Histone modifiers, YY1 and p300, regulate the expression of cartilage-specific gene, chondromodulin-I, in mesenchymal stem cells

Abstract

Elucidating the regulatory mechanism for tissue-specific gene expression is key to understanding the differentiation process. The chondromodulin-I gene (ChM-I) is a cartilage-specific gene, the expression of which is regulated by the transcription factor, Sp3. The binding of Sp3 to the core-promoter region is regulated by the methylation status of the Sp3-binding motif as we reported previously. In this study, we have investigated the molecular mechanisms of the down-regulation of ChM-I expression in mesenchymal stem cells (MSCs) and normal mesenchymal tissues other than cartilage. The core-promoter region of cells in bone and peripheral nerve tissues was hypermethylated, whereas the methylation status in cells of other tissues including MSCs did not differ from that in cells of cartilage, suggesting the presence of inhibitory mechanisms other than DNA methylation. We found that a transcriptional repressor, YY1, negatively regulated the expression of ChM-I by recruiting histone deacetylase and thus inducing the deacetylation of associated histones. As for a positive regulator, we found that a transcriptional co-activator, p300, bound to the core-promoter region with Sp3, inducing the acetylation of histone. Inhibition of YY1 in combination with forced expression of p300 and Sp3 restored the expression of ChM-I in cells with a hypomethylated promoter region, but not in cells with hypermethylation. These results suggested that the expression of tissue-specific genes is regulated in two steps; reversible down-regulation by transcriptional repressor complex and tight down-regulation via DNA methylation.

FIGURE 1.

DNA methylation and histone deacetylation down-regulate the expression of ChM-I in a cell type-specific manner. A, expression of the ChM-I and Sp3 genes in primary cultured mesenchymal cells. B, methylation status of the core-promoter region of ChM-I. The methylation of each CpG site was analyzed in 10 alleles by bisulfite genomic sequencing. The y-axis indicates the fraction of methylated alleles and the x-axis indicates the position of each CpG site relative to the transcription start site. Methylation status in hPC and NHOST treated with a demethylating reagent (5-aza-dC, 1 μm for 96 h) were also shown. C, ChIP-qPCR assay for the modification of histones in primary cultured mesenchymal cells. Open box, acetylated H3K9; closed box, dimethylated H3K9; gray box, Pan H3. Histone modification in hBMSC treated with an HDAC inhibitor (MS-275, 1 μm for 24 h) were also shown. The y-axis represents fold enrichment relative to input. D, expression of ChM-I after treatment with 5-aza-dC and/or MS-275.

FIGURE 2.

Binding of YY1 and p300 determined the modification of H3K9 and the mRNA expression of ChM-I in mesenchymal tissues. A, methylation status of the core-promoter region of the ChM-I. DNA extracted from normal tissue was analyzed by bisufite genomic sequencing. B, genomic structure of the core-promoter region of ChM-I. CpG sites from −262 to +71 were marked as indicated, and the transcription start site is indicated by an arrow. Two overlapping YY1-binding motifs (−344 to −347 and −342 to −346), and an Sp3-binding motifs (−56 to −48) are also indicated. A DNA fragment for the ChIP assay was amplified by primer 1 (−446 to −425) and primer 2 (+70 to +91). GR3 (−357 to −337) and GR4 (−86 to −44) were OND probes used in the EMSA for YY1 and p300, respectively. ChIP-qPCR assay for the binding of transcriptional regulators in primary cultured cells (C) and cells of normal tissues (D). The y-axis represents fold enrichment relative to input. E, ChIP-qPCR assay for the modification of histones in normal tissues.

FIGURE 3.

YY1 bound to the regulatory region of ChM-I and decreased the promoter activity of ChM-I. A, ChIP-qPCR assay for YY1 and HDAC2. B, luciferase reporter assay. a, DNA fragment encompassing −446 to +86 was cloned into a reporter vector containing the luciferase gene (PGV-B-f1). The black box indicates the location of the consensus sequence for the YY1-binding motif. b, PGV-B-f1-mt contains mutations in the YY1-binding motif (arrowhead), and PGV-B-f1-del lacked the YY1-binding motifs (c). Each reporter vector was co-transfected with empty vector (pCEP) or the YY1 expression vector (pCEP-YY1) into ANOS. The fold-increase was calculated based on empty vector activity. C, expression of endogenous ChM-I in hPCs transfected with the YY1 expression vector. The expression of ChM-I was semi-quantified taking the value for endogenous expression as 1.0 and is demonstrated at the top. D, ChIP-qPCR assay of hPCs transfected with the YY1 expression vector with or without MS275 treatment (1 μm for 24 h). Forced expression of YY1 in hPCs changed the modification of the H3 tail from acetylation to dimethylation.

FIGURE 4.

p300 binds to the core promoter region and enhances transcription. A, ChIP-qPCR assay of p300, Sp3 and acetylated H3. B, luciferase reporter assay. The reporter construct was described in the legend for Fig. 3B, and co-transfected with empty vector (pcDNA3) or the p300 expression vector (pcDNA3-p300) into ANOS (a), Saos2 (b), or TAKAO (c). The fold-increase was calculated based on empty vector activity. C, down-regulation of ChM-I gene expression by siRNA for Sp3 and/or p300. siRNAs for Sp3 and/or p300 were transfected into ANOS, and the expression of ChM-I, Sp3, and p300 was analyzed by RT-PCR. The expression of ChM-I was semi-quantified taking the value for endogenous expression as 1.0 and is demonstrated at the top. D, ChIP-qPCR assay for the modification of H3K9 (D) and for the binding of transcription regulators (E): cross-linked DNA-protein complexes were prepared from ANOS treated with or without siRNA for p300 and used for ChIP-qPCR assay.

FIGURE 5.

Promoter activity of ChM-I was promoted by Sp3 and p300, but completely inhibited by YY1 in primary cultured cells. The luciferase reporter vector containing the core-promoter fragment of the ChM-I gene (PGV-B-f1) was co-transfected with YY1, p300 and/or Sp3 expression vectors into hMSCs (A), hPCs (B), NHOSTs (C), and hPAs (D). The fold-increase was calculated based on empty vector activity.

FIGURE 6.

Induction of ChM-I expression in ChM-I-negative primary cultured cells by modification of regulators. A–C, primary-cultured cells were transfected with a combination of the siRNA for YY1, p300 expression vector, and Sp3 expression vector, and mRNA level of ChM-1, YY1, p300, and Sp3 gene were analyzed by semi-quantitative RT-PCR (lower panel). The expression of the ChM-I was further analyzed by quantitative RT-PCR and digitalized (upper panel). A, hMSCs; C, hPAs; E, NHOSTs. ChIP-qPCR assay: B, hMSCs; D, hPAs; F, NHOSTs. Cross-linked DNA-protein complexes were prepared from primary cultured cells treated with or without siRNA for YY1 and used for ChIP-qPCR assay for the modificationf H3K9 (left panels) and for the binding of transcription regulators (right panels).