The proto-oncoprotein FBI-1 interacts with MBD3 to recruit the Mi-2/NuRD-HDAC complex and BCoR and to silence p21WAF/CDKN1A by DNA methylation

Abstract

The tumour-suppressor gene CDKN1A (encoding p21Waf/Cip1) is thought to be epigenetically repressed in cancer cells. FBI-1 (ZBTB7A) is a proto-oncogenic transcription factor repressing the alternative reading frame and p21WAF/CDKN1A genes of the p53 pathway. FBI-1 interacts directly with MBD3 (methyl-CpG–binding domain protein 3) in the nucleus. We demonstrated that FBI-1 binds both non-methylated and methylated DNA and that MBD3 is recruited to the CDKN1A promoter through its interaction with FBI-1, where it enhances transcriptional repression by FBI-1. FBI-1 also interacts with the co-repressors nuclear receptor corepressor (NCoR), silencing mediator for retinoid and thyroid receptors (SMRT) and BCL-6 corepressor (BCoR) to repress transcription. MBD3 regulates a molecular interaction between the co-repressor and FBI-1. MBD3 decreases the interaction between FBI-1 and NCoR/SMRT but increases the interaction between FBI-1 and BCoR. Because MBD3 is a subunit of the Mi-2 autoantigen (Mi-2)/nucleosome remodelling and histone deacetylase (NuRD)-HDAC complex, FBI-1 recruits the Mi-2/NuRD-HDAC complex via MBD3. BCoR interacts with the Mi-2/NuRD-HDAC complex, DNMTs and HP1. MBD3 and BCoR play a significant role in the recruitment of the Mi-2/NuRD-HDAC complex– and the NuRD complex–associated proteins, DNMTs and HP. By recruiting DNMTs and HP1, Mi-2/NuRD-HDAC complex appears to play key roles in epigenetic repression of CDKN1A by DNA methylation.

Role of Promyelocytic Leukemia Zinc Finger (PLZF) in Cell Proliferation and Cyclin-dependent Kinase Inhibitor 1A (p21WAF/CDKN1A) Gene Repression

“…Plasmids, Antibodies, and Reagents-Various promoter-reporter gene fusion plasmids and pcDNA3-p53, pcDNA3-Sp1, and co-repressor expression vectors were either reported elsewhere or prepared by us (4,5). The pcDNA3.1-PLZF plasmid was prepared by cloning a full-length cDNA into pcDNA3.1 (Invitrogen).…”

“…The reporter fusion plasmids pGL2-ARF-Luc, pGL2-MDM2-Luc, pGL2-TP53-Luc, pG13-Luc, pG5-5x(GC-box)-Luc, and pGL2-CDKN1A-Luc, as well as pcDNA3.1-PLZF, pcDNA3-p53, and pCMVLacZ in various combinations were transiently transfected into cell lines using Lipofectamine Plus reagent (Invitrogen) and analyzed as described elsewhere (4,5) Knock-down of PLZF mRNA by siRNA-The following two siRNA against PLZF mRNA were designed and purchased from Bioneer (Taejeon, Korea): PLZF siRNA-1, sense, 5Ј-GGC CAAC-CAGAUGCGGCUGUU-3Ј, and antisense, 5Ј-UUCCGGUUGG-UCUACGCCGAC-3Ј; and PLZF siRNA-2, sense, 5Ј-GAUGUU-UGAGAUCCUCUUCUU-3Ј, and antisense, 5Ј-UUCUACAAA-CUCUAGGAGAAG-3Ј. The siRNA (200 pmol) was transfected into HEK293 cells using Lipofectamine RNAiMAX (Invitrogen).…”

“…In these cells, the molecular interaction between PLZF and either p53 or Sp1 at the endogenous CDKN1A promoter and histone modification at the CDKN1A proximal promoter was analyzed using the standard ChIP assay protocol, as described elsewhere (4,5).…”

Promyelocytic Leukemia Zinc Finger-Retinoic Acid Receptor α (PLZF-RARα), an Oncogenic Transcriptional Repressor of Cyclin-dependent Kinase Inhibitor 1A (p21WAF/CDKN1A) and Tumor Protein p53 (TP53) Genes

“…Interestingly, the methylated DNA-binding protein MBD3, which is one of the subunits of the Mi-2⅐NuRD-HDAC3 complex, was found to be associated with the proximal promoter of CDNK1A (35). PLZF-RAR␣ interacts with MBD3 and recruits the Mi-2⅐NuRD-HDAC3 complex and the NuRD-associated DNMT1/ 3b and HP1, which eventually leads to CDKN1A promoter DNA methylation.…”

Nongenetic stochastic expansion of JAK2V617F-homozygous subclones in polycythemia vera?

“…Interestingly, both GATAD2B and BCOR are transcriptional repressors associated with the Mi-2/NuRD complex, 8,9 raising the possibility that aberrant histone deacetylation is advantageous to JAK2V617F-homozygous cells. However, both mutations were also present in a minor JAK2V617F-homozygous subclone and cannot account for the dominance of the larger clones.…”