Faculty Bibliography

Polycomb group proteins are critical to maintaining gene repression established during Drosophila development. Part of this group forms the PRC2 complex containing Ez that catalyzes di- and trimethylation of histone H3 lysine 27 (H3K37me2/3), marks repressive to transcription. We report that the mammalian homologs Ezh1 and Ezh2 form similar PRC2 complexes but exhibit contrasting repressive roles. While PRC2-Ezh2 catalyzes H3K27me2/3 and its knockdown affects global H3K27me2/3 levels, PRC2-Ezh1 performs this function weakly. In accordance, Ezh1 knockdown was ineffectual on global H3K27me2/3 levels. Instead, PRC2-Ezh1 directly and robustly represses transcription from chromatinized templates and compacts chromatin in the absence of the methyltransferase cofactor SAM, as evidenced by electron microscopy. Ezh1 targets a subset of Ezh2 genes, yet Ezh1 is more abundant in nonproliferative adult organs while Ezh2 expression is tightly associated with proliferation, as evidenced when analyzing aging mouse kidney. These results might reflect subfunctionalization of a PcG protein during evolution

The multisubunit Sin3 corepressor complex regulates gene transcription through deacetylation of nucleosomes. However, the full range of Sin3 activities and targets is not well understood. Here, we have investigated genome-wide binding of mouse Sin3 and RBP2 as well as histone modifications and nucleosome positioning as a function of myogenic differentiation. Remarkably, we find that Sin3 complexes spread immediately downstream of the transcription start site on repressed and transcribed genes during differentiation. We show that RBP2 is part of a Sin3 complex and that on a subset of E2F4 target genes, the coordinated activity of Sin3 and RBP2 leads to deacetylation, demethylation, and repositioning of nucleosomes. Our work provides evidence for coordinated binding of Sin3, chromatin modifications, and chromatin remodeling within discrete regulatory regions, suggesting a model in which spreading of Sin3 binding is ultimately linked to permanent gene silencing on a subset of E2F4 target genes

Primary cilia are nonmotile organelles implicated in signaling and sensory functions. Understanding how primary cilia assemble could shed light on the many human diseases caused by mutations in ciliary proteins. The centrosomal protein CP110 is known to suppress ciliogenesis through an unknown mechanism. Here, we report that CP110 interacts with CEP290--a protein whose deficiency is implicated in human ciliary disease--in a discrete complex separable from other CP110 complexes involved in regulating the centrosome cycle. Ablation of CEP290 prevents ciliogenesis without affecting centrosome function or cell-cycle progression. Interaction with CEP290 is absolutely required for the ability of CP110 to suppress primary cilia formation. Furthermore, CEP290 and CP110 interact with Rab8a, a small GTPase required for cilia assembly. Depletion of CEP290 interferes with localization of Rab8a to centrosomes and cilia. Our results suggest that CEP290 cooperates with Rab8a to promote ciliogenesis and that this function is antagonized by CP110

Mdm2, as the most important negative regulator of p53, plays an important homeostatic role in regulating cell division and the cellular response to DNA damage, oncogenic insult and other forms of cellular stress. We discovered that the DNA damaging agent adriamycin (doxorubicin) induces a novel aberrantly spliced Mdm2 mRNA which incorporates 108 bp of intronic sequence not normally found in the Mdm2 mature mRNA. Accordingly, we term this Mdm2 splice variant Mdm2(+108). Importantly, this insertion introduces in-frame nonsense codons, thus encoding a profoundly truncated mdm2 protein lacking the C-terminal RING finger domain and the E3 ubiquitin ligase activity. A wide range of pharmacological testing revealed that Mdm2(+108) is induced, in mouse and rat cells, in specific response to Adriamycin and actinomycin D, but not other modes of DNA damage. Meanwhile, antibodies against the N-terminal region of mdm2 reveal a marked reduction in detectable mdm2 protein upon Adriamycin treatment, while p53 accumulates to strikingly high levels. We thus conclude that this alternative spicing of Mdm2 may be an important mechanism to facilitate massive accumulation of p53 in response to genotoxic agents

Cyclin A is a key regulator of DNA replication and mitosis, and cyclin A/Cdk2 activity is critical for progression of cells from G(1)/S to M phase of the cell cycle. There is abundant evidence that cyclin A is predominantly localized to, and functions in, the nucleus, and a number of nuclear cyclin A/Cdk2 substrates have been identified. Evidence supporting the presence of cyclin A and its associated activity in the cytoplasm have also been reported, but the biological significance of this cyclin/Cdk pool during cell cycle progression remains controversial. Recently, we identified and characterized a new cyclin A/Cdk2 substrate named SCAPER which is localized to the endoplasmic reticulum. By sequestering cyclin A/Cdk2, SCAPER is capable of directing the activity of this kinase complex away from the nucleus and regulating cyclin A/Cdk2 equilibrium in distinct subcellular compartments. This work paves new avenues for understanding the role of cytoplasmic cyclin A/Cdk2 and its potential contribution to cancer and tumor formation

Sgo1 plays a key role in protecting sister chromatid cohesion during mitosis. In this issue of Developmental Cell, Wang et al. describe a shorter splice variant of Sgo1 (sSgo1) that functions specifically in centriole cohesion. sSgo1 may be the 'glue' that holds paired centrioles together in an engaged state before their disengagement in late mitosis

Characterization of the transcriptional regulatory network of the normal cell cycle is essential for understanding the perturbations that lead to cancer. However, the complete set of cycling genes in primary cells has not yet been identified. Here, we report the results of genome-wide expression profiling experiments on synchronized primary human foreskin fibroblasts across the cell cycle. Using a combined experimental and computational approach to deconvolve measured expression values into 'single-cell' expression profiles, we were able to overcome the limitations inherent in synchronizing nontransformed mammalian cells. This allowed us to identify 480 periodically expressed genes in primary human foreskin fibroblasts. Analysis of the reconstructed primary cell profiles and comparison with published expression datasets from synchronized transformed cells reveals a large number of genes that cycle exclusively in primary cells. This conclusion was supported by both bioinformatic analysis and experiments performed on other cell types. We suggest that this approach will help pinpoint genetic elements contributing to normal cell growth and cellular transformation

In the current issue of Cancer Cell, Hallstrom et al. show that a subset of targets of the growth regulatory transcription factor E2F1 are repressed by a serum-induced PI3K activation, explaining how apoptosis can be suppressed while simultaneously engaging a proliferation program

The retinoblastoma tumor suppressor protein (pRb) is involved in mitotic exit, promoting the arrest of myoblasts, and myogenic differentiation. However, it is unclear how permanent cell cycle exit is maintained in differentiated muscle. Using RNA interference, expression profiling, and chromatin immunoprecipitations, we show that pRb is essential for cell cycle exit and the differentiation of myoblasts and is also uniquely required to maintain this arrest in myotubes. Remarkably, we also uncover a function for the pRb-related proteins p107 and p130 as enforcers of a G2/M phase checkpoint that prevents progression into mitosis in cells that have lost pRb. We further demonstrate that pRb effects permanent cell cycle exit in part by maintaining trimethylation of histone H3 lysine 27 (H3K27) on cell cycle genes. H3K27 trimethylation silences other genes, including Cyclin D1, in a pRb-independent but polycomb-dependent manner. Thus, our data distinguish two distinct chromatin-based regulatory mechanisms that lead to terminal differentiation

The E2F family of proteins was identified on the basis of its role in promoting the G0 to S phase transition. Research over the past several years has unveiled considerable complexity within the family, with numerous studies pointing to delegation of function for distinct family members. More recent studies highlighted in this review have expanded this picture, suggesting ways in which E2F target gene expression is refined during cell cycle progression by facilitating the acquisition of promoter-specific histone modifications. E2F associated co-activators promote activating histone marks while recruitment of co-repressors associated with E2Fs and the pRB family leads to accretion of inhibitory histone modifications that provoke chromatin compaction.