RESUMO
Promoters and enhancers are sites of transcription initiation (TSSs) and carry specific histone modifications, including H3K4me1, H3K4me3, and H3K27ac. Yet, the principles governing the boundaries of such regulatory elements are still poorly characterized. Alu elements are good candidates for a boundary function, being highly abundant in gene-rich regions, while essentially excluded from regulatory elements. Here, we show that the interval ranging from TSS to first upstream Alu, accommodates all H3K4me3 and most H3K27ac marks, while excluding DNA methylation. Remarkably, the average length of these intervals greatly varies in-between tissues, being longer in stem- and shorter in immune-cells. The very shortest TSS-to-first-Alu intervals were observed at promoters active in T-cells, particularly at immune genes, where first-Alus were traversed by RNA polymerase II transcription, while accumulating H3K4me1 signal. Finally, DNA methylation at first-Alus was found to evolve with age, regressing from young to middle-aged, then recovering later in life. Thus, the first-Alus upstream of TSSs appear as dynamic boundaries marking the transition from DNA methylation to active histone modifications at regulatory elements, while also participating in the recording of immune gene transcriptional events by positioning H3K4me1-modified nucleosomes.
Assuntos
Código das Histonas , Sequências Reguladoras de Ácido Nucleico , Epigênese Genética , Epigenômica , Regiões Promotoras GenéticasRESUMO
Several autoimmune diseases including multiple sclerosis (MS) cause increased transcription of endogenous retroviruses (HERVs) normally repressed by heterochromatin. In parallel, HERV-derived sequences were reported to drive gene expression. Here, we have examined a possible link between promoter and enhancer divergent transcription and the production of HERV transcripts. We find that HERV-derived sequences are in general counter-selected at regulatory regions, a counter-selection that is strongest in brain tissues while very moderate in stem cells. By exposing T cells to the pesticide dieldrin, we further found that a series of HERV-driven enhancers otherwise active only at stem cell stages can be reactivated by stress. This in part relies on peptidylarginine deiminase activity, possibly participating in the reawakening of silenced enhancers. Likewise, usage of HERV-driven enhancers was increased in myelin-reactive T cells from patients with MS, correlating with activation of nearby genes at several sites. Altogether, we propose that HERV-driven enhancers constitute a reservoir of auxiliary enhancers transiently induced by stress while chronically active in diseases like MS.
Assuntos
Retrovirus Endógenos/genética , Esclerose Múltipla/genética , Esclerose Múltipla/imunologia , Sequências Reguladoras de Ácido Nucleico/genética , Linfócitos T/metabolismo , Adulto , Estudos de Casos e Controles , Células Cultivadas , Feminino , Regulação Viral da Expressão Gênica/fisiologia , Humanos , Células Jurkat , Masculino , Pessoa de Meia-Idade , Esclerose Múltipla/metabolismo , Esclerose Múltipla/virologia , Linfócitos T/patologiaRESUMO
HP1 proteins are best known as markers of heterochromatin and gene silencing. Yet, they are also RNA-binding proteins and the HP1γ/CBX3 family member is present on transcribed genes together with RNA polymerase II, where it regulates co-transcriptional processes such as alternative splicing. To gain insight in the role of the RNA-binding activity of HP1γ in transcriptionally active chromatin, we have captured and analysed RNAs associated with this protein. We find that HP1γ is specifically targeted to hexameric RNA motifs and coincidentally transposable elements of the SINE family. As these elements are abundant in introns, while essentially absent from exons, the HP1γ RNA association tethers unspliced pre-mRNA to chromatin via the intronic regions and limits the usage of intronic cryptic splice sites. Thus, our data unveil novel determinants in the relationship between chromatin and co-transcriptional splicing.
Assuntos
Precursores de RNA , Splicing de RNA , Processamento Alternativo/genética , Íntrons/genética , Precursores de RNA/genética , Precursores de RNA/metabolismo , Splicing de RNA/genética , Proteínas de Ligação a RNARESUMO
DNA methylation (meDNA) is a modulator of alternative splicing, and splicing perturbations are involved in tumorigenesis nearly as frequently as DNA mutations. However, the impact of meDNA on tumorigenesis via splicing-mediated mechanisms has not been thoroughly explored. Here, we found that HCT116 colon carcinoma cells inactivated for the DNA methylases DNMT1/3b undergo a partial epithelial to mesenchymal transition associated with increased CD44 variant exon skipping. These skipping events are directly mediated by the loss of intragenic meDNA and the chromatin factors MBD1/2/3 and HP1γ and are also linked to phosphorylation changes in elongating RNA polymerase II. The role of meDNA in alternative splicing was confirmed by using the dCas9/DNMT3b tool. We further tested whether the meDNA level could have predictive value in the MCF10A model for breast cancer progression and in patients with acute lymphoblastic leukemia (B ALL). We found that a small number of differentially spliced genes, mostly involved in splicing and signal transduction, are correlated with the local modulation of meDNA. Our observations suggest that, although DNA methylation has multiple avenues to affect alternative splicing, its indirect effect may also be mediated through alternative splicing isoforms of these meDNA sensors.
Assuntos
Processamento Alternativo , Metilação de DNA , Regulação Neoplásica da Expressão Gênica , Receptores de Hialuronatos/genética , Neoplasias da Mama/genética , Neoplasias da Mama/metabolismo , Carcinogênese/genética , Linhagem Celular Tumoral , Homólogo 5 da Proteína Cromobox , Proteínas Cromossômicas não Histona/metabolismo , DNA (Citosina-5-)-Metiltransferase 1/genética , DNA (Citosina-5-)-Metiltransferases/genética , Proteínas de Ligação a DNA/metabolismo , Transição Epitelial-Mesenquimal , Éxons , Feminino , Células HeLa , Código das Histonas , Humanos , Receptores de Hialuronatos/metabolismo , Leucemia-Linfoma Linfoblástico de Células Precursoras/genética , Leucemia-Linfoma Linfoblástico de Células Precursoras/metabolismo , RNA Polimerase II/metabolismo , Fatores de Transcrição/metabolismo , Transcrição Gênica , DNA Metiltransferase 3BRESUMO
Several studies propose an influence of chromatin on pre-mRNA splicing, but it is still unclear how widespread and how direct this phenomenon is. We find here that when assembled in vivo, the U2 snRNP co-purifies with a subset of chromatin-proteins, including histones and remodeling complexes like SWI/SNF. Yet, an unbiased RNAi screen revealed that the outcome of splicing is influenced by a much larger variety of chromatin factors not all associating with the spliceosome. The availability of this broad range of chromatin factors impacting splicing further unveiled their very context specific effect, resulting in either inclusion or skipping, depending on the exon under scrutiny. Finally, a direct assessment of the impact of chromatin on splicing using an in vitro co-transcriptional splicing assay with pre-mRNAs transcribed from a nucleosomal template, demonstrated that chromatin impacts nascent pre-mRNP in their competence for splicing. Altogether, our data show that numerous chromatin factors associated or not with the spliceosome can affect the outcome of splicing, possibly as a function of the local chromatin environment that by default interferes with the efficiency of splicing.
RESUMO
Old long-lived proteins contain dehydroalanine (Dha) and dehydrobutyrine (Dhb), two amino acids engendered by dehydration of serines and threonines, respectively. Although these residues have a suspected role in protein cross-linking and aggregation, their direct implication has yet to be determined. Here, we have taken advantage of the ability of the enteropathogen Shigella to convert the phosphothreonine residue of the pT-X-pY consensus sequence of ERK and p38 into Dhb and followed the impact of dehydration on the fate of the two MAPKs. To that end, we have generated the first antibodies recognizing Dhb-modified proteins and allowing tracing them as they form. We showed that Dhb modifications accumulate in a long-lasting manner in Shigella-infected cells, causing subsequent formation of covalent cross-links of MAPKs. Moreover, the Dhb signal correlates precisely with the activation of the Shigella type III secretion apparatus, thus evidencing injectisome activity. This observation is the first to document a causal link between Dhb formation and protein cross-linking in live cells. Detection of eliminylation is a new avenue to phosphoproteome regulation in eukaryotes that will be instrumental for the development of type III secretion inhibitors.
Assuntos
Proteínas de Bactérias/metabolismo , Carbono-Oxigênio Liases/metabolismo , Sistema de Sinalização das MAP Quinases , Shigella/enzimologia , Treonina/química , Alanina/análogos & derivados , Alanina/química , Aminobutiratos/química , Animais , Anticorpos/química , Células CACO-2 , Linhagem Celular , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Células HeLa , Humanos , Camundongos , Ligação Proteica , Proteômica , Especificidade por Substrato , Sistemas de Secreção Tipo III , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismoRESUMO
HP1 proteins are transcriptional regulators that, like histones, are targets for post-translational modifications defining an HP1-mediated subcode. HP1γ has multiple phosphorylation sites, including serine 83 (S83) that marks it to sites of active transcription. In a guinea pig model for Shigella enterocolitis, we observed that the defective type III secretion mxiD Shigella flexneri strain caused more HP1γ phosphorylation in the colon than the wild-type strain. Shigella interferes with HP1 phosphorylation by injecting the phospholyase OspF. This effector interacts with HP1γ and alters its phosphorylation at S83 by inactivating ERK and consequently MSK1, a downstream kinase. MSK1 that here arises as a novel HP1γ kinase, phosphorylates HP1γ at S83 in the context of an MSK1-HP1γ complex, and thereby favors its accumulation on its target genes. Genome-wide transcriptome analysis reveals that this mechanism is linked to up-regulation of proliferative gene and fine-tuning of immune gene expression. Thus, in addition to histones, bacteria control host transcription by modulating the activity of HP1 proteins, with potential implications in transcriptional reprogramming at the mucosal barrier.
Assuntos
Proteínas da Membrana Bacteriana Externa/metabolismo , Carbono-Oxigênio Liases/metabolismo , Proteínas Cromossômicas não Histona/metabolismo , Disenteria Bacilar/metabolismo , Enterocolite/metabolismo , Shigella flexneri/metabolismo , Transcriptoma , Animais , Proteínas da Membrana Bacteriana Externa/genética , Carbono-Oxigênio Liases/genética , Células Cultivadas , Homólogo 5 da Proteína Cromobox , Proteínas Cromossômicas não Histona/genética , Modelos Animais de Doenças , Disenteria Bacilar/genética , Disenteria Bacilar/patologia , Enterocolite/genética , Enterocolite/patologia , MAP Quinases Reguladas por Sinal Extracelular/genética , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Estudo de Associação Genômica Ampla , Cobaias , Camundongos , Camundongos Mutantes , Fosforilação , Proteínas Quinases S6 Ribossômicas 90-kDa/genética , Proteínas Quinases S6 Ribossômicas 90-kDa/metabolismo , Shigella flexneri/genéticaRESUMO
Alternative splicing is the main source of proteome diversity. Here, we have investigated how alternative splicing affects the function of two human histone methyltransferases (HMTase): G9A and SUV39H2. We show that exon 10 in G9A and exon 3 in SUV39H2 are alternatively included in a variety of tissues and cell lines, as well as in a different species. The production of these variants is likely tightly regulated because both constitutive and alternative splicing factors control their splicing profiles. Based on this evidence, we have assessed the link between the inclusion of these exons and the activity of both enzymes. We document that these HMTase genes yield several protein isoforms, which are likely issued from alternative splicing regulation. We demonstrate that inclusion of SUV39H2 exon 3 is a determinant of the stability, the sub-nuclear localization, and the HMTase activity. Genome-wide expression analysis further revealed that alternative inclusion of SUV39H2 exon 3 differentially modulates the expression of target genes. Our data also suggest that a variant of G9A may display a function that is independent of H3K9 methylation. Our work emphasizes that expression and function of genes are not collinear; therefore alternative splicing must be taken into account in any functional study.
Assuntos
Processamento Alternativo , Metilases de Modificação do DNA/genética , Linhagem Celular , Metilases de Modificação do DNA/metabolismo , HumanosRESUMO
Ectopic repression of retinoic acid (RA) receptor target genes by PML/RARA and PLZF/RARA fusion proteins through aberrant recruitment of nuclear corepressor complexes drives cellular transformation and acute promyelocytic leukemia (APL) development. In the case of PML/RARA, this repression can be reversed through treatment with all-trans RA (ATRA), leading to leukemic remission. However, PLZF/RARA ectopic repression is insensitive to ATRA, resulting in persistence of the leukemic diseased state after treatment, a phenomenon that is still poorly understood. Here we show that, like PML/RARA, PLZF/RARA expression leads to recruitment of the Polycomb-repressive complex 2 (PRC2) Polycomb group (PcG) complex to RA response elements. However, unlike PML/RARA, PLZF/RARA directly interacts with the PcG protein Bmi-1 and forms a stable component of the PRC1 PcG complex, resulting in PLZF/RARA-dependent ectopic recruitment of PRC1 to RA response elements. Upon treatment with ATRA, ectopic recruitment of PRC2 by either PML/RARA or PLZF/RARA is lost, whereas PRC1 recruited by PLZF/RARA remains, resulting in persistent RA-insensitive gene repression. We further show that Bmi-1 is essential for the PLZF/RARA cellular transformation property and implicates a central role for PRC1 in PLZF/RARA-mediated myeloid leukemic development.
Assuntos
Transformação Celular Neoplásica , Leucemia/fisiopatologia , Proteínas de Fusão Oncogênica/metabolismo , Proteínas Repressoras/metabolismo , Antineoplásicos/farmacologia , Cromatina/metabolismo , Humanos , Proteínas Nucleares/metabolismo , Complexo Repressor Polycomb 1 , Proteínas do Grupo Polycomb , Ligação Proteica/efeitos dos fármacos , Estrutura Terciária de Proteína , Proteínas Proto-Oncogênicas/metabolismo , Tretinoína/farmacologia , Células U937RESUMO
Heterochromatin protein 1 (HP1) proteins are chromatin-bound transcriptional regulators. While their chromodomain binds histone H3 methylated on lysine 9, their chromoshadow domain associates with the H3 histone fold in a region involved in chromatin remodeling. Here, we show that phosphorylation at histone H3 threonine 45 and serine 57 within this latter region differentially affects binding of the three mammalian HP1 isoforms HP1α, HP1ß and HP1γ. Both phosphorylation events are dependent on the activity of the DYRK1A kinase that antagonizes HP1-mediated transcriptional repression and participates in abnormal activation of cytokine genes in Down's syndrome-associated megakaryoblastic leukemia.
Assuntos
Proteínas Cromossômicas não Histona/metabolismo , Regulação da Expressão Gênica/genética , Histonas/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Tirosina Quinases/metabolismo , Western Blotting , Linhagem Celular Tumoral , Imunoprecipitação da Cromatina , Homólogo 5 da Proteína Cromobox , Éxons/genética , Humanos , Immunoblotting , Análise de Sequência com Séries de Oligonucleotídeos , Fosforilação , Regiões Promotoras Genéticas/genética , Ligação Proteica , Isoformas de Proteínas/metabolismo , Reação em Cadeia da Polimerase em Tempo Real , Quinases DyrkRESUMO
Dynamic epigenetic modifications play a key role in mediating the expression of genes required for neuronal development. We previously identified nitric oxide (NO) as a signaling molecule that mediates S-nitrosylation of histone deacetylase 2 (HDAC2) and epigenetic changes in neurons. Here, we show that HDAC2 nitrosylation regulates neuronal radial migration during cortical development. Bead-array analysis performed in the developing cortex revealed that brahma (Brm), a subunit of the ATP-dependent chromatin-remodeling complex BRG/brahma-associated factor, is one of the genes regulated by S-nitrosylation of HDAC2. In the cortex, expression of a mutant form of HDAC2 that cannot be nitrosylated dramatically inhibits Brm expression. Our study identifies NO and HDAC2 nitrosylation as part of a signaling pathway that regulates cortical development and the expression of Brm in neurons.
Assuntos
Movimento Celular , Montagem e Desmontagem da Cromatina , Histona Desacetilase 2/metabolismo , Neurônios/citologia , Óxido Nítrico/metabolismo , Fatores de Transcrição/metabolismo , Animais , Separação Celular , Córtex Cerebral/citologia , Córtex Cerebral/metabolismo , Eletroporação , Feminino , Citometria de Fluxo , Camundongos , Gravidez , Transdução de SinaisRESUMO
Multiple Sclerosis (MS) is an autoimmune disease associated with abnormal expression of a subset of cytokines, resulting in inappropriate T-lymphocyte activation and uncontrolled immune response. A key issue in the field is the need to understand why these cytokines are transcriptionally activated in the patients. Here, we have examined several transcription units subject to pathological reactivation in MS, including the TNFα and IL8 cytokine genes and also several Human Endogenous RetroViruses (HERVs). We find that both the immune genes and the HERVs require the heterochromatin protein HP1α for their transcriptional repression. We further show that the Peptidylarginine Deiminase 4 (PADI4), an enzyme with a suspected role in MS, weakens the binding of HP1α to tri-methylated histone H3 lysine 9 by citrullinating histone H3 arginine 8. The resulting de-repression of both cytokines and HERVs can be reversed with the PADI-inhibitor Cl-amidine. Finally, we show that in peripheral blood mononuclear cells (PBMCs) from MS patients, the promoters of TNFα, and several HERVs share a deficit in HP1α recruitment and an augmented accumulation of histone H3 with a double citrulline 8 tri-methyl lysine 9 modifications. Thus, our study provides compelling evidence that HP1α and PADI4 are regulators of both immune genes and HERVs, and that multiple events of transcriptional reactivation in MS patients can be explained by the deficiency of a single mechanism of gene silencing.
Assuntos
Proteínas Cromossômicas não Histona , Histonas , Hidrolases , Esclerose Múltipla , Adulto , Homólogo 5 da Proteína Cromobox , Proteínas Cromossômicas não Histona/genética , Proteínas Cromossômicas não Histona/metabolismo , Citrulina/metabolismo , Retrovirus Endógenos/genética , Retrovirus Endógenos/metabolismo , Feminino , Regulação da Expressão Gênica/efeitos dos fármacos , Células HEK293 , Histonas/genética , Histonas/metabolismo , Humanos , Hidrolases/genética , Hidrolases/metabolismo , Imunidade Inata/genética , Interleucina-8/genética , Interleucina-8/metabolismo , Leucócitos Mononucleares/efeitos dos fármacos , Ativação Linfocitária/genética , Células MCF-7 , Masculino , Pessoa de Meia-Idade , Esclerose Múltipla/genética , Esclerose Múltipla/metabolismo , Ornitina/análogos & derivados , Ornitina/farmacologia , Proteína-Arginina Desiminase do Tipo 4 , Desiminases de Arginina em Proteínas , Linfócitos T/metabolismo , Fator de Necrose Tumoral alfa/genética , Fator de Necrose Tumoral alfa/metabolismoRESUMO
Malaria parasites have a complex life cycle, during which they undergo significant biological changes to adapt to different hosts and changing environments. Plasmodium falciparum, the species responsible for the deadliest form of human malaria, maintains this complex life cycle with a relatively small number of genes. Alternative splicing (AS) is an important post-transcriptional mechanisms that enables eukaryotic organisms to expand their protein repertoire out of relatively small number of genes. SR proteins are major regulators of AS in higher eukaryotes. Nevertheless, the regulation of splicing as well as the AS machinery in Plasmodium spp. are still elusive. Here, we show that PfSR1, a putative P. falciparum SR protein, can mediate RNA splicing in vitro. In addition, we show that PfSR1 functions as an AS factor in mini-gene in vivo systems similar to the mammalian SR protein SRSF1. Expression of PfSR1-myc in P. falciparum shows distinct patterns of cellular localization during intra erythrocytic development. Furthermore, we determine that the predicted RS domain of PfSR1 is essential for its localization to the nucleus. Finally, we demonstrate that proper regulation of pfsr1 is required for parasite proliferation in human RBCs and over-expression of pfsr1 influences AS activity of P. falciparum genes in vivo.
Assuntos
Processamento Alternativo , Eritrócitos/parasitologia , Proteínas Nucleares/metabolismo , Plasmodium falciparum/genética , Proteínas de Protozoários/metabolismo , Proteínas de Ligação a RNA/metabolismo , Linhagem Celular , Humanos , Sinais de Localização Nuclear , Proteínas Nucleares/química , Proteínas Nucleares/genética , Plasmodium falciparum/crescimento & desenvolvimento , Plasmodium falciparum/metabolismo , Proteínas de Protozoários/química , Proteínas de Protozoários/genética , Precursores de RNA/metabolismo , RNA Mensageiro/metabolismo , Proteínas de Ligação a RNA/química , Proteínas de Ligação a RNA/genética , Fatores de Processamento de Serina-ArgininaRESUMO
HP1α/CBX5 is an epigenetic regulator with a suspected role in multiple sclerosis (MS). Here, using high-depth RNA sequencing on monocytes, we identified a subset of MS patients with reduced CBX5 expression, correlating with progressive stages of the disease and extensive transcriptomic alterations. Examination of rare non-coding RNA species in these patients revealed impaired maturation/degradation of U snRNAs and enhancer RNAs, indicative of reduced activity of the Integrator, a complex with suspected links to increased MS risk. At protein-coding genes, compromised Integrator activity manifested in reduced pre-mRNA splicing efficiency and altered expression of genes regulated by RNA polymerase II pause-release. Inactivation of Cbx5 in the mouse mirrored most of these transcriptional defects and resulted in hypersensitivity to experimental autoimmune encephalomyelitis. Collectively, our observations suggested a major contribution of the Integrator complex in safeguarding against transcriptional anomalies characteristic of MS, with HP1α/CBX5 emerging as an unexpected regulator of this complex's activity. These findings bring novel insights into the transcriptional aspects of MS and provide potential new criteria for patient stratification.
Assuntos
Homólogo 5 da Proteína Cromobox , Encefalomielite Autoimune Experimental , Esclerose Múltipla , Transcriptoma , Humanos , Esclerose Múltipla/genética , Esclerose Múltipla/metabolismo , Animais , Camundongos , Transcriptoma/genética , Feminino , Encefalomielite Autoimune Experimental/genética , Encefalomielite Autoimune Experimental/metabolismo , Masculino , Adulto , Proteínas Cromossômicas não Histona/metabolismo , Proteínas Cromossômicas não Histona/genética , Pessoa de Meia-Idade , Splicing de RNA/genética , Regulação da Expressão Gênica , Monócitos/metabolismo , RNA Nuclear Pequeno/genética , RNA Nuclear Pequeno/metabolismo , Camundongos Endogâmicos C57BL , Perfilação da Expressão Gênica/métodosRESUMO
Alternative splicing allows for one gene to encode multiple proteins. This mechanism is regulated by dedicated splicing factors. However, recent data have shown that these factors contact the RNA polymerase II as well as transcription factors and chromatin remodeling enzymes present inside the coding region of the gene. These observations favor a model where cotranscriptional splice decisions are assisted by factors recruited at the promoter or by the elongating polymerase. We also suggest that chromatin could function as an RNA-binding matrix displaying the immature transcripts to the spliceosomes.
Assuntos
Cromatina/genética , Splicing de RNA/genética , Transcrição Gênica/genética , Animais , Humanos , RNA Mensageiro/genéticaRESUMO
Brm is an ATPase subunit of the SWI/SNF chromatin-remodelling complex. Previously, we identified a novel hotspot mutation in Brm in human skin cancer, which is caused by exposure to ultraviolet radiation (UVR). As SWI/SNF is involved in DNA repair, we investigated whether Brm-/- mice had enhanced photocarcinogenesis. P53+/- and Brm-/-p53+/- mice were also examined as the p53 tumor suppressor gene is mutated early during human skin carcinogenesis. Mice were exposed to a low-dose irradiation protocol that caused few skin tumors in wild-type mice. Brm-/- mice with both p53 alleles intact had an increased incidence of skin and ocular tumors compared to Brm+/+p53+/+ controls. Brm loss in p53+/- mice did not further enhance skin or ocular cancer incidence beyond the increased photocarcinogenesis in p53+/- mice. However, the skin tumors that arose early in Brm-/- p53+/- mice had a higher growth rate. Brm-/- did not prevent UVR-induced apoptotic sunburn cell formation, which is a protective response. Unexpectedly, Brm-/- inhibited UVR-induced immunosuppression, which would be predicted to reduce rather than enhance photocarcinogenesis. In conclusion, the absence of Brm increased skin and ocular photocarcinogenesis. Even when one allele of p53 is lost, Brm has additional tumor suppressing capability.
Assuntos
Neoplasias Oculares/fisiopatologia , Neoplasias Induzidas por Radiação/fisiopatologia , Neoplasias Cutâneas/fisiopatologia , Fatores de Transcrição/deficiência , Raios Ultravioleta/efeitos adversos , Animais , Apoptose , Relação Dose-Resposta à Radiação , Neoplasias Oculares/genética , Neoplasias Oculares/patologia , Feminino , Predisposição Genética para Doença/genética , Incidência , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Neoplasias Induzidas por Radiação/genética , Neoplasias Induzidas por Radiação/patologia , Neoplasias Cutâneas/genética , Neoplasias Cutâneas/patologia , Fatores de Transcrição/genética , Fatores de Transcrição/fisiologia , Proteína Supressora de Tumor p53/deficiência , Proteína Supressora de Tumor p53/genética , Proteína Supressora de Tumor p53/fisiologiaRESUMO
The heterochromatin-enriched HP1 proteins play a critical role in regulation of transcription. These proteins contain two related domains known as the chromo- and the chromoshadow-domain. The chromo-domain binds histone H3 tails methylated on lysine 9. However, in vivo and in vitro experiments have shown that the affinity of HP1 proteins to native methylated chromatin is relatively poor and that the opening of chromatin occurring during DNA replication facilitates their binding to nucleosomes. These observations prompted us to investigate whether HP1 proteins have additional histone binding activities, envisioning also affinity for regions potentially occluded by the nucleosome structure. We find that the chromoshadow-domain interacts with histone H3 in a region located partially inside the nucleosomal barrel at the entry/exit point of the nucleosome. Interestingly, this region is also contacted by the catalytic subunits of the human SWI/SNF complex. In vitro, efficient SWI/SNF remodeling requires this contact and is inhibited in the presence of HP1 proteins. The antagonism between SWI/SNF and HP1 proteins is also observed in vivo on a series of interferon-regulated genes. Finally, we show that SWI/SNF activity favors loading of HP1 proteins to chromatin both in vivo and in vitro. Altogether, our data suggest that HP1 chromoshadow-domains can benefit from the opening of nucleosomal structures to bind chromatin and that HP1 proteins use this property to detect and arrest unwanted chromatin remodeling.
Assuntos
DNA Helicases/metabolismo , Heterocromatina/metabolismo , Histonas/metabolismo , Proteínas Nucleares/metabolismo , Fatores de Transcrição/metabolismo , Sequência de Bases , Linhagem Celular Tumoral , Cromatina/metabolismo , Imunoprecipitação da Cromatina , Proteínas Cromossômicas não Histona/metabolismo , DNA Helicases/química , Primers do DNA , Humanos , Proteínas Nucleares/química , Reação em Cadeia da Polimerase , Conformação Proteica , Interferência de RNA , Fatores de Transcrição/químicaRESUMO
Gene silencing via heterochromatin formation plays a major role in cell differentiation and maintenance of homeostasis. Here we report the identification and characterization of a novel heterochromatinization factor in vertebrates, bromo adjacent homology domain-containing protein 1 (BAHD1). This nuclear protein interacts with HP1, MBD1, HDAC5, and several transcription factors. Through electron and immunofluorescence microscopy studies, we show that BAHD1 overexpression directs HP1 to specific nuclear sites and promotes the formation of large heterochromatic domains, which lack acetyl histone H4 and are enriched in H3 trimethylated at lysine 27 (H3K27me3). Furthermore, ectopically expressed BAHD1 colocalizes with the heterochromatic inactive X chromosome (Xi). The BAH domain is required for BAHD1 colocalization with H3K27me3, but not with the Xi chromosome. As highlighted by whole genome microarray analysis of BAHD1 knockdown cells, BAHD1 represses several proliferation and survival genes, in particular the insulin-like growth factor II gene (IGF2). When overexpressed, BAHD1 specifically binds the CpG-rich P3 promoter of IGF2, which increases MBD1 and HDAC5 targeting at this locus. This region contains DNA-binding sequences for the transcription factor SP1, with which BAHD1 coimmunoprecipitates. Collectively, these findings provide evidence that BAHD1 acts as a silencer by recruiting at specific promoters a set of proteins that coordinate heterochromatin assembly.
Assuntos
Proteínas Cromossômicas não Histona/fisiologia , Inativação Gênica , Heterocromatina/química , Proteínas Nucleares/genética , Proteínas Nucleares/fisiologia , Cromatina/química , Mapeamento Cromossômico , Ilhas de CpG , Heterocromatina/metabolismo , Histonas/química , Humanos , Fator de Crescimento Insulin-Like II/metabolismo , Lisina/química , Microscopia de Fluorescência/métodos , Modelos Genéticos , Ligação Proteica , Estrutura Terciária de Proteína , Transcrição GênicaRESUMO
Influenza virus transcription is catalyzed by the viral RNA-polymerase (FluPol) through a cap-snatching activity. The snatching of the cap of cellular mRNA by FluPol is preceded by its binding to the flexible C-terminal domain (CTD) of the RPB1 subunit of RNA-polymerase II (Pol II). To better understand how FluPol brings the 3'-end of the genomic RNAs in close proximity to the host-derived primer, we hypothesized that FluPol may recognize additional Pol II subunits/domains to ensure cap-snatching. Using binary complementation assays between the Pol II and influenza A FluPol subunits and their structural domains, we revealed an interaction between the N-third domain of PB2 and RPB4. This interaction was confirmed by a co-immunoprecipitation assay and was found to occur with the homologous domains of influenza B and C FluPols. The N-half domain of RPB4 was found to be critical in this interaction. Punctual mutants generated at conserved positions between influenza A, B, and C FluPols in the N-third domain of PB2 exhibited strong transcriptional activity defects. These results suggest that FluPol interacts with several domains of Pol II (the CTD to bind Pol II), initiating host transcription and a second transcription on RPB4 to locate FluPol at the proximity of the 5'-end of nascent host mRNA.
Assuntos
Influenza Humana , Orthomyxoviridae , Humanos , Orthomyxoviridae/genética , RNA Polimerase II/metabolismo , RNA Mensageiro/genética , RNA Viral/genética , RNA Viral/metabolismo , RNA Polimerase Dependente de RNA/genética , Transcrição Viral , Replicação ViralRESUMO
Gephyrin (GPHN) regulates the clustering of postsynaptic components at inhibitory synapses and is involved in pathophysiology of neuropsychiatric disorders. Here, we uncover an extensive diversity of GPHN transcripts that are tightly controlled by splicing during mouse and human brain development. Proteomic analysis reveals at least a hundred isoforms of GPHN incorporated at inhibitory Glycine and gamma-aminobutyric acid A receptors containing synapses. They exhibit different localization and postsynaptic clustering properties, and altering the expression level of one isoform is sufficient to affect the number, size, and density of inhibitory synapses in cerebellar Purkinje cells. Furthermore, we discovered that splicing defects reported in neuropsychiatric disorders are carried by multiple alternative GPHN transcripts, demonstrating the need for a thorough analysis of the GPHN transcriptome in patients. Overall, we show that alternative splicing of GPHN is an important genetic variation to consider in neurological diseases and a determinant of the diversity of postsynaptic inhibitory synapses.