RESUMO
Chromatin organization plays a crucial role in gene regulation by controlling the accessibility of DNA to transcription machinery. While significant progress has been made in understanding the regulatory role of clock proteins in circadian rhythms, how chromatin organization affects circadian rhythms remains poorly understood. Here, we employed ATAC-seq (Assay for Transposase-Accessible Chromatin with Sequencing) on FAC-sorted Drosophila clock neurons to assess genome-wide chromatin accessibility at dawn and dusk over the circadian cycle. We observed significant oscillations in chromatin accessibility at promoter and enhancer regions of hundreds of genes, with enhanced accessibility either at dusk or dawn, which correlated with their peak transcriptional activity. Notably, genes with enhanced accessibility at dusk were enriched with E-box motifs, while those more accessible at dawn were enriched with VRI/PDP1-box motifs, indicating that they are regulated by the core circadian feedback loops, PER/CLK and VRI/PDP1, respectively. Further, we observed a complete loss of chromatin accessibility rhythms in per01 null mutants, with chromatin consistently accessible at both dawn and dusk, underscoring the critical role of Period protein in driving chromatin compaction during the repression phase at dawn. Together, this study demonstrates the significant role of chromatin organization in circadian regulation, revealing how the interplay between clock proteins and chromatin structure orchestrates the precise timing of biological processes throughout the day. This work further implies that variations in chromatin accessibility might play a central role in the generation of diverse circadian gene expression patterns in clock neurons.
Assuntos
Cromatina , Ritmo Circadiano , Proteínas de Drosophila , Drosophila melanogaster , Animais , Cromatina/genética , Cromatina/metabolismo , Ritmo Circadiano/genética , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/genética , Regulação da Expressão Gênica , Transcrição Gênica , Proteínas CLOCK/genética , Proteínas CLOCK/metabolismo , Neurônios/metabolismo , Neurônios/fisiologia , Regiões Promotoras Genéticas , Proteínas Circadianas Period/genética , Proteínas Circadianas Period/metabolismo , Relógios Circadianos/genética , Drosophila/genética , Elementos Facilitadores Genéticos , Fatores de Transcrição de Zíper de Leucina BásicaRESUMO
BACKGROUND: The radiation of mammals at the extinction of the dinosaurs produced a plethora of new forms-as diverse as bats, dolphins, and elephants-in only 10-20 million years. Behind the scenes, adaptation to new niches is accompanied by extensive innovation in large families of genes that allow animals to contact the environment, including chemosensors, xenobiotic enzymes, and immune and barrier proteins. Genes in these "outward-looking" families are allelically diverse among humans and exhibit tissue-specific and sometimes stochastic expression. RESULTS: Here, we show that these tandem arrays of outward-looking genes occupy AT-biased isochores and comprise the "tissue-specific" gene class that lack CpG islands in their promoters. Models of mammalian genome evolution have not incorporated the sharply different functions and transcriptional patterns of genes in AT- versus GC-biased regions. To examine the relationship between gene family expansion, sequence content, and allelic diversity, we use population genetic data and comparative analysis. First, we find that AT bias can emerge during evolutionary expansion of gene families in cis. Second, human genes in AT-biased isochores or with GC-poor promoters experience relatively low rates of de novo point mutation today but are enriched for non-synonymous variants. Finally, we find that isochores containing gene clusters exhibit low rates of recombination. CONCLUSIONS: Our analyses suggest that tolerance of non-synonymous variation and low recombination are two forces that have produced the depletion of GC bases in outward-facing gene arrays. In turn, high AT content exerts a profound effect on their chromatin organization and transcriptional regulation.
Assuntos
Quirópteros , Isocoros , Animais , Humanos , Mamíferos/genética , Quirópteros/genética , Aclimatação , AlelosRESUMO
Chromatin organization plays a crucial role in gene regulation by controlling the accessibility of DNA to transcription machinery. While significant progress has been made in understanding the regulatory role of clock proteins in circadian rhythms, how chromatin organization affects circadian rhythms remains poorly understood. Here, we employed ATAC-seq (Assay for Transposase-Accessible Chromatin with Sequencing) on FAC-sorted Drosophila clock neurons to assess genome-wide chromatin accessibility over the circadian cycle. We observed significant circadian oscillations in chromatin accessibility at promoter and enhancer regions of hundreds of genes, with enhanced accessibility either at dusk or dawn, which correlated with their peak transcriptional activity. Notably, genes with enhanced accessibility at dusk were enriched with E-box motifs, while those more accessible at dawn were enriched with VRI/PDP1-box motifs, indicating that they are regulated by the core circadian feedback loops, PER/CLK and VRI/PDP1, respectively. Further, we observed a complete loss of chromatin accessibility rhythms in per01 null mutants, with chromatin consistently accessible throughout the circadian cycle, underscoring the critical role of Period protein in driving chromatin compaction during the repression phase. Together, this study demonstrates the significant role of chromatin organization in circadian regulation, revealing how the interplay between clock proteins and chromatin structure orchestrates the precise timing of biological processes throughout the day. This work further implies that variations in chromatin accessibility might play a central role in the generation of diverse circadian gene expression patterns in clock neurons.
RESUMO
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.
Assuntos
Encéfalo/metabolismo , Proteínas de Drosophila/genética , Drosophila melanogaster/genética , Regulação da Expressão Gênica , Proteínas do Tecido Nervoso/genética , Neurônios/metabolismo , Elementos Reguladores de Transcrição/genética , Fatores de Transcrição/genética , Animais , Animais Geneticamente Modificados , Encéfalo/citologia , Mecanismo Genético de Compensação de Dose , Feminino , Estudo de Associação Genômica Ampla/métodos , Genômica/métodos , Masculino , Neurônios/citologia , RNA-Seq/métodos , Fatores SexuaisRESUMO
The intermediate filament protein keratin 14 (K14) provides vital structural support in basal keratinocytes of epidermis. Recent studies evidenced a role for K14-dependent disulfide bonding in the organization and dynamics of keratin IFs in skin keratinocytes. Here we report that knock-in mice harboring a cysteine-to-alanine substitution at Krt14's codon 373 (C373A) exhibit alterations in disulfide-bonded K14 species and a barrier defect secondary to enhanced proliferation, faster transit time and altered differentiation in epidermis. A proteomics screen identified 14-3-3 as K14 interacting proteins. Follow-up studies showed that YAP1, a transcriptional effector of Hippo signaling regulated by 14-3-3sigma in skin keratinocytes, shows aberrant subcellular partitioning and function in differentiating Krt14 C373A keratinocytes. Residue C373 in K14, which is conserved in a subset of keratins, is revealed as a novel regulator of keratin organization and YAP function in early differentiating keratinocytes, with an impact on cell mechanics, homeostasis and barrier function in epidermis.
Assuntos
Proteínas 14-3-3/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Proteínas de Ciclo Celular/metabolismo , Epiderme/metabolismo , Queratina-14/metabolismo , Proteínas 14-3-3/fisiologia , Proteínas Adaptadoras de Transdução de Sinal/fisiologia , Animais , Proteínas de Ciclo Celular/fisiologia , Epiderme/fisiologia , Epiderme/ultraestrutura , Feminino , Técnicas de Introdução de Genes , Homeostase , Queratina-14/fisiologia , Queratinócitos/metabolismo , Queratinócitos/fisiologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Microscopia Eletrônica de Transmissão , Proteínas de Sinalização YAPRESUMO
Chromatin insulators are DNA-protein complexes that regulate chromatin structure and gene expression in a wide range of organisms. These complexes also harbor enhancer blocking and barrier activities. Increasing evidence suggests that RNA molecules are integral components of insulator complexes. However, how these RNA molecules are involved in insulator function remains unclear. The Drosophila RNA-binding protein Shep associates with the gypsy insulator complex and inhibits insulator activities. By mutating key residues in the RRM domains, we generated a Shep mutant protein incapable of RNA-binding, and this mutant lost the ability to inhibit barrier activity. In addition, we found that one of many wildtype Shep isoforms but not RRM mutant Shep was sufficient to repress enhancer blocking activities. Finally, wildtype Shep rescued synthetic lethality of shep, mod(mdg4) double-mutants and developmental defects of shep mutant neurons, whereas mutant Shep failed to do so. These results indicate that the RNA-binding ability of Shep is essential for its ability to antagonize insulator activities and promote neuronal maturation. Our findings suggest that regulation of insulator function by RNA-binding proteins relies on RNA-mediated interactions.