RESUMO
Histone posttranslational modifications (PTMs) have crucial roles in a multitude of cellular processes, and their aberrant levels have been linked with numerous diseases, including cancer. Although histone PTM investigations have focused so far on methylations and acetylations, alternative long-chain acylations emerged as new dimension, as they are linked to cellular metabolic states and affect gene expression through mechanisms distinct from those regulated by acetylation. Mass spectrometry is the most powerful, comprehensive, and unbiased method to study histone PTMs. However, typical mass spectrometry-based protocols for histone PTM analysis do not allow the identification of naturally occurring propionylation and butyrylation. Here, we present improved state-of-the-art sample preparation and analysis protocols to quantitate these classes of modifications. After testing different derivatization methods coupled to protease digestion, we profiled common histone PTMs and histone acylations in seven mouse tissues and human normal and tumor breast clinical samples, obtaining a map of propionylations and butyrylations found in different tissue contexts. A quantitative histone PTM analysis also revealed a contribution of histone acylations in discriminating different tissues, also upon perturbation with antibiotics, and breast cancer samples from the normal counterpart. Our results show that profiling only classical modifications is limiting and highlight the importance of using sample preparation methods that allow the analysis of the widest possible spectrum of histone modifications, paving the way for deeper insights into their functional significance in cellular processes and disease states.
Assuntos
Neoplasias da Mama , Histonas , Processamento de Proteína Pós-Traducional , Histonas/metabolismo , Humanos , Animais , Camundongos , Neoplasias da Mama/metabolismo , Feminino , Espectrometria de Massas/métodos , Acilação , Especificidade de Órgãos , Acetilação , Proteômica/métodosRESUMO
BACKGROUND: The continuous proliferation of intestinal stem cells followed by their tightly regulated differentiation to epithelial cells is essential for the maintenance of the gut epithelial barrier and its functions. How these processes are tuned by diet and gut microbiome is an important, but poorly understood question. Dietary soluble fibers, such as inulin, are known for their ability to impact the gut bacterial community and gut epithelium, and their consumption has been usually associated with health improvement in mice and humans. In this study, we tested the hypothesis that inulin consumption modifies the composition of colonic bacteria and this impacts intestinal stem cells functions, thus affecting the epithelial structure. METHODS: Mice were fed with a diet containing 5% of the insoluble fiber cellulose or the same diet enriched with an additional 10% of inulin. Using a combination of histochemistry, host cell transcriptomics, 16S microbiome analysis, germ-free, gnotobiotic, and genetically modified mouse models, we analyzed the impact of inulin intake on the colonic epithelium, intestinal bacteria, and the local immune compartment. RESULTS: We show that the consumption of inulin diet alters the colon epithelium by increasing the proliferation of intestinal stem cells, leading to deeper crypts and longer colons. This effect was dependent on the inulin-altered gut microbiota, as no modulations were observed in animals deprived of microbiota, nor in mice fed cellulose-enriched diets. We also describe the pivotal role of γδ T lymphocytes and IL-22 in this microenvironment, as the inulin diet failed to induce epithelium remodeling in mice lacking this T cell population or cytokine, highlighting their importance in the diet-microbiota-epithelium-immune system crosstalk. CONCLUSION: This study indicates that the intake of inulin affects the activity of intestinal stem cells and drives a homeostatic remodeling of the colon epithelium, an effect that requires the gut microbiota, γδ T cells, and the presence of IL-22. Our study indicates complex cross kingdom and cross cell type interactions involved in the adaptation of the colon epithelium to the luminal environment in steady state. Video Abstract.
Assuntos
Microbioma Gastrointestinal , Inulina , Humanos , Animais , Camundongos , Inulina/farmacologia , Dieta , Fibras na Dieta , Celulose , Epitélio , Comunicação CelularRESUMO
Severe malaria can manifest itself with a variety of well-recognized clinical phenotypes that are highly predictive of death - severe anaemia, coma (cerebral malaria), multiple organ failure, and respiratory distress. The reasons why an infected individual develops one pathology rather than another remain poorly understood. Here we use distinct rodent models of infection to show that the host microbiota is a contributing factor for the development of respiratory distress syndrome and host mortality in the context of malaria infections (malaria-associated acute respiratory distress syndrome, MA-ARDS). We show that parasite sequestration in the lung results in sustained immune activation. Subsequent production of the anti-inflammatory cytokine IL-10 by T cells compromises microbial control, leading to severe lung disease. Notably, bacterial clearance with linezolid, an antibiotic commonly used in the clinical setting to control lung-associated bacterial infections, prevents MA-ARDS-associated lethality. Thus, we propose that the host's anti-inflammatory response to limit tissue damage can result in loss of microbial control, which promotes MA-ARDS. This must be considered when intervening against life-threatening respiratory complications.
Assuntos
Malária , Microbiota , Síndrome do Desconforto Respiratório , Animais , Modelos Animais de Doenças , Pulmão/patologia , Malária/complicações , Malária/parasitologia , Plasmodium berghei/fisiologiaRESUMO
Alternative histone acylations integrate gene expression with cellular metabolic states. Recent measurements of cellular acyl-coenzyme A (acyl-CoA) pools highlight the potential that histone post-translational modifications (PTMs) contribute directly to the regulation of metabolite pools. A metabolite-centric view throws new light onto roles and evolution of histone PTMs.
Assuntos
Cromatina , Histonas , Acil Coenzima A/metabolismo , Acilação , Histonas/metabolismo , Processamento de Proteína Pós-TraducionalRESUMO
BACKGROUND: How intestinal epithelial cells interact with the microbiota and how this is regulated at the gene expression level are critical questions. Smarcad1 is a conserved chromatin remodeling factor with a poorly understood tissue function. As this factor is highly expressed in the stem and proliferative zones of the intestinal epithelium, we explore its role in this tissue. RESULTS: Specific deletion of Smarcad1 in the mouse intestinal epithelium leads to colitis resistance and substantial changes in gene expression, including a striking increase of expression of several genes linked to innate immunity. Absence of Smarcad1 leads to changes in chromatin accessibility and significant changes in histone H3K9me3 over many sites, including genes that are differentially regulated upon Smarcad1 deletion. We identify candidate members of the gut microbiome that elicit a Smarcad1-dependent colitis response, including members of the poorly understood TM7 phylum. CONCLUSIONS: Our study sheds light onto the role of the chromatin remodeling machinery in intestinal epithelial cells in the colitis response and shows how a highly conserved chromatin remodeling factor has a distinct role in anti-microbial defense. This work highlights the importance of the intestinal epithelium in the colitis response and the potential of microbial species as pharmacological and probiotic targets in the context of inflammatory diseases.
Assuntos
Colite/genética , DNA Helicases/fisiologia , Regulação da Expressão Gênica , Mucosa Intestinal/metabolismo , Animais , Colite/microbiologia , DNA Helicases/genética , DNA Helicases/metabolismo , Deleção de Genes , Histonas/metabolismo , Camundongos , Microbiota , Elementos Reguladores de TranscriçãoRESUMO
BACKGROUND: The microbiota in the human gut are an important component of normal physiology that has co-evolved from the earliest multicellular organisms. Therefore, it is unsurprising that there is intimate crosstalk between the microbial world in the gut and the host. Genome regulation through microbiota-host interactions not only affects the host's immunity, but also metabolic health and resilience against cancer. Chromatin dynamics of the host epithelium involving histone modifications and other facets of the epigenetic machinery play an important role in this process. SCOPE OF REVIEW: This review discusses recent findings relevant to how chromatin dynamics shape the crosstalk between the microbiota and its host, with a special focus on the role of histone modifications. MAJOR CONCLUSIONS: Host-microbiome interactions are important evolutionary drivers and are thus expected to be hardwired into and mould the epigenetic machinery in multicellular organisms. Microbial-derived short-chain fatty acids (SCFA) are dominant determinants of microbiome-host interactions, and the inhibition of histone deacetylases (HDACs) by SCFA is a key mechanism in this process. The discovery of alternative histone acylations, such as crotonylation, in addition to the canonical histone acetylation reveals a new layer of complexity in this crosstalk.
Assuntos
Cromatina/metabolismo , Epigenoma/genética , Microbioma Gastrointestinal/genética , Cromatina/genética , Epigênese Genética/genética , Epigenômica/métodos , Microbioma Gastrointestinal/fisiologia , Código das Histonas , Histona Desacetilases/genética , Histonas/genética , Histonas/metabolismo , Interações entre Hospedeiro e Microrganismos/genética , Interações entre Hospedeiro e Microrganismos/fisiologia , Humanos , Microbiota/genéticaRESUMO
The intestinal epithelium undergoes constant regeneration driven by intestinal stem cells. How old age affects the transcriptome in this highly dynamic tissue is an important, but poorly explored question. Using transcriptomics on sorted intestinal stem cells and adult enterocytes, we identified candidate genes, which change expression on aging. Further validation of these on intestinal epithelium of multiple middle-aged versus old-aged mice highlighted the consistent up-regulation of the expression of the gene encoding chemokine receptor Ccr2, a mediator of inflammation and several disease processes. We observed also increased expression of Strc, coding for stereocilin, and dramatically decreased expression of Rps4l, coding for a ribosome subunit. Ccr2 and Rps4l are located close to the telomeric regions of chromosome 9 and 6, respectively. As only few genes were differentially expressed and we did not observe significant protein level changes of identified ageing markers, our analysis highlights the overall robustness of murine intestinal epithelium gene expression to old age.
Assuntos
Expressão Gênica/genética , Mucosa Intestinal/fisiologia , Intestinos/fisiologia , Transcriptoma/genética , Envelhecimento/genética , Animais , Enterócitos/fisiologia , Perfilação da Expressão Gênica/métodos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Células-Tronco/fisiologiaRESUMO
Antibiotic-induced dysbiosis is a key factor predisposing intestinal infection by Clostridium difficile. Here, we show that interventions that restore butyrate intestinal levels mitigate clinical and pathological features of C. difficile-induced colitis. Butyrate has no effect on C. difficile colonization or toxin production. However, it attenuates intestinal inflammation and improves intestinal barrier function in infected mice, as shown by reduced intestinal epithelial permeability and bacterial translocation, effects associated with the increased expression of components of intestinal epithelial cell tight junctions. Activation of the transcription factor HIF-1 in intestinal epithelial cells exerts a protective effect in C. difficile-induced colitis, and it is required for butyrate effects. We conclude that butyrate protects intestinal epithelial cells from damage caused by C. difficile toxins via the stabilization of HIF-1, mitigating local inflammatory response and systemic consequences of the infection.
Assuntos
Butiratos/administração & dosagem , Clostridioides difficile/patogenicidade , Colite/prevenção & controle , Fator 1 Induzível por Hipóxia/metabolismo , Administração Oral , Animais , Antibacterianos/farmacologia , Butiratos/farmacologia , Clostridioides difficile/metabolismo , Colite/etiologia , Colite/microbiologia , Células Epiteliais/citologia , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/metabolismo , Ácidos Graxos Voláteis/metabolismo , Humanos , Insulina/administração & dosagem , Mucosa Intestinal/citologia , Mucosa Intestinal/patologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Microbiota/efeitos dos fármacos , Permeabilidade/efeitos dos fármacos , Junções Íntimas/metabolismo , Toxinas Biológicas/toxicidade , Triglicerídeos/administração & dosagemRESUMO
Metastatic progression is a major cause of mortality in cervical cancers, but factors regulating migratory and pre-metastatic cell populations remain poorly understood. Here, we sought to assess whether a SUV39H1-low chromatin state promotes migratory cell populations in cervical cancers, using meta-analysis of data from The Cancer Genome Atlas (TCGA), immunohistochemistry, genomics and functional assays. Cervical cancer cells sorted based on migratory ability in vitro have low levels of SUV39H1 protein, and SUV39H1 knockdown in vitro enhanced cervical cancer cell migration. Further, TCGA SUV39H1-low tumours correlated with poor clinical outcomes and showed gene expression signatures of cell migration. SUV39H1 expression was examined within biopsies, and SUV39H1low cells within tumours also demonstrated migratory features. Next, to understand genome scale transcriptional and chromatin changes in migratory populations, cell populations sorted based on migration in vitro were examined using RNA-Seq, along with ChIP-Seq for H3K9me3, the histone mark associated with SUV39H1. Migrated populations showed SUV39H1-linked migratory gene expression signatures, along with broad depletion of H3K9me3 across gene promoters. We show for the first time that a SUV39H1-low chromatin state associates with, and promotes, migratory populations in cervical cancers. Our results posit SUV39H1-low cells as key populations for prognosis estimation and as targets for novel therapies.
Assuntos
Movimento Celular , Metiltransferases/fisiologia , Proteínas Repressoras/fisiologia , Neoplasias do Colo do Útero/patologia , Linhagem Celular Tumoral , Cromatina , Feminino , Técnicas de Silenciamento de Genes , Histonas/metabolismo , Humanos , Metiltransferases/genética , Metástase Neoplásica , Proteínas Repressoras/genética , Resultado do TratamentoRESUMO
Class I histone deacetylases (HDACs) are efficient histone decrotonylases, broadening the enzymatic spectrum of these important (epi-)genome regulators and drug targets. Here, we describe an in vitro approach to assaying class I HDACs with different acyl-histone substrates, including crotonylated histones and expand this to examine the effect of inhibitors and estimate kinetic constants.
RESUMO
BACKGROUND: Aging is characterized by loss of function of the adaptive immune system, but the underlying causes are poorly understood. To assess the molecular effects of aging on B cell development, we profiled gene expression and chromatin features genome-wide, including histone modifications and chromosome conformation, in bone marrow pro-B and pre-B cells from young and aged mice. RESULTS: Our analysis reveals that the expression levels of most genes are generally preserved in B cell precursors isolated from aged compared with young mice. Nonetheless, age-specific expression changes are observed at numerous genes, including microRNA encoding genes. Importantly, these changes are underpinned by multi-layered alterations in chromatin structure, including chromatin accessibility, histone modifications, long-range promoter interactions, and nuclear compartmentalization. Previous work has shown that differentiation is linked to changes in promoter-regulatory element interactions. We find that aging in B cell precursors is accompanied by rewiring of such interactions. We identify transcriptional downregulation of components of the insulin-like growth factor signaling pathway, in particular downregulation of Irs1 and upregulation of Let-7 microRNA expression, as a signature of the aged phenotype. These changes in expression are associated with specific alterations in H3K27me3 occupancy, suggesting that Polycomb-mediated repression plays a role in precursor B cell aging. CONCLUSIONS: Changes in chromatin and 3D genome organization play an important role in shaping the altered gene expression profile of aged precursor B cells. Components of the insulin-like growth factor signaling pathways are key targets of epigenetic regulation in aging in bone marrow B cell precursors.
Assuntos
Envelhecimento/genética , Linfócitos B/metabolismo , Cromatina/química , Epigênese Genética , Somatomedinas/fisiologia , Transcriptoma , Envelhecimento/imunologia , Animais , Linfócitos B/imunologia , Regulação para Baixo , Genoma , Masculino , Camundongos Endogâmicos C57BL , Transdução de Sinais/genética , Células-Tronco/imunologia , Células-Tronco/metabolismoRESUMO
The recently discovered histone post-translational modification crotonylation connects cellular metabolism to gene regulation. Its regulation and tissue-specific functions are poorly understood. We characterize histone crotonylation in intestinal epithelia and find that histone H3 crotonylation at lysine 18 is a surprisingly abundant modification in the small intestine crypt and colon, and is linked to gene regulation. We show that this modification is highly dynamic and regulated during the cell cycle. We identify class I histone deacetylases, HDAC1, HDAC2, and HDAC3, as major executors of histone decrotonylation. We show that known HDAC inhibitors, including the gut microbiota-derived butyrate, affect histone decrotonylation. Consistent with this, we find that depletion of the gut microbiota leads to a global change in histone crotonylation in the colon. Our results suggest that histone crotonylation connects chromatin to the gut microbiota, at least in part, via short-chain fatty acids and HDACs.
Assuntos
Crotonatos/metabolismo , Ácidos Graxos Voláteis/fisiologia , Histona Desacetilases/metabolismo , Histonas/metabolismo , Mucosa Intestinal/metabolismo , Acilação , Animais , Ciclo Celular , Colo/metabolismo , Colo/microbiologia , Microbioma Gastrointestinal , Células HCT116 , Inibidores de Histona Desacetilases , Humanos , Masculino , Camundongos Endogâmicos C57BL , Processamento de Proteína Pós-TraducionalRESUMO
Improved treatment for major depressive disorder (MDD) remains elusive because of the limited understanding of its underlying biological mechanisms. It is likely that stress-induced maladaptive transcriptional regulation in limbic neural circuits contributes to the development of MDD, possibly through epigenetic factors that regulate chromatin structure. We establish that persistent upregulation of the ACF (ATP-utilizing chromatin assembly and remodeling factor) ATP-dependent chromatin-remodeling complex, occurring in the nucleus accumbens of stress-susceptible mice and depressed humans, is necessary for stress-induced depressive-like behaviors. We found that altered ACF binding after chronic stress was correlated with altered nucleosome positioning, particularly around the transcription start sites of affected genes. These alterations in ACF binding and nucleosome positioning were associated with repressed expression of genes implicated in susceptibility to stress. Together, our findings identify the ACF chromatin-remodeling complex as a critical component in the development of susceptibility to depression and in regulating stress-related behaviors.
Assuntos
Montagem e Desmontagem da Cromatina , Depressão/metabolismo , Estresse Psicológico , Animais , Proteínas Cromossômicas não Histona , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Fatores de Transcrição/genética , Fatores de Transcrição/fisiologiaRESUMO
Endocytosis is essential for uptake of many substances into the cell, but how it links to nutritional signalling is poorly understood. Here, we show a new role for endocytosis in regulating the response to low phosphate in Schizosaccharomyces pombe. Loss of function of myosin I (Myo1), Sla2/End4 or Arp2, proteins involved in the early steps of endocytosis, led to increased proliferation in low-phosphate medium compared to controls. We show that once cells are deprived of phosphate they undergo a quiescence response that is dependent on the endocytic function of Myo1. Transcriptomic analysis revealed a wide perturbation of gene expression with induction of stress-regulated genes upon phosphate starvation in wild-type but not Δmyo1 cells. Thus, endocytosis plays a pivotal role in mediating the cellular response to nutrients, bridging the external environment and internal molecular functions of the cell.
Assuntos
Endocitose/fisiologia , Regulação Fúngica da Expressão Gênica/fisiologia , Cadeias Pesadas de Miosina/metabolismo , Fosfatos/metabolismo , Proteínas de Schizosaccharomyces pombe/metabolismo , Schizosaccharomyces/metabolismo , Transdução de Sinais/fisiologia , Proteína 2 Relacionada a Actina/genética , Proteína 2 Relacionada a Actina/metabolismo , Deleção de Genes , Cadeias Pesadas de Miosina/genética , Schizosaccharomyces/genética , Proteínas de Schizosaccharomyces pombe/genética , TranscriptomaRESUMO
Budding yeast centromeres are sequence-defined point centromeres and are, unlike in many other organisms, not embedded in heterochromatin. Here we show that Fun30, a poorly understood SWI/SNF-like chromatin remodeling factor conserved in humans, promotes point centromere function through the formation of correct chromatin architecture at centromeres. Our determination of the genome-wide binding and nucleosome positioning properties of Fun30 shows that this enzyme is consistently enriched over centromeres and that a majority of CENs show Fun30-dependent changes in flanking nucleosome position and/or CEN core micrococcal nuclease accessibility. Fun30 deletion leads to defects in histone variant Htz1 occupancy genome-wide, including at and around most centromeres. FUN30 genetically interacts with CSE4, coding for the centromere-specific variant of histone H3, and counteracts the detrimental effect of transcription through centromeres on chromosome segregation and suppresses transcriptional noise over centromere CEN3. Previous work has shown a requirement for fission yeast and mammalian homologs of Fun30 in heterochromatin assembly. As centromeres in budding yeast are not embedded in heterochromatin, our findings indicate a direct role of Fun30 in centromere chromatin by promoting correct chromatin architecture.
Assuntos
Centrômero/genética , Proteínas Cromossômicas não Histona , Proteínas de Ligação a DNA , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae , Fatores de Transcrição , Montagem e Desmontagem da Cromatina/genética , Proteínas Cromossômicas não Histona/genética , Proteínas Cromossômicas não Histona/metabolismo , Segregação de Cromossomos/genética , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Heterocromatina/genética , Histonas/genética , Humanos , Cinetocoros , Nucleossomos/genética , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismoRESUMO
Disruption of chromatin organization during replication poses a major challenge to the maintenance and integrity of genome organization. It creates the need to accurately reconstruct the chromatin landscape following DNA duplication but there is little mechanistic understanding of how chromatin based modifications are restored on newly synthesized DNA. ATP-dependent chromatin remodeling activities serve multiple roles during replication and recent work underscores their requirement in the maintenance of proper chromatin organization. A new component of chromatin replication, the SWI/SNF-like chromatin remodeler SMARCAD1, acts at replication sites to facilitate deacetylation of newly assembled histones. Deacetylation is a pre-requisite for the restoration of epigenetic signatures in heterochromatin regions following replication. In this way, SMARCAD1, in concert with histone modifying activities and transcriptional repressors, reinforces epigenetic instructions to ensure that silenced loci are correctly perpetuated in each replication cycle. The emerging concept is that remodeling of nucleosomes is an early event imperative to promote the re-establishment of histone modifications following DNA replication.
Assuntos
Cromatina/química , Reparo do DNA , Replicação do DNA , Regulação Enzimológica da Expressão Gênica , Heterocromatina/química , Nucleossomos/química , Acetilação , Trifosfato de Adenosina/química , Cromatina/genética , Montagem e Desmontagem da Cromatina , DNA Helicases/química , DNA Helicases/genética , Ativação Enzimática , Epigênese Genética , Inativação Gênica , Heterocromatina/genética , Histonas/química , Humanos , Nucleossomos/genética , Mapeamento de Interação de ProteínasRESUMO
Epigenetic marks such as posttranslational histone modifications specify the functional states of underlying DNA sequences, though how they are maintained after their disruption during DNA replication remains a critical question. We identify the mammalian SWI/SNF-like protein SMARCAD1 as a key factor required for the re-establishment of repressive chromatin. The ATPase activity of SMARCAD1 is necessary for global deacetylation of histones H3/H4. In this way, SMARCAD1 promotes methylation of H3K9, the establishment of heterochromatin, and faithful chromosome segregation. SMARCAD1 associates with transcriptional repressors including KAP1, histone deacetylases HDAC1/2 and the histone methyltransferase G9a/GLP and modulates the interaction of HDAC1 and KAP1 with heterochromatin. SMARCAD1 directly interacts with PCNA, a central component of the replication machinery, and is recruited to sites of DNA replication. Our findings suggest that chromatin remodeling by SMARCAD1 ensures that silenced loci, such as pericentric heterochromatin, are correctly perpetuated.