Functional analysis of the breast cancer associated transcriptional repressor PLU-1/JARID1B

The PLU-1/JARID1B gene, which is upregulated in breast cancers, encodes for a 1,544-amino-acid multidomain protein that is exclusively localised to the nucleus. 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 [1, 2].

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.

Co-immunolocalisation and/or co-immunoprecipitation of PLU-1/JARID1B with HDACs were carried out using anti Myc/HisA antibodies or an antiserum (αPLU-1-C) against PLU-1/Jarid1B after transient transfection of Cos and MCF7 cells with expression vectors coding for Myc or HisA tagged proteins. Direct interactions of PLU-1/JARID1B expressed from a baculovirus with in vitro translated HDACs were also demonstrated. In vitro mutagenesis and reporter assays were also used. HB2 and MCF7 cells were subjected to microarray using the Affymetrix gene chip HG-U133A after transduction with a recombinant adenovirus or silencing the endogenous gene using a short hairpin RNA (shRNA) expression vector (Imgenex). ChIP assays were carried out using the αPLU-1-C specific antiserum or an antibody against the acetylated form of Histone H3. PCR-assisted DNA binding selection from a random pool of oligonucleotides was carried out using in vitro translated full-length PLU-1/JARID1B and GST-PLU-1-ARID.

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 using co-immunoprecipitation assays and binding of PLU-1/JARID1B to in vitro translated HDACs. Two PHD domains in PLU-1 were shown to be crucial for binding to a domain in the N-terminal region of HDAC4 and for the transcriptional repression. Approximately 100 target genes were identified by microarray analysis after overexpressing or silencing the human PLU-1/JARID1B gene in human mammary epithelial cells using adenovirus and RNA interference systems, respectively. Most of the candidate genes were downregulated by PLU-1/JARID1B overexpression, including the mellathionein (MT) genes, the BRCA1 gene, and genes involved in the regulation of the spindle and G2/M checkpoints such as BUBR1, BUB3, STK6, TTK, CDC2 and Cyclin B1. ChIP assays confirmed that the MT1H, MT1F and MT1X genes are direct transcriptional targets of PLU-1/JARID1B, and that PLU-1/JARID1B affects the level of acetylation of the promoter of the MT1H gene. Some other candidate genes such as BRCA1 may be downregulated indirectly. The PLU-1/JARID1B ARID domain preferentially binds to a GCACA motif, a putative consensus sequence that is abundant in MT promoters.

The downregulation of the metallothionein genes, checkpoint genes and BRCA1 by PLU-1/JARID1B overexpression is of great interest and could be highly relevant to any role this protein plays in the development and progression of breast cancer.