Despite biochemical and genetic data suggesting that E2F and pRB (pocket protein) families regulate transcription via chromatin-modifying factors, the precise mechanisms underlying gene regulation by these protein families have not yet been defined in a physiological setting. In this study, we have investigated promoter occupancy in wild-type and pocket protein-deficient primary cells. We show that corepressor complexes consisting of histone deacetylase (HDAC1) and mSin3B were specifically recruited to endogenous E2F-regulated promoters in quiescent cells. These complexes dissociated from promoters once cells reached late G 1 , coincident with gene activation. Interestingly, recruitment of HDAC1 complexes to promoters depended absolutely on p107 and p130, and required an intact E2F-binding site. In contrast, mSin3B recruitment to certain promoters did not require p107 or p130, suggesting that recruitment of this corepressor can occur via E2F-dependent and -independent mechanisms. Remarkably, loss of pRB had no effect on HDAC1 or mSin3B recruitment. p107/p130 deficiency triggered a dramatic loss of E2F4 nuclear localization as well as transcriptional derepression, which is suggested by nucleosome mapping studies to be the result of localized hyperacetylation of nucleosomes proximal to E2F-binding sites. Taken together, these findings show that p130 escorts E2F4 into the nucleus and, together with corepressor complexes that contain mSin3B and/or HDAC1, directly represses transcription from target genes as cells withdraw from the cell cycle.

Triple-negative breast cancers (TNBCs) have poor prognosis and lack targeted therapies. Interrogation of genomic datasets identified increased PIM1 copy number-driven expression in TNBC. RNA interference in breast cancer and non-malignant mammary epithelial cell models revealed a PIM1 dependency in TNBC cells for proliferation and apoptotic protection, absent in non-malignant cells. PIM1 knockdown reduced BCL2 expression, and dynamic BH3 profiling analysis revealed that PIM1 prevents mitochondrial-mediated apoptosis in TNBC cell lines. PIM1 expression associates with several MYC-transcriptional signatures and promotes cell population growth through regulation of c-MYC and transcription of MYC-targets, including MCL1. The pan-PIM kinase inhibitor AZD1208 inhibited growth and sensitized TNBC cell lines, xenografts and patient-derived xenografts to standard of care chemotherapy. We identify PIM1 as a malignant cell-selective target in TNBC, illustrating relationships with MYC activation, regulation of anti-apoptotic BCL2 and MCL1, established TNBC oncogenic proteins SHP2 and EPHA2 and cell cycle inhibitor p27. Finally we identify a potential use of PIM1 inhibitors to abrogate TNBC's high threshold to TNBC standard of care chemotherapy induced apoptotic cell death.

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) ...