Steven Catchpole

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 G1, 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.

Using in vitro and in vivo experimental systems and in situ analysis, we show that growth hormone (GH) is secreted locally by normal human mammary epithelial cells upon progesterone stimulation. GH increases proliferation of a subset of cells that express growth hormone receptor (GHR) and have functional properties of stem and early progenitor cells. In 72% of ductal carcinoma in situ lesions, an expansion of the cell population that expresses GHR was observed, suggesting that GH signaling may contribute to breast cancer development.

Transcription from the B-myb (MybL2 gene) promoter is strictly cell cycle-regulated by repression mediated through an E2F site during G(0)/early G(1). We report here the characterization of a corepressor site (downstream repression site (DRS)) required for this activity that is closely linked to the E2F site. Systematic mutagenesis of the DRS enabled a consensus to be derived, and it is notable that this sequence is compatible with cell cycle gene homology region sequences associated with cell cycle-dependent elements in the cyclin A, cdc2, and CDC25C promoters. The B-myb promoter is inappropriately active during G(0) in mouse embryo fibroblasts lacking the p107 and p130 pocket proteins, and we show that the ability of transfected p107 and p130 to re-impose repression on the promoter is dependent on the DRS. In contrast, transfected Rb was unable to repress the B-myb promoter. Consistent with the notion that Rb.E2F complexes are unable to bind the B-myb promoter E2F site in vivo, footprinting showed that this site is unoccupied in cells lacking p107 and p130. Chromatin immunoprecipitation assays showed a requirement for the DRS in recruiting p107 and p130 complexes to the B-myb promoter, indicating that in vivo the DRS governs the occupancy of the adjacent E2F site by transcriptional repressors.

Post‑translational modification of histones serve a crucial role in the control of gene transcription. Trimethylation of lysine 4 on histone 3 is associated with transcription activation. There are currently six known methylases and six known demethylases that can control the methylation status of this site. Lysine demethylase 5B (KDM5B) is one such demethylase, which can repress gene expression. In particular KDM5B has been found to be overexpressed in a number of cancer types, and small‑molecular weight inhibitors of its demethylase activity have been identified. Previous characterisation of <em>Kdm5b</em> knock‑out mice has revealed that this genotype leads to either embryonic or neonatal lethality. However, the ΔA‑T rich interaction domain (ΔARID)‑KDM5B strain of mice, which have the ARID domain and five amino acids within the Jumonji (Jmj)N domain spliced out from KDM5B, remain viable and fertile. In the present study, ΔARID‑KDM5B was found to have no demethylase activity as determined by <em>in vitro</em> demethylase assays and by immunofluorescence in transfected Cos‑1 cells. Furthermore, molecular dynamic simulations revealed conformational changes within the ΔARID‑KDM5B structure compared with that in WT‑KDM5B, particularly in the JmjC domain, which is responsible for the catalytic activity of WT‑KDM5B. This supports the experimental data that shows the loss of demethylase activity. Since <em>Kdm5b</em> knock‑out mice show varying degrees of lethality, these data suggest that KDM5B serves a crucial function in development in a manner that is independent of its demethylase activity.