ABSTRACT
The PLU-1/JARID1B nuclear protein, which is upregulated in breast cancers, belongs to the ARID family of DNA binding proteins and has strong transcriptional repression activity. To identify the target genes regulated by PLU-1/JARID1B, we overexpressed or silenced the human PLU-1/JARID1B gene in human mammary epithelial cells by using adenovirus and RNA interference systems, respectively, and then applied microarray analysis to identify candidate genes. A total of 100 genes showed inversely correlated differential expression in the two systems. Most of the candidate genes were downregulated by the overexpression of PLU-1/JARID1B, including the MT genes, the tumor suppressor gene BRCA1, and genes involved in the regulation of the M phase of the mitotic cell cycle. Chromatin immunoprecipitation assays confirmed that the metallothionein 1H (MT1H), -1F, and -1X genes are direct transcriptional targets of PLU-1/JARID1B in vivo. Furthermore, the level of trimethyl H3K4 of the MT1H promoter was increased following silencing of PLU-1/JARID1B. Both the PLU-1/JARID1B protein and the ARID domain selectively bound CG-rich DNA. The GCACA/C motif, which is abundant in metallothionein promoters, was identified as a consensus binding sequence of the PLU-1/JARID1B ARID domain. As expected from the microarray data, cells overexpressing PLU-1/JARID1B have an impaired G(2)/M checkpoint. Our study provides insight into the molecular function of the breast cancer-associated transcriptional repressor PLU-1/JARID1B.
Subject(s)
DNA-Binding Proteins/metabolism , Gene Expression Regulation , Nuclear Proteins/metabolism , Repressor Proteins/metabolism , Transcription, Genetic , Animals , BRCA1 Protein/genetics , BRCA1 Protein/metabolism , Base Sequence , Cell Cycle/physiology , Cells, Cultured , DNA-Binding Proteins/genetics , Epithelial Cells/cytology , Epithelial Cells/physiology , Gene Expression Profiling , Gene Silencing , Humans , Jumonji Domain-Containing Histone Demethylases , Mammary Glands, Human/anatomy & histology , Metallothionein/genetics , Metallothionein/metabolism , Molecular Sequence Data , Nuclear Proteins/genetics , Oligonucleotide Array Sequence Analysis , Promoter Regions, Genetic , RNA Interference , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/metabolism , Repressor Proteins/genetics , Spindle Apparatus/metabolismABSTRACT
The PLU-1/JARID1B nuclear protein, which is expressed in a high proportion of breast cancers, but shows restricted expression elsewhere, belongs to the ARID family of proteins, known to play important roles in development, differentiation, transcriptional regulation and chromatin remodeling. PLU-1/JARID1B is a strong transcriptional repressor, and here we show that the protein localizes in MAD bodies when cotransfected with class IIa histone deacetylases (HDACs) or N-CoR. Direct binding to class I and class IIa HDACs is demonstrated, while the interaction with N-CoR appears to be indirect. The domains involved in the HDAC4-PLU-1/JARID1B interaction were investigated in detail, and the data show that 2 PHD domains in PLU-1/JARID1B, which are involved in transcriptional repression, are also crucial for binding to a domain in the 5' region of HDAC4, overlapping the MEF-2 binding region. Physiological relevance of this interaction in the mammary gland is suggested from the observation that HDAC4 and PLU-1/JARID1B are coexpressed in the pregnant and involuting mouse mammary gland and are both silenced at lactation. Significantly, the expression of both proteins is seen in breast cancers.
