Biomedical subjects

Nicole J Francis

Publications and source records attributed to Nicole J Francis.

6 recordsLinked to original sources

PubMed2005

Analysis of a polycomb group protein defines regions that link repressive activity on nucleosomal templates to in vivo function.

Polycomb group (PcG) genes propagate patterns of transcriptional repression throughout development. The products of several such genes are part of Polycomb repressive complex 1 (PRC1), which inhibits chromatin remodeling and transcription in vitro. Genetic and biochemical studies suggest the product of the Posterior sex combs (Psc) gene plays a central role in both PcG-mediated gene repression in vivo and PRC1 activity in vitro. To dissect the relationship between the in vivo and in vitro activities of Psc, we identified the lesions associated with 11 genetically characterized Psc mutations and asked how the corresponding mutant proteins affect Psc activity on nucleosomal templates in vitro. Analysis of both single-mutant Psc proteins and recombinant complexes containing mutant protein revealed that Psc encodes at least two functions, complex formation and the inhibition of remodeling and transcription, which require different regions of the protein. There is an excellent correlation between the in vivo phenotypes of mutant Psc alleles and the structure and in vitro activities of the corresponding proteins, suggesting that the in vitro activities of PRC1 reflect essential functions of Psc in vivo.

Animals↗

PubMed2004

Chromatin compaction by a polycomb group protein complex.

Polycomb group proteins preserve body patterning through development by maintaining transcriptional silencing of homeotic genes. A long-standing hypothesis is that silencing involves creating chromatin structure that is repressive to gene transcription. We demonstrate by electron microscopy that core components of Polycomb Repressive Complex 1 induce compaction of defined nucleosomal arrays. Compaction by Polycomb proteins requires nucleosomes but not histone tails. Each Polycomb complex can compact about three nucleosomes. A region of Posterior Sex Combs that is important for gene silencing in vivo is also important for chromatin compaction, linking the two activities. This mechanism of chromatin compaction might be central to stable gene silencing by the Polycomb group.

Chromatin↗

PubMed2004

Propagation of silencing; recruitment and repression of naive chromatin in trans by polycomb repressed chromatin.

The Polycomb group (PcG) proteins maintain stable and heritable repression of homeotic genes. Typically, Polycomb response elements (PRE) that direct PcG repression are located at great distances (10s of kb) from the promoters of PcG-repressed genes, and it is not known how these PREs can communicate with promoters over such distances. Using Class II mouse PRC core complexes (mPCCs) assembled from recombinant subunits, we investigated how PcG complexes might bridge distant chromosomal regions. Like native and recombinant Drosophila Class II complexes, mPCC represses chromatin remodeling and transcription. Interestingly, mPCC bound to one polynucleosome template can recruit a second template from solution and renders it refractory to transcription and chromatin remodeling. A Drosophila PRC core complex (dPCC) also is able to recruit a second template. Posterior sex combs (PSC), a subunit of dPCC, inhibits chromatin remodeling and transcription efficiently but requires assembly with dRING1 to recruit chromatin. Thus, repression and template bridging require different subunits of PcG complexes, suggesting that long-range effects may be mechanistically distinct from repression.

Animals↗

PubMed2004

Requirement for sex comb on midleg protein interactions in Drosophila polycomb group repression.

The Drosophila Sex Comb on Midleg (SCM) protein is a transcriptional repressor of the Polycomb group (PcG). Although genetic studies establish SCM as a crucial PcG member, its molecular role is not known. To investigate how SCM might link to PcG complexes, we analyzed the in vivo role of a conserved protein interaction module, the SPM domain. This domain is found in SCM and in another PcG protein, Polyhomeotic (PH), which is a core component of Polycomb repressive complex 1 (PRC1). SCM-PH interactions in vitro are mediated by their respective SPM domains. Yeast two-hybrid and in vitro binding assays were used to isolate and characterize >30 missense mutations in the SPM domain of SCM. Genetic rescue assays showed that SCM repressor function in vivo is disrupted by mutations that impair SPM domain interactions in vitro. Furthermore, overexpression of an isolated, wild-type SPM domain produced PcG loss-of-function phenotypes in flies. Coassembly of SCM with a reconstituted PRC1 core complex shows that SCM can partner with PRC1. However, gel filtration chromatography showed that the bulk of SCM is biochemically separable from PH in embryo nuclear extracts. These results suggest that SCM, although not a core component of PRC1, interacts and functions with PRC1 in gene silencing.

Amino Acid Sequence↗

PubMed2002

Histone methyltransferase activity of a Drosophila Polycomb group repressor complex.

Polycomb group (PcG) proteins maintain transcriptional repression during development, likely by creating repressive chromatin states. The Extra Sex Combs (ESC) and Enhancer of Zeste [E(Z)] proteins are partners in an essential PcG complex, but its full composition and biochemical activities are not known. A SET domain in E(Z) suggests this complex might methylate histones. We purified an ESC-E(Z) complex from Drosophila embryos and found four major subunits: ESC, E(Z), NURF-55, and the PcG repressor, SU(Z)12. A recombinant complex reconstituted from these four subunits methylates lysine-27 of histone H3. Mutations in the E(Z) SET domain disrupt methyltransferase activity in vitro and HOX gene repression in vivo. These results identify E(Z) as a PcG protein with enzymatic activity and implicate histone methylation in PcG-mediated silencing.

Animals↗

PubMed2002

Native and recombinant polycomb group complexes establish a selective block to template accessibility to repress transcription in vitro.

Polycomb group (PcG) proteins are responsible for stable repression of homeotic gene expression during Drosophila melanogaster development. They are thought to stabilize chromatin structure to prevent transcription, though how they do this is unknown. We have established an in vitro system in which the PcG complex PRC1 and a recombinant PRC1 core complex (PCC) containing only PcG proteins are able to repress transcription by both RNA polymerase II and by T7 RNA polymerase. We find that assembly of the template into nucleosomes enhances repression by PRC1 and PCC. The subunit Psc is able to inhibit transcription on its own. PRC1- and PCC-repressed templates remain accessible to Gal4-VP16 binding, and incubation of the template with HeLa nuclear extract before the addition of PCC eliminates PCC repression. These results suggest that PcG proteins do not merely prohibit all transcription machinery from binding the template but instead likely inhibit specific steps in the transcription reaction.

Animals↗