Ashkan Golshani

The Paf1 Complex Is Required for Histone H3 Methylation by COMPASS and Dot1p Linking Transcriptional Elongation to Histone Methylation

Methylation of histone proteins is one of their many modifications that affect chromatin structure and regulate gene expression. Methylation of histone H3 on lysines 4 and 79, catalyzed by the Set1-containing complex COMPASS and Dot1p, respectively, is required for silencing of expression of genes located near chromosome telomeres in yeast. We report that the Paf1 protein complex, which is associated with the elongating RNA polymerase II, is required for methylation of lysines 4 and 79 of histone H3 and for silencing of expression of a telomere-associated gene. We show that the Paf1 complex is required for recruitment of the COMPASS methyltransferase to RNA polymerase II and that the subunits of these complexes interact physically and genetically. Collectively, our results suggest that the Paf1 complex is required for histone H3 methylation, therefore linking transcriptional elongation to chromatin methylation.

Methylation of Histone H3 by Set2 in Saccharomyces cerevisiae Is Linked to Transcriptional Elongation by RNA Polymerase II

ABSTRACT Set2 methylates Lys36 of histone H3. We show here that yeast Set2 copurifies with RNA polymerase II (RNAPII). Chromatin immunoprecipitation analyses demonstrated that Set2 and histone H3 Lys36 methylation are associated with the coding regions of several genes that were tested and correlate with active transcription. Both depend, as well, on the Paf1 elongation factor complex. The C terminus of Set2, which contains a WW domain, is also required for effective Lys36 methylation. Deletion of CTK1, encoding an RNAPII CTD kinase, prevents Lys36 methylation and Set2 recruitment, suggesting that methylation may be triggered by contact of the WW domain or C terminus of Set2 with Ser2-phosphorylated CTD. A set2 deletion results in slight sensitivity to 6-azauracil and much less β-galactosidase produced by a reporter plasmid, resulting from a defect in transcription. In synthetic genetic array (SGA) analysis, synthetic growth defects were obtained when a set2 deletion was combined with deletions of all five components of the Paf1 complex, the chromodomain elongation factor Chd1, the putative elongation factor Soh1, the Bre1 or Lge1 components of the histone H2B ubiquitination complex, or the histone H2A variant Htz1. SET2 also interacts genetically with components of the Set1 and Set3 complexes, suggesting that Set1, Set2, and Set3 similarly affect transcription by RNAPII.

Bre1, an E3 ubiquitin ligase required for recruitment and substrate selection of Rad6 at a promoter.

Ubiquitination of histone H2B catalyzed by Rad6 is required for methylation of histone H3 by COMPASS. We identified Bre1 as the probable E3 for Rad6s role in transcription. Bre1 contains a C3HC4 (RING) finger and is present with Rad6 in a complex. The RING finger of Bre1 is required for ubiquitination of histone H2B, methylation of lysine 4 and 79 of H3 and for telomeric silencing. Chromatin immunoprecipitation experiments indicated that both Rad6 and Bre1 are recruited to a promoter. Bre1 is essential for this recruitment of Rad6 and is dedicated to the transcriptional pathway of Rad6. These results suggest that Bre1 is the likely E3 enzyme that directs Rad6 to modify chromatin and ultimately to affect gene expression.

Novel function discovery with GeneMANIA: a new integrated resource for gene function prediction in Escherichia coli

MOTIVATION The model bacterium Escherichia coli is among the best studied prokaryotes, yet nearly half of its proteins are still of unknown biological function. This is despite a wealth of available large-scale physical and genetic interaction data. To address this, we extended the GeneMANIA function prediction web application developed for model eukaryotes to support E.coli. RESULTS We integrated 48 distinct E.coli functional interaction datasets and used the GeneMANIA algorithm to produce thousands of novel functional predictions and prioritize genes for further functional assays. Our analysis achieved cross-validation performance comparable to that reported for eukaryotic model organisms, and revealed new functions for previously uncharacterized genes in specific bioprocesses, including components required for cell adhesion, iron-sulphur complex assembly and ribosome biogenesis. The GeneMANIA approach for network-based function prediction provides an innovative new tool for probing mechanisms underlying bacterial bioprocesses. CONTACT gary.bader@utoronto.ca; mohan.babu@uregina.ca SUPPLEMENTARY INFORMATION Supplementary data are available at Bioinformatics online.