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The PLU-1/JARID1B gene, which is upregulated in breast cancers, belongs to the ARID family of DNA binding proteins. The protein contains several conserved domains, including the ARID DNA binding domain, both N and C jumonji domains, three PHD domains and putative nuclear localisation signals, indicating that it could regulate the transcription of specific genes either through direct binding or through other transcription factors. By reporter assay we have previously shown that PLU-1/JARID1B has a strong transcriptional repression activity. In this study, we aim to identify the target genes regulated by PLU-1/ JARID1B and the possible mechanism of PLU-1/JARID1B-mediated transcriptional regulation. We showed that PLU-1/JARID1B binds to chromatin and the nuclear matrix and localises in MAD bodies when co-transfected with Class IIa histone deacetylases (HDACs) or N-CoR. Direct binding to Class I and Class IIa HDACs is demonstrated by using co-immunoprecipitation assays and binding by PLU-1/JARID1B of in vitro translated HDACs. Two PHD domains in PLU-1 are crucial for binding to a domain in the N-terminal region of HDAC4 and for the transcriptional repression. Target genes regulated by PLU-1/JARID1B were identified by microarray analysis after over-expressing or silencing the human PLU-1/JARID1B gene in human mammary epithelial cells using adenovirus and RNA interference systems, respectively. A total of 100 genes showed an inversely correlated differential expression in the two systems. Most of the candidate genes were down-regulated by PLU-1/JARID1B over-expression, including the mellathionein (MT) genes, the tumor suppressor gene BRCA1, and genes involved in the regulation of the M-phase of the mitotic cell cycle. Chromatin immuno-precipitation assays confirmed that the MT1H, -1F and -1X genes are direct transcriptional targets of PLU-1/JARID1B in vivo. We showed that the PLU-1/JARID1B ARID domain preferentially binds CG rich DNA in a sequence-specific manner and that the GCACA motif, which appears to be a consensus sequence bound by PLU-1/JARID1B, are abundant in MT promoters. We also showed that PLU-1/JARID1B affects the level of acetylation of the promoter of the metallothionein 1H gene. Furthermore cells overexpressing PLU-1/JARID1B showed an attenuation of the G2/M block induced both by nocodazole and CdCl2 treatment. The downregulation of the metallothionein genes, checkpoint genes and BRCA1 as well as the attenuation of G2/M checkpoint by PLU-1/JARID1B overexpression are of great interest and could be highly relevant to any role this protein plays in the development and progression of breast cancer.

98th AACR Annual Meeting-- Apr 14-18, 2007; Los Angeles, CA