No 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 cancerassociated transcriptional repressor PLU-1/JARID1B.The PLU-1 nuclear protein is expressed in most primary breast cancers and breast cancer cell lines (4, 46), while its expression in normal adult tissues is largely restricted to testes (46), where it is expressed in spermatogonia and in specific stages of meiosis (48). However, significant expression is also seen in the murine pregnant mammary gland (4) and in the embryonic mammary bud (4, 47). Thus, in addition to being elevated in breast cancer, PLU-1 is involved in the development and differentiation of the mammary gland.The PLU-1 gene encodes a 1,544-amino-acid multidomain protein that is exclusively localized to the nucleus. Sequence homology analysis shows that it contains several conserved domains, including the ARID DNA binding domain (AT-richinteracting domain), plant homeodomain/leukemia-associated protein domains (PHD domains), Jumonji domains, and putative nuclear localization signals (46). The ARID domain, the N-terminal and C-terminal Jumonji domains (JmJN and JmJC), two of the plant homeodomain domains, and a novel Trp/Tyr/Phe/Cys domain (the PLU domain), which overlaps the JmJC domain (62), are conserved in the four members of the JARID1 subfamily of the larger family of ARID proteins (15 proteins to date) (86). Although their sequence homology is high, these proteins appear to have diverse functions, with JARID1A (RBP2) being involved in activating transcription from nuclear receptors (14), PLU-1 (now referred to as PLU-1/JARID1B) being a strong transcriptional repressor (75, 88), and JARID1C (the SMCX gene) ...

Abstract. The four members of the JARID1/KDM5 family of proteins, a sub-group of the larger ARID (AT rich DNA binding domain) family, have been shown to demethylate trimethylated lysine 4 on histone 3 (H3K4me3), a chromatin mark associated with actively transcribed genes. In some lower organisms a single homologue of JARID1 is found, and functions of the four proteins found in mice and humans may be specific or overlapping. To investigate the function of the JARID1B protein we examined the effects of deletion of the gene in mice. Systemic knock out of Jarid1b resulted in early embryonic lethality, whereas mice not expressing the related Jarid1A gene are viable and fertile. A second mouse strain expressing a Jarid1b gene with the ARID domain deleted was viable and fertile but displayed a mammary phenotype, where terminal end bud development and side branching was delayed at puberty and in early pregnancy. Since development of terminal end buds are completely dependent on signalling from the estrogen receptor (ERα), we investigated the expression of a target gene (progesterone receptor) in the ∆ARID mouse and found levels to be reduced as compared to wild-type. JARID1B is widely expressed in ER + breast cancers and breast cancer cell lines, and interaction with ERα was demonstrated by co-immunoprecipitations in cells transfected with tagged ERα and JARID1B genes. Down-regulation of expression of JARID1B using shRNAi in MCF-7 cells resulted in a dramatic decrease in E2 stimulated tumour growth in nude mice. The data demonstrate a specific role for Jarid1B in early embryonic development, in the development and differentiation of the normal mammary gland, and in estrogen induced growth of ER + breast cancer.

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 0 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. ' 2007 Wiley-Liss, Inc.Key words: PLU-1/JARID1B; breast cancer; transcriptional repression; histone deacetylasesThe PLU-1 gene was identified in a differential screen for genes that were down-regulated when HER2 signaling was inhibited. 1 However, PLU-1 has since been demonstrated to be expressed in 90% of breast carcinomas, regardless of the level of HER2 expression, and appears to be associated with malignant progression.2 In normal human adult tissues, the highest levels of expression of PLU-1 mRNA are seen in testis 1,3 with low levels of expression being observed in placenta, lymph node, and thymus.2 In the normal mammary gland, mPlu-1 mRNA is expressed in the embryo in the developing mammary bud, and at pregnancy in the adult, 4 suggesting a role in proliferation in the developing and differentiating tissue.The PLU-1 protein sequence contains several well-conserved domains known to be involved in protein interactions, gene regulation, and chromatin remodeling. These include 3 PHD domains, 5,6 the ARID domain (AT-rich interacting domain), 7 and 2 potential hormone-binding motifs. 8 The novel Trp/Tyr/Cys domain (overlapping the JmjC domain and renamed the PLU domain 9 ) interacts with a conserved motif found in 2 unrelated transcription factors (BF1 and PAX9).10 Thus, the protein could be recruited to DNA directly through the ARID DNA binding domain, or through other transcription factors.Within the larger ARID family of proteins, PLU-1 has been placed in the JARID1 subclass which contains 4 proteins, characterized by the presence of the JmjN and JmjC domains.11 Consequently, PLU-1 is now referred to as PLU-1/JARID1B. The 4 members of the JARID1 subclass show high conservation of sequence, but appear to have different profiles of expression and different functions. The RBBP2 (JARID1A) protein contains the canonical LXCXC domain...

PLU-1 is a novel breast cancer associated nuclear protein containing highly conserved domains including the PLU domain, putative DNA/chromatin binding motifs, and PHD/LAP domains. Here we report the cloning of the mouse homologue (Plu-1), and document its expression in adult tissues, mammary tumours and the embryo. The overall homology with human PLU-1 is 94% at the protein level, with almost 100% identity in the conserved domains, suggesting functional conservation. As with human PLU-1 the expression of Plu-1 in adult tissues is restricted, with high expression being seen only in testis, while expression in mammary tumours from c-neu transgenic mice is high. Plu-1 is also differentially expressed in the adult mammary gland. In the developing embryo Plu-1 is expressed in a temporally restricted fashion with tissue specific expression being limited to parts of the developing brain, whisker follicle, mammary bud, thymus, limbs, intervertebral disc, olfactory epithelium, teeth, eye, and stomach. The temporal and spatial expression patterns of the transcription factors Bf-1 and Pax9, recently found to bind to PLU-1 through the PLU domain overlap with Plu-1 expression during development. Thus Plu-1 appears to play an important role in mouse embryonic development which may involve interaction with Pax9 and Bf-1.

PLU-1, a large multi-domain nuclear protein with strong transcriptional repression activity, is a member of the ARID family of DNA binding proteins. In previous studies, high levels of expression of Plu-1 mRNA and PLU-1 protein were detected in breast cancers, while expression in normal adult tissues was detected only in the testis, ovary and transiently in the mammary gland of the pregnant female. Due to its high levels of expression in the testis and to its specific relationship to cancer, PLU-1 has been proposed to belong to the family of testis-cancer antigens. In this study we attempted to determine putative functions for PLU-1 during spermatogenesis. To address this, we analysed the pattern of expression and localisation of this protein in mouse testicular cells during postnatal development and adulthood. Using in situ hybridisation and immunostaining of testis sections we show that Plu-1 mRNA and PLU-1 protein are both highly expressed in the mitotic spermatogonia. Expression is reduced dramatically in the early prophase I stages (zygotene, leptotene), but reappears at pachytene and is still detectable in diplotene cells. We also found that PLU-1 localises diffusely over the nucleus, which indicates a potential chromatin binding ability of this protein. Consistent with this notion, we found that PLU-1 is present in the chromatin fraction in biochemical cell fractionation experiments using both somatic and meiotic cells. Our data point to a role for PLU-1 in meiotic transcription, which may be restricted to certain meiotic stages and may be mediated by the ability of this protein to associate with the chromatin.

Basement membranes, specialized extracellular matrices composed of collagens, laminins, and proteoglycans, form thin, continuous sheetlike structures that separate epithelial tissues from adjacent connective tissues. The crossing of basement membranes by cancer cells is a crucial aspect of metastasis-it must occur in order that cancer cells can invade lymphatic or blood vessels during dissemination and also when they penetrate into the target organ tissue where they will eventually colonize to form secondary tumors. The assay system described in this chapter utilizes the solubilized basement membrane preparation Matrigel™ and measures the ability of cells to attach to the matrix, invade into and through the matrix, and migrate towards a chemoattractant. It is technically straightforward and requires no specialist equipment and provides a useful tool for assessing the invasive ability of cancer cells, exploring the functional role of specific cell surface molecules/receptors in this process and screening for inhibitors of invasive ability, thus contributing to current knowledge of the molecular events occurring during the invasive process.

No abstract