The methyl-CpG binding protein MBD1 is required for PML-RARalpha function

Abstract

PML-RARalpha induces a block of hematopoietic differentiation and acute promyelocytic leukemia. This block is based on its capacity to inactivate target genes by recruiting histone deacetylase (HDAC) and DNA methyltransferase activities. Here we report that MBD1, a member of a conserved family of proteins able to bind methylated DNA, cooperates with PML-RARalpha in transcriptional repression and cellular transformation. PML-RARalpha recruits MBD1 to its target promoter through an HDAC3-mediated mechanism. Binding of HDAC3 and MBD1 is not confined to the promoter region but instead is spread over the locus. Knock-down of HDAC3 expression by RNA interference in acute promyelocytic leukemia cells alleviates PML-RAR-induced promoter silencing. We further demonstrate that retroviral expression of dominant-negative mutants of MBD1 in hematopoietic precursors compromises the ability of PML-RARalpha to block their differentiation and thus restored cell differentiation. Our results demonstrate that PML-RARalpha functions by recruiting an HDAC3-MBD1 complex that contributes to the establishment and maintenance of the silenced chromatin state.

Fig. 1.

MBD1 synergizes with PML-RARα to repress RARβ2 promoter activity. (A) Effect of MBD1 on PML-RARα-mediated repression of RARβ2. 293T cells were transfected with a reporter construct containing the human RARβ2 promoter upstream of the luciferase cDNA (0.5 μg) and expression vectors for PML-RARα (10 ng and 1 μg) and MBD1 (10 ng and 500 ng). Where indicated, the reporter construct was methylated in vitro with SssI before addition. Error bars represent the standard deviation from the mean for triplicate experiments. (B) Effect of DNA methylation and HDAC inhibitors on transcriptional repression by MBD1. TSA (100 nM) or 5-Aza-dC (1 μM), or a combination of these, was added 20 and 36 h, respectively, before the reporter assay was performed as described in A. Error bars represent the standard deviation from the mean for triplicate experiments. (C) Structure of MBD1. (D) Effect of the mutated MBD1 on PML-RARα-mediated repression. Luciferase assay was performed as in A by using expression plasmids for PML-RARα (10 ng), MBD1 (100 ng), and MBD1 mutants (100 ng). Error bars represent the standard deviation from the mean for triplicate experiments. (Inset) Wild-type and mutant forms of MBD1 are expressed at equal levels.

Fig. 2.

HDAC3 bridges PML-RARα and MBD1. (A) Interaction between PML-RARα and HDAC3. 293T cells were transfected with PML-RARα and FLAG-HDAC3 expression vectors, and extracts were immunoprecipitated with anti-FLAG antibody. Western blots of input lysate or of immunoprecipitates were analyzed by using antisera against RARα or FLAG. Note that Flag-HADC3 comigrates with a nonspecific band (indicated by an asterisk) but is clearly seen above background levels. (B) Interaction between MBD1 and HDAC3. Cells were transfected with MBD1, MBD1-dm (MBD1 R22A/I527R), and FLAG-HDAC3 expression vectors, and extracts were immunoprecipitated with anti-FLAG antibody. Immunocomplexes were detected by Western blot as in A.(C) Interaction between MBD1 and PML-RARα. Cells were transfected with MBD1, MBD1-dm, and PML-RARα expression vectors, and extracts were immunoprecipitated with anti-PML (PGM3) antibody. (D) RA disrupts the interaction between PML-RARα and MBD1. 293T cells were transfected with MBD1 and PML-RARα expression vectors, and extracts underwent immunoprecipitation with an anti-PML (PGM3) antibody. Where indicated, cells were treated with RA (1 mM) 5 h before the immunoprecipitation was performed. (E and F) Endogenous interaction among PML-RARα, HDAC3, and MBD1. Cell extracts from NB4 were immunoprecipitated by using either PGM3 or anti-MBD1 antibody.

Fig. 3.

Binding of PML-RARα and corepressors to the endogenous RARβ2 promoter. (A) Western blot analysis of total cell lysates derived from mock and HDAC3 interference RNA APL cells. Human NB4 leukemic cells were infected with a retroviral construct generating HDAC3-specific small hairpin RNA (pRS HDAC3) or the empty vector (pRS) and selected with puromycin for 3 days. Equal amounts of cell extract from mock and interference RNA cells were blotted with antibodies indicated. (B) Knock-down of HDAC3 in APL cells affects RARβ2 H3 acetylation levels. HDAC3 interference RNA cells (pRS HDAC3) or control cell (pRS) were subjected to ChIP analysis, as indicated. The promoter of RARβ2 was amplified with real-time PCR. Error bars indicate the standard deviation obtained from three independent experiments. (C) Knock-down of HDAC3 in APL cells affects RARβ2 promoter activity. Total RNA was prepared from cells as in A, and RARb2 gene expression was analyzed relative to GAPDH control by quantitative real-time PCR. Results are expressed as the mean ± SEM of two independent experiments performed in duplicate. (D) RA induces release of the PML-RARα corerepressor complex. NB4 cells were treated or not with RA (1 μM) for 24 h and then subjected to ChIP analysis, as indicated. The promoter of RARβ2 was amplified with real-time PCR. Error bars indicate the standard deviation obtained from three to five independent experiments. (E) PML-RARα recruits MBD1 to the RARβ25′ region. U937-PR9 cells were treated with Zn (0 h, 4 h, and 24 h) and then subjected to ChIP analysis. The promoter region and exon of RARβ2 were amplified with real-time PCR. Error bars indicate the standard deviation obtained from three independent experiments.

Fig. 4.

MBD1 cooperates with PML-RARα-mediated block of hematopoietic differentiation. (A) U937-PR9 cells, after retroviral infection with MBD1 and MBD1 mutants or empty vector, were treated or not with Zn for 16 h, as indicated. Infected cells were treated either with vitamin D and TGFβ (blue bars) or with vehicle alone (ethanol, white bars). Cell differentiation was evaluated by quantitative expression of CD14 antigen (11). Error bars represent the standard deviation from the mean for triplicate experiments. (B) Model of promoter repression and activation mechanisms in leukemia. The oncoprotein PML-RARα binds to a well defined DNA sequence (5) and recruits NCoR, which in turn serves as platform for the interaction with HDAC3 and corepressors. The N-terminal region of HDAC3 is additionally responsible for the interaction with the TRD of MBD1. Similarly, PML establishes interaction with DNMTs (9). The activity of these corepressors leads to hypoacetylation of histone tails, DNA methylation (depicted by green lollipop), and transcriptional silencing. Methylated CpGs are potential docking sites for MBD1, which can in turn recruit further repressor enzymes. The progression wave of the proposed mechanism might “close” the chromatin structure and influence neighboring genes. Administration of RA, alone or in combination with TSA/5-Aza-dC, induces release of the corepressor complex and promotes recruiting of the coactivators containing histone acetyltransferases (HAT) and ATP-dependent chromatin remodeling activity (40).