The PLU-1 gene is expressed at the level of message in breast cancers and breast cancer cell lines and shows restricted expression in normal adult tissues with the exception of testis. The predicted protein sequence contains several domains, including the PLU domain, which is shared by other proteins involved in transcription and/or development. We have developed a polyclonal antiserum to a C-terminal fragment of the PLU-1 protein, which shows little homology to other family members. Immunohistochemic analysis with the antiserum ␣-PLU-1C confirmed the nuclear localisation of PLU-1. ␣-PLU-1C also reacted with the mouse homologue of PLU-1 (mPlu-1) but not with the closest family member, RBP2. Using Western blot analysis, PLU-1 was shown to be well expressed in breast cancers and breast cancer cell lines, while it was not detected in a range of normal adult tissues. Our results suggest that the PLU-1 protein may belong to the class of testis/cancer antigens. © 2002 Wiley-Liss, Inc. Key words: breast cancer: PLU-1 protein; testis/cancer antigenThe PLU-1 gene was identified as being downregulated in a human mammary epithelial cell line (MTSV1-7) transfected with HER2/c-erbB2, 1 after treatment with the 4D5/HERCEPTIN antibody, which inhibits c-erbB2 signaling. 2 However, in situ hybridisation and Northern blot analyses showed that PLU-1 mRNA is expressed in most breast cancers and breast cancer cell lines, regardless of the level of c-erbB2. 3 However, Northern blot analysis of total mRNA from normal human adult tissues showed high levels of expression in testis and detectable levels in placenta, but levels were undetectable in the other tissues examined.Following our own report on the identification and characterisation of PLU-1, other investigators reported on the cloning of cDNAs of splice and transcriptional variants of the PLU-1 gene. These were referred to as RBP2-H1 4 and RBBP2H1A, 5 respectively. The nomenclature of the splice and transcriptional variants of PLU-1 reflects the high homology between the predicted protein sequence of PLU-1 and the RBP2 6 in conserved domains, particularly in the novel PLU domain. 3,7 PLU-1 binds to a conserved consensus sequence in 2 transcriptions factors through the novel PLU domain. 8 Additionally, PLU-1 was a potent transcriptional repressor and may be involved in gene regulation in breast cancer. We also defined the sequence of mPlu-1 cDNA, which shows at the amino acid level an overall homology with human PLU-1 of 94% and almost 100% identity within the conserved domains. 9 The very conserved sequence of PLU-1 from human to mouse in combination with an almost identical expression pattern in adult tissues and breast cancers in both species suggests a conserved function in breast cancer.The later studies by Vogt et al. 4 and Kashuba et al. 5 on the RNA variants of PLU-1 examined mRNA expression using polyAϩ RNA and probes that would pick up all 3 transcripts. Expression was reported to be less restricted in normal tissues than we observed using total RNA. However, as we repo...

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...

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The TFAP2C transcription factor has been shown to downregulate transcription of the universal cell cycle inhibitor p21 cip (CDKN1A). In examining the mechanism of TFAP2C-mediated repression, we have identified a ternary complex at the proximal promoter containing TFAP2C, the oncoprotein Myc, and the trimethylated lysine 4 of histone H3 (H3K4me3) demethylase, KDM5B. We demonstrated that while TFAP2C and Myc can downregulate the CDKN1A promoter independently, KDM5B acts as a corepressor dependent on the other two proteins. All three factors collaborate for optimal CDKN1A repression, which requires the AP-2 binding site at ؊111/؊103 and KDM5B demethylase activity. Silencing of TFAP2C-KDM5B-Myc led to increased H3K4me3 at the endogenous promoter and full induction of CDKN1A expression. Coimmunoprecipitation assays showed that TFAP2C and Myc associate with distinct domains of KDM5B and the TFAP2C C-terminal 270 amino acids (aa) are required for Myc and KDM5B interaction. Overexpression of all three proteins resulted in forced S-phase entry and attenuation of checkpoint activation, even in the presence of chemotherapy drugs. Since each protein has been linked to poor prognosis in breast cancer, our findings suggest that the TFAP2C-Myc-KDM5B complex promotes cell cycle progression via direct CDKN1A repression, thereby contributing to tumorigenesis and therapy failure.

Overexpression of the activator protein (AP)-2gamma transcription factor in breast tumours has been identified as an independent predictor of poor outcome and failure of hormone therapy. To understand further the function of AP-2gamma in breast carcinoma, we have used an RNA interference and gene expression profiling strategy with the MCF-7 cell line as a model. Gene expression changes between control and silenced cells implicate AP-2gamma in the control of cell cycle progression and developmental signalling. A function for AP-2gamma in cell cycle control was verified using flow cytometry: AP-2gamma silencing led to a partial G1/S arrest and induction of the cyclin-dependent kinase inhibitor, p21cip/CDKN1A. Reporter and chromatin immunoprecipitation assays demonstrated a direct, functional interaction by AP-2gamma at the CDKN1A proximal promoter. AP-2gamma silencing coincided with acquisition of an active chromatin conformation at the CDKN1A locus and increased gene expression. These data provide a mechanism whereby AP-2gamma overexpression can promote breast epithelial proliferation and, coupled with previously published data, suggest how loss of oestrogen regulation of AP-2gamma may contribute to the failure of hormone therapy in patients.

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