RESUMO
Studies in three mouse models of breast cancer identified profound discrepancies between cell-autonomous and systemic Akt1- or Akt2-inducible deletion on breast cancer tumorigenesis and metastasis. Although systemic Akt1 deletion inhibits metastasis, cell-autonomous Akt1 deletion does not. Single-cell mRNA sequencing revealed that systemic Akt1 deletion maintains the pro-metastatic cluster within primary tumors but ablates pro-metastatic neutrophils. Systemic Akt1 deletion inhibits metastasis by impairing survival and mobilization of tumor-associated neutrophils. Importantly, either systemic or neutrophil-specific Akt1 deletion is sufficient to inhibit metastasis of Akt-proficient tumors. Thus, Akt1-specific inhibition could be therapeutic for breast cancer metastasis regardless of primary tumor origin. Systemic Akt2 deletion does not inhibit and exacerbates mammary tumorigenesis and metastasis, but cell-autonomous Akt2 deletion prevents breast cancer tumorigenesis by ErbB2. Elevated circulating insulin level induced by Akt2 systemic deletion hyperactivates tumor Akt, exacerbating ErbB2-mediated tumorigenesis, curbed by pharmacological reduction of the elevated insulin.
Assuntos
Neoplasias Mamárias Animais/enzimologia , Neoplasias Mamárias Animais/patologia , Proteínas Proto-Oncogênicas c-akt/metabolismo , Animais , Carcinogênese/patologia , Feminino , Deleção de Genes , Humanos , Insulina/metabolismo , Isoenzimas/metabolismo , Metástase Neoplásica , Neutrófilos/metabolismo , Receptor ErbB-2/metabolismoRESUMO
The canonical role of the transcription factor E2F is to control the expression of cell cycle genes by binding to the E2F sites in their promoters. However, the list of putative E2F target genes is extensive and includes many metabolic genes, yet the significance of E2F in controlling the expression of these genes remains largely unknown. Here, we used the CRISPR/Cas9 technology to introduce point mutations in the E2F sites upstream of five endogenous metabolic genes in Drosophila melanogaster. We found that the impact of these mutations on both the recruitment of E2F and the expression of the target genes varied, with the glycolytic gene, Phosphoglycerate kinase (Pgk), being mostly affected. The loss of E2F regulation on the Pgk gene led to a decrease in glycolytic flux, tricarboxylic acid cycle intermediates levels, adenosine triphosphate (ATP) content, and an abnormal mitochondrial morphology. Remarkably, chromatin accessibility was significantly reduced at multiple genomic regions in PgkΔE2F mutants. These regions contained hundreds of genes, including metabolic genes that were downregulated in PgkΔE2F mutants. Moreover, PgkΔE2F animals had shortened life span and exhibited defects in high-energy consuming organs, such as ovaries and muscles. Collectively, our results illustrate how the pleiotropic effects on metabolism, gene expression, and development in the PgkΔE2F animals underscore the importance of E2F regulation on a single E2F target, Pgk.
Assuntos
Proteínas de Drosophila , Drosophila , Fatores de Transcrição E2F , Fosfoglicerato Quinase , Animais , Cromatina , Drosophila/genética , Fatores de Transcrição E2F/genética , Fosfoglicerato Quinase/genética , Fosfoglicerato Quinase/metabolismo , Regiões Promotoras Genéticas , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismoRESUMO
Polymorphism of transcription factor 7-like 2 (TCF7L2) has a link with type 2 diabetes mellitus (T2DM) through ß cell dysfunction that causes defect in blood glucose homeostasis. This case-control study recruited 67 T2DM as cases and 65 age-matched healthy individuals as controls to determine whether the polymorphism rs12255372 (G > T) in the TCF7L2 gene have an association with T2DM in Bangladeshi population. Genomic DNA was purified from peripheral whole blood sample and direct Sanger sequencing was done for genotyping of SNP. Bivariate logistic regression was done to find out the association between genetic variant and T2DM. In our study, the minor T allele frequency was significantly more frequent in T2DM group than healthy controls (29.1% vs. 16.9%). After adjusting with confounding factors, heterozygous-genotype GT had higher odds of developing T2DM (OR 2.4; 95% CI: 1.0-5.5; p value = 0.04) and in dominant model, having SNP in TCF7L2 increased the risk of T2DM 2.3 times (95% CI: 1.0-5.2; p value = 0.04). In interaction model, genetic susceptible SNP cases interacted significantly with increasing age and BMI, female gender, and having family history of diabetes mellitus to develop T2DM (pinteraction < 0.001). Having minor T allele either in heterozygous or homozygous variant form of rs12255372 (G > T) TCF7L2 had significant association with T2DM. In conclusion, TCF7L2 gene variant increases risk of developing T2DM among the Bangladeshi population.
Assuntos
Diabetes Mellitus Tipo 2 , Feminino , Humanos , Estudos de Casos e Controles , Diabetes Mellitus Tipo 2/epidemiologia , Diabetes Mellitus Tipo 2/genética , Predisposição Genética para Doença , Polimorfismo de Nucleotídeo Único/genética , Fator 1 de Transcrição de Linfócitos T/genética , Proteína 2 Semelhante ao Fator 7 de Transcrição/genéticaRESUMO
DYRK1A is a dosage-sensitive protein kinase that fulfills key roles during development and in tissue homeostasis, and its dysregulation results in human pathologies. DYRK1A is present in both the nucleus and cytoplasm of mammalian cells, although its nuclear function remains unclear. Genome-wide analysis of DYRK1A-associated loci reveals that the kinase is recruited preferentially to promoters of genes actively transcribed by RNA polymerase II (RNAPII), which are functionally associated with translation, RNA processing, and cell cycle. DYRK1A-bound promoter sequences are highly enriched in a conserved palindromic motif, which is necessary to drive DYRK1A-dependent transcriptional activation. DYRK1A phosphorylates the C-terminal domain (CTD) of RNAPII at Ser2 and Ser5. Depletion of DYRK1A results in reduced association of RNAPII at the target promoters as well as hypophosphorylation of the RNAPII CTD along the target gene bodies. These results are consistent with DYRK1A being a transcriptional regulator by acting as a CTD kinase.
Assuntos
Regiões Promotoras Genéticas , Proteínas Quinases/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Tirosina Quinases/metabolismo , RNA Mensageiro/metabolismo , Sítios de Ligação , Linhagem Celular Tumoral , Núcleo Celular/genética , Células HeLa , Humanos , Sequências Repetidas Invertidas , Dados de Sequência Molecular , Fosforilação , Proteínas Serina-Treonina Quinases/química , Proteínas Serina-Treonina Quinases/genética , Proteínas Tirosina Quinases/química , Proteínas Tirosina Quinases/genética , RNA Polimerase II , Serina/metabolismo , Transcrição Gênica , Quinases DyrkRESUMO
The retinoblastoma tumor suppressor protein pRb restricts cell growth through inhibition of cell cycle progression. Increasing evidence suggests that pRb also promotes differentiation, but the mechanisms are poorly understood, and the key question remains as to how differentiation in tumor cells can be enhanced in order to diminish their aggressive potential. Previously, we identified the histone demethylase KDM5A (lysine [K]-specific demethylase 5A), which demethylates histone H3 on Lys4 (H3K4), as a pRB-interacting protein counteracting pRB's role in promoting differentiation. Here we show that loss of Kdm5a restores differentiation through increasing mitochondrial respiration. This metabolic effect is both necessary and sufficient to induce the expression of a network of cell type-specific signaling and structural genes. Importantly, the regulatory functions of pRB in the cell cycle and differentiation are distinct because although restoring differentiation requires intact mitochondrial function, it does not necessitate cell cycle exit. Cells lacking Rb1 exhibit defective mitochondria and decreased oxygen consumption. Kdm5a is a direct repressor of metabolic regulatory genes, thus explaining the compensatory role of Kdm5a deletion in restoring mitochondrial function and differentiation. Significantly, activation of mitochondrial function by the mitochondrial biogenesis regulator Pgc-1α (peroxisome proliferator-activated receptor γ-coactivator 1α; also called PPARGC1A) a coactivator of the Kdm5a target genes, is sufficient to override the differentiation block. Overexpression of Pgc-1α, like KDM5A deletion, inhibits cell growth in RB-negative human cancer cell lines. The rescue of differentiation by loss of KDM5A or by activation of mitochondrial biogenesis reveals the switch to oxidative phosphorylation as an essential step in restoring differentiation and a less aggressive cancer phenotype.
Assuntos
Diferenciação Celular/genética , Regulação da Expressão Gênica no Desenvolvimento/genética , Mitocôndrias/enzimologia , Mitocôndrias/genética , Proteína do Retinoblastoma/genética , Proteína 2 de Ligação ao Retinoblastoma/metabolismo , Animais , Ciclo Celular , Linhagem Celular Tumoral , Células Cultivadas , Feminino , Fibroblastos/citologia , Fibroblastos/enzimologia , Humanos , Camundongos , Proteínas Mitocondriais/genética , Coativador 1-alfa do Receptor gama Ativado por Proliferador de Peroxissomo , Proteína do Retinoblastoma/metabolismo , Proteína 2 de Ligação ao Retinoblastoma/genética , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismoRESUMO
The receptor tyrosine kinase (RTK) pathway plays an essential role in development and disease by controlling cell proliferation and differentiation. Here, we profile the Drosophila larval brain by single-cell RNA-sequencing and identify Amalgam (Ama), which encodes a cell adhesion protein of the immunoglobulin IgLON family, as regulating the RTK pathway activity during glial cell development. Depletion of Ama reduces cell proliferation, affects glial cell type composition and disrupts the blood-brain barrier (BBB), which leads to hemocyte infiltration and neuronal death. We show that Ama depletion lowers RTK activity by upregulating Sprouty (Sty), a negative regulator of the RTK pathway. Knockdown of Ama blocks oncogenic RTK signaling activation in the Drosophila glioma model and halts malignant transformation. Finally, knockdown of a human ortholog of Ama, LSAMP, results in upregulation of SPROUTY2 in glioblastoma cell lines, suggesting that the relationship between Ama and Sty is conserved.
Assuntos
Proteínas de Drosophila/genética , Drosophila , Imunoglobulinas/genética , Proteínas de Membrana/genética , Animais , Encéfalo/metabolismo , Drosophila/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Larva/metabolismo , Proteínas de Membrana/metabolismo , Neuroglia/metabolismo , Receptores Proteína Tirosina Quinases/genética , Receptores Proteína Tirosina Quinases/metabolismoRESUMO
The cyclin-dependent kinase inhibitor p27Kip1 (p27) also behaves as a transcriptional repressor. Data showing that the p300/CBP-associated factor (PCAF) acetylates p27 inducing its degradation suggested that PCAF and p27 could collaborate in the regulation of transcription. However, this possibility remained to be explored. We analyzed here the transcriptional programs regulated by PCAF and p27 in the colon cancer cell line HCT116 by chromatin immunoprecipitation sequencing (ChIP-seq). We identified 269 protein-encoding genes that contain both p27 and PCAF binding sites being the majority of these sites different for PCAF and p27. PCAF or p27 knock down revealed that both regulate the expression of these genes, PCAF as an activator and p27 as a repressor. The double knock down of PCAF and p27 strongly reduced their expression indicating that the activating role of PCAF overrides the repressive effect of p27. We also observed that the transcription factor Pax5 interacts with both p27 and PCAF and that the knock down of Pax5 induces the expression of p27/PCAF target genes indicating that it also participates in the transcriptional regulation mediated by p27/PCAF. In summary, we report here a previously unknown mechanism of transcriptional regulation mediated by p27, Pax5 and PCAF.
Assuntos
Inibidor de Quinase Dependente de Ciclina p27/fisiologia , Regulação da Expressão Gênica , Fator de Transcrição PAX5/fisiologia , Fatores de Transcrição de p300-CBP/fisiologia , Animais , Sítios de Ligação , Linhagem Celular Tumoral , Células Cultivadas , Cromatina/metabolismo , Células HCT116 , Humanos , Células MCF-7 , Camundongos , Ligação Proteica , Proteínas/genética , Análise Serial de Tecidos , Transcrição GênicaRESUMO
The E2F family of transcription factors regulates the expression of both genes associated with cell proliferation and genes that regulate cell death. The net outcome is dependent on cellular context and tissue environment. The mir-11 gene is located in the last intron of the Drosophila E2F1 homolog gene dE2f1, and its expression parallels that of dE2f1. Here, we investigated the role of miR-11 and found that miR-11 specifically modulated the proapoptotic function of its host gene, dE2f1. A mir-11 mutant was highly sensitive to dE2F1-dependent, DNA damage-induced apoptosis. Consistently, coexpression of miR-11 in transgenic animals suppressed dE2F1-induced apoptosis in multiple tissues, while exerting no effect on dE2F1-driven cell proliferation. Importantly, miR-11 repressed the expression of the proapoptotic genes reaper (rpr) and head involution defective (hid), which are directly regulated by dE2F1 upon DNA damage. In addition to rpr and hid, we identified a novel set of cell death genes that was also directly regulated by dE2F1 and miR-11. Thus, our data support a model in which the coexpression of miR-11 limits the proapoptotic function of its host gene, dE2f1, upon DNA damage by directly modulating a dE2F1-dependent apoptotic transcriptional program.
Assuntos
Apoptose/genética , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/embriologia , Drosophila melanogaster/genética , Regulação da Expressão Gênica no Desenvolvimento , MicroRNAs/metabolismo , Fatores de Transcrição/metabolismo , Animais , Animais Geneticamente Modificados , Linhagem Celular , Proliferação de Células , Dano ao DNA/genética , Proteínas de Drosophila/genética , Células HeLa , Humanos , MicroRNAs/genética , Mutação/genética , Neuropeptídeos/metabolismo , Fatores de Transcrição/genéticaRESUMO
The Hippo signaling pathway regulates organ size homeostasis, while its inactivation leads to severe hyperplasia in flies and mammals. The transcriptional coactivator Yorkie (Yki) mediates transcriptional output of the Hippo signaling. Yki lacks a DNA-binding domain and is recruited to its target promoters as a complex with DNA-binding proteins such as Scalloped (Sd). In spite of recent progress, an open question in the field is the mechanism through which the Yki/Sd transcriptional signature is defined. Here, we report that Yki/Sd synergizes with and requires the transcription factor dE2F1 to induce a specific transcriptional program necessary to bypass the cell cycle exit. We show that Yki/Sd and dE2F1 bind directly to the promoters of the Yki/Sd-dE2F1 shared target genes and activate their expression in a strong cooperative manner. Consistently, RBF, a negative regulator of dE2F1, negates this synergy and limits the overall level of expression of the Yki/Sd-dE2F1 target genes. Significantly, dE2F1 is needed for Yki/Sd-dependent full activation of these target genes, and a de2f1 mutation strongly blocks yki-induced proliferation in vivo. Thus, the Yki transcriptional program is determined through functional interactions with other transcription factors directly at target promoters. We suggest that such functional interactions would influence Yki activity and help diversify the transcriptional output of the Hippo pathway.
Assuntos
Ciclo Celular/genética , Proteínas de Drosophila/fisiologia , Fator de Transcrição E2F1/fisiologia , Proteínas Nucleares/fisiologia , Transativadores/fisiologia , Fatores de Transcrição/fisiologia , Transcrição Gênica/genética , Animais , Animais Geneticamente Modificados , Ciclo Celular/fisiologia , Divisão Celular/genética , Divisão Celular/fisiologia , Proliferação de Células , Células Cultivadas , Análise por Conglomerados , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/genética , Drosophila melanogaster/metabolismo , Fator de Transcrição E2F1/genética , Fator de Transcrição E2F1/metabolismo , Embrião não Mamífero , Perfilação da Expressão Gênica , Regulação da Expressão Gênica no Desenvolvimento , Análise em Microsséries , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Transdução de Sinais/genética , Transativadores/genética , Transativadores/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Proteínas de Sinalização YAPRESUMO
The importance of microRNAs in gene expression and disease is well recognized. However, what is less appreciated is that almost half of miRNA genes are organized in polycistronic clusters and are therefore coexpressed. The mir-11â¼998 cluster consists of two miRNAs, miR-11 and miR-998. Here, we describe a novel layer of regulation that links the processing and expression of miR-998 to the presence of the mir-11 gene. We show that the presence of miR-11 in the pri-miRNA is required for processing by Drosha, and deletion of mir-11 prevents the expression of miR-998. Replacing mir-11 with an unrelated miRNA rescued miR-998 expression in vivo and in vitro, as did expressing miR-998 from a shorter, more canonical miRNA scaffold. The embedded regulation of miR-998 is functionally important because unchecked miR-998 expression in the absence of miR-11 resulted in pleiotropic developmental defects. This novel regulation of expression of miRNAs within a cluster is not limited to the mir-11â¼998 cluster and, thus, likely reflects the more general cis-regulation of expression of individual miRNAs. Collectively, our results uncover a novel layer of regulation within miRNA clusters that tempers the functions of the individual miRNAs. Unlinking their expression has the potential to change the expression of multiple miRNA targets and shift a biological response.
Assuntos
Regulação da Expressão Gênica , MicroRNAs/genética , Alelos , Animais , Células Cultivadas , DrosophilaRESUMO
The importance of microRNAs in the regulation of various aspects of biology and disease is well recognized. However, what remains largely unappreciated is that a significant number of miRNAs are embedded within and are often co-expressed with protein-coding host genes. Such a configuration raises the possibility of a functional interaction between a miRNA and the gene it resides in. This is exemplified by the Drosophila melanogaster dE2f1 gene that harbors two miRNAs, mir-11 and mir-998, within its last intron. miR-11 was demonstrated to limit the proapoptotic function of dE2F1 by repressing cell death genes that are directly regulated by dE2F1, however the biological role of miR-998 was unknown. Here we show that one of the functions of miR-998 is to suppress dE2F1-dependent cell death specifically in rbf mutants by elevating EGFR signaling. Mechanistically, miR-998 operates by repressing dCbl, a negative regulator of EGFR signaling. Significantly, dCbl is a critical target of miR-998 since dCbl phenocopies the effects of miR-998 on dE2f1-dependent apoptosis in rbf mutants. Importantly, this regulation is conserved, as the miR-998 seed family member miR-29 repressed c-Cbl, and enhanced MAPK activity and wound healing in mammalian cells. Therefore, the two intronic miRNAs embedded in the dE2f1 gene limit the apoptotic function of dE2f1, but operate in different contexts and act through distinct mechanisms. These results also illustrate that examining an intronic miRNA in the context of its host's function can be valuable in elucidating the biological function of the miRNA, and provide new information about the regulation of the host gene itself.
Assuntos
Diferenciação Celular/genética , Proteínas de Drosophila/metabolismo , Fatores de Transcrição E2F/metabolismo , Receptores ErbB/metabolismo , MicroRNAs/metabolismo , Receptores de Peptídeos de Invertebrados/metabolismo , Animais , Apoptose , Proteínas de Drosophila/genética , Drosophila melanogaster , Fatores de Transcrição E2F/genética , Receptores ErbB/genética , Regulação da Expressão Gênica no Desenvolvimento , Íntrons/genética , MicroRNAs/genética , Proteínas Proto-Oncogênicas c-cbl/metabolismo , Receptores de Peptídeos de Invertebrados/genética , Transdução de Sinais/genética , Fatores de TranscriçãoRESUMO
Epstein-Barr virus (EBV) infects and transforms human primary B cells inducing indefinite proliferation. To investigate the potential participation of chromatin mechanisms during the EBV-mediated transformation of resting B cells we performed an analysis of global changes in histone modifications. We observed a remarkable decrease and redistribution of heterochromatin marks including H4K20me3, H3K27me3 and H3K9me3. Loss of H4K20me3 and H3K9me3 occurred at constitutive heterochromatin repeats. For H3K27me3 and H3K9me3, comparison of ChIP-seq data revealed a decrease in these marks in thousands of genes, including clusters of HOX and ZNF genes, respectively. Moreover, DNase-seq data comparison between resting and EBV-transformed B cells revealed increased endonuclease accessibility in thousands of genomic sites. We observed that both loss of H3K27me3 and increased accessibility are associated with transcriptional activation. These changes only occurred in B cells transformed with EBV and not in those stimulated to proliferate with CD40L/IL-4, despite their similarities in the cell pathways involved and proliferation rates. In fact, B cells infected with EBNA-2 deficient EBV, which have much lower proliferation rates, displayed similar decreases for heterochromatic histone marks. Our study describes a novel phenomenon related to transformation of B cells, and highlights its independence of the pure acquisition of proliferation.
Assuntos
Linfócitos B/virologia , Herpesvirus Humano 4/fisiologia , Heterocromatina/metabolismo , Transformação Genética , Linfócitos B/citologia , Linfócitos B/metabolismo , Proliferação de Células , Histonas/metabolismo , HumanosRESUMO
Seven linker histone H1 variants are present in human somatic cells with distinct prevalence across cell types. Despite being key structural components of chromatin, it is not known whether the different variants have specific roles in the regulation of nuclear processes or are differentially distributed throughout the genome. Using variant-specific antibodies to H1 and hemagglutinin (HA)-tagged recombinant H1 variants expressed in breast cancer cells, we have investigated the distribution of six H1 variants in promoters and genome-wide. H1 is depleted at promoters depending on its transcriptional status and differs between variants. Notably, H1.2 is less abundant than other variants at the transcription start sites of inactive genes, and promoters enriched in H1.2 are different from those enriched in other variants and tend to be repressed. Additionally, H1.2 is enriched at chromosomal domains characterized by low guanine-cytosine (GC) content and is associated with lamina-associated domains. Meanwhile, other variants are associated with higher GC content, CpG islands and gene-rich domains. For instance, H1.0 and H1X are enriched at gene-rich chromosomes, whereas H1.2 is depleted. In short, histone H1 is not uniformly distributed along the genome and there are differences between variants, H1.2 being the one showing the most specific pattern and strongest correlation with low gene expression.
Assuntos
Neoplasias da Mama/genética , Histonas/análise , Neoplasias da Mama/química , Linhagem Celular Tumoral , Ilhas de CpG , Feminino , Regulação Neoplásica da Expressão Gênica , Genômica , Histonas/genética , Humanos , Regiões Promotoras Genéticas , Sítio de Iniciação de Transcrição , Transcrição Gênica , Ativação TranscricionalRESUMO
MicroRNAs (miRNAs) have negative effects on gene expression and are major players in cell function in normal and pathological conditions. Epstein-Barr virus (EBV) infection of resting B lymphocytes results in their growth transformation and associates with different B cell lymphomas. EBV-mediated B cell transformation involves large changes in gene expression, including cellular miRNAs. We performed miRNA expression analysis in growth transformation of EBV-infected B cells. We observed predominant downregulation of miRNAs and upregulation of a few miRNAs. We observed similar profiles of miRNA expression in B cells stimulated with CD40L/IL-4, and those infected with EBNA-2- and LMP-1-deficient EBV particles, suggesting the implication of the NF-kB pathway, common to all four situations. In fact, the NF-kB subunit p65 associates with the transcription start site (TSS) of both upregulated and downregulated miRNAs following EBV infection This occurs together with changes at histone H3K27me3 and histone H3K4me3. Inhibition of the NF-kB pathway impairs changes in miRNA expression, NF-kB binding and changes at the above histone modifications near the TSS of these miRNA genes. Changes in expression of these miRNAs also occurred in diffuse large B cell lymphomas (DLBCL), which are strongly NF-kB dependent. Our results highlight the relevance of the NF-kB pathway in epigenetically mediated miRNA control in B cell transformation and DLBCL.
Assuntos
Linfócitos B/virologia , Transformação Celular Viral/genética , Epigênese Genética , Herpesvirus Humano 4/fisiologia , Linfoma de Células B/virologia , MicroRNAs/metabolismo , NF-kappa B/metabolismo , Linfócitos B/metabolismo , Linhagem Celular Tumoral , Células Cultivadas , Humanos , Linfoma de Células B/genética , Linfoma de Células B/metabolismo , Transcrição GênicaRESUMO
B lymphopoiesis is the result of several cell-commitment, lineage-choice, and differentiation processes. Every differentiation step is characterized by the activation of a new, lineage-specific, genetic program and the extinction of the previous one. To date, the central role of specific transcription factors in positively regulating these distinct differentiation processes to acquire a B cell-specific genetic program is well established. However, the existence of specific transcriptional repressors responsible for the silencing of lineage inappropriate genes remains elusive. Here we addressed the molecular mechanism behind repression of non-lymphoid genes in B cells. We report that the histone deacetylase HDAC7 was highly expressed in pre-B cells but dramatically down-regulated during cellular lineage conversion to macrophages. Microarray analysis demonstrated that HDAC7 re-expression interfered with the acquisition of the gene transcriptional program characteristic of macrophages during cell transdifferentiation; the presence of HDAC7 blocked the induction of key genes for macrophage function, such as immune, inflammatory, and defense response, cellular response to infections, positive regulation of cytokines production, and phagocytosis. Moreover, re-introduction of HDAC7 suppressed crucial functions of macrophages, such as the ability to phagocytose bacteria and to respond to endotoxin by expressing major pro-inflammatory cytokines. To gain insight into the molecular mechanisms mediating HDAC7 repression in pre-B cells, we undertook co-immunoprecipitation and chromatin immunoprecipitation experimental approaches. We found that HDAC7 specifically interacted with the transcription factor MEF2C in pre-B cells and was recruited to MEF2 binding sites located at the promoters of genes critical for macrophage function. Thus, in B cells HDAC7 is a transcriptional repressor of undesirable genes. Our findings uncover a novel role for HDAC7 in maintaining the identity of a particular cell type by silencing lineage-inappropriate genes.
Assuntos
Transdiferenciação Celular/genética , Histona Desacetilases/genética , Linfopoese , Macrófagos/citologia , Células Precursoras de Linfócitos B/citologia , Linfócitos B/citologia , Linfócitos B/metabolismo , Sítios de Ligação , Diferenciação Celular , Linhagem da Célula , Regulação para Baixo , Histona Desacetilases/metabolismo , Humanos , Proteínas de Domínio MADS/metabolismo , Fatores de Transcrição MEF2 , Macrófagos/metabolismo , Células Mieloides/citologia , Células Mieloides/metabolismo , Fatores de Regulação Miogênica/metabolismo , Células Precursoras de Linfócitos B/metabolismo , Regiões Promotoras GenéticasRESUMO
Epigenetic regulation underlies the robust changes in gene expression that occur during development. How precisely epigenetic enzymes contribute to development and differentiation processes is largely unclear. Here we show that one of the enzymes that removes the activating epigenetic mark of trimethylated lysine 4 on histone H3, lysine (K)-specific demethylase 5A (KDM5A), reinforces the effects of the retinoblastoma (RB) family of transcriptional repressors on differentiation. Global location analysis showed that KDM5A cooccupies a substantial portion of target genes with the E2F4 transcription factor. During ES cell differentiation, knockout of KDM5A resulted in derepression of multiple genomic loci that are targets of KDM5A, denoting a direct regulatory function. In terminally differentiated cells, common KDM5A and E2F4 gene targets were bound by the pRB-related protein p130, a DREAM complex component. KDM5A was recruited to the transcription start site regions independently of E2F4; however, it cooperated with E2F4 to promote a state of deepened repression at cell cycle genes during differentiation. These findings reveal a critical role of H3K4 demethylation by KDM5A in the transcriptional silencing of genes that are suppressed by RB family members in differentiated cells.
Assuntos
Diferenciação Celular/genética , Fator de Transcrição E2F4/metabolismo , Genes cdc/genética , Proteínas Repressoras/metabolismo , Proteína 2 de Ligação ao Retinoblastoma/metabolismo , Animais , Imunoprecipitação da Cromatina , Células-Tronco Embrionárias/metabolismo , Histonas/metabolismo , Humanos , Lisina/metabolismo , Metilação , Camundongos , Camundongos Endogâmicos C57BL , Complexos Multiproteicos/metabolismo , Ligação Proteica , Células U937RESUMO
Adverse childhood experiences (ACEs) are potentially traumatic events that occur before 18 years of age. Studies emphasize the importance of childhood adversity as a risk factor for developing non-communicable diseases, including type-2 diabetes mellitus (T2DM) in adulthood. This case-control study involved 137 patients with T2DM and 134 non-diabetic adults of both genders (mean age 46.9 and 45.7 years, respectively). In addition to collecting socio-demographic, behavioral, and anthropological data, a 10-item ACE scale was utilized to gather information regarding childhood adversities, while perceived stress was assessed using the perceived stress scale-4. Fasting and 2-hour post glucose load blood sugar levels, HbA1c, and fasting lipid profiles were measured. Both univariable and multivariable binary logistic regression analyses were performed to investigate whether ACE is a potential risk factor for T2DM, with a significance level of 0.05. Around two-thirds of T2DM patients reported having ACE scores of 4 or higher, with the mean ACE score significantly higher in the case group than in the control group (3.96 vs. 3.34; p<0.0001). The logistic regression analysis found that T2DM was linked to female gender, hypertension, dyslipidemia, family history of DM, higher perceived stress, and a higher ACE score of 4 and above. After adjusting for confounding factors, individuals with an ACE score of 4 or higher had a significantly greater risk of developing T2DM (OR: 2.24; 95% CI 1.238-4.061). The study revealed a significant association between higher ACE scores and an increased risk of developing T2DM. As a recommendation, further investigation into the epigenetic mechanisms underlying this relationship is warranted.
RESUMO
TPO (Thyroid Peroxidase) is known to be one of the major genes involved in congenital hypothyroid patients with thyroid dyshormonogenesis. The present study aims to validate high-resolution melting (HRM) curve analysis as a substitute method for Sanger sequencing, focusing on the frequently observed non-synonymous mutations c.1117G>T, c.1193G>C, and c.2173A>C in the TPO gene in patients from Bangladesh. We enrolled 36 confirmed cases of congenital hypothyroid patients with dyshormonogenesis to establish the HRM method. Blood specimens were collected, and DNA was extracted followed by PCR and Sanger sequencing. Among the 36 specimens, 20 were pre-sequenced, and variants were characterized through Sanger sequencing. Following pre-sequencing, the 20 pre-sequenced specimens underwent real-time PCR-HRM curve analysis to determine the proper HRM condition for separating the three variations from the wild-type state into heterozygous and homozygous states. Furthermore, 16 unknown specimens were subjected to HRM analysis to validate the method. This method demonstrated a sensitivity and specificity of 100 percent in accurately discerning wild-type alleles from both homozygous and heterozygous states of c.1117G>T (23/36; 63.8%), c.1193G>C (30/36; 83.3%), and c.2173A>C (23/36; 63.8%) variants frequently encountered among 36 Bangladeshi patients. The HRM data was found to be similar to the sequencing result, thus confirming the validity of the HRM approach for TPO gene variant detection. In conclusion, HRM-based molecular technique targeting variants c.1117G>T, c.1193G>C, and c.2173A>C could be used as a high throughput, rapid, reliable, and cost-effective screening approach for the detection of all common mutations in TPO gene in Bangladeshi patients with dyshormonogenesis.
Assuntos
Hipotireoidismo Congênito , Humanos , Bangladesh , Hipotireoidismo Congênito/diagnóstico , Hipotireoidismo Congênito/genética , Mutação , DNA , Reação em Cadeia da Polimerase em Tempo RealRESUMO
Autoimmune rheumatic diseases are complex disorders, whose etiopathology is attributed to a crosstalk between genetic predisposition and environmental factors. Both variants of autoimmune susceptibility genes and environment are involved in the generation of aberrant epigenetic profiles in a cell-specific manner, which ultimately result in dysregulation of expression. Furthermore, changes in miRNA expression profiles also cause gene dysregulation associated with aberrant phenotypes. In rheumatoid arthritis, several cell types are involved in the destruction of the joints, synovial fibroblasts being among the most important. In this study we performed DNA methylation and miRNA expression screening of a set of rheumatoid arthritis synovial fibroblasts and compared the results with those obtained from osteoarthritis patients with a normal phenotype. DNA methylation screening allowed us to identify changes in novel key target genes like IL6R, CAPN8 and DPP4, as well as several HOX genes. A significant proportion of genes undergoing DNA methylation changes were inversely correlated with expression. miRNA screening revealed the existence of subsets of miRNAs that underwent changes in expression. Integrated analysis highlighted sets of miRNAs that are controlled by DNA methylation, and genes that are regulated by DNA methylation and are targeted by miRNAs with a potential use as clinical markers. Our study enabled the identification of novel dysregulated targets in rheumatoid arthritis synovial fibroblasts and generated a new workflow for the integrated analysis of miRNA and epigenetic control.
Assuntos
Artrite Reumatoide/genética , Metilação de DNA , Fibroblastos/metabolismo , Perfilação da Expressão Gênica , MicroRNAs/genética , Artrite Reumatoide/patologia , Ilhas de CpG/genética , Fibroblastos/patologia , Predisposição Genética para Doença , Humanos , Análise de Sequência com Séries de Oligonucleotídeos , Osteoartrite/genética , Osteoartrite/patologia , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Membrana Sinovial/metabolismo , Membrana Sinovial/patologiaRESUMO
N6-methyladenosine (m6A) is the most abundant internal modification on eukaryotic mRNAs. Demethylation of m6A on mRNA is catalyzed by the enzyme fat mass and obesity-associated protein (FTO), a member of the nonheme Fe(II) and 2-oxoglutarate (2-OG)-dependent family of dioxygenases. FTO activity and m6A-mRNA are dysregulated in multiple diseases including cancers, yet endogenous signaling molecules that modulate FTO activity have not been identified. Here we show that nitric oxide (NO) is a potent inhibitor of FTO demethylase activity by directly binding to the catalytic iron center, which causes global m6A hypermethylation of mRNA in cells and results in gene-specific enrichment of m6A on mRNA of NO-regulated transcripts. Both cell culture and tumor xenograft models demonstrated that endogenous NO synthesis can regulate m6A-mRNA levels and transcriptional changes of m6A-associated genes. These results build a direct link between NO and m6A-mRNA regulation and reveal a novel signaling mechanism of NO as an endogenous regulator of the epitranscriptome.