2017 Theses Doctoral
Making Memories: Modes and Mechanisms of Gene Silencing by the Polycomb Repressive Complexes in Drosophila
Fundamental to the development of metazoa and plants is the capacity of cells to respond to transient intrinsic and extrinsic signals with permanent changes in gene expression that control cellular fates. A paradigmatic example of this process is observed in the case of the conserved, master regulatory HOX genes. The HOX genes are activated early in embryogenesis in combinatorial ON/OFF codes of expression, which act to specify and maintain segment identity in animals. In Drosophila, the “choice” of HOX code is controlled by transiently expressed transcription factors, while the “memory” of that choice is maintained in all future descendant cells through the action of the evolutionarily ancient Polycomb Group (PcG) gene family. The products of the PcG genes function in large, multimeric enzyme complexes known as Polycomb Repressive Complexes (PRCs) that are targeted to the HOX loci by cis-acting Polycomb Response Elements (PREs). Anchored at PREs, the PRCs catalyze a variety of chromatin modifications, most notably the trimethylation of histone H3 at lysine 27 (H3K27me3) by PRC2. These chromatin modifications are thought to both carry the memory of the HOX OFF code through DNA replication and to maintain transcriptional repression.
In addition to the HOX genes, the PcG regulates hundreds of other important developmental control genes, the majority of which do not adopt heritable patterns of ON/OFF expression but instead are more dynamically expressed. This poses the question of how PcG activities control such diverse modes of gene expression.
To investigate how PRE-anchored PRCs maintain heritable patterns of HOX gene expression, we have generated a transgenic lacZ reporter of the classical HOX gene Ultrabithorax (Ubx). Composed of minimal Ubx enhancer and promoter elements required to recapitulate the regulation of the native Ubx gene, the transgene contains the Ubx PRE embedded within a genetically labeled Flp-out cassette. H3K27me3 is deposited throughout the transgene in a manner that depends on the presence of the PRE. By excising the PRE in the cells of the wing imaginal disc where the transgene, like native Ubx, is heritably repressed, we are able to monitor the consequences of the loss of PRE-anchored PRC2 on both the inheritance of H3K27me3 and OFF state. We demonstrate that loss of the OFF state following PRE excision is correlated with the cell division-coupled dilution of H3K27me3. Further, by directly manipulating PRC2 activity of the H3K27 substrate, we demonstrate a causal relationship between the dilution of H3K27me3 nucleosomes and the number of times a cell can divide while maintaining the OFF state following PRE excision.
In addition, we identified Ubx-lacZ transgene insertions that deviate from the classical patterns of heritable expression characteristic of the HOX genes in novel and informative ways. Our analysis of these insertions supports the view that PcG dependent chromatin modifications impose a quantitative rather than qualitative repressive influence on a gene’s promoter, with the promoter’s activity being determined within the context of other regulatory inputs. Similarly, contrary to classical view, we demonstrate that transcription too plays a quantitative role in determining whether or not a HOX locus adopts the heritable ON state.
Together, our work suggests that the activities of the PcG confer a generic repressive influence on target loci. We posit that this influence is capable of maintaining heritable patterns of repression, as in the case of the HOX genes, because these loci have undergone stringent selection against enhancers capable of overcoming the repression mediated by the PcG. The absence of such strong activating inputs, together with the capacity of H3K27me3 to confer locus specific memory of the OFF state, allows for heritable patterns of repression. We propose that this is a special, albeit essential, attribute of the HOX genes. In contrast, most target genes have evolved to integrate repressive PcG chromatin modifications within the context of activating inputs that can override them. In these contexts, we propose that the PcG performs the role of a more general repressor, ensuring that repression is only overridden in those cells receiving peak activating cues. In this way the system may perform an essential role in conferring spatial and temporal robustness to gene expression programs.
- Coleman_columbia_0054D_13744.pdf binary/octet-stream 124 MB Download File
More About This Work
- Academic Units
- Genetics and Development
- Thesis Advisors
- Struhl, Gary
- Ph.D., Columbia University
- Published Here
- January 27, 2017