RESUMO
Most common genetic variants associated with disease are located in non-coding regions of the genome. One mechanism by which they function is through altering transcription factor (TF) binding. In this study, we explore how genetic variation is connected to differences in the regulatory landscape of livers from C57BL/6J and 129S1/SvImJ mice fed either chow or a high-fat diet. To identify sites where regulatory variation affects TF binding and nearby gene expression, we employed an integrative analysis of H3K27ac ChIP-seq (active enhancers), ATAC-seq (chromatin accessibility) and RNA-seq (gene expression). We show that, across all these assays, the genetically driven (i.e. strain-specific) differences in the regulatory landscape are more pronounced than those modified by diet. Most notably, our analysis revealed that differentially accessible regions (DARs, N = 29635, FDR < 0.01 and fold change > 50%) are almost always strain-specific and enriched with genetic variation. Moreover, proximal DARs are highly correlated with differentially expressed genes. We also show that TF binding is affected by genetic variation, which we validate experimentally using ChIP-seq for TCF7L2 and CTCF. This study provides detailed insights into how non-coding genetic variation alters the gene regulatory landscape, and demonstrates how this can be used to study the regulatory variation influencing TF binding.
Assuntos
Cromatina , Regulação da Expressão Gênica , Camundongos , Animais , Cromatina/genética , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos , Variação GenéticaRESUMO
BACKGROUND: Obesity is a worldwide epidemic characterized by adipose tissue (AT) inflammation. AT is also a source of extracellular vesicles (EVs) that have recently been implicated in disorders related to metabolic syndrome. However, our understanding of mechanistic aspect of obesity's impact on EV secretion from human AT remains limited. METHODS: We investigated EVs from human Simpson Golabi Behmel Syndrome (SGBS) adipocytes, and from AT as well as plasma of subjects undergoing bariatric surgery. SGBS cells were treated with TNFα, palmitic acid, and eicosapentaenoic acid. Various analyses, including nanoparticle tracking analysis, electron microscopy, high-resolution confocal microscopy, and gas chromatography-mass spectrometry, were utilized to study EVs. Plasma EVs were analyzed with imaging flow cytometry. RESULTS: EVs from mature SGBS cells differed significantly in size and quantity compared to preadipocytes, disagreeing with previous findings in mouse adipocytes and indicating that adipogenesis promotes EV secretion in human adipocytes. Inflammatory stimuli also induced EV secretion, and altered EV fatty acid (FA) profiles more than those of cells, suggesting the role of EVs as rapid responders to metabolic shifts. Visceral AT (VAT) exhibited higher EV secretion compared to subcutaneous AT (SAT), with VAT EV counts positively correlating with plasma triacylglycerol (TAG) levels. Notably, the plasma EVs of subjects with obesity contained a higher number of adiponectin-positive EVs than those of lean subjects, further demonstrating higher AT EV secretion in obesity. Moreover, plasma EV counts of people with obesity positively correlated with body mass index and TNF expression in SAT, connecting increased EV secretion with AT expansion and inflammation. Finally, EVs from SGBS adipocytes and AT contained TAGs, and EV secretion increased despite signs of less active lipolytic pathways, indicating that AT EVs could be involved in the mobilization of excess lipids into circulation. CONCLUSIONS: We are the first to provide detailed FA profiles of human AT EVs. We report that AT EV secretion increases in human obesity, implicating their role in TAG transport and association with adverse metabolic parameters, thereby emphasizing their role in metabolic disorders. These findings promote our understanding of the roles that EVs play in human AT biology and metabolic disorders.
Assuntos
Adipócitos , Tecido Adiposo , Vesículas Extracelulares , Inflamação , Obesidade , Humanos , Vesículas Extracelulares/metabolismo , Obesidade/metabolismo , Obesidade/patologia , Adipócitos/metabolismo , Inflamação/patologia , Inflamação/metabolismo , Tecido Adiposo/metabolismo , Tecido Adiposo/patologia , Metabolismo dos Lipídeos , Feminino , Masculino , Adulto , Ácidos Graxos/metabolismoRESUMO
Glucocorticoid receptor (GR) is an essential transcription factor (TF), controlling metabolism, development and immune responses. SUMOylation regulates chromatin occupancy and target gene expression of GR in a locus-selective manner, but the mechanism of regulation has remained elusive. Here, we identify the protein network around chromatin-bound GR by using selective isolation of chromatin-associated proteins and show that the network is affected by receptor SUMOylation, with several nuclear receptor coregulators and chromatin modifiers preferring interaction with SUMOylation-deficient GR and proteins implicated in transcriptional repression preferring interaction with SUMOylation-competent GR. This difference is reflected in our chromatin binding, chromatin accessibility and gene expression data, showing that the SUMOylation-deficient GR is more potent in binding and opening chromatin at glucocorticoid-regulated enhancers and inducing expression of target loci. Blockage of SUMOylation by a SUMO-activating enzyme inhibitor (ML-792) phenocopied to a large extent the consequences of GR SUMOylation deficiency on chromatin binding and target gene expression. Our results thus show that SUMOylation modulates the specificity of GR by regulating its chromatin protein network and accessibility at GR-bound enhancers. We speculate that many other SUMOylated TFs utilize a similar regulatory mechanism.
Assuntos
Cromatina/metabolismo , Receptores de Glucocorticoides/metabolismo , Sumoilação , Sítios de Ligação , Regulação da Expressão Gênica , Células HEK293 , Humanos , Correpressor 1 de Receptor Nuclear/metabolismo , Coativador 1 de Receptor Nuclear , Mapeamento de Interação de Proteínas , Proteínas Modificadoras Pequenas Relacionadas à Ubiquitina/metabolismo , Sumoilação/efeitos dos fármacosRESUMO
In many species, mate choice continues after the mating via female- or egg-derived biochemical factors that induce selective changes in sperm pre-fertilization physiology and behaviour. Recent studies have indicated that gamete-mediated mate choice likely occurs also in humans, but the mechanistic basis of the process has remained virtually unexplored. Here, we investigated whether female-induced modifications in sperm protein SUMOylation (post-translational modification of the proteome) could serve as a novel mechanism for gamete-mediated mate choice in humans. We treated the sperm of ten males with the oocyte-surrounding bioactive liquid (follicular fluid) of five females and investigated motility, viability and global protein SUMOylation status of the sperm in all (n = 50) of these male-female combinations (full-factorial design). All the measured sperm traits were affected by male-female combinations, and sperm protein SUMOylation status was also negatively associated with sperm motility. Furthermore, our results indicate that female-induced sperm protein SUMOylation is selective, potentially allowing females to increase sperm motility in some males, whereas decreasing it in the others. Consequently, our findings suggest that follicular fluid may non-randomly modify the structure and function of sperm proteome and in this way facilitate gamete-mediated mate choice in humans and possibly many other species. However, due to the relatively low number of female subjects and their potential infertility problems, our results should be replicated with larger subset of fully fertile women.
Assuntos
Motilidade dos Espermatozoides , Sumoilação , Feminino , Fertilização , Células Germinativas , Humanos , Masculino , Espermatozoides/fisiologiaRESUMO
Infertility is assumed to arise exclusively from male- and female-dependent pathological factors. However, recent studies have indicated that reproductive failure may also result from the reproductive incompatibility of the partners. Selection against such incompatibilities likely occurs via female-derived reproductive secretions, including follicular fluid (FF), that mediate gamete-level mate choice towards the sperm of specific males. To facilitate potential development of diagnostic tests for human reproductive incompatibility, we examined whether sperm physiological response to female serum indicate male-female compatibility in the presence of FF. We performed a full-factorial experiment, in which the sperm of 10 males were treated with the FF and serum of 6 healthy females. We found that sperm motility and viability in both biofluids were highly similar and that in 70% of the males, sperm serum treatment predicted male-female compatibility. We also identified male human leucocyte antigen (HLA) alleles and female (FF and serum) anti-HLA antibodies and tested whether the number of allele-antibody matches predict sperm physiological response to female fluids. However, no association was found between measured sperm traits and the number of allele-antibody matches. Overall, the present results may open novel possibilities for the future development of reproductive incompatibility tests and may pave the way towards more accurate infertility diagnostics and treatments.
Assuntos
Infertilidade , Motilidade dos Espermatozoides , Feminino , Células Germinativas , Humanos , Masculino , Reprodução , Espermatozoides/fisiologiaRESUMO
Extracellular vesicles (EVs) function as conveyors of fatty acids (FAs) and other bioactive lipids and can modulate the gene expression and behavior of target cells. EV lipid composition influences the fluidity and stability of EV membranes and reflects the availability of lipid mediator precursors. Fibroblast-like synoviocytes (FLSs) secrete EVs that transport hyaluronic acid (HA). FLSs play a central role in inflammation, pannus formation, and cartilage degradation in joint diseases, and EVs have recently emerged as potential mediators of these effects. The aim of the present study was to follow temporal changes in HA and EV secretion by normal FLSs, and to characterize the FA profiles of FLSs and EVs during proliferation. The methods used included nanoparticle tracking analysis, confocal laser scanning microscopy, sandwich-type enzyme-linked sorbent assay, quantitative PCR, and gas chromatography. The expression of hyaluronan synthases 1-3 in FLSs and HA concentrations in conditioned media decreased during cell proliferation. This was associated with elevated proportions of 20:4n-6 and total n-6 polyunsaturated FAs (PUFAs) in high-density cells, reductions in n-3/n-6 PUFA ratios, and up-regulation of cluster of differentiation 44, tumor necrosis factor α, peroxisome proliferator-activated receptor (PPAR)-α, and PPAR-γ. Compared to the parent FLSs, 16:0, 18:0, and 18:1n-9 were enriched in the EV fraction. EV counts decreased during cell growth, and 18:2n-6 in EVs correlated with the cell count. To conclude, FLS proliferation was featured by increased 20:4n-6 proportions and reduced n-3/n-6 PUFA ratios, and FAs with a low degree of unsaturation were selectively transferred from FLSs into EVs. These FA modifications have the potential to affect membrane fluidity, biosynthesis of lipid mediators, and inflammatory processes in joints, and could eventually provide tools for translational studies to counteract cartilage degradation in inflammatory joint diseases.
Assuntos
Vesículas Extracelulares , Sinoviócitos , Vesículas Extracelulares/metabolismo , Ácidos Graxos/metabolismo , Ácidos Graxos Insaturados/metabolismo , Fibroblastos/metabolismo , Humanos , Hialuronan Sintases/genética , Hialuronan Sintases/metabolismo , Ácido Hialurônico/metabolismo , PPAR gama/metabolismo , Sinoviócitos/metabolismoRESUMO
Hypoxia-inducible factor (HIF), an αß dimer, is the master regulator of oxygen homeostasis with hundreds of hypoxia-inducible target genes. Three HIF isoforms differing in the oxygen-sensitive α subunit exist in vertebrates. While HIF-1 and HIF-2 are known transcription activators, HIF-3 has been considered a negative regulator of the hypoxia response pathway. However, the human HIF3A mRNA is subject to complex alternative splicing. It was recently shown that the long HIF-3α variants can form αß dimers that possess transactivation capacity. Here, we show that overexpression of the long HIF-3α2 variant induces the expression of a subset of genes, including the erythropoietin (EPO) gene, while simultaneous downregulation of all HIF-3α variants by siRNA targeting a shared HIF3A region leads to downregulation of EPO and additional genes. EPO mRNA and protein levels correlated with HIF3A silencing and HIF-3α2 overexpression. Chromatin immunoprecipitation analyses showed that HIF-3α2 binding associated with canonical hypoxia response elements in the promoter regions of EPO. Luciferase reporter assays showed that the identified HIF-3α2 chromatin-binding regions were sufficient to promote transcription by all three HIF-α isoforms. Based on these data, HIF-3α2 is a transcription activator that directly regulates EPO expression.
Assuntos
Proteínas Reguladoras de Apoptose/metabolismo , Eritropoetina/metabolismo , Proteínas Repressoras/metabolismo , Proteínas Reguladoras de Apoptose/antagonistas & inibidores , Proteínas Reguladoras de Apoptose/genética , Proteína Morfogenética Óssea 6/genética , Proteína Morfogenética Óssea 6/metabolismo , Proteína C-Reativa/genética , Proteína C-Reativa/metabolismo , Hipóxia Celular , Linhagem Celular Tumoral , Cromatina/metabolismo , Dimerização , Eritropoetina/análise , Eritropoetina/genética , Transportador de Glucose Tipo 1/genética , Transportador de Glucose Tipo 1/metabolismo , Humanos , Regiões Promotoras Genéticas , Ligação Proteica , Isoformas de Proteínas/antagonistas & inibidores , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Interferência de RNA , Splicing de RNA , RNA Interferente Pequeno/metabolismo , Proteínas Repressoras/antagonistas & inibidores , Proteínas Repressoras/genética , Componente Amiloide P Sérico/genética , Componente Amiloide P Sérico/metabolismo , Ativação TranscricionalRESUMO
Inflammatory processes and androgen signaling are critical for the growth of prostate cancer (PC), the most common cancer among males in Western countries. To understand the importance of potential interplay between pro-inflammatory and androgen signaling for gene regulation, we have interrogated the crosstalk between androgen receptor (AR) and NF-κB, a key transcriptional mediator of inflammatory responses, by utilizing genome-wide chromatin immunoprecipitation sequencing and global run-on sequencing in PC cells. Co-stimulation of LNCaP cells with androgen and pro-inflammatory cytokine TNFα invoked a transcriptome which was very distinct from that induced by either stimulation alone. The altered transcriptome that included gene programs linked to cell migration and invasiveness was orchestrated by significant remodeling of NF-κB and AR cistrome and enhancer landscape. Although androgen multiplied the NF-κB cistrome and TNFα restrained the AR cistrome, there was no general reciprocal tethering of the AR to the NF-κB on chromatin. Instead, redistribution of FOXA1, PIAS1 and PIAS2 contributed to the exposure of latent NF-κB chromatin-binding sites and masking of AR chromatin-binding sites. Taken together, concomitant androgen and pro-inflammatory signaling significantly remodels especially the NF-κB cistrome, reprogramming the PC cell transcriptome in fashion that may contribute to the progression of PC.
Assuntos
Androgênios/metabolismo , Regulação Neoplásica da Expressão Gênica , Mediadores da Inflamação/metabolismo , NF-kappa B/metabolismo , Neoplasias da Próstata/genética , Neoplasias da Próstata/metabolismo , Receptores Androgênicos/metabolismo , Transcriptoma , Linhagem Celular Tumoral , Cromatina/genética , Cromatina/metabolismo , Análise por Conglomerados , Citocinas/metabolismo , Elementos Facilitadores Genéticos , Perfilação da Expressão Gênica , Fator 3-alfa Nuclear de Hepatócito/metabolismo , Humanos , Masculino , Proteínas Inibidoras de STAT Ativados/metabolismoRESUMO
Androgen receptor (AR) is a ligand-activated transcription factor that plays a central role in the development and growth of prostate carcinoma. PIAS1 is an AR- and SUMO-interacting protein and a putative transcriptional coregulator overexpressed in prostate cancer. To study the importance of PIAS1 for the androgen-regulated transcriptome of VCaP prostate cancer cells, we silenced its expression by RNAi. Transcriptome analyses revealed that a subset of the AR-regulated genes is significantly influenced, either activated or repressed, by PIAS1 depletion. Interestingly, PIAS1 depletion also exposed a new set of genes to androgen regulation, suggesting that PIAS1 can mask distinct genomic loci from AR access. In keeping with gene expression data, silencing of PIAS1 attenuated VCaP cell proliferation. ChIP-seq analyses showed that PIAS1 interacts with AR at chromatin sites harboring also SUMO2/3 and surrounded by H3K4me2; androgen exposure increased the number of PIAS1-occupying sites, resulting in nearly complete overlap with AR chromatin binding events. PIAS1 interacted also with the pioneer factor FOXA1. Of note, PIAS1 depletion affected AR chromatin occupancy at binding sites enriched for HOXD13 and GATA motifs. Taken together, PIAS1 is a genuine chromatin-bound AR coregulator that functions in a target gene selective fashion to regulate prostate cancer cell growth.
Assuntos
Cromatina/metabolismo , Regulação Neoplásica da Expressão Gênica , Neoplasias da Próstata/genética , Proteínas Inibidoras de STAT Ativados/metabolismo , Receptores Androgênicos/metabolismo , Proteínas Modificadoras Pequenas Relacionadas à Ubiquitina/metabolismo , Sítios de Ligação , Linhagem Celular Tumoral , Proliferação de Células , Fator 3-alfa Nuclear de Hepatócito/metabolismo , Humanos , Masculino , Neoplasias da Próstata/enzimologia , Neoplasias da Próstata/metabolismo , Neoplasias da Próstata/patologia , Ubiquitina-Proteína Ligases/metabolismoRESUMO
Androgen receptor (AR) plays an important regulatory role in prostate cancer. AR's transcriptional activity is regulated by androgenic ligands, but also by post-translational modifications, such as SUMOylation. To study the role of AR SUMOylation in genuine chromatin environment, we compared androgen-regulated gene expression and AR chromatin occupancy in PC-3 prostate cancer cell lines stably expressing wild-type (wt) or doubly SUMOylation site-mutated AR (AR-K386R,K520R). Our genome-wide gene expression analyses reveal that the SUMOylation modulates the AR function in a target gene and pathway selective manner. The transcripts that are differentially regulated by androgen and SUMOylation are linked to cellular movement, cell death, cellular proliferation, cellular development and cell cycle. Fittingly, SUMOylation mutant AR cells proliferate faster and are more sensitive to apoptosis. Moreover, ChIP-seq analyses show that the SUMOylation can modulate the chromatin occupancy of AR on many loci in a fashion that parallels their differential androgen-regulated expression. De novo motif analyses reveal that FOXA1, C/EBP and AP-1 motifs are differentially enriched at the wtAR- and the AR-K386R,K520R-preferred genomic binding positions. Taken together, our data indicate that SUMOylation does not simply repress the AR activity, but it regulates AR's interaction with the chromatin and the receptor's target gene selection.
Assuntos
Regulação Neoplásica da Expressão Gênica , Neoplasias da Próstata/genética , Receptores Androgênicos/metabolismo , Sumoilação , Apoptose , Linhagem Celular Tumoral , Proliferação de Células , Células HEK293 , Humanos , Masculino , Neoplasias da Próstata/metabolismo , Neoplasias da Próstata/patologia , Transcrição GênicaRESUMO
Vitamin D modulates innate and adaptive immunity, the molecular mechanisms of which we aim to understand under human in vivo conditions. Therefore, we designed the study VitDHiD (NCT03537027) as a human investigation, in which 25 healthy individuals were supplemented with a single vitamin D3 bolus (80,000 IU). Transcriptome-wide differential gene expression analysis of peripheral blood mononuclear cells (PBMCs), which were isolated directly before and 24 h after supplementation, identified 452 genes significantly (FDR < 0.05) responding to vitamin D. In vitro studies using PBMCs from the same individuals confirmed 138 of these genes as targets of 1α,25-dihydroxyvitamin D3. A subset of the 91 most regulated in vivo vitamin D target genes indicated focal adhesion as the major pathway being upregulated by vitamin D3 supplementation of healthy individuals. Differences in the individual-specific responsiveness of in vivo vitamin D target genes in relation to the increase of the person's vitamin D status allowed a segregation of the VitDHiD participants into 9 high, 12 mid and 4 low responders. The expression profile of nearly 600 genes elucidate the difference between high and low vitamin D responders, the most prominent of which is the HLA-C (major histocompatibility complex, class I, C) gene.
Assuntos
Adesões Focais , Leucócitos Mononucleares , Vitamina D , Adulto , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Adulto Jovem , Colecalciferol/farmacologia , Suplementos Nutricionais , Adesões Focais/efeitos dos fármacos , Perfilação da Expressão Gênica , Voluntários Saudáveis , Leucócitos Mononucleares/metabolismo , Leucócitos Mononucleares/efeitos dos fármacos , Leucócitos Mononucleares/imunologia , Transcriptoma/efeitos dos fármacos , Regulação para Cima/efeitos dos fármacos , Vitamina D/farmacologiaRESUMO
The inflammatory tumor microenvironment (TME) is a key driver for tumor-promoting processes. Tumor-associated macrophages are one of the main immune cell types in the TME and their increased density is related to poor prognosis in prostate cancer. Here, we investigated the influence of pro-inflammatory (M1) and immunosuppressive (M2) macrophages on prostate cancer lineage plasticity. Our findings reveal that M1 macrophage secreted factors upregulate genes related to stemness while downregulating genes associated with androgen response in prostate cancer cells. The expression of cancer stem cell (CSC) plasticity markers NANOG, KLF4, SOX2, OCT4, and CD44 was stimulated by the secreted factors from M1 macrophages. Moreover, AR and its target gene PSA were observed to be suppressed in LNCaP cells treated with secreted factors from M1 macrophages. Inhibition of NFκB signaling using the IKK16 inhibitor resulted in downregulation of NANOG, SOX2, and CD44 and CSC plasticity. Our study highlights that the secreted factors from M1 macrophages drive prostate cancer cell plasticity by upregulating the expression of CSC plasticity markers through NFκB signaling pathway.
Assuntos
Receptores de Hialuronatos , Fator 4 Semelhante a Kruppel , Macrófagos , NF-kappa B , Proteína Homeobox Nanog , Células-Tronco Neoplásicas , Neoplasias da Próstata , Fatores de Transcrição SOXB1 , Transdução de Sinais , Masculino , Humanos , Neoplasias da Próstata/patologia , Neoplasias da Próstata/metabolismo , Neoplasias da Próstata/genética , Proteína Homeobox Nanog/metabolismo , Proteína Homeobox Nanog/genética , Fatores de Transcrição SOXB1/metabolismo , Fatores de Transcrição SOXB1/genética , Receptores de Hialuronatos/metabolismo , Receptores de Hialuronatos/genética , Células-Tronco Neoplásicas/metabolismo , Células-Tronco Neoplásicas/patologia , Fator 4 Semelhante a Kruppel/metabolismo , NF-kappa B/metabolismo , Linhagem Celular Tumoral , Macrófagos/metabolismo , Regulação para Cima , Microambiente Tumoral/imunologia , Plasticidade Celular/genética , Regulação Neoplásica da Expressão Gênica , Macrófagos Associados a Tumor/metabolismo , Macrófagos Associados a Tumor/imunologia , Animais , CamundongosRESUMO
Treatment-induced neuroendocrine prostate cancer (t-NEPC) is a lethal subtype of castration-resistant prostate cancer resistant to androgen receptor (AR) inhibitors. Our study unveils that AR suppresses the neuronal development protein dihydropyrimidinase-related protein 5 (DPYSL5), providing a mechanism for neuroendocrine transformation under androgen deprivation therapy. Our unique CRPC-NEPC cohort, comprising 135 patient tumor samples, including 55 t-NEPC patient samples, exhibits a high expression of DPYSL5 in t-NEPC patient tumors. DPYSL5 correlates with neuroendocrine-related markers and inversely with AR and PSA. DPYSL5 overexpression in prostate cancer cells induces a neuron-like phenotype, enhances invasion, proliferation, and upregulates stemness and neuroendocrine-related markers. Mechanistically, DPYSL5 promotes prostate cancer cell plasticity via EZH2-mediated PRC2 activation. Depletion of DPYSL5 decreases proliferation, induces G1 phase cell cycle arrest, reverses neuroendocrine phenotype, and upregulates luminal genes. In conclusion, DPYSL5 plays a critical role in regulating prostate cancer cell plasticity, and we propose the AR/DPYSL5/EZH2/PRC2 axis as a driver of t-NEPC progression.
Assuntos
Neoplasias da Próstata , Masculino , Humanos , Neoplasias da Próstata/tratamento farmacológico , Neoplasias da Próstata/genética , Neoplasias da Próstata/metabolismo , Antagonistas de Androgênios , Próstata/patologia , Hidrolases , Proteínas Associadas aos Microtúbulos , Proteína Potenciadora do Homólogo 2 de Zeste/genéticaRESUMO
The nuclear receptor vitamin D receptor (VDR) is known to associate with two vitamin D response element (VDRE) containing chromatin regions of the insulin-like growth factor binding protein 3 (IGFBP3) gene. In non-malignant MCF-10A human mammary cells, we show that the natural VDR ligand 1α,25-dihydroxyvitamin D(3) (1α,25(OH)(2)D(3)) causes cyclical IGFBP3 mRNA accumulation with a periodicity of 60 min, while in the presence of the potent VDR agonist Gemini the mRNA is continuously accumulated. Accordingly, VDR also showed cyclical ligand-dependent association with the chromatin regions of both VDREs. Histone deacetylases (HDACs) play an important role both in VDR signalling and in transcriptional cycling. From the 11 HDAC gene family members, only HDAC4 and HDAC6 are up-regulated in a cyclical fashion in response to 1α,25(OH)(2)D(3), while even these two genes do not respond to Gemini. Interestingly, HDAC4 and HDAC6 proteins show cyclical VDR ligand-induced association with both VDRE regions of the IGFBP3 gene, which coincides with histone H4 deacetylation on these regions. Moreover, combined silencing of HDAC4 and HDAC6 abolishes the cycling of the IGFBP3 gene. We assume that due to more efficient VDR interaction, Gemini induces longer lasting chromatin activation and therefore no transcriptional cycling but monotonically increasing IGFBP3 mRNA. In conclusion, 1α,25(OH)(2)D(3) regulates IGFBP3 transcription through short-term cyclical association of VDR, HDAC4 and HDAC6 to both VDRE-containing chromatin regions.
Assuntos
Calcitriol/farmacologia , Regulação da Expressão Gênica , Proteína 3 de Ligação a Fator de Crescimento Semelhante à Insulina/genética , Elemento de Resposta à Vitamina D , Calcitriol/análogos & derivados , Linhagem Celular , Cromatina/metabolismo , Desacetilase 6 de Histona , Histona Desacetilases/genética , Histona Desacetilases/metabolismo , Humanos , Proteína 3 de Ligação a Fator de Crescimento Semelhante à Insulina/biossíntese , Masculino , Pessoa de Meia-Idade , Periodicidade , Interferência de RNA , RNA Mensageiro/metabolismo , Receptores de Calcitriol/metabolismo , Proteínas Repressoras/antagonistas & inibidores , Proteínas Repressoras/genética , Proteínas Repressoras/metabolismo , Transcrição GênicaRESUMO
BACKGROUND: The histone demethylase GASC1 (JMJD2C) is an epigenetic factor suspected of involvement in development of different cancers, including breast cancer. It is thought to be overexpressed in the more aggressive breast cancer types based on mRNA expression studies on cell lines and meta analysis of human breast cancer sets. This study aimed to evaluate the prognostic and predictive value of GASC1 for women with invasive breast cancer. METHODS: All the 355 cases were selected from a cohort enrolled in the Kuopio Breast Cancer Project between April 1990 and December 1995. The expression of GASC1 was studied by immunohistochemistry (IHC) on tissue microarrays. Additionally relative GASC1 mRNA expression was measured from available 57 cases. RESULTS: In our material, 56% of the cases were GASC1 negative and 44% positive in IHC staining. Women with GASC1 negative tumors had two years shorter breast cancer specific survival and time to relapse than the women with GASC1 positive tumors (p=0.017 and p=0.034 respectively). The majority of GASC1 negative tumors were ductal cases (72%) of higher histological grade (84% of grade II and III altogether). When we evaluated estrogen receptor negative and progesterone receptor negative cases separately, there was 2 times more GASC1 negative than GASC1 positive tumors in each group (chi2, p= 0.033 and 0.001 respectively). In the HER2 positive cases, there was 3 times more GASC1 negative cases than GASC1 positives (chi2, p= 0.029). Patients treated with radiotherapy (n=206) and hormonal treatment (n=62) had better breast cancer specific survival, when they were GASC1 positive (Cox regression: HR=0.49, p=0.007 and HR=0.33, p=0.015, respectively). The expression of GASC1 mRNA was in agreement with the protein analysis. CONCLUSIONS: This study indicates that the GASC1 is both a prognostic and a predictive factor for women with invasive breast cancer. GASC1 negativity is associated with tumors of more aggressive histopathological types (ductal type, grade II and III, ER negative, PR negative). Patients with GASC1 positive tumors have better breast cancer specific survival and respond better to radiotherapy and hormonal treatment.
Assuntos
Biomarcadores Tumorais/análise , Neoplasias da Mama/enzimologia , Carcinoma Ductal de Mama/enzimologia , Histona Desmetilases com o Domínio Jumonji/biossíntese , Antineoplásicos/uso terapêutico , Neoplasias da Mama/mortalidade , Neoplasias da Mama/patologia , Carcinoma Ductal de Mama/mortalidade , Carcinoma Ductal de Mama/patologia , Intervalo Livre de Doença , Moduladores de Receptor Estrogênico/uso terapêutico , Feminino , Humanos , Imuno-Histoquímica , Histona Desmetilases com o Domínio Jumonji/análise , Estimativa de Kaplan-Meier , Pessoa de Meia-Idade , Prognóstico , Modelos de Riscos Proporcionais , RNA Mensageiro/análise , Radioterapia , Análise Serial de TecidosRESUMO
Seminal plasma (SP) plays a crucial role in reproduction and contains a large number of proteins, many of which may potentially modify sperm functionality. To evaluate the effects of SP identity and its protein composition on human sperm function, we treated the sperm of several males with either their own or multiple foreign SPs in all possible sperm-SP combinations (full-factorial design). Then we recorded sperm motility and viability in these combinations and investigated whether the sperm performance is dependent on sperm and SP identity (or their interaction). Finally, we studied whether the above-mentioned sperm traits are affected by the abundance of three SP proteins, dipeptidyl peptidase-4 (DPP4), neutral endopeptidase (NEP), and aminopeptidase N (APN). The identity of the SP donor affected sperm swimming velocity, viability, and the proportion of hyperactivated sperm, but males' own SP was not consistently more beneficial for sperm than foreign SPs. Furthermore, we show that sperm performance is also partly affected by the interaction between sperm and SP donor. Finally, we found that DPP4 and NEP levels in SP were positively associated with sperm swimming velocity and hyperactivation. Taken together, our results highlight the importance of seminal plasma as a potential source of biomarkers for diagnostics and therapeutic interventions for male-derived infertility.
Assuntos
Infertilidade Masculina , Sêmen , Dipeptidil Peptidase 4/metabolismo , Humanos , Infertilidade Masculina/metabolismo , Masculino , Neprilisina/metabolismo , Sêmen/metabolismo , Motilidade dos Espermatozoides , Espermatozoides/metabolismoRESUMO
Primary 1alpha,25-dihydroxyvitamin D(3) (1alpha,25(OH)(2)D(3))-responding genes are controlled by the vitamin D receptor (VDR) binding to specific sites (VDREs) that are located within the regulatory regions of these genes. According to previous studies, the gene encoding 25-dihydroxyvitamin D(3) 24-hydroxylase, CYP24, which is the strongest known 1alpha,25(OH)(2)D(3)-responsive gene, has multiple VDREs that locate within the proximal and the distal promoter. However, it has remained unclear, what is the biological role of these regions and how they participate in the regulation of transcription. In this study, we found a different CYP24 expression profile in normal (MCF-10A) and malignant (MCF-7) human mammary cells. Moreover, CYP24 mRNA showed to be three times more stable in MCF-7 cells than in MCF-10A cells. We studied the mechanism of this difference using expression profiling, quantitative chromatin immunoprecipitation and chromosome conformation capture assays. Interestingly, the number of functional VDREs was higher in MCF-7 cells than in MCF-10A cells. Three functional VDREs in MCF-7 cells are connected to linear mRNA accumulation, whereas only one VDRE seems to lead to stepwise CYP24 mRNA accumulation in MCF-10A cells. The distal VDREs were involved in transcriptional regulation via ligand-dependent, dynamic chromatin looping, which brings cyclically the distal elements together either individually or simultaneously next to the transcription start site. In conclusion, our data suggest that in comparison to normal cells, clearing of 1alpha,25(OH)(2)D(3) is enhanced in malignant cells due to differences in transcriptional regulation of CYP24 and metabolism of CYP24 mRNA.
Assuntos
Neoplasias da Mama/metabolismo , Mama/metabolismo , Regulação Neoplásica da Expressão Gênica , Receptores de Calcitriol/metabolismo , Esteroide Hidroxilases/metabolismo , Vitamina D/metabolismo , Linhagem Celular Tumoral , Núcleo Celular/metabolismo , Cromatina/química , Epigênese Genética , Humanos , Ligantes , Regiões Promotoras Genéticas , RNA Mensageiro/metabolismo , Fatores de Tempo , Vitamina D/química , Vitamina D3 24-HidroxilaseRESUMO
Glucocorticoid (GC) receptor (GR) is a key transcription factor (TF) that regulates vital metabolic and anti-inflammatory processes. We have identified BCL6 corepressor (BCOR) as a dexamethasone-stimulated interaction partner of GR. BCOR is a component of non-canonical polycomb repressor complex 1.1 (ncPCR1.1) and linked to different developmental disorders and cancers, but the role of BCOR in GC signaling is poorly characterized. Here, using ChIP-seq we show that, GC induces genome-wide redistribution of BCOR chromatin binding towards GR-occupied enhancers in HEK293 cells. As assessed by RNA-seq, depletion of BCOR altered the expression of hundreds of GC-regulated genes, especially the ones linked to TNF signaling, GR signaling and cell migration pathways. Biotinylation-based proximity mapping revealed that GR and BCOR share several interacting partners, including nuclear receptor corepressor NCOR1. ChIP-seq showed that the NCOR1 co-occurs with both BCOR and GR on a subset of enhancers upon GC treatment. Simultaneous depletion of BCOR and NCOR1 influenced GR target gene expression in a combinatorial and gene-specific manner. Finally, we show using live cell imaging that the depletion of BCOR together with NCOR1 markedly enhances cell migration. Collectively, our data suggest BCOR as an important gene and pathway selective coregulator of GR transcriptional activity.
Assuntos
Proteínas Proto-Oncogênicas/metabolismo , Receptores de Glucocorticoides/genética , Proteínas Repressoras/metabolismo , Sítios de Ligação , Movimento Celular/genética , Proliferação de Células/genética , Imunoprecipitação da Cromatina , Dexametasona/farmacologia , Elementos Facilitadores Genéticos , Regulação da Expressão Gênica/efeitos dos fármacos , Células HEK293 , Humanos , Correpressor 1 de Receptor Nuclear/genética , Correpressor 1 de Receptor Nuclear/metabolismo , Mapas de Interação de Proteínas , Proteínas Proto-Oncogênicas/genética , Receptores de Glucocorticoides/metabolismo , Proteínas Repressoras/genéticaRESUMO
Treatment of prostate cancer confronts resistance to androgen receptor (AR)-targeted therapies. AR-associated coregulators and chromatin proteins hold a great potential for novel therapy targets. Here, we employed a powerful chromatin-directed proteomics approach termed ChIP-SICAP to uncover the composition of chromatin protein network, the chromatome, around endogenous AR in castration resistant prostate cancer (CRPC) cells. In addition to several expected AR coregulators, the chromatome contained many nuclear proteins not previously associated with the AR. In the context of androgen signaling in CRPC cells, we further investigated the role of a known AR-associated protein, a chromatin remodeler SMARCA4 and that of SIM2, a transcription factor without a previous association with AR. To understand their role in chromatin accessibility and AR target gene expression, we integrated data from ChIP-seq, RNA-seq, ATAC-seq and functional experiments. Despite the wide co-occurrence of SMARCA4 and AR on chromatin, depletion of SMARCA4 influenced chromatin accessibility and expression of a restricted set of AR target genes, especially those involved in cell morphogenetic changes in epithelial-mesenchymal transition. The depletion also inhibited the CRPC cell growth, validating SMARCA4's functional role in CRPC cells. Although silencing of SIM2 reduced chromatin accessibility similarly, it affected the expression of a much larger group of androgen-regulated genes, including those involved in cellular responses to external stimuli and steroid hormone stimulus. The silencing also reduced proliferation of CRPC cells and tumor size in chick embryo chorioallantoic membrane assay, further emphasizing the importance of SIM2 in CRPC cells and pointing to the functional relevance of this potential prostate cancer biomarker in CRPC cells. Overall, the chromatome of AR identified in this work is an important resource for the field focusing on this important drug target.
Assuntos
Receptores Androgênicos , Animais , Embrião de Galinha , Masculino , Próstata , ProteômicaRESUMO
The anti-proliferative effects of histone deacetylase (HDAC) inhibitors and 1alpha,25-dihydroxyvitamin D3 [1alpha,25(OH)2D3] converge via the interaction of un-liganded vitamin D receptor (VDR) with co-repressors recruiting multiprotein complexes containing HDACs and via the induction of cyclin-dependent kinase inhibitor (CDKI) genes of the INK4 and Cip/Kip family. We investigated the effects of the HDAC inhibitor Trichostatin A (TSA) and 1alpha,25(OH)2D3 on the proliferation and CDKI gene expression in malignant and non-malignant mammary epithelial cell lines. TSA induced the INK4-family genes p18 and p19, whereas the Cip/Kip family gene p21 was stimulated by 1alpha,25(OH)2D3. Chromatin immunoprecipitation and RNA inhibition assays showed that the co-repressor NCoR1 and some HDAC family members complexed un-liganded VDR and repressed the basal level of CDKI genes, but their role in regulating CDKI gene expression by TSA and 1alpha,25(OH)2D3 were contrary. HDAC3 and HDAC7 attenuated 1alpha,25(OH)2D3-dependent induction of the p21 gene, for which NCoR1 is essential. In contrast, TSA-mediated induction of the p18 gene was dependent on HDAC3 and HDAC4, but was opposed by NCoR1 and un-liganded VDR. This suggests that the attenuation of the response to TSA by NCoR1 or that to 1alpha,25(OH)2D3 by HDACs can be overcome by their combined application achieving maximal induction of anti-proliferative target genes.