RESUMO
Biomolecules incur damage during stress conditions, and damage partitioning represents a vital survival strategy for cells. Here, we identified a distinct stress granule (SG), marked by dsRNA helicase DHX9, which compartmentalizes ultraviolet (UV)-induced RNA, but not DNA, damage. Our FANCI technology revealed that DHX9 SGs are enriched in damaged intron RNA, in contrast to classical SGs that are composed of mature mRNA. UV exposure causes RNA crosslinking damage, impedes intron splicing and decay, and triggers DHX9 SGs within daughter cells. DHX9 SGs promote cell survival and induce dsRNA-related immune response and translation shutdown, differentiating them from classical SGs that assemble downstream of translation arrest. DHX9 modulates dsRNA abundance in the DHX9 SGs and promotes cell viability. Autophagy receptor p62 is activated and important for DHX9 SG disassembly. Our findings establish non-canonical DHX9 SGs as a dedicated non-membrane-bound cytoplasmic compartment that safeguards daughter cells from parental RNA damage.
Assuntos
RNA , Grânulos de Estresse , Citoplasma , RNA Mensageiro/genética , Estresse Fisiológico , Humanos , Células HeLaRESUMO
In diploid organisms, biallelic gene expression enables the production of adequate levels of mRNA1,2. This is essential for haploinsufficient genes, which require biallelic expression for optimal function to prevent the onset of developmental disorders1,3. Whether and how a biallelic or monoallelic state is determined in a cell-type-specific manner at individual loci remains unclear. MSL2 is known for dosage compensation of the male X chromosome in flies. Here we identify a role of MSL2 in regulating allelic expression in mammals. Allele-specific bulk and single-cell analyses in mouse neural progenitor cells revealed that, in addition to the targets showing biallelic downregulation, a class of genes transitions from biallelic to monoallelic expression after MSL2 loss. Many of these genes are haploinsufficient. In the absence of MSL2, one allele remains active, retaining active histone modifications and transcription factor binding, whereas the other allele is silenced, exhibiting loss of promoter-enhancer contacts and the acquisition of DNA methylation. Msl2-knockout mice show perinatal lethality and heterogeneous phenotypes during embryonic development, supporting a role for MSL2 in regulating gene dosage. The role of MSL2 in preserving biallelic expression of specific dosage-sensitive genes sets the stage for further investigation of other factors that are involved in allelic dosage compensation in mammalian cells, with considerable implications for human disease.
Assuntos
Alelos , Regulação da Expressão Gênica , Ubiquitina-Proteína Ligases , Animais , Feminino , Masculino , Camundongos , Metilação de DNA , Mecanismo Genético de Compensação de Dose , Desenvolvimento Embrionário , Elementos Facilitadores Genéticos , Haploinsuficiência , Histonas/metabolismo , Camundongos Knockout , Regiões Promotoras Genéticas , Fatores de Transcrição/metabolismo , Ubiquitina-Proteína Ligases/deficiência , Ubiquitina-Proteína Ligases/genética , Ubiquitina-Proteína Ligases/metabolismoRESUMO
Confinement of the X chromosome to a territory for dosage compensation is a prime example of how subnuclear compartmentalization is used to regulate transcription at the megabase scale. In Drosophila melanogaster, two sex-specific non-coding RNAs (roX1 and roX2) are transcribed from the X chromosome. They associate with the male-specific lethal (MSL) complex1, which acetylates histone H4 lysine 16 and thereby induces an approximately twofold increase in expression of male X-linked genes2,3. Current models suggest that X-over-autosome specificity is achieved by the recognition of cis-regulatory DNA high-affinity sites (HAS) by the MSL2 subunit4,5. However, HAS motifs are also found on autosomes, indicating that additional factors must stabilize the association of the MSL complex with the X chromosome. Here we show that the low-complexity C-terminal domain (CTD) of MSL2 renders its recruitment to the X chromosome sensitive to roX non-coding RNAs. roX non-coding RNAs and the MSL2 CTD form a stably condensed state, and functional analyses in Drosophila and mammalian cells show that their interactions are crucial for dosage compensation in vivo. Replacing the CTD of mammalian MSL2 with that from Drosophila and expressing roX in cis is sufficient to nucleate ectopic dosage compensation in mammalian cells. Thus, the condensing nature of roX-MSL2CTD is the primary determinant for specific compartmentalization of the X chromosome in Drosophila.
Assuntos
Compartimento Celular , Proteínas de Ligação a DNA/metabolismo , Proteínas de Drosophila/metabolismo , Drosophila/citologia , Drosophila/genética , RNA/metabolismo , Fatores de Transcrição/metabolismo , Cromossomo X/genética , Cromossomo X/metabolismo , Animais , Compartimento Celular/genética , Linhagem Celular , Proteínas de Ligação a DNA/química , Drosophila/metabolismo , Proteínas de Drosophila/química , Feminino , Humanos , Masculino , Camundongos , Conformação de Ácido Nucleico , RNA/genética , Fatores de Transcrição/químicaRESUMO
Nucleosomal organization at gene promoters is critical for transcription, with a nucleosome-depleted region (NDR) at transcription start sites (TSSs) being required for transcription initiation. How NDRs and the precise positioning of the +1 nucleosomes are maintained on active genes remains unclear. Here, we report that the Drosophila nonspecific lethal (NSL) complex is necessary to maintain this stereotypical nucleosomal organization at promoters. Upon NSL1 depletion, nucleosomes invade the NDRs at TSSs of NSL-bound genes. NSL complex member NSL3 binds to TATA-less promoters in a sequence-dependent manner. The NSL complex interacts with the NURF chromatin remodeling complex and is necessary and sufficient to recruit NURF to target promoters. Not only is the NSL complex essential for transcription, but it is required for accurate TSS selection for genes with multiple TSSs. Furthermore, loss of the NSL complex leads to an increase in transcriptional noise. Thus, the NSL complex establishes a canonical nucleosomal organization that enables transcription and determines TSS fidelity.
Assuntos
Proteínas de Drosophila/metabolismo , Drosophila melanogaster/genética , Nucleossomos/genética , Transcrição Gênica/genética , Animais , Proteínas de Drosophila/genética , Drosophila melanogaster/metabolismo , Proteínas Nucleares , Regiões Promotoras Genéticas/genética , Fatores de Transcrição/genética , Iniciação da Transcrição Genética , Proteínas de Transporte VesicularRESUMO
Here, we report the biochemical characterization of the nonspecific lethal (NSL) complex (NSL1, NSL2, NSL3, MCRS2, MBD-R2, and WDS) that associates with the histone acetyltransferase MOF in both Drosophila and mammals. Chromatin immunoprecipitation-Seq analysis revealed association of NSL1 and MCRS2 with the promoter regions of more than 4000 target genes, 70% of these being actively transcribed. This binding is functional, as depletion of MCRS2, MBD-R2, and NSL3 severely affects gene expression genome wide. The NSL complex members bind to their target promoters independently of MOF. However, depletion of MCRS2 affects MOF recruitment to promoters. NSL complex stability is interdependent and relies mainly on the presence of NSL1 and MCRS2. Tethering of NSL3 to a heterologous promoter leads to robust transcription activation and is sensitive to the levels of NSL1, MCRS2, and MOF. Taken together, we conclude that the NSL complex acts as a major transcriptional regulator in Drosophila.
Assuntos
Proteínas de Drosophila/metabolismo , Drosophila/metabolismo , Histona Acetiltransferases/metabolismo , Proteínas Nucleares/metabolismo , Animais , Drosophila/genética , Proteínas de Drosophila/genética , Genoma de Inseto , Histona Acetiltransferases/genética , Complexos Multiproteicos/genética , Complexos Multiproteicos/metabolismo , Proteínas Nucleares/genética , Regiões Promotoras Genéticas , Ligação Proteica , Transcrição GênicaRESUMO
MOF is the major histone H4 lysine 16-specific (H4K16) acetyltransferase in mammals and Drosophila. In flies, it is involved in the regulation of X-chromosomal and autosomal genes as part of the MSL and the NSL complexes, respectively. While the function of the MSL complex as a dosage compensation regulator is fairly well understood, the role of the NSL complex in gene regulation is still poorly characterized. Here we report a comprehensive ChIP-seq analysis of four NSL complex members (NSL1, NSL3, MBD-R2, and MCRS2) throughout the Drosophila melanogaster genome. Strikingly, the majority (85.5%) of NSL-bound genes are constitutively expressed across different cell types. We find that an increased abundance of the histone modifications H4K16ac, H3K4me2, H3K4me3, and H3K9ac in gene promoter regions is characteristic of NSL-targeted genes. Furthermore, we show that these genes have a well-defined nucleosome free region and broad transcription initiation patterns. Finally, by performing ChIP-seq analyses of RNA polymerase II (Pol II) in NSL1- and NSL3-depleted cells, we demonstrate that both NSL proteins are required for efficient recruitment of Pol II to NSL target gene promoters. The observed Pol II reduction coincides with compromised binding of TBP and TFIIB to target promoters, indicating that the NSL complex is required for optimal recruitment of the pre-initiation complex on target genes. Moreover, genes that undergo the most dramatic loss of Pol II upon NSL knockdowns tend to be enriched in DNA Replication-related Element (DRE). Taken together, our findings show that the MOF-containing NSL complex acts as a major regulator of housekeeping genes in flies by modulating initiation of Pol II transcription.
Assuntos
Proteínas de Drosophila , Drosophila melanogaster/genética , Histona-Lisina N-Metiltransferase/genética , Proteínas Nucleares , RNA Polimerase II , Fatores de Transcrição , Acetilação , Animais , Sítios de Ligação , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/metabolismo , Regulação da Expressão Gênica , Genoma de Inseto , Histona-Lisina N-Metiltransferase/metabolismo , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Regiões Promotoras Genéticas , Ligação Proteica , RNA Polimerase II/genética , RNA Polimerase II/metabolismo , Fator de Transcrição TFIIB/genética , Fator de Transcrição TFIIB/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Proteínas de Transporte VesicularRESUMO
Members of the NSL histone acetyltransferase complex are involved in multiorgan developmental syndromes. While the NSL complex is known for its importance in early development, its role in fully differentiated cells remains enigmatic. Using a kidney-specific model, we discovered that deletion of NSL complex members KANSL2 or KANSL3 in postmitotic podocytes led to catastrophic kidney dysfunction. Systematic comparison of two primary differentiated cell types reveals the NSL complex as a master regulator of intraciliary transport genes in both dividing and nondividing cells. NSL complex ablation led to loss of cilia and impaired sonic hedgehog pathway in ciliated fibroblasts. By contrast, nonciliated podocytes responded with altered microtubule dynamics and obliterated podocyte functions. Finally, overexpression of wild-type but not a double zinc finger (ZF-ZF) domain mutant of KANSL2 rescued the transcriptional defects, revealing a critical function of this domain in NSL complex assembly and function. Thus, the NSL complex exhibits bifurcation of functions to enable diversity of specialized outcomes in differentiated cells.
Assuntos
Núcleo Celular , Proteínas Hedgehog , Proteínas Hedgehog/genética , Regulação da Expressão Gênica , Diferenciação Celular/genética , FibroblastosRESUMO
Mutations in chromatin-modifying complexes and metabolic enzymes commonly underlie complex human developmental syndromes affecting multiple organs. A major challenge is to determine how disease-causing genetic lesions cause deregulation of homeostasis in unique cell types. Here we show that neural-specific depletion of three members of the non-specific lethal (NSL) chromatin complex-Mof, Kansl2 or Kansl3-unexpectedly leads to severe vascular defects and brain haemorrhaging. Deregulation of the epigenetic landscape induced by the loss of the NSL complex in neural cells causes widespread metabolic defects, including an accumulation of free long-chain fatty acids (LCFAs). Free LCFAs induce a Toll-like receptor 4 (TLR4)-NFκB-dependent pro-inflammatory signalling cascade in neighbouring vascular pericytes that is rescued by TLR4 inhibition. Pericytes display functional changes in response to LCFA-induced activation that result in vascular breakdown. Our work establishes that neurovascular function is determined by the neural metabolic environment.
Assuntos
Núcleo Celular/patologia , Cromatina/metabolismo , Histona Acetiltransferases/fisiologia , Inflamação/patologia , Neovascularização Patológica/patologia , Neurônios/patologia , Pericitos/patologia , Animais , Encéfalo/citologia , Encéfalo/metabolismo , Núcleo Celular/metabolismo , Cromatina/genética , Ácidos Graxos/metabolismo , Feminino , Feto/citologia , Feto/metabolismo , Humanos , Inflamação/metabolismo , Masculino , Metaboloma , Camundongos Endogâmicos C57BL , Camundongos Knockout , Neovascularização Patológica/metabolismo , Neurônios/metabolismo , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Pericitos/metabolismoRESUMO
Since the generation of cell-type specific knockout models, the importance of inter-cellular communication between neural, vascular, and microglial cells during neural development has been increasingly appreciated. However, the extent of communication between these major cell populations remains to be systematically mapped. Here, we describe EMBRACE (embryonic brain cell extraction using FACS), a method to simultaneously isolate neural, mural, endothelial, and microglial cells to more than 94% purity in â¼4 h. Utilizing EMBRACE we isolate, transcriptionally analyze, and build a cell-cell communication map of the developing mouse brain. We identify 1,710 unique ligand-receptor interactions between neural, endothelial, mural, and microglial cells in silico and experimentally confirm the APOE-LDLR, APOE-LRP1, VTN-KDR, and LAMA4-ITGB1 interactions in the E14.5 brain. We provide our data via the searchable "Brain interactome explorer", available at https://mpi-ie.shinyapps.io/braininteractomeexplorer/. Together, this study provides a comprehensive map that reveals the richness of communication within the developing brain.
RESUMO
Proper gene expression requires coordinated interplay among transcriptional coactivators, transcription factors and the general transcription machinery. We report here that MSL1, a central component of the dosage compensation complex in Drosophila melanogaster and Drosophila virilis, displays evolutionarily conserved sex-independent binding to promoters. Genetic and biochemical analyses reveal a functional interaction of MSL1 with CDK7, a subunit of the Cdk-activating kinase (CAK) complex of the general transcription factor TFIIH. Importantly, MSL1 depletion leads to decreased phosphorylation of Ser5 of RNA polymerase II. In addition, we demonstrate that MSL1 is a phosphoprotein, and transgenic flies expressing MSL1 phosphomutants show mislocalization of the histone acetyltransferase MOF and histone H4 K16 acetylation, thus ultimately causing male lethality due to a failure of dosage compensation. We propose that, by virtue of its interaction with components of the general transcription machinery, MSL1 exists in different phosphorylation states, thereby modulating transcription in flies.
Assuntos
Quinases Ciclina-Dependentes/metabolismo , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/metabolismo , Proteínas Nucleares/metabolismo , RNA Polimerase II/metabolismo , Fatores de Transcrição/metabolismo , Animais , Animais Geneticamente Modificados , Cromatina/genética , Cromatina/metabolismo , Quinases Ciclina-Dependentes/genética , Mecanismo Genético de Compensação de Dose , Proteínas de Drosophila/genética , Drosophila melanogaster/genética , Feminino , Deleção de Genes , Masculino , Mutação , Proteínas Nucleares/genética , Fosforilação , Regiões Promotoras Genéticas , Ligação Proteica , Serina/metabolismo , Fatores de Transcrição/genética , Ativação Transcricional , Quinase Ativadora de Quinase Dependente de CiclinaRESUMO
The histone H4 lysine 16 (H4K16)-specific acetyltransferase MOF is part of two distinct complexes involved in X chromosome dosage compensation and autosomal transcription regulation. Here we show that the MOF chromobarrel domain is essential for H4K16 acetylation throughout the Drosophila genome and is required for spreading of the male-specific lethal (MSL) complex on the X chromosome. The MOF chromobarrel domain directly interacts with nucleic acids and potentiates MOF's enzymatic activity after chromatin binding, making it a unique example of a chromo-like domain directly controlling acetylation activity in vivo. We also show that the Drosophila-specific N terminus of MOF has evolved to perform sex-specific functions. It modulates nucleosome binding and HAT activity and controls MSL complex assembly, thus regulating MOF function in dosage compensation. We propose that MOF has been especially tailored to achieve tight regulation of its enzymatic activity and enable its dual role on X and autosomes.
Assuntos
Proteínas de Drosophila/metabolismo , Drosophila melanogaster/enzimologia , Genoma de Inseto , Histona Acetiltransferases/metabolismo , Histonas/metabolismo , Proteínas Nucleares/metabolismo , Acetilação , Animais , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Proteínas de Drosophila/química , Proteínas de Drosophila/genética , Drosophila melanogaster/genética , Feminino , Histona Acetiltransferases/química , Histona Acetiltransferases/genética , Histonas/genética , Masculino , Proteínas Nucleares/química , Proteínas Nucleares/genética , Estrutura Terciária de Proteína , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Cromossomo X/genética , Cromossomo X/metabolismoRESUMO
The male-specific lethal (MSL) complex is required for dosage compensation in Drosophila melanogaster, and analogous complexes exist in mammals. We report structures of binary complexes of mammalian MSL3 and the histone acetyltransferase (HAT) MOF with consecutive segments of MSL1. MSL1 interacts with MSL3 as an extended chain forming an extensive hydrophobic interface, whereas the MSL1-MOF interface involves electrostatic interactions between the HAT domain and a long helix of MSL1. This structure provides insights into the catalytic mechanism of MOF and enables us to show analogous interactions of MOF with NSL1. In Drosophila, selective disruption of Msl1 interactions with Msl3 or Mof severely affects Msl1 targeting to the body of dosage-compensated genes and several high-affinity sites, without affecting promoter binding. We propose that Msl1 acts as a scaffold for MSL complex assembly to achieve specific targeting to the X chromosome.
Assuntos
Mecanismo Genético de Compensação de Dose , Histona Acetiltransferases/química , Histona Acetiltransferases/metabolismo , Sequência de Aminoácidos , Animais , Linhagem Celular , Proteínas Cromossômicas não Histona , Cristalografia por Raios X , Proteínas de Ligação a DNA , Drosophila/metabolismo , Proteínas de Drosophila/metabolismo , Histona Acetiltransferases/genética , Humanos , Masculino , Camundongos , Modelos Moleculares , Dados de Sequência Molecular , Mutagênese , Proteínas Nucleares/metabolismo , Conformação Proteica , Alinhamento de Sequência , Fatores de Transcrição/química , Fatores de Transcrição/metabolismoRESUMO
Dosage compensation in Drosophila is dependent on MSL proteins and involves hypertranscription of the male X chromosome, which ensures equal X-linked gene expression in both sexes. Here, we report the purification of enzymatically active MSL complexes from Drosophila embryos, Schneider cells, and human HeLa cells. We find a stable association of the histone H4 lysine 16-specific acetyltransferase MOF with the RNA/protein containing MSL complex as well as with an evolutionary conserved complex. We show that the MSL complex interacts with several components of the nuclear pore, in particular Mtor/TPR and Nup153. Strikingly, knockdown of Mtor or Nup153 results in loss of the typical MSL X-chromosomal staining and dosage compensation in Drosophila male cells but not in female cells. These results reveal an unexpected physical and functional connection between nuclear pore components and chromatin regulation through MSL proteins, highlighting the role of nucleoporins in gene regulation in higher eukaryotes.
Assuntos
Mecanismo Genético de Compensação de Dose , Proteínas de Drosophila/metabolismo , Drosophila/genética , Regulação da Expressão Gênica , Poro Nuclear/metabolismo , Proteínas Nucleares/metabolismo , Fatores de Transcrição/metabolismo , Transcrição Gênica , Acetiltransferases , Animais , Animais Geneticamente Modificados , Linhagem Celular , Cromatografia de Afinidade , Drosophila/embriologia , Drosophila/metabolismo , Proteínas de Drosophila/química , Evolução Molecular , Feminino , Células HeLa , Humanos , Masculino , Espectrometria de Massas , Complexo de Proteínas Formadoras de Poros Nucleares/metabolismo , Complexo de Proteínas Formadoras de Poros Nucleares/fisiologia , Proteínas Nucleares/química , Proteínas Quinases/metabolismo , Proteínas Quinases/fisiologia , Serina-Treonina Quinases TOR , Fatores de Transcrição/química , Cromossomo X/genéticaRESUMO
Dosage compensation ensures equal expression of X-linked genes in males and females. In Drosophila, equalization is achieved by hypertranscription of the male X chromosome. This process requires an RNA/protein containing dosage compensation complex (DCC). Here we use RNA interference of individual DCC components to define the order of complex assembly in Schneider cells. We show that interaction of MOF with MSL-3 leads to specific acetylation of MSL-3 at a single lysine residue adjacent to one of its chromodomains. We observe that localization of MSL-3 to the X chromosome is RNA dependent and acetylation sensitive. We find that the acetylation status of MSL-3 determines its interaction with roX2 RNA. Furthermore, we find that RPD3 interacts with MSL-3 and that MSL-3 can be deacetylated by the RPD3 complex. We propose that regulated acetylation of MSL-3 may provide a mechanistic explanation for spreading of the dosage compensation complex along the male X chromosome.