaffold is repeat-rich, non-coding, and derived predominantly from introns of nascent transcripts. Insoluble, repeat-rich (C0T-1) RNA co-distributes with known scaffold proteins including scaffold attachment factor A (SAF-A), and distribution of these components inversely correlates with chromatin compaction in normal and experimentally manipulated nuclei. We further show that RNA is required for SAF-A to interact with chromatin and for enrichment of structurally embedded "scaffold attachment regions" prevalent in euchromatin. Collectively, the results indicate that long nascent transcripts contribute a dynamic structural role that promotes the open architecture of active chromosome territories.
Assuntos
Cromatina/metabolismo , Matriz Nuclear/metabolismo , RNA não Traduzido/metabolismo , Animais , Linhagem Celular , Núcleo Celular/fisiologia , Cromatina/genética , Cromossomos/genética , Cromossomos/metabolismo , Eucromatina/metabolismo , Humanos , Camundongos , Matriz Nuclear/genética , RNA/genética , RNA/metabolismo , RNA Longo não Codificante/genética , RNA não Traduzido/genética , Transcrição Gênica/genéticaRESUMO
The formation of silenced and condensed heterochromatin foci involves enrichment of heterochromatin protein 1 (HP1). HP1 can bridge chromatin segments and form liquid droplets, but the biophysical principles underlying heterochromatin compartmentalization in the cell nucleus are elusive. Here, we assess mechanistically relevant features of pericentric heterochromatin compaction in mouse fibroblasts. We find that (1) HP1 has only a weak capacity to form liquid droplets in living cells; (2) the size, global accessibility, and compaction of heterochromatin foci are independent of HP1; (3) heterochromatin foci lack a separated liquid HP1 pool; and (4) heterochromatin compaction can toggle between two "digital" states depending on the presence of a strong transcriptional activator. These findings indicate that heterochromatin foci resemble collapsed polymer globules that are percolated with the same nucleoplasmic liquid as the surrounding euchromatin, which has implications for our understanding of chromatin compartmentalization and its functional consequences.
Assuntos
Cromatina/genética , Proteínas Cromossômicas não Histona/genética , Eucromatina/genética , Heterocromatina/genética , Animais , Homólogo 5 da Proteína Cromobox , Fibroblastos , CamundongosRESUMO
Spatially and functionally distinct domains of heterochromatin and euchromatin play important roles in the maintenance of chromosome stability and regulation of gene expression, but a comprehensive knowledge of their composition is lacking. Here, we develop a strategy for the isolation of native Schizosaccharomyces pombe heterochromatin and euchromatin fragments and analyze their composition by using quantitative mass spectrometry. The shared and euchromatin-specific proteomes contain proteins involved in DNA and chromatin metabolism and in transcription, respectively. The heterochromatin-specific proteome includes all proteins with known roles in heterochromatin formation and, in addition, is enriched for subsets of nucleoporins and inner nuclear membrane (INM) proteins, which associate with different chromatin domains. While the INM proteins are required for the integrity of the nucleolus, containing ribosomal DNA repeats, the nucleoporins are required for aggregation of heterochromatic foci and epigenetic inheritance. The results provide a comprehensive picture of heterochromatin-associated proteins and suggest a role for specific nucleoporins in heterochromatin function.
Assuntos
Núcleo Celular/metabolismo , Montagem e Desmontagem da Cromatina/fisiologia , Cromatina/metabolismo , Heterocromatina/metabolismo , DNA Ribossômico/metabolismo , Epigênese Genética/fisiologia , Eucromatina/metabolismo , Complexo de Proteínas Formadoras de Poros Nucleares/metabolismo , Proteômica/métodos , Schizosaccharomyces/metabolismo , Proteínas de Schizosaccharomyces pombe/metabolismo , Transcrição Gênica/fisiologiaRESUMO
Сhromatin is critical for genome compaction and gene expression. On a coarse scale, the genome is divided into euchromatin, which harbors the majority of genes and is enriched in active chromatin marks, and heterochromatin, which is gene-poor but repeat-rich. The conserved molecular hallmark of heterochromatin is the H3K9me3 modification, which is associated with gene silencing. We found that in Drosophila, deposition of most of the H3K9me3 mark depends on SUMO and the SUMO ligase Su(var)2-10, which recruits the histone methyltransferase complex SetDB1/Wde. In addition to repressing repeats, H3K9me3 influences expression of both hetero- and euchromatic host genes. High H3K9me3 levels in heterochromatin are required to suppress spurious transcription and ensure proper gene expression. In euchromatin, a set of conserved genes is repressed by Su(var)2-10/SetDB1-induced H3K9 trimethylation, ensuring tissue-specific gene expression. Several components of heterochromatin are themselves repressed by this pathway, providing a negative feedback mechanism to ensure chromatin homeostasis.
Assuntos
Proteínas de Drosophila/metabolismo , Regulação da Expressão Gênica/genética , Proteínas Inibidoras de STAT Ativados/metabolismo , Proteínas Modificadoras Pequenas Relacionadas à Ubiquitina/metabolismo , Animais , Proteínas Cromossômicas não Histona/metabolismo , Metilação de DNA/genética , Proteínas de Drosophila/genética , Drosophila melanogaster/genética , Eucromatina/metabolismo , Retroalimentação Fisiológica , Expressão Gênica/genética , Inativação Gênica/fisiologia , Heterocromatina/genética , Heterocromatina/metabolismo , Histona-Lisina N-Metiltransferase/genética , Histona-Lisina N-Metiltransferase/metabolismo , Histonas/genética , Histonas/metabolismo , Ligases/genética , Metiltransferases/genética , Proteínas Inibidoras de STAT Ativados/genética , Proteínas Repressoras/metabolismo , Proteínas Modificadoras Pequenas Relacionadas à Ubiquitina/genéticaRESUMO
H3K9me3-dependent heterochromatin is critical for the silencing of repeat-rich pericentromeric regions and also has key roles in repressing lineage-inappropriate protein-coding genes in differentiation and development. Here, we investigate the molecular consequences of heterochromatin loss in cells deficient in both SUV39H1 and SUV39H2 (Suv39DKO), the major mammalian histone methyltransferase enzymes that catalyze heterochromatic H3K9me3 deposition. We reveal a paradoxical repression of protein-coding genes in Suv39DKO cells, with these differentially expressed genes principally in euchromatic (Tn5-accessible, H3K4me3- and H3K27ac-marked) rather than heterochromatic (H3K9me3-marked) or polycomb (H3K27me3-marked) regions. Examination of the three-dimensional (3D) nucleome reveals that transcriptomic dysregulation occurs in euchromatic regions close to the nuclear periphery in 3D space. Moreover, this transcriptomic dysregulation is highly correlated with altered 3D genome organization in Suv39DKO cells. Together, our results suggest that the nuclear lamina-tethering of Suv39-dependent H3K9me3 domains provides an essential scaffold to support euchromatic genome organization and the maintenance of gene transcription for healthy cellular function.
Assuntos
Eucromatina , Heterocromatina , Histona-Lisina N-Metiltransferase , Histonas , Metiltransferases , Transcrição Gênica , Animais , Camundongos , Linhagem Celular , Eucromatina/metabolismo , Eucromatina/genética , Regulação da Expressão Gênica , Heterocromatina/metabolismo , Heterocromatina/genética , Histona-Lisina N-Metiltransferase/metabolismo , Histona-Lisina N-Metiltransferase/genética , Histonas/metabolismo , Histonas/genética , Metiltransferases/metabolismo , Metiltransferases/genética , Proteínas Repressoras/metabolismo , Proteínas Repressoras/genéticaRESUMO
Euchromatin is an accessible phase of genetic material containing genes that encode proteins with increased expression levels. The structure of euchromatin in vitro has been described as a 30-nm fiber formed from ordered nucleosome arrays. However, recent advances in microscopy have revealed an in vivo euchromatin architecture that is much more disordered, characterized by variable-length linker DNA and sporadic nucleosome clusters. In this work, we develop a theoretical model to elucidate factors contributing to the disordered in vivo architecture of euchromatin. We begin by developing a 1D model of nucleosome positioning that captures the interactions between bound epigenetic reader proteins to predict the distribution of DNA linker lengths between adjacent nucleosomes. We then use the predicted linker lengths to construct 3D chromatin configurations consistent with the physical properties of DNA within the nucleosome array, and we evaluate the distribution of nucleosome cluster sizes in those configurations. Our model reproduces experimental cluster-size distributions, which are dramatically influenced by the local pattern of epigenetic marks and the concentration of reader proteins. Based on our model, we attribute the disordered arrangement of euchromatin to the heterogeneous binding of reader proteins and subsequent short-range interactions between bound reader proteins on adjacent nucleosomes. By replicating experimental results with our physics-based model, we propose a mechanism for euchromatin organization in the nucleus that impacts gene regulation and the maintenance of epigenetic marks.
Assuntos
Epigênese Genética , Eucromatina , Nucleossomos , Nucleossomos/metabolismo , Nucleossomos/genética , Eucromatina/metabolismo , Eucromatina/genética , DNA/metabolismo , DNA/químicaRESUMO
Although mutation rates have been extensively studied, variation in mutation rates throughout the genome is poorly understood. To understand patterns of genetic variation, it is important to understand how mutation rates vary. Chromatin modifications may be an important factor in determining variation in mutation rates in eukaryotic genomes. To study variation in mutation rates, we performed a mutation accumulation (MA) experiment in the filamentous fungus Neurospora crassa and sequenced the genomes of the 40 MA lines that had been propagated asexually for approximately 1015 [Formula: see text] mitoses. We detected 1322 mutations in total and observed that the mutation rate was higher in regions of low GC, in domains of H3K9 trimethylation, in centromeric regions, and in domains of H3K27 trimethylation. The rate of single-nucleotide mutations in euchromatin was [Formula: see text] In contrast, the mutation rate in H3K9me3 domains was 10-fold higher: 2.43 [Formula: see text] We also observed that the spectrum of single-nucleotide mutations was different between H3K9me3 and euchromatic domains. Our statistical model of mutation rate variation predicted a moderate amount of extant genetic variation, suggesting that the mutation rate is an important factor in determining levels of natural genetic variation. Furthermore, we characterized mutation rates of structural variants, complex mutations, and the effect of local sequence context on the mutation rate. Our study highlights that chromatin modifications are associated with mutation rates, and accurate evolutionary inferences should take variation in mutation rates across the genome into account.
Assuntos
Neurospora crassa , Neurospora crassa/genética , Mutagênese , Mutação , Taxa de Mutação , Eucromatina , NucleotídeosRESUMO
Atf1, the fission yeast homolog of activation transcription factor-2 (ATF-2), contributes to heterochromatin formation. However, the role of ATF-2 in chromatin assembly in higher organisms remains unknown. This study reveals that Drosophila ATF-2 (dATF-2) is required for heterochromatin assembly, whereas the stress-induced phosphorylation of dATF-2, via Mekk1-p38, disrupts heterochromatin. The dATF-2 protein colocalized with HP1, not only on heterochromatin but also at specific loci in euchromatin. Heat shock or osmotic stress induced phosphorylation of dATF-2 and resulted in its release from heterochromatin. This heterochromatic disruption was an epigenetic event that was transmitted to the next generation in a non-Mendelian fashion. When embryos were exposed to heat stress over multiple generations, the defective chromatin state was maintained over multiple successive generations, though it gradually returned to the normal state. The results suggest a mechanism by which the effects of stress are inherited epigenetically via the regulation of a tight chromatin structure.
Assuntos
Fator 2 Ativador da Transcrição/metabolismo , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/crescimento & desenvolvimento , Drosophila melanogaster/genética , Heterocromatina/metabolismo , Fator 2 Ativador da Transcrição/análise , Fator 2 Ativador da Transcrição/genética , Animais , Proteínas de Drosophila/análise , Proteínas de Drosophila/genética , Drosophila melanogaster/metabolismo , Epigenômica , Eucromatina/metabolismo , Feminino , Heterocromatina/química , MAP Quinase Quinase Quinase 1/metabolismo , Masculino , Mutagênese , Fosforilação , Transdução de Sinais , Estresse Fisiológico , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismoRESUMO
The recent discovery of N6-methyladenine (N6-mA) in mammalian genomes suggests that it may serve as an epigenetic regulatory mechanism1. However, the biological role of N6-mA and the molecular pathways that exert its function remain unclear. Here we show that N6-mA has a key role in changing the epigenetic landscape during cell fate transitions in early development. We found that N6-mA is upregulated during the development of mouse trophoblast stem cells, specifically at regions of stress-induced DNA double helix destabilization (SIDD)2-4. Regions of SIDD are conducive to topological stress-induced unpairing of the double helix and have critical roles in organizing large-scale chromatin structures3,5,6. We show that the presence of N6-mA reduces the in vitro interactions by more than 500-fold between SIDD and SATB1, a crucial chromatin organizer that interacts with SIDD regions. Deposition of N6-mA also antagonizes SATB1 function in vivo by preventing its binding to chromatin. Concordantly, N6-mA functions at the boundaries between euchromatin and heterochromatin to restrict the spread of euchromatin. Repression of SIDD-SATB1 interactions mediated by N6-mA is essential for gene regulation during trophoblast development in cell culture models and in vivo. Overall, our findings demonstrate an unexpected molecular mechanism for N6-mA function via SATB1, and reveal connections between DNA modification, DNA secondary structures and large chromatin domains in early embryonic development.
Assuntos
Adenina/análogos & derivados , DNA/química , DNA/metabolismo , Desenvolvimento Embrionário , Proteínas de Ligação à Região de Interação com a Matriz/antagonistas & inibidores , Adenina/metabolismo , Animais , Pareamento de Bases , Desenvolvimento Embrionário/genética , Eucromatina/genética , Eucromatina/metabolismo , Feminino , Humanos , Masculino , Proteínas de Ligação à Região de Interação com a Matriz/genética , Proteínas de Ligação à Região de Interação com a Matriz/metabolismo , Camundongos , Células-Tronco/citologia , Células-Tronco/metabolismo , Termodinâmica , Trofoblastos/citologiaRESUMO
Chromatin three-dimensional (3D) organization inside the cell nucleus determines the separation of euchromatin and heterochromatin domains. Their segregation results in the definition of active and inactive chromatin compartments, whereby the local concentration of associated proteins, RNA and DNA results in the formation of distinct subnuclear structures. Thus, chromatin domains spatially confined in a specific 3D nuclear compartment are expected to share similar epigenetic features and biochemical properties, in terms of accessibility and solubility. Based on this rationale, we developed the 4f-SAMMY-seq to map euchromatin and heterochromatin based on their accessibility and solubility, starting from as little as 10 000 cells. Adopting a tailored bioinformatic data analysis approach we reconstruct also their 3D segregation in active and inactive chromatin compartments and sub-compartments, thus recapitulating the characteristic properties of distinct chromatin states. A key novelty of the new method is the capability to map both the linear segmentation of open and closed chromatin domains, as well as their compartmentalization in one single experiment.
Assuntos
Eucromatina , Heterocromatina , Heterocromatina/química , Heterocromatina/metabolismo , Eucromatina/química , Eucromatina/metabolismo , Eucromatina/genética , Humanos , Cromatina/química , Cromatina/metabolismo , Cromatina/genética , Núcleo Celular/genética , Núcleo Celular/química , Núcleo Celular/metabolismo , DNA/química , DNA/metabolismo , AnimaisRESUMO
In Drosophila, a group of zinc finger architectural proteins recruits the CP190 protein to the chromatin, an interaction that is essential for the functional activity of promoters and insulators. In this study, we describe a new architectural C2H2 protein called Madf and Zinc-Finger Protein 1 (Mzfp1) that interacts with CP190. Mzfp1 has an unusual structure that includes six C2H2 domains organized in a C-terminal cluster and two tandem MADF domains. Mzfp1 predominantly binds to housekeeping gene promoters located in both euchromatin and heterochromatin genome regions. In vivo mutagenesis studies showed that Mzfp1 is an essential protein, and both MADF domains and the CP190 interaction region are required for its functional activity. The C2H2 cluster is sufficient for the specific binding of Mzfp1 to regulatory elements, while the second MADF domain is required for Mzfp1 recruitment to heterochromatin. Mzfp1 binds to the proximal part of the Fub boundary that separates regulatory domains of the Ubx and abd-A genes in the Bithorax complex. Mzfp1 participates in Fub functions in cooperation with the architectural proteins Pita and Su(Hw). Thus, Mzfp1 is a new architectural C2H2 protein involved in the organization of active promoters and insulators in Drosophila.