Analysis of chromatin organization and epigenetic gene regulation in health and disease

We study epigenetic regulators using genetics, genomics and proteomics. The factors we study include the Polycomb and Trithorax Groups in both flies and humans, and chromatin-associated fusion oncoproteins such as BRD4-NUT and MOZ-TIF2 in human cancers. The common thread is that each is strongly implicated in the creation of active or silent chromatin domains that are integral to the fidelity of gene regulation. One serious obstacle to understanding the interactions of such factors with additional proteins and RNAs on chromatin has been the trade-off between removal from the DNA, to allow purification, and the resultant loss of interactions with key partners in function. Therefore, we have adapted a crosslinking approach that allows us to affinity-purify fragmented chromatin with protein and RNAs attached, to avoid disruption of interactions that may only occur on DNA. After reversal of crosslinks, the DNA, protein, histone peptides, and RNA fractions can be separately analyzed using comprehensive sequencing and mass spectrometry. Our current results are providing us with a rich and comprehensive view of key epigenetic complexes bound to their chromatin templates.

One example is our recent work with the highly conserved Polycomb group (PcG) regulators in Drosophila. Our proteomic analyses have led my lab to propose a model in which the Polycomb Repressive Complex 1 (PRC1) and classical co-activators form ‘bivalent' protein complexes on transcriptionally poised developmental genes. We speculate that these function as ‘master switches' that are responsive to the amount of local acetylation or deacetylation activities recruited by cell type-specific DNA binding factors, leading to stable but reversible activation or repression, respectively. Our model is based on chromatin crosslinking, affinity purification, and mass spectrometry experiments, in which Drosophila PRC1 strongly interacts with classical co-activators, dBRD4 and dMOZ. We are currently exploring potential parallels in mammalian development.