RESUMO
BACKGROUND: The precise origin of newly formed ACTA2+ (alpha smooth muscle actin-positive) cells appearing in nonmuscularized vessels in the context of pulmonary hypertension is still debatable although it is believed that they predominantly derive from preexisting vascular smooth muscle cells (VSMCs). METHODS: Gli1Cre-ERT2; tdTomatoflox mice were used to lineage trace GLI1+ (glioma-associated oncogene homolog 1-positive) cells in the context of pulmonary hypertension using 2 independent models of vascular remodeling and reverse remodeling: hypoxia and cigarette smoke exposure. Hemodynamic measurements, right ventricular hypertrophy assessment, flow cytometry, and histological analysis of thick lung sections followed by state-of-the-art 3-dimensional reconstruction and quantification using Imaris software were used to investigate the contribution of GLI1+ cells to neomuscularization of the pulmonary vasculature. RESULTS: The data show that GLI1+ cells are abundant around distal, nonmuscularized vessels during steady state, and this lineage contributes to around 50% of newly formed ACTA2+ cells around these normally nonmuscularized vessels. During reverse remodeling, cells derived from the GLI1+ lineage are largely cleared in parallel to the reversal of muscularization. Partial ablation of GLI1+ cells greatly prevented vascular remodeling in response to hypoxia and attenuated the increase in right ventricular systolic pressure and right heart hypertrophy. Single-cell RNA sequencing on sorted lineage-labeled GLI1+ cells revealed an Acta2high fraction of cells with pathways in cancer and MAPK (mitogen-activated protein kinase) signaling as potential players in reprogramming these cells during vascular remodeling. Analysis of human lung-derived material suggests that GLI1 signaling is overactivated in both group 1 and group 3 pulmonary hypertension and can promote proliferation and myogenic differentiation. CONCLUSIONS: Our data highlight GLI1+ cells as an alternative cellular source of VSMCs in pulmonary hypertension and suggest that these cells and the associated signaling pathways represent an important therapeutic target for further studies.
Assuntos
Hipertensão Pulmonar , Remodelação Vascular , Proteína GLI1 em Dedos de Zinco , Animais , Proteína GLI1 em Dedos de Zinco/metabolismo , Proteína GLI1 em Dedos de Zinco/genética , Camundongos , Hipertensão Pulmonar/metabolismo , Hipertensão Pulmonar/fisiopatologia , Hipertensão Pulmonar/patologia , Músculo Liso Vascular/metabolismo , Músculo Liso Vascular/patologia , Miócitos de Músculo Liso/metabolismo , Miócitos de Músculo Liso/patologia , Camundongos Endogâmicos C57BL , Artéria Pulmonar/metabolismo , Artéria Pulmonar/patologia , Artéria Pulmonar/fisiopatologia , Camundongos Transgênicos , Masculino , Humanos , Hipóxia/metabolismo , Hipóxia/fisiopatologiaRESUMO
Transcription factor RBPJ is the central component in Notch signal transduction and directly forms a coactivator complex together with the Notch intracellular domain (NICD). While RBPJ protein levels remain constant in most tissues, dynamic expression of Notch target genes varies depending on the given cell-type and the Notch activity state. To elucidate dynamic RBPJ binding genome-wide, we investigated RBPJ occupancy by ChIP-Seq. Surprisingly, only a small set of the total RBPJ sites show a dynamic binding behavior in response to Notch signaling. Compared to static RBPJ sites, dynamic sites differ in regard to their chromatin state, binding strength and enhancer positioning. Dynamic RBPJ sites are predominantly located distal to transcriptional start sites (TSSs), while most static sites are found in promoter-proximal regions. Importantly, gene responsiveness is preferentially associated with dynamic RBPJ binding sites and this static and dynamic binding behavior is repeatedly observed across different cell types and species. Based on the above findings we used a machine-learning algorithm to predict Notch responsiveness with high confidence in different cellular contexts. Our results strongly support the notion that the combination of binding strength and enhancer positioning are indicative of Notch responsiveness.
Assuntos
Elementos Facilitadores Genéticos , Receptores Notch , Animais , Humanos , Camundongos , Sítios de Ligação , Cromatina/metabolismo , Cromatina/genética , Sequenciamento de Cromatina por Imunoprecipitação , Regulação da Expressão Gênica , Genômica/métodos , Aprendizado de Máquina , Regiões Promotoras Genéticas , Ligação Proteica , Receptores Notch/metabolismo , Receptores Notch/genética , Transdução de Sinais/genética , Sítio de Iniciação de TranscriçãoRESUMO
How cytokine-driven changes in chromatin topology are converted into gene regulatory circuits during inflammation still remains unclear. Here, we show that interleukin (IL)-1α induces acute and widespread changes in chromatin accessibility via the TAK1 kinase and NF-κB at regions that are highly enriched for inflammatory disease-relevant SNPs. Two enhancers in the extended chemokine locus on human chromosome 4 regulate the IL-1α-inducible IL8 and CXCL1-3 genes. Both enhancers engage in dynamic spatial interactions with gene promoters in an IL-1α/TAK1-inducible manner. Microdeletions of p65-binding sites in either of the two enhancers impair NF-κB recruitment, suppress activation and biallelic transcription of the IL8/CXCL2 genes, and reshuffle higher-order chromatin interactions as judged by i4C interactome profiles. Notably, these findings support a dominant role of the IL8 "master" enhancer in the regulation of sustained IL-1α signaling, as well as for IL-8 and IL-6 secretion. CRISPR-guided transactivation of the IL8 locus or cross-TAD regulation by TNFα-responsive enhancers in a different model locus supports the existence of complex enhancer hierarchies in response to cytokine stimulation that prime and orchestrate proinflammatory chromatin responses downstream of NF-κB.
Assuntos
Cromatina/metabolismo , Elementos Facilitadores Genéticos/genética , Regulação da Expressão Gênica/efeitos dos fármacos , Mediadores da Inflamação/metabolismo , Interleucina-1alfa/farmacologia , MAP Quinase Quinase Quinases/metabolismo , NF-kappa B/metabolismo , Sítios de Ligação , Células Cultivadas , Quimiocinas/metabolismo , Cromatina/química , Cromatina/genética , Células HeLa , Humanos , MAP Quinase Quinase Quinases/genética , NF-kappa B/genética , Transdução de Sinais , Fator de Necrose Tumoral alfa/farmacologiaRESUMO
Idiopathic pulmonary fibrosis (IPF) is a chronic human disease with persistent destruction of lung parenchyma. Transforming growth factor-ß1 (TGF-ß1) signaling plays a pivotal role in the initiation and pathogenesis of IPF. As shown herein, TGF-ß1 signaling down-regulated not only peroxisome biogenesis but also the metabolism of these organelles in human IPF fibroblasts. In vitro cell culture observations in human fibroblasts and human lung tissue indicated that peroxisomal biogenesis and metabolic proteins were significantly down-regulated in the lung of 1-month-old transgenic mice expressing a constitutively active TGF-ß type I receptor kinase (ALK5). The peroxisome biogenesis protein peroxisomal membrane protein Pex13p (PEX13p) as well as the peroxisomal lipid metabolic enzyme peroxisomal acyl-coenzyme A oxidase 1 (ACOX1) and antioxidative enzyme catalase were highly up-regulated in TGF-ß type II receptor and Smad3 knockout mice. This study reports a novel mechanism of peroxisome biogenesis and metabolic regulation via TGF-ß1-Smad signaling: interaction of the Smad3 transcription factor with the PEX13 gene in chromatin immunoprecipitation-on-chip assay as well as in a bleomycin-induced pulmonary fibrosis model applied to TGF-ß type II receptor knockout mice. Taken together, data from this study suggest that TGF-ß1 participates in regulation of peroxisomal biogenesis and metabolism via Smad-dependent signaling, opening up novel strategies for the development of therapeutic approaches to inhibit progression of pulmonary fibrosis patients with IPF.
Assuntos
Fibrose Pulmonar Idiopática , Fator de Crescimento Transformador beta1 , Camundongos , Animais , Humanos , Lactente , Fator de Crescimento Transformador beta1/metabolismo , Camundongos Transgênicos , Receptor do Fator de Crescimento Transformador beta Tipo II/genética , Receptor do Fator de Crescimento Transformador beta Tipo II/metabolismo , Fibrose Pulmonar Idiopática/induzido quimicamente , Fibrose Pulmonar Idiopática/genética , Fibrose Pulmonar Idiopática/metabolismo , Pulmão/patologia , Bleomicina/efeitos adversos , Fibroblastos/metabolismo , Camundongos KnockoutRESUMO
Signal transduction pathways often involve transcription factors that promote activation of defined target gene sets. The transcription factor RBPJ is the central player in Notch signaling and either forms an activator complex with the Notch intracellular domain (NICD) or a repressor complex with corepressors like KYOT2/FHL1. The balance between these two antagonizing RBPJ-complexes depends on the activation state of the Notch receptor regulated by cell-to-cell interaction, ligand binding and proteolytic cleavage events. Here, we depleted RBPJ in mature T-cells lacking active Notch signaling and performed RNA-Seq, ChIP-Seq and ATAC-seq analyses. RBPJ depletion leads to upregulation of many Notch target genes. Ectopic expression of NICD1 activates several Notch target genes and enhances RBPJ occupancy. Based on gene expression changes and RBPJ occupancy we define four different clusters, either RBPJ- and/or Notch-regulated genes. Importantly, we identify early (Hes1 and Hey1) and late Notch-responsive genes (IL2ra). Similarly, to RBPJ depletion, interfering with transcriptional repression by squelching with cofactor KYOT2/FHL1, leads to upregulation of Notch target genes. Taken together, RBPJ is not only an essential part of the Notch co-activator complex but also functions as a repressor in a Notch-independent manner.
Assuntos
Proteína de Ligação a Sequências Sinal de Recombinação J de Imunoglobina , Receptores Notch , Linfócitos T , Regulação da Expressão Gênica , Proteína de Ligação a Sequências Sinal de Recombinação J de Imunoglobina/genética , Proteína de Ligação a Sequências Sinal de Recombinação J de Imunoglobina/metabolismo , Receptores Notch/genética , Receptores Notch/metabolismo , Transdução de Sinais , Linfócitos T/metabolismoRESUMO
The Notch pathway transmits signals between neighboring cells to elicit downstream transcriptional programs. Notch is a major regulator of cell fate specification, proliferation, and apoptosis, such that aberrant signaling leads to a pleiotropy of human diseases, including developmental disorders and cancers. The pathway signals through the transcription factor CSL (RBPJ in mammals), which forms an activation complex with the intracellular domain of the Notch receptor and the coactivator Mastermind. CSL can also function as a transcriptional repressor by forming complexes with one of several different corepressor proteins, such as FHL1 or SHARP in mammals and Hairless in Drosophila. Recently, we identified L3MBTL3 as a bona fide RBPJ-binding corepressor that recruits the repressive lysine demethylase LSD1/KDM1A to Notch target genes. Here, we define the RBPJ-interacting domain of L3MBTL3 and report the 2.06 Å crystal structure of the RBPJ-L3MBTL3-DNA complex. The structure reveals that L3MBTL3 interacts with RBPJ via an unusual binding motif compared to other RBPJ binding partners, which we comprehensively analyze with a series of structure-based mutants. We also show that these disruptive mutations affect RBPJ and L3MBTL3 function in cells, providing further insights into Notch mediated transcriptional regulation.
Assuntos
Proteínas de Ligação a DNA , Regulação da Expressão Gênica , Proteína de Ligação a Sequências Sinal de Recombinação J de Imunoglobina , Animais , Humanos , Proteínas de Ligação a DNA/metabolismo , Epigênese Genética , Histona Desmetilases/genética , Proteína de Ligação a Sequências Sinal de Recombinação J de Imunoglobina/genética , Proteína de Ligação a Sequências Sinal de Recombinação J de Imunoglobina/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/genética , Proteínas com Domínio LIM/metabolismo , Proteínas Musculares/genética , Ligação Proteica , Receptores Notch/genética , Receptores Notch/metabolismoRESUMO
Adaptation of bacteria to changes in their environment is often accomplished by changes of the transcriptome. While we learned a lot on the impact of transcriptional regulation in bacterial adaptation over the last decades, much less is known on the role of ribonucleases. This study demonstrates an important function of the endoribonuclease RNase E in the adaptation to different growth conditions. It was shown previously that RNase E activity does not influence the doubling time of the facultative phototroph Rhodobacter sphaeroides during chemotrophic growth, however, it has a strong impact on phototrophic growth. To better understand the impact of RNase E on phototrophic growth, we now quantified gene expression by RNA-seq and mapped 5' ends during chemotrophic growth under high oxygen or low oxygen levels and during phototrophic growth in the wild type and a mutant expressing a thermosensitive RNase E. Based on the RNase E-dependent expression pattern, the RNAs could be grouped into different classes. A strong effect of RNase E on levels of RNAs for photosynthesis genes was observed, in agreement with poor growth under photosynthetic conditions. RNase E cleavage sites and 5' ends enriched in the rnets mutant were differently distributed among the gene classes. Furthermore, RNase E affects the level of RNAs for important transcription factors thus indirectly affecting the expression of their regulons. As a consequence, RNase E has an important role in the adaptation of R. sphaeroides to different growth conditions. [Figure: see text].
Assuntos
Proteínas de Bactérias , Endorribonucleases , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Endorribonucleases/genética , Bactérias/metabolismo , OxigênioRESUMO
Given the intimate link between inflammation and dysregulated cell proliferation in cancer, we investigated cytokine-triggered gene expression in different cell cycle stages. Transcriptome analysis revealed that G1 release through cyclin-dependent kinase 6 (CDK6) and CDK4 primes and cooperates with the cytokine-driven gene response. CDK6 physically and functionally interacts with the NF-κB subunit p65 in the nucleus and is found at promoters of many transcriptionally active NF-κB target genes. CDK6 recruitment to distinct chromatin regions of inflammatory genes was essential for proper loading of p65 to its cognate binding sites and for the function of p65 coactivators, such as TRIP6. Furthermore, cytokine-inducible nuclear translocation and chromatin association of CDK6 depends on the kinase activity of TAK1 and p38. These results have widespread biological implications, as aberrant CDK6 expression or activation that is frequently observed in human tumors modulates NF-κB to shape the cytokine and chemokine repertoires in chronic inflammation and cancer.
Assuntos
Cromatina/metabolismo , Quinase 6 Dependente de Ciclina/fisiologia , NF-kappa B/genética , Ciclo Celular/genética , Quinase 4 Dependente de Ciclina/genética , Quinase 4 Dependente de Ciclina/metabolismo , Quinase 4 Dependente de Ciclina/fisiologia , Quinase 6 Dependente de Ciclina/análise , Quinase 6 Dependente de Ciclina/metabolismo , Regulação da Expressão Gênica , Células HeLa , Humanos , Interleucina-1/metabolismo , Interleucina-1/fisiologia , Interleucina-8/genética , Interleucina-8/metabolismo , MAP Quinase Quinase Quinases/genética , MAP Quinase Quinase Quinases/metabolismo , MAP Quinase Quinase Quinases/fisiologia , Regiões Promotoras Genéticas , Fator de Transcrição RelA/genética , Fator de Transcrição RelA/metabolismo , Fator de Transcrição RelA/fisiologia , Fator de Necrose Tumoral alfa/metabolismo , Fator de Necrose Tumoral alfa/fisiologia , Proteínas Quinases p38 Ativadas por Mitógeno/genética , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo , Proteínas Quinases p38 Ativadas por Mitógeno/fisiologiaRESUMO
Aberrant Notch signaling plays a pivotal role in T-cell acute lymphoblastic leukemia (T-ALL) and chronic lymphocytic leukemia (CLL). Amplitude and duration of the Notch response is controlled by ubiquitin-dependent proteasomal degradation of the Notch1 intracellular domain (NICD1), a hallmark of the leukemogenic process. Here, we show that HDAC3 controls NICD1 acetylation levels directly affecting NICD1 protein stability. Either genetic loss-of-function of HDAC3 or nanomolar concentrations of HDAC inhibitor apicidin lead to downregulation of Notch target genes accompanied by a local reduction of histone acetylation. Importantly, an HDAC3-insensitive NICD1 mutant is more stable but biologically less active. Collectively, these data show a new HDAC3- and acetylation-dependent mechanism that may be exploited to treat Notch1-dependent leukemias.
Assuntos
Histona Desacetilases/metabolismo , Leucemia/metabolismo , Receptor Notch1/metabolismo , Transdução de Sinais , Animais , Linhagem Celular , Linhagem Celular Tumoral , Inibidores de Histona Desacetilases/farmacologia , Humanos , Leucemia/enzimologia , Lisina/metabolismo , Camundongos , Mutação , Peptídeos Cíclicos/farmacologia , Estabilidade Proteica , Receptor Notch1/química , Receptor Notch1/genéticaRESUMO
Urinary tract infections are common and costly diseases affecting millions of people. Uropathogenic Escherichia coli (UPEC) is a primary cause of these infections and has developed multiple strategies to avoid the host immune response. Here, we dissected the molecular mechanisms underpinning UPEC inhibition of inflammatory cytokine in vitro and in vivo. We found that UPEC infection simulates nuclear factor-κB activation but does not result in transcription of cytokine genes. Instead, UPEC-mediated suppression of the metabolic enzyme ATP citrate lyase results in decreased acetyl-CoA levels, leading to reduced H3K9 histone acetylation in the promotor region of CXCL8. These effects were dependent on the UPEC virulence factor α-hemolysin and were reversed by exogenous acetate. In a murine cystitis model, prior acetate supplementation rapidly resolved UPEC-elicited immune responses and improved tissue recovery. Thus, upon infection, UPEC rearranges host cell metabolism to induce chromatin remodeling processes that subvert expression of host innate immune response genes.
Assuntos
Citocinas/imunologia , Infecções por Escherichia coli , Proteínas Hemolisinas , Infecções Urinárias , Escherichia coli Uropatogênica , Acetilação , Animais , Citocinas/genética , Infecções por Escherichia coli/imunologia , Proteínas de Escherichia coli/metabolismo , Proteínas Hemolisinas/metabolismo , Histonas/metabolismo , Interações Hospedeiro-Patógeno , Humanos , Imunidade Inata , Camundongos , Infecções Urinárias/imunologia , Escherichia coli Uropatogênica/metabolismo , Fatores de Virulência/metabolismoRESUMO
Notch signaling is an evolutionarily conserved signal transduction pathway that is essential for metazoan development. Upon ligand binding, the Notch intracellular domain (NOTCH ICD) translocates into the nucleus and forms a complex with the transcription factor RBPJ (also known as CBF1 or CSL) to activate expression of Notch target genes. In the absence of a Notch signal, RBPJ acts as a transcriptional repressor. Using a proteomic approach, we identified L3MBTL3 (also known as MBT1) as a novel RBPJ interactor. L3MBTL3 competes with NOTCH ICD for binding to RBPJ In the absence of NOTCH ICD, RBPJ recruits L3MBTL3 and the histone demethylase KDM1A (also known as LSD1) to the enhancers of Notch target genes, leading to H3K4me2 demethylation and to transcriptional repression. Importantly, in vivo analyses of the homologs of RBPJ and L3MBTL3 in Drosophila melanogaster and Caenorhabditis elegans demonstrate that the functional link between RBPJ and L3MBTL3 is evolutionarily conserved, thus identifying L3MBTL3 as a universal modulator of Notch signaling in metazoans.
Assuntos
Proteínas de Ligação a DNA/genética , Proteínas de Drosophila/genética , Histona Desmetilases/genética , Proteína de Ligação a Sequências Sinal de Recombinação J de Imunoglobina/genética , Neuroglia/metabolismo , Receptores Notch/genética , Animais , Evolução Biológica , Caenorhabditis elegans/genética , Caenorhabditis elegans/metabolismo , Linhagem Celular Tumoral , Sequência Conservada , Proteínas de Ligação a DNA/metabolismo , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/genética , Drosophila melanogaster/metabolismo , Regulação da Expressão Gênica , Histona Desmetilases/metabolismo , Histonas/genética , Histonas/metabolismo , Humanos , Proteína de Ligação a Sequências Sinal de Recombinação J de Imunoglobina/metabolismo , Neuroglia/citologia , Ligação Proteica , Domínios Proteicos , Receptores Notch/metabolismo , Transcrição Gênica , Técnicas do Sistema de Duplo-HíbridoRESUMO
Histone variants differ in amino acid sequence, expression timing and genomic localization sites from canonical histones and convey unique functions to eukaryotic cells. Their tightly controlled spatial and temporal deposition into specific chromatin regions is accomplished by dedicated chaperone and/or remodeling complexes. While quantitatively identifying the chaperone complexes of many human H2A variants by using mass spectrometry, we also found additional members of the known H2A.Z chaperone complexes p400/TIP60/NuA4 and SRCAP. We discovered JAZF1, a nuclear/nucleolar protein, as a member of a p400 sub-complex containing MBTD1 but excluding ANP32E. Depletion of JAZF1 results in transcriptome changes that affect, among other pathways, ribosome biogenesis. To identify the underlying molecular mechanism contributing to JAZF1's function in gene regulation, we performed genome-wide ChIP-seq analyses. Interestingly, depletion of JAZF1 leads to reduced H2A.Z acetylation levels at > 1000 regulatory sites without affecting H2A.Z nucleosome positioning. Since JAZF1 associates with the histone acetyltransferase TIP60, whose depletion causes a correlated H2A.Z deacetylation of several JAZF1-targeted enhancer regions, we speculate that JAZF1 acts as chromatin modulator by recruiting TIP60's enzymatic activity. Altogether, this study uncovers JAZF1 as a member of a TIP60-containing p400 chaperone complex orchestrating H2A.Z acetylation at regulatory regions controlling the expression of genes, many of which are involved in ribosome biogenesis.
Assuntos
Proteínas Correpressoras/metabolismo , Proteínas de Ligação a DNA/metabolismo , Histonas/metabolismo , Sequências Reguladoras de Ácido Nucleico , Acetilação , Linhagem Celular , Montagem e Desmontagem da Cromatina , Biologia Computacional/métodos , DNA Helicases/metabolismo , Elementos Facilitadores Genéticos , Regulação da Expressão Gênica , Genômica/métodos , Humanos , Íntrons , Lisina Acetiltransferase 5/metabolismo , Chaperonas Moleculares/metabolismo , Complexos Multiproteicos , Ligação Proteica , Ribossomos , Fatores de Transcrição/metabolismoRESUMO
In mammalian cells, the MYC oncoprotein binds to thousands of promoters. During mitogenic stimulation of primary lymphocytes, MYC promotes an increase in the expression of virtually all genes. In contrast, MYC-driven tumour cells differ from normal cells in the expression of specific sets of up- and downregulated genes that have considerable prognostic value. To understand this discrepancy, we studied the consequences of inducible expression and depletion of MYC in human cells and murine tumour models. Changes in MYC levels activate and repress specific sets of direct target genes that are characteristic of MYC-transformed tumour cells. Three factors account for this specificity. First, the magnitude of response parallels the change in occupancy by MYC at each promoter. Functionally distinct classes of target genes differ in the E-box sequence bound by MYC, suggesting that different cellular responses to physiological and oncogenic MYC levels are controlled by promoter affinity. Second, MYC both positively and negatively affects transcription initiation independent of its effect on transcriptional elongation. Third, complex formation with MIZ1 (also known as ZBTB17) mediates repression of multiple target genes by MYC and the ratio of MYC and MIZ1 bound to each promoter correlates with the direction of response.
Assuntos
Regulação para Baixo/genética , Regulação Neoplásica da Expressão Gênica/genética , Genes myc/genética , Neoplasias/genética , Transcriptoma , Regulação para Cima/genética , Animais , Sítios de Ligação , Linhagem Celular Tumoral , Elementos E-Box/genética , Humanos , Fatores de Transcrição Kruppel-Like/metabolismo , Camundongos , Proteínas Nucleares/metabolismo , Regiões Promotoras Genéticas/genética , Proteínas Inibidoras de STAT Ativados/metabolismo , Proteínas Proto-Oncogênicas c-myc/genética , Proteínas Proto-Oncogênicas c-myc/metabolismo , RNA Polimerase II/metabolismo , Ubiquitina-Proteína LigasesRESUMO
The two paralogous zinc finger factors CTCF and CTCFL differ in expression such that CTCF is ubiquitously expressed, whereas CTCFL is found during spermatogenesis and in some cancer types in addition to other cell types. Both factors share the highly conserved DNA binding domain and are bound to DNA sequences with an identical consensus. In contrast, both factors differ substantially in the number of bound sites in the genome. Here, we addressed the molecular features for this binding specificity. In contrast to CTCF we found CTCFL highly enriched at 'open' chromatin marked by H3K27 acetylation, H3K4 di- and trimethylation, H3K79 dimethylation and H3K9 acetylation plus the histone variant H2A.Z. CTCFL is enriched at transcriptional start sites and regions bound by transcription factors. Consequently, genes deregulated by CTCFL are highly cell specific. In addition to a chromatin-driven choice of binding sites, we determined nucleotide positions critical for DNA binding by CTCFL, but not by CTCF.
Assuntos
Fator de Ligação a CCCTC/metabolismo , Cromatina/metabolismo , Proteínas de Ligação a DNA/metabolismo , Animais , Sequência de Bases , Sítios de Ligação , Linhagem Celular , DNA/química , Humanos , Células K562 , Camundongos , Células NIH 3T3RESUMO
A fundamental as yet incompletely understood feature of Notch signal transduction is a transcriptional shift from repression to activation that depends on chromatin regulation mediated by transcription factor RBP-J and associated cofactors. Incorporation of histone variants alter the functional properties of chromatin and are implicated in the regulation of gene expression. Here, we show that depletion of histone variant H2A.Z leads to upregulation of canonical Notch target genes and that the H2A.Z-chaperone TRRAP/p400/Tip60 complex physically associates with RBP-J at Notch-dependent enhancers. When targeted to RBP-J-bound enhancers, the acetyltransferase Tip60 acetylates H2A.Z and upregulates Notch target gene expression. Importantly, the Drosophila homologs of Tip60, p400 and H2A.Z modulate Notch signaling response and growth in vivo. Together, our data reveal that loading and acetylation of H2A.Z are required to assure tight control of canonical Notch activation.
Assuntos
Regulação da Expressão Gênica , Histonas/genética , Receptores Notch/genética , Transdução de Sinais/genética , Acetilação , Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Adenosina Trifosfatases/genética , Adenosina Trifosfatases/metabolismo , Animais , Linhagem Celular , Linhagem Celular Tumoral , Células HEK293 , Células HeLa , Histonas/metabolismo , Humanos , Proteína de Ligação a Sequências Sinal de Recombinação J de Imunoglobina/genética , Proteína de Ligação a Sequências Sinal de Recombinação J de Imunoglobina/metabolismo , Lisina Acetiltransferase 5/genética , Lisina Acetiltransferase 5/metabolismo , Camundongos Knockout , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Receptores Notch/metabolismoRESUMO
Coronavirus replication takes place in the host cell cytoplasm and triggers inflammatory gene expression by poorly characterized mechanisms. To obtain more insight into the signals and molecular events that coordinate global host responses in the nucleus of coronavirus-infected cells, first, transcriptome dynamics was studied in human coronavirus 229E (HCoV-229E)-infected A549 and HuH7 cells, respectively, revealing a core signature of upregulated genes in these cells. Compared to treatment with the prototypical inflammatory cytokine interleukin(IL)-1, HCoV-229E replication was found to attenuate the inducible activity of the transcription factor (TF) NF-κB and to restrict the nuclear concentration of NF-κB subunits by (i) an unusual mechanism involving partial degradation of IKKß, NEMO and IκBα and (ii) upregulation of TNFAIP3 (A20), although constitutive IKK activity and basal TNFAIP3 expression levels were shown to be required for efficient virus replication. Second, we characterized actively transcribed genomic regions and enhancers in HCoV-229E-infected cells and systematically correlated the genome-wide gene expression changes with the recruitment of Ser5-phosphorylated RNA polymerase II and prototypical histone modifications (H3K9ac, H3K36ac, H4K5ac, H3K27ac, H3K4me1). The data revealed that, in HCoV-infected (but not IL-1-treated) cells, an extensive set of genes was activated without inducible p65 NF-κB being recruited. Furthermore, both HCoV-229E replication and IL-1 were shown to upregulate a small set of genes encoding immunomodulatory factors that bind p65 at promoters and require IKKß activity and p65 for expression. Also, HCoV-229E and IL-1 activated a common set of 440 p65-bound enhancers that differed from another 992 HCoV-229E-specific enhancer regions by distinct TF-binding motif combinations. Taken together, the study shows that cytoplasmic RNA viruses fine-tune NF-κB signaling at multiple levels and profoundly reprogram the host cellular chromatin landscape, thereby orchestrating the timely coordinated expression of genes involved in multiple signaling, immunoregulatory and metabolic processes.
Assuntos
Cromatina/fisiologia , Coronavirus Humano 229E , Infecções por Coronavirus/genética , NF-kappa B/metabolismo , Transcriptoma , Linhagem Celular , Imunoprecipitação da Cromatina , Regulação da Expressão Gênica , Humanos , Immunoblotting , Microdissecção e Captura a Laser , Microscopia de Fluorescência , Análise de Sequência com Séries de Oligonucleotídeos , Reação em Cadeia da PolimeraseRESUMO
Centrosomal 190 kDa protein (CP190) is a promoter binding factor, mediates long-range interactions in the context of enhancer-promoter contacts and in chromosomal domain formation. All Drosophila insulator proteins bind CP190 suggesting a crucial role in insulator function. CP190 has major effects on chromatin, such as depletion of nucleosomes, high nucleosomal turnover and prevention of heterochromatin expansion. Here, we searched for enzymes, which might be involved in CP190 mediated chromatin changes. Eighty percent of the genomic binding sites of the histone acetyltransferase Gcn5 are colocalizing with CP190 binding. Depletion of CP190 reduces Gcn5 binding to chromatin. Binding dependency was further supported by Gcn5 mediated co-precipitation of CP190. Gcn5 is known to activate transcription by histone acetylation. We used the dCas9 system to target CP190 or Gcn5 to a Polycomb repressed and H3K27me3 marked gene locus. Both, CP190 as well as Gcn5, activate this locus, thus supporting the model that CP190 recruits Gcn5 and thereby activates chromatin.
Assuntos
Cromatina/metabolismo , Proteínas de Drosophila/metabolismo , Histona Acetiltransferases/metabolismo , Proteínas Associadas aos Microtúbulos/metabolismo , Proteínas Nucleares/metabolismo , Ativação Transcricional , Animais , Sítios de Ligação , Células Cultivadas , Drosophila/genética , Drosophila/metabolismoRESUMO
Histone chaperones prevent promiscuous histone interactions before chromatin assembly. They guarantee faithful deposition of canonical histones and functionally specialized histone variants into chromatin in a spatial- and temporally-restricted manner. Here, we identify the binding partners of the primate-specific and H3.3-related histone variant H3.Y using several quantitative mass spectrometry approaches, and biochemical and cell biological assays. We find the HIRA, but not the DAXX/ATRX, complex to recognize H3.Y, explaining its presence in transcriptionally active euchromatic regions. Accordingly, H3.Y nucleosomes are enriched in the transcription-promoting FACT complex and depleted of repressive post-translational histone modifications. H3.Y mutational gain-of-function screens reveal an unexpected combinatorial amino acid sequence requirement for histone H3.3 interaction with DAXX but not HIRA, and for H3.3 recruitment to PML nuclear bodies. We demonstrate the importance and necessity of specific H3.3 core and C-terminal amino acids in discriminating between distinct chaperone complexes. Further, chromatin immunoprecipitation sequencing experiments reveal that in contrast to euchromatic HIRA-dependent deposition sites, human DAXX/ATRX-dependent regions of histone H3 variant incorporation are enriched in heterochromatic H3K9me3 and simple repeat sequences. These data demonstrate that H3.Y's unique amino acids allow a functional distinction between HIRA and DAXX binding and its consequent deposition into open chromatin.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas de Ciclo Celular/genética , Chaperonas de Histonas/genética , Código das Histonas , Histonas/genética , Proteínas Nucleares/genética , Fatores de Transcrição/genética , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Sequência de Aminoácidos , Animais , Sítios de Ligação , Proteínas de Ciclo Celular/metabolismo , Linhagem Celular Transformada , Cromatina/química , Cromatina/metabolismo , Proteínas Correpressoras , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Células Epiteliais/citologia , Células Epiteliais/metabolismo , Células HeLa , Proteínas de Grupo de Alta Mobilidade/genética , Proteínas de Grupo de Alta Mobilidade/metabolismo , Chaperonas de Histonas/metabolismo , Histonas/metabolismo , Humanos , Células-Tronco Mesenquimais/citologia , Células-Tronco Mesenquimais/metabolismo , Repetições de Microssatélites , Chaperonas Moleculares , Proteínas Nucleares/metabolismo , Nucleossomos/genética , Nucleossomos/metabolismo , Cultura Primária de Células , Ligação Proteica , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Fatores de Transcrição/metabolismo , Transcrição Gênica , Fatores de Elongação da Transcrição/genética , Fatores de Elongação da Transcrição/metabolismoRESUMO
Insulators are multiprotein-DNA complexes that regulate the nuclear architecture. The Drosophila CP190 protein is a cofactor for the DNA-binding insulator proteins Su(Hw), CTCF, and BEAF-32. The fact that CP190 has been found at genomic sites devoid of either of the known insulator factors has until now been unexplained. We have identified two DNA-binding zinc-finger proteins, Pita, and a new factor named ZIPIC, that interact with CP190 in vivo and in vitro at specific interaction domains. Genomic binding sites for these proteins are clustered with CP190 as well as with CTCF and BEAF-32. Model binding sites for Pita or ZIPIC demonstrate a partial enhancer-blocking activity and protect gene expression from PRE-mediated silencing. The function of the CTCF-bound MCP insulator sequence requires binding of Pita. These results identify two new insulator proteins and emphasize the unifying function of CP190, which can be recruited by many DNA-binding insulator proteins.
Assuntos
Cromatina/genética , Proteínas de Ligação a DNA/metabolismo , Proteínas de Drosophila/metabolismo , Proteínas Associadas aos Microtúbulos/metabolismo , Proteínas Nucleares/metabolismo , Fatores de Transcrição/metabolismo , Dedos de Zinco , Animais , Sítios de Ligação , Cromatina/metabolismo , Mapeamento Cromossômico , Proteínas de Ligação a DNA/genética , Drosophila/citologia , Drosophila/genética , Proteínas de Drosophila/genética , Expressão Gênica , Estudos de Associação Genética , Genômica , Proteínas Associadas aos Microtúbulos/genética , Proteínas Nucleares/genética , Regiões Promotoras Genéticas , Fatores de Transcrição/genéticaRESUMO
Much of spermatid differentiation takes place in the absence of active transcription, but in the early phase, large amounts of mRNA are synthesized, translationally repressed, and stored. Most nucleosomal histones are then degraded, and chromatin is repackaged by protamines. For both transcription and the histone-to-protamine transition in differentiating spermatids, chromatin must be opened. This raises the question of whether two different processes exist. It is conceivable that for initiation of the histone-to-protamine transition, the already accessible, actively transcribed chromatin regions are utilized or vice versa. We analyzed the enrichment of different canonical TATA-box-binding, protein-associated factors and their variants in murine spermatids, diverse bromodomain-containing proteins, and components of the Polycomb repressive complexes PRC1 and PRC2 using quantitative PCR. We compared the enrichment of corresponding proteins in human and murine spermatids and analyzed the time frame of postmeiotic transcription and expression of histones, transition proteins, and protamines in human and murine spermatids using immunohistology. We correlated the expression of different transcription factors and bromodomain-containing proteins and the pattern of acetylated histones to active transcription and to the histone-to-protamine transition in both human and murine spermatids. Our findings suggest that differentiating spermatids use both common and specific features to open chromatin first for transcription and subsequently for histone-to-protamine transition.