Nicole J. Francis
Mechanisms of Epigenetic Inheritance by Polycomb Group Proteins
My lab aims to understand the nature of epigenetic information and how it is inherited. We use biochemical methods to study how Drosophila Polycomb Group proteins affect chromatin, and how changes in chromatin can be inherited.
Cellular differentiation involves the stepwise establishment of cell-type specific genetic programs in proliferating cell lineages; once established, these programs can be stable for the lifetime of an organism. Yet, signals that direct differentiation can be transient so that other mechanisms, such as those encoded by the Polycomb Group (PcG) and trithorax Group (trxG) genes, are used to maintain the gene expression patterns that encode cellular identities. These epigenetic mechanisms are thought to involve changes in chromatin structure that fix gene expression patterns in a manner that is inherited through cell division. This allows the expression pattern of a gene to reflect the ancestry of the cell it resides in. For example, during the first few hours of development in Drosophila, precise patterns of HOX gene expression along the anterior-posterior axis are established by the actions of the Gap and pair-rule genes. This pattern of expression encodes the identity of body segments; thus if HOX genes are inappropriately expressed in more anterior segments, those segments form posterior instead of anterior structures. This can happen either because the correct HOX gene expression pattern is not established initially, or because the pattern is not maintained. The maintenance of HOX gene repression requires the PcG, while maintenance of active transcription requires the trxG. In mammals PcG genes are also important for neural and hematopoietic development and stem cell self-renewal, and they are misregulated in several cancers.
PcG proteins function in large complexes that modify chromatin structure and repress transcription. Two PcG complexes have been characterized, Polycomb Repressive Complex 1 (PRC1), and ESC-E(Z). They have distinct biochemical activities towards chromatin and collaborate to maintain gene repression. PRC1 alters chromatin structure in a manner that inhibits both ATP-dependent chromatin remodeling and transcription. ESC-E(Z) methylates histone H3 on lysine 27, perhaps providing a stable mark for PcG repressed chromatin.
Characterization of PcG complexes has begun to uncover how PcG proteins might use chromatin structure to block transcription, but does not provide an explanation for how these PcG dependent chromatin structures are inherited in each cell cycle. Both DNA replication and mitosis involve changes in chromatin structure, which might interfere with PcG protein actions. It therefore seems likely that there are specific mechanisms operating as chromatin progresses through the cell cycle to ensure that PcG-mediated repression is maintained. Indeed, regulation of single genes involves more than 15 PcG gene products, some of which might be specifically involved in inheritance. Our goal is to reconstitute steps in epigenetic inheritance in vitro to dissect the biochemical mechanisms involved. Replication of chromatin in the presence or absence of PcG proteins is carried out in cell free systems, and the effect of DNA replication on PcG activities determined. We aim to determine requirements for duplication of PcG dependent chromatin structures, and whether inheritance is mechanistically separable from transcriptional repression.A second project in the lab is the characterization of chromatin structure alterations induced by one PcG complex, PRC1. Data obtained using a four-protein recombinant core of this complex suggests that each PcG complex clusters 3-4 nucleosomes, leading to compaction of nucleosomal arrays. One way this could occur is if the complex has multiple nucleosome binding sites; we will test this idea and identify the nucleosome binding site(s) in the complex. The clustering of nucleosomes by PcG complexes might represent a novel way of folding chromatin, which will be determined by comparing the orientation of nucleosomes in PcG-compacted chromatin to those in chromatin compacted by histone H1.