Transcriptional Regulation, Chromatin, and the Retinoblastoma Protein

(Blais et al., JCB 2007; Balciunaite et al., MCB 2005; Cam et al., 2004; Ren et al., 2002)

The Dynlacht laboratory uses a combination of biochemical, genomic, and computational methods to dissect the role that the retinoblastoma tumor suppressor protein (pRb) family plays in effecting chromatin modifications that underlie gene repression during cell cycle progression and myogenic differentiation. The mammalian cell cycle is driven in part by periodic gene expression, and the E2F transcription factor family plays a major role in driving cell cycle-dependent gene expression. E2F activity is held in check by members of the retinoblastoma tumor suppressor (pRB) family, so-called pocket proteins, comprised of pRB, p107, and p130. Our efforts over the past decade have been aimed at trying to decipher the underlying transcriptional regulatory mechanisms that govern transcription during the cell cycle. As a first approach, we have used a combination of chromatin immunoprecipitation and microarrays, termed ChIP-on-chip, to identify the compendium of genes bound by pocket proteins and E2F (Image 1). Importantly, this approach can, in principle, be used to identify the targets of any DNA-binding factor under any condition, and we are using this methodology to understand how E2F and pRB drives gene expression in cells that have exited the cell cycle in the process of becoming senescent or terminally differentiated. We are particularly interested in the role of pRB family in myogenic and osteogenic differentiation.

Image 1: Using ChIP-on-chip to identify targets of transcription factors and the pRB tumor suppressor family

We have also shown that recruitment of E2F and pRB proteins provoke dramatic chromatin modifications and chromatin remodeling. We have identified chromatin modifications and epigenetic changes that occur during cell cycle progression and terminal differentiation. These modifications, which include histone acetylation and methylation, can be studied using on a genomic level using ChIP-on-chip to discern general principles of epigenetic regulation. Using such genome-wide methods and computational biology, we can map nucleosome positions and all histone modifications and thereby attempt to understand the “codes” that underlie transcriptional control during the cell cycle and terminal differentiation. Our recent studies have uncovered one histone modification, methylation of histone H3 lysine 27, that could play an important role in maintenance of the terminally differentiated state, and this modification appears to depend on the presence of pRB. Such methylation is important for the irreversibility of cell cycle exit in this setting (Image 2). Our efforts are currently directed toward understanding the mechanisms underlying the pRB-dependent histone modification.

Image 2: pRB-dependent chromatin modifications lead to irreversible cell cycle exit and maintenance of the terminally differentiated state