RESUMO
The bone remodeling process is crucial for titanium (Ti) osseointegration and involves the crosstalk between osteoclasts and osteoblasts. Considering the high osteogenic potential of Ti with nanotopography (Ti Nano) and that osteoclasts inhibit osteoblast differentiation, we hypothesized that nanotopography attenuate the osteoclast-induced disruption of osteoblast differentiation. Osteoblasts were co-cultured with osteoclasts on Ti Nano and Ti Control and non-co-cultured osteoblasts were used as control. Gene expression analysis using RNAseq showed that osteoclasts downregulated the expression of osteoblast marker genes and upregulated genes related to histone modification and chromatin organization in osteoblasts grown on both Ti surfaces. Osteoclasts also inhibited the mRNA and protein expression of osteoblast markers, and such effect was attenuated by Ti Nano. Also, osteoclasts increased the protein expression of H3K9me2, H3K27me3 and EZH2 in osteoblasts grown on both Ti surfaces. ChIP assay revealed that osteoclasts increased accumulation of H3K27me3 that represses the promoter regions of Runx2 and Alpl in osteoblasts grown on Ti Control, which was reduced by Ti Nano. In conclusion, these data show that despite osteoclast inhibition of osteoblasts grown on both Ti Control and Ti Nano, the nanotopography attenuates the osteoclast-induced disruption of osteoblast differentiation by preventing the increase of H3K27me3 accumulation that represses the promoter regions of some key osteoblast marker genes. These findings highlight the epigenetic mechanisms triggered by nanotopography to protect osteoblasts from the deleterious effects of osteoclasts, which modulate the process of bone remodeling and may benefit the osseointegration of Ti implants.
Assuntos
Osteoclastos , Titânio , Histonas/metabolismo , Metilação , Osteoblastos , Osteoclastos/metabolismo , Propriedades de Superfície , Titânio/farmacologiaRESUMO
Cell therapy is a valuable strategy for the replacement of bone grafts and repair bone defects, and mesenchymal stem cells (MSCs) are the most frequently used cells. This study was designed to genetically edit MSCs to overexpress bone morphogenetic protein 9 (BMP-9) using Clustered Regularly Interspaced Short Palindromic Repeats/associated nuclease Cas9 (CRISPR-Cas9) technique to generate iMSCs-VPRBMP-9+, followed by in vitro evaluation of osteogenic potential and in vivo enhancement of bone formation in rat calvaria defects. Overexpression of BMP-9 was confirmed by its gene expression and protein expression, as well as its targets Hey-1, Bmpr1a, and Bmpr1b, Dlx-5, and Runx2 and protein expression of SMAD1/5/8 and pSMAD1/5/8. iMSCs-VPRBMP-9+ displayed significant changes in the expression of a panel of genes involved in TGF-ß/BMP signaling pathway. As expected, overexpression of BMP-9 increased the osteogenic potential of MSCs indicated by increased gene expression of osteoblastic markers Runx2, Sp7, Alp, and Oc, higher ALP activity, and matrix mineralization. Rat calvarial bone defects treated with injection of iMSCs-VPRBMP-9+ exhibited increased bone formation and bone mineral density when compared with iMSCs-VPR- and phosphate buffered saline (PBS)-injected defects. This is the first study to confirm that CRISPR-edited MSCs overexpressing BMP-9 effectively enhance bone formation, providing novel options for exploring the capability of genetically edited cells to repair bone defects.
Assuntos
Fator 2 de Diferenciação de Crescimento , Células-Tronco Mesenquimais , Osteogênese , Animais , Sistemas CRISPR-Cas , Diferenciação Celular , Células Cultivadas , Fator 2 de Diferenciação de Crescimento/genética , Células-Tronco Mesenquimais/citologia , Osteogênese/genética , RatosRESUMO
Epigenetic control is critical for the regulation of gene transcription in mammalian cells. Among the most important epigenetic mechanisms are those associated with posttranslational modifications of chromosomal histone proteins, which modulate chromatin structure and increased accessibility of promoter regulatory elements for competency to support transcription. A critical histone mark is trimethylation of histone H3 at lysine residue 27 (H3K27me3), which is mediated by Ezh2, the catalytic subunit of the polycomb group complex PRC2 to repress transcription. Treatment of cells with the active vitamin D metabolite 1,25(OH)2 D3 , results in transcriptional activation of the CYP24A1 gene, which encodes a 24-hydroxylase enzyme, that is, essential for physiological control of vitamin D3 levels. We report that the Ezh2-mediated deposition of H3K27me3 at the CYP24A1 gene promoter is a requisite regulatory component during transcriptional silencing of this gene in osteoblastic cells in the absence of 1,25(OH)2 D3 . 1,25(OH)2 D3 dependent transcriptional activation of the CYP24A1 gene is accompanied by a rapid release of Ezh2 from the promoter, together with the binding of the H3K27me3-specific demethylase Utx/Kdm6a and thereby subsequent erasing of the H3K27me3 mark. Importantly, we find that these changes in H3K27me3 enrichment at the CYP24A1 gene promoter are highly dynamic, as this modification is rapidly reacquired following the withdrawal of 1,25(OH)2 D3 .
Assuntos
Colecalciferol/genética , Proteína Potenciadora do Homólogo 2 de Zeste/genética , Osteossarcoma/genética , Vitamina D3 24-Hidroxilase/genética , Animais , Linhagem Celular Tumoral , Epigênese Genética/genética , Regulação da Expressão Gênica no Desenvolvimento/genética , Código das Histonas/genética , Humanos , Osteoblastos/metabolismo , Osteossarcoma/patologia , Regiões Promotoras Genéticas/genética , Processamento de Proteína Pós-Traducional/genética , Ratos , Ativação Transcricional/genéticaRESUMO
In bone cells vitamin D dependent regulation of gene expression principally occurs through modulation of gene transcription. Binding of the active vitamin D metabolite, 1,25-dihydroxy vitamin D3 (1,25(OH)2 D3 ) to the vitamin D receptor (VDR) induces conformational changes in its C-terminal domain enabling competency for interaction with physiologically relevant coactivators, including SRC-1. Consequently, regulatory complexes can be assembled that support intrinsic enzymatic activities with competency to posttranslationally modify chromatin histones at target genomic sequences to epigenetically alter transcription. Here we examine specific transitions in representation and/or enrichment of epigenetic histone marks during 1,25(OH)2 D3 mediated upregulation of CYP24A1 gene expression in osteoblastic cells. This gene encodes the 24-hydroxylase enzyme, essential for biological control of vitamin D levels. We demonstrate that as the CYP24A1 gene promoter remains transcriptionally silent, there is enrichment of H4R3me2s together with its "writing" enzyme PRMT5 and decreased abundance of the istone H3 and H4 acetylation, H3R17me2a, and H4R3me2a marks as well as of their corresponding "writers." Exposure of osteoblastic cells to 1,25(OH)2 D3 stimulates the recruitment of a VDR/SRC-1 containing complex to the CYP24A1 promoter to mediate increased H3/H4 acetylation. VDR/SRC-1 binding occurs concomitant with the release of PRMT5 and the recruitment of the arginine methyltransferases CARM1 and PRMT1 to catalyze the deposition of the H3R17me2a and H4R3me2a marks, respectively. Our results indicate that these dynamic transitions of histone marks at the CYP24A1 promoter, provide a "chromatin context" that is transcriptionally competent for activation of the CYP24A1 gene in osteoblastic cells in response to 1,25(OH)2 D3 .
Assuntos
Proteína-Arginina N-Metiltransferases/genética , Receptores de Calcitriol/genética , Transcrição Gênica , Vitamina D3 24-Hidroxilase/genética , Colecalciferol/genética , Cromatina/genética , Epigênese Genética/genética , Regulação da Expressão Gênica no Desenvolvimento/genética , Código das Histonas/genética , Histonas/genética , Humanos , Osteoblastos/metabolismo , Regiões Promotoras Genéticas/genética , Ligação Proteica/genética , Proteínas Repressoras/genética , Ativação Transcricional/genéticaRESUMO
The major role of integrins is to mediate cell adhesion but some of them are involved in the osteoblasts-titanium (Ti) interactions. In this study, we investigated the participation of integrins in osteoblast differentiation induced by Ti with nanotopography (Ti-Nano) and with microtopography (Ti-Micro). By using a PCR array, we observed that, compared with Ti-Micro, Ti-Nano upregulated the expression of five integrins in mesenchymal stem cells, including integrin ß3, which increases osteoblast differentiation. Silencing integrin ß3, using CRISPR-Cas9, in MC3T3-E1 cells significantly reduced the osteoblast differentiation induced by Ti-Nano in contrast to the effect on T-Micro. Concomitantly, integrin ß3 silencing downregulated the expression of integrin αv, the parent chain that combines with other integrins and several components of the Wnt/ß-catenin and BMP/Smad signaling pathways, all involved in osteoblast differentiation, only in cells cultured on Ti-Nano. Taken together, our results showed the key role of integrin ß3 in the osteogenic potential of Ti-Nano but not of Ti-Micro. Additionally, we propose a novel mechanism to explain the higher osteoblast differentiation induced by Ti-Nano that involves an intricate regulatory network triggered by integrin ß3 upregulation, which activates the Wnt and BMP signal transductions. © 2019 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 107A: 1303-1313, 2019.
Assuntos
Diferenciação Celular , Integrina beta3/metabolismo , Nanoestruturas/química , Osteoblastos/metabolismo , Titânio/química , Via de Sinalização Wnt , Animais , Linhagem Celular , Masculino , Camundongos , Osteoblastos/citologia , Ratos , Ratos WistarRESUMO
Expression of Runx2/p57 is a hallmark of the osteoblast-lineage identity. Although several regulators that control the expression of Runx2/p57 during osteoblast-lineage commitment have been identified, the epigenetic mechanisms that sustain this expression in differentiated osteoblasts remain to be completely determined. Here, we assess epigenetic mechanisms associated with Runx2/p57 gene transcription in differentiating MC3T3 mouse osteoblasts. Our results show that an enrichment of activating histone marks at the Runx2/p57 P1 promoter is accompanied by the simultaneous interaction of Wdr5 and Utx proteins, both are components of COMPASS complexes. Knockdown of Wdr5 and Utx expression confirms the activating role of both proteins at the Runx2-P1 promoter. Other chromatin modifiers that were previously described to regulate Runx2/p57 transcription in mesenchymal precursor cells (Ezh2, Prmt5, and Jarid1b proteins) were not found to contribute to Runx2/p57 transcription in full-committed osteoblasts. We also determined the presence of additional components of COMPASS complexes at the Runx2/p57 promoter, evidencing that the Mll2/COMPASS- and Mll3/COMPASS-like complexes bind to the P1 promoter in osteoblastic cells expressing Runx2/p57 to modulate the H3K4me1 to H3K4me3 transition.
Assuntos
Subunidade alfa 1 de Fator de Ligação ao Core/genética , Histona Desmetilases/genética , Histonas/genética , Peptídeos e Proteínas de Sinalização Intracelular/genética , Osteoblastos/metabolismo , Células 3T3 , Animais , Diferenciação Celular/fisiologia , Subunidade alfa 1 de Fator de Ligação ao Core/metabolismo , Epigênese Genética/genética , Regulação da Expressão Gênica/fisiologia , Histona Desmetilases/metabolismo , Histonas/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Camundongos , Osteoblastos/citologia , Transcrição GênicaRESUMO
RUNX1 a member of the family of runt related transcription factors (RUNX), is essential for hematopoiesis. The expression of RUNX1 gene is controlled by two promoters; the distal P1 promoter and the proximal P2 promoter. Several isoforms of RUNX1 mRNA are generated through the use of both promoters and alternative splicing. These isoforms not only differs in their temporal expression pattern but also exhibit differences in tissue specificity. The RUNX1 isoforms derived from P2 are expressed in a variety of tissues, but expression of P1-derived isoform is restricted to cells of hematopoietic lineage. However, the control of hematopoietic-cell specific expression is poorly understood. Here we report regulation of P1-derived RUNX1 mRNA by RUNX1 protein. In silico analysis of P1 promoter revealed presence of two evolutionary conserved RUNX motifs, 0.6kb upstream of the transcription start site, and three RUNX motifs within 170bp of the 5'UTR. Transcriptional contribution of these RUNX motifs was studied in myeloid and T-cells. RUNX1 genomic fragment containing all sites show very low basal activity in both cell types. Mutation or deletion of RUNX motifs in the UTR enhances basal activity of the RUNX1 promoter. Chromatin immunoprecipitation revealed that RUNX1 protein is recruited to these sites. Overexpression of RUNX1 in non-hematopoietic cells results in a dose dependent activation of the RUNX1 P1 promoter. We also demonstrate that RUNX1 protein regulates transcription of endogenous RUNX1 mRNA in T-cell. Finally we show that SCL transcription factor is recruited to regions containing RUNX motifs in the promoter and the UTR and regulates activity of the RUNX1 P1 promoter in vitro. Thus, multiple lines of evidence show that RUNX1 protein regulates its own gene transcription.
Assuntos
Subunidade alfa 2 de Fator de Ligação ao Core/genética , Subunidade alfa 2 de Fator de Ligação ao Core/metabolismo , Regulação da Expressão Gênica , Regiões Promotoras Genéticas , Transcrição Gênica , Regiões 5' não Traduzidas , Sequência de Bases , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Sítios de Ligação , Linhagem Celular Tumoral , Humanos , Dados de Sequência Molecular , Mutação , Motivos de Nucleotídeos , Ligação Proteica , Proteínas Proto-Oncogênicas/metabolismo , RNA Mensageiro , Alinhamento de Sequência , Proteína 1 de Leucemia Linfocítica Aguda de Células T , Ativação TranscricionalRESUMO
Transcription factor Runx2 controls bone development and osteoblast differentiation by regulating expression of a significant number of bone-related target genes. Here, we report that transcriptional activation and repression of the Runx2 gene via its osteoblast-specific P1 promoter (encoding mRNA for the Runx2/p57 isoform) is accompanied by selective deposition and elimination of histone marks during differentiation of mesenchymal cells to the osteogenic and myoblastic lineages. These epigenetic profiles are mediated by key components of the Trithorax/COMPASS-like and Polycomb group complexes together with histone arginine methylases like PRMT5 and lysine demethylases like JARID1B/KDM5B. Importantly, knockdown of the H3K4me2/3 demethylase JARID1B, but not of the demethylases UTX and NO66, prevents repression of the Runx2 P1 promoter during myogenic differentiation of mesenchymal cells. The epigenetically forced expression of Runx2/p57 and osteocalcin, a classical bone-related target gene, under myoblastic-differentiation is accompanied by enrichment of the H3K4me3 and H3K27ac marks at the Runx2 P1 promoter region. Our results identify JARID1B as a key component of a potent epigenetic switch that controls mesenchymal cell fate into myogenic and osteogenic lineages.
Assuntos
Subunidade alfa 1 de Fator de Ligação ao Core/genética , Proteínas de Ligação a DNA/metabolismo , Epigênese Genética , Histona Desmetilases com o Domínio Jumonji/metabolismo , Osteoblastos/citologia , Animais , Diferenciação Celular , Linhagem Celular , Linhagem da Célula , Histonas/metabolismo , Humanos , Camundongos , Osteoblastos/metabolismo , Regiões Promotoras GenéticasRESUMO
The chromatin remodeling complex SWI/SNF and the transcription factor C/EBPß play critical roles in osteoblastic cells as they jointly control transcription of a number of bone-related target genes. The largest C/EBPß isoform, LAP*, possesses a short additional N-terminal domain that has been proposed to mediate the interaction of this factor with SWI/SNF in myeloid cells. Here we examine the requirement of a functional N-terminus in C/EBPß-LAP* for binding SWI/SNF and for recruiting this complex to the Ric-8B gene to mediate transcriptional repression. We find that both C/EBPß-LAP* and SWI/SNF simultaneously bind to the Ric-8B promoter in differentiating osteoblasts that repress Ric-8B expression. This decreased expression of Ric-8B is not accompanied by significant changes in histone acetylation at the Ric-8B gene promoter sequence. A single aminoacid change at the C/EBPß-LAP* N-terminus (R3L) that inhibits C/EBPß-LAP*-SWI/SNF interaction, also prevents SWI/SNF recruitment to the Ric-8B promoter as well as C/EBPß-LAP*-dependent repression of the Ric-8B gene. Inducible expression of the C/EBPß-LAP*R3L protein in stably transfected osteoblastic cells demonstrates that this mutant protein binds to C/EBPß-LAP*-target promoters and competes with the endogenous C/EBPß factor. Together our results indicate that a functional N-terminus in C/EBPß-LAP* is required for interacting with SWI/SNF and for Ric-8B gene repression in osteoblasts.
Assuntos
Proteína beta Intensificadora de Ligação a CCAAT/metabolismo , Montagem e Desmontagem da Cromatina , Proteínas Cromossômicas não Histona/metabolismo , Fatores de Troca do Nucleotídeo Guanina/metabolismo , Proteínas Nucleares/metabolismo , Osteoblastos/metabolismo , Fatores de Transcrição/metabolismo , Transcrição Gênica , Células 3T3 , Acetilação , Animais , Sítios de Ligação , Proteína beta Intensificadora de Ligação a CCAAT/genética , Linhagem Celular Tumoral , Proliferação de Células , Regulação para Baixo , Fatores de Troca do Nucleotídeo Guanina/genética , Histonas/metabolismo , Camundongos , Mutação , Proteínas Nucleares/genética , Osteocalcina/metabolismo , Regiões Promotoras Genéticas , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas , Isoformas de Proteínas , Ratos , TransfecçãoRESUMO
The Runx2 factor is an essential component of the regulatory mechanisms that control transcription during skeletogenesis. Runx2/p57 expression in osteoblastic cells is controlled by the P1 promoter, which is recognized by key regulators of osteoblast differentiation including homeodomain factors and Wnt- and BMP-signaling mediators. Here, we report that the transcription factor C/EBPß up-regulates Runx2/p57 expression by directly binding to the Runx2 P1 promoter in mesenchymal, pre-osteoblastic, and osteoblastic cells. This C/EBPß-mediated up-regulation is principally dependent on C/EBP site II that is located within the first 180 bp of the proximal P1 promoter region and is highly conserved among mouse, rat, and human Runx2 genes. Our studies reveal how the C/EBPß factor, known to have a key role during osteogenesis, contributes to regulating the expression of Runx2, the master regulator of osteoblast differentiation.
Assuntos
Proteína beta Intensificadora de Ligação a CCAAT/metabolismo , Subunidade alfa 1 de Fator de Ligação ao Core/genética , Regulação da Expressão Gênica , Osteoblastos/metabolismo , Regiões Promotoras Genéticas , Transcrição Gênica , Animais , Sequência de Bases , Linhagem Celular , Humanos , Células-Tronco Mesenquimais/metabolismo , Camundongos , Dados de Sequência Molecular , Ratos , Regulação para CimaRESUMO
1alpha,25-dihydroxy vitamin D(3) (vitamin D(3)) has an important role during osteoblast differentiation as it directly modulates the expression of key bone-related genes. Vitamin D(3) binds to the vitamin D(3) receptor (VDR), a member of the superfamily of nuclear receptors, which in turn interacts with transcriptional activators to target this regulatory complex to specific sequence elements within gene promoters. Increasing evidence demonstrates that the architectural organization of the genome and regulatory proteins within the eukaryotic nucleus support gene expression in a physiological manner. Previous reports indicated that the VDR exhibits a punctate nuclear distribution that is significantly enhanced in cells grown in the presence of vitamin D(3). Here, we demonstrate that in osteoblastic cells, the VDR binds to the nuclear matrix in a vitamin D(3)-dependent manner. This interaction of VDR with the nuclear matrix occurs rapidly after vitamin D(3) addition and does not require a functional VDR DNA-binding domain. Importantly, nuclear matrix-bound VDR colocalizes with its transcriptional coactivator DRIP205/TRAP220/MED1 which is also matrix bound. Together these results indicate that after ligand stimulation the VDR rapidly enters the nucleus and associates with the nuclear matrix preceding vitamin D(3)-transcriptional upregulation.
Assuntos
Calcitriol/metabolismo , Matriz Nuclear/metabolismo , Osteoblastos/metabolismo , Receptores de Calcitriol/metabolismo , Animais , Sítios de Ligação , Linhagem Celular Tumoral , Subunidade alfa 1 de Fator de Ligação ao Core/metabolismo , DNA/metabolismo , Humanos , Ligantes , Subunidade 1 do Complexo Mediador/metabolismo , Camundongos , Mutação , Ligação Proteica , Estrutura Terciária de Proteína , Ratos , Receptores de Calcitriol/genética , Proteínas Recombinantes de Fusão/metabolismo , Ativação Transcricional , Transdução GenéticaRESUMO
Bone formation and osteoblast differentiation require the functional expression of the Runx2/Cbfbeta heterodimeric transcription factor complex. Runx2 is also a suppressor of proliferation in osteoblasts by attenuating cell cycle progression in G(1). Runx2 levels are modulated during the cell cycle, which are maximal in G(1) and minimal beyond the G(1)/S phase transition (S, G(2), and M phases). It is not known whether Cbfbeta gene expression is cell cycle controlled in preosteoblasts nor how Runx2 or Cbfbeta are regulated during the cell cycle in bone cancer cells. We investigated Runx2 and Cbfbeta gene expression during cell cycle progression in MC3T3-E1 osteoblasts, as well as ROS17/2.8 and SaOS-2 osteosarcoma cells. Runx2 protein levels are reduced as expected in MC3T3-E1 cells arrested in late G(1) (by mimosine) or M phase (by nocodazole), but not in cell cycle arrested osteosarcoma cells. Cbfbeta protein levels are cell cycle independent in both osteoblasts and osteosarcoma cells. In synchronized MC3T3-E1 osteoblasts progressing from late G1 or mitosis, Runx2 levels but not Cbfbeta levels are cell cycle regulated. However, both factors are constitutively elevated throughout the cell cycle in osteosarcoma cells. Proteasome inhibition by MG132 stabilizes Runx2 protein levels in late G(1) and S in MC3T3-E1 cells, but not in ROS17/2.8 and SaOS-2 osteosarcoma cells. Thus, proteasomal degradation of Runx2 is deregulated in osteosarcoma cells. We propose that cell cycle control of Runx2 gene expression is impaired in osteosarcomas and that this deregulation may contribute to the pathogenesis of osteosarcoma.
Assuntos
Ciclo Celular/fisiologia , Subunidade alfa 1 de Fator de Ligação ao Core/metabolismo , Subunidade beta de Fator de Ligação ao Core/metabolismo , Regulação Neoplásica da Expressão Gênica/fisiologia , Osteossarcoma/metabolismo , Animais , Linhagem Celular , Linhagem Celular Tumoral , Subunidade alfa 1 de Fator de Ligação ao Core/genética , Subunidade beta de Fator de Ligação ao Core/genética , Inibidores de Cisteína Proteinase , Fase G1/fisiologia , Expressão Gênica/genética , Humanos , Leupeptinas/farmacologia , Camundongos , Mitose/fisiologia , Osteoblastos/citologia , Osteoblastos/metabolismo , Osteossarcoma/patologia , Complexo de Endopeptidases do Proteassoma/metabolismo , Inibidores de Proteassoma , Ratos , Ubiquitinação/efeitos dos fármacosRESUMO
The Runx2 transcription factor is essential for skeletal development as it regulates expression of several key bone-related genes. Multiple lines of evidence indicate that expression of the Runx2/p57 isoform in osteoblasts is controlled by the distal P1 promoter. Alterations of chromatin structure are often associated with transcription and can be mediated by members of the SWI/SNF family of chromatin remodeling complexes, or by transcriptional coactivators that possess enzymatic activities that covalently modify structural components of the chromatin. Here, we report that a specific chromatin remodeling process at the proximal region (residues -400 to 35) of the Runx2 gene P1 promoter accompanies transcriptional activity in osteoblasts. This altered chromatin organization is reflected by the presence of two DNase I hypersensitive sites that span key regulatory elements for Runx2/p57 transcription. Chromatin remodeling and transcription of the Runx2 gene are associated with elevated levels of histone acetylation at the P1 promoter region and binding of active RNA polymerase II and are independent of the activity of the SWI/SNF chromatin remodeling complex. Changes in chromatin organization at the P1 promoter are stimulated during differentiation of C2C12 mesenchymal cells to the osteoblastic lineage by treatment with BMP2. Together, our results support a model in which changes in chromatin organization occur at very early stages of mesenchymal differentiation to facilitate subsequent expression of the Runx2/p57 isoform in osteoblastic cells.
Assuntos
Proteínas Cromossômicas não Histona/metabolismo , Subunidade alfa 1 de Fator de Ligação ao Core/metabolismo , Desoxirribonucleases/metabolismo , Histonas/metabolismo , Regiões Promotoras Genéticas , Fatores de Transcrição/metabolismo , Transcrição Gênica , Acetilação , Animais , Proteína Morfogenética Óssea 2/genética , Proteína Morfogenética Óssea 2/metabolismo , Diferenciação Celular/fisiologia , Linhagem Celular , Montagem e Desmontagem da Cromatina , Proteínas Cromossômicas não Histona/genética , Subunidade alfa 1 de Fator de Ligação ao Core/genética , Regulação da Expressão Gênica , Histonas/genética , Camundongos , Osteoblastos/citologia , Osteoblastos/fisiologia , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Fatores de Transcrição/genéticaRESUMO
The RUNX1/AML1 gene is the most frequent target for chromosomal translocation, and often identified as a site for reciprocal rearrangement of chromosomes 8 and 21 in patients with acute myelogenous leukemia. Virtually all chromosome translocations in leukemia show no consistent homologous sequences at the breakpoint regions. However, specific chromatin elements (DNase I and topoisomerase II cleavage) have been found at the breakpoints of some genes suggesting that structural motifs are determinant for the double strand DNA-breaks. We analyzed the chromatin organization at intron 5 of the RUNX1 gene where all the sequenced breakpoints involved in t(8;21) have been mapped. Using chromatin immunoprecipitation assays we show that chromatin organization at intron 5 of the RUNX1 gene is different in HL-60 and HeLa cells. Two distinct features mark the intron 5 in cells expressing RUNX1: a complete lack or significantly reduced levels of Histone H1 and enrichment of hyperacetylated histone H3. Strikingly, induction of DNA damage resulted in formation of t(8;21) in HL-60 but not in HeLa cells. Taken together, our results suggest that H1 depletion and/or histone H3 hyperacetylation may have a linkage with an increase susceptibility of specific chromosomal regions to undergo translocations.
Assuntos
Cromatina/metabolismo , Quebra Cromossômica , Cromossomos Humanos Par 21/genética , Cromossomos Humanos Par 8/genética , Subunidade alfa 2 de Fator de Ligação ao Core/genética , Translocação Genética/genética , Acetilação , Cromatina/química , Células HL-60 , Células HeLa , Histonas/metabolismo , Humanos , Íntrons/genética , Ligação ProteicaRESUMO
Vitamin D is a principal modulator of skeletal gene expression, thus necessitating an understanding of interfaces between the activity of this steroid hormone and regulatory cascades that are functionally linked to the regulation of skeletal genes. Physiologic responsiveness requires combinatorial control, whereas co-regulatory proteins determine the specificity of biologic responsiveness to physiologic cues. It is becoming increasingly evident that regulatory complexes containing the vitamin D receptor are dynamic rather than static. Temporal and spatial modifications in the composition of these complexes provide a mechanism for integrating regulatory signals to support positive or negative control through synergism and antagonism. Compartmentalization of components of vitamin D control in nuclear microenvironments supports the integration of regulatory activities, perhaps by establishing thresholds for protein activity in time frames that are consistent with the execution of regulatory signaling.
Assuntos
Desenvolvimento Ósseo/genética , Regulação da Expressão Gênica no Desenvolvimento , Vitamina D/fisiologia , Animais , Núcleo Celular/metabolismo , Humanos , Modelos Biológicos , Complexos Multiproteicos/fisiologia , Fatores de Transcrição/fisiologiaRESUMO
Binding of 1alpha,25-dihydroxy vitamin D(3) to the C-terminal ligand-binding domain (LBD) of its receptor (VDR) induces a conformational change that enables interaction of VDR with transcriptional coactivators such as members of the p160/SRC family or the DRIP (vitamin D receptor-interacting complex)/Mediator complex. These interactions are critical for VDR-mediated transcriptional enhancement of target genes. The p160/SRC members contain intrinsic histone acetyl transferase (HAT) activities that remodel chromatin at promoter regulatory regions, and the DRIP/Mediator complex may establish a molecular bridge between the VDR complex and the basal transcription machinery. Here, we have analyzed the rate of recruitment of these coactivators to the bone-specific osteocalcin (OC) gene in response to short and long exposures to 1alpha,25-dihydroxy vitamin D3. We report that in intact osteoblastic cells VDR, in association with SRC-1, rapidly binds to the OC promoter in response to the ligand. The recruitment of SRC-1 correlates with maximal transcriptional enhancement of the OC gene at 4 h and with increased histone acetylation at the OC promoter. In contrast to other 1alpha,25-dihydroxy vitamin D3-enhanced genes, binding of the DRIP205 subunit, which anchors the DRIP/Mediator complex to the VDR, is detected at the OC promoter only after several hours of incubation with 1alpha,25-dihydroxy vitamin D(3), concomitant with the release of SRC-1. Together, our results support a model where VDR preferentially recruits SRC-1 to enhance bone-specific OC gene transcription.
Assuntos
Regulação da Expressão Gênica/efeitos dos fármacos , Histona Acetiltransferases/metabolismo , Osteocalcina/genética , Regiões Promotoras Genéticas/genética , Receptores de Calcitriol/metabolismo , Fatores de Transcrição/metabolismo , Transcrição Gênica/efeitos dos fármacos , Vitamina D/análogos & derivados , Animais , Subunidade 1 do Complexo Mediador , Modelos Genéticos , Coativador 1 de Receptor Nuclear , Osteoblastos/efeitos dos fármacos , Osteoblastos/enzimologia , Osteoblastos/metabolismo , Ligação Proteica/efeitos dos fármacos , RNA Polimerase II/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Ratos , Regulação para Cima/efeitos dos fármacos , Vitamina D/farmacologiaRESUMO
Chromatin organization within the nuclear compartment is a fundamental mechanism to regulate the expression of eukaryotic genes. During the last decade, a number of nuclear protein complexes with the ability to remodel chromatin and regulate gene transcription have been reported. Among these complexes is the SWI/SNF family, which alters chromatin structure in an ATP-dependent manner. A considerable effort has been made to understand the molecular mechanisms by which SWI/SNF catalyzes nucleosome remodeling. However, limited attention has been dedicated to studying the role of the DNA sequence in this remodeling process. Therefore, in this minireview, we discuss the contribution of nucleosome positioning and nucleosome excluding sequences to the targeting and activity of SWI/SNF complexes. This discussion includes results from our group using the rat osteocalcin gene promoter as a model. Based on these results, we postulate a model for chromatin remodeling and transcriptional activation of this gene in osteoblastic cells.
Assuntos
Sequência de Bases , Montagem e Desmontagem da Cromatina , Proteínas Cromossômicas não Histona/metabolismo , DNA , Nucleossomos , Fatores de Transcrição/metabolismo , Animais , DNA/genética , DNA/metabolismo , Modelos Genéticos , Nucleossomos/metabolismo , Nucleossomos/ultraestrutura , Osteocalcina/genética , Regiões Promotoras Genéticas , Transcrição GênicaRESUMO
Upon ligand binding the 1alpha,25-dihydroxy Vitamin D3 receptor (VDR) undergoes a conformational change that allows interaction with coactivator proteins including p160/SRC family members and the multimeric DRIP complex through the DRIP205 subunit. Casein kinase II (CKII) phosphorylates VDR both in vitro and in vivo at serine 208 within the hinge domain. This phosphorylation does not affect the ability of VDR to bind DNA, but increases its ability to transactivate target promoters. Here, we have analyzed whether phosphorylation of VDR by CKII modulates the ability of VDR to interact with coactivators in vitro. We find that both mutation of serine 208 to aspartic acid (VDRS208D) or phosphorylation of VDR by CKII enhance the interaction of VDR with DRIP205 in the presence of 1alpha,25-dihydroxy Vitamin D3. We also find that the mutation VDRS208D neither affects the ability of this protein to bind DNA nor to interact with SRC-1 and RXRalpha. Together, our results indicate that phosphorylation of VDR at serine 208 contributes to modulate the affinity of VDR for the DRIP complex and therefore may have a role in vivo regulating VDR-mediated transcriptional enhancement.
Assuntos
Fosfosserina/metabolismo , Receptores de Calcitriol/metabolismo , Transativadores/metabolismo , Mutação/genética , Ligação Proteica , Receptores de Calcitriol/genéticaRESUMO
Changes in local chromatin structure accompany transcriptional activation of eukaryotic genes. In vivo these changes in chromatin organization can be catalyzed by ATP-dependent chromatin-remodeling complexes, such as SWI/SNF. These complexes alter the tight wrapping of DNA in the nucleosomes and can facilitate the mobilization of the histone octamer to adjacent DNA segments, leaving promoter regulatory elements exposed for transcription factor binding. To gain understanding of how the activity of SWI/SNF complexes may be modulated by the different DNA sequences within a natural promoter, we have reconstituted nucleosomes containing promoter segments of the transcriptionally active cell type-specific osteocalcin (OC) gene and determined how they affect the directional movements of the nucleosomes. Our results indicate that SWI/SNF complexes induce octamer sliding to preferential positions in the OC promoter, leading to a nucleosomal organization that resembles that described in intact cells expressing the OC gene. Our studies demonstrate that the position of the histone octamer is primarily determined by sequences within the OC promoter that include or exclude nucleosomes. We propose that these sequences are critical components of the regulatory mechanisms that mediate expression of this tissue-specific gene.
Assuntos
Montagem e Desmontagem da Cromatina/fisiologia , Proteínas Cromossômicas não Histona/química , Nucleossomos/metabolismo , Osteocalcina/genética , Osteocalcina/metabolismo , Regiões Promotoras Genéticas/fisiologia , Fatores de Transcrição/química , Animais , Linhagem Celular Tumoral , Proteínas Cromossômicas não Histona/fisiologia , Regulação da Expressão Gênica/genética , Nucleossomos/genética , Osteocalcina/biossíntese , Ratos , Fatores de Transcrição/fisiologiaRESUMO
Vitamin D serves as a principal modulator of skeletal gene transcription, thus necessitating an understanding of interfaces between the activity of this steroid hormone and regulatory cascades that are functionally linked to the regulation of skeletal genes. Physiological responsiveness requires combinatorial control where coregulatory proteins determine the specificity of biological responsiveness to physiological cues. It is becoming increasingly evident that the regulatory complexes containing the vitamin D receptor are dynamic rather than static. Temporal and spatial modifications in the composition of these complexes provide a mechanism for integrating regulatory signals to support positive or negative control through synergism and antagonism. Compartmentalization of components of vitamin D control in nuclear microenvironments supports the integration of regulatory activities, perhaps by establishing thresholds for protein activity in time frames that are consistent with the execution of regulatory signaling.