Anti-RTF1 monoclonal antibody, clone FQS24726

Conserved from yeast to humans, the Paf1 complex participates in a number of diverse processes including transcriptional initiation and polyadenylation. This complex typically includes five proteins: Paf1, Rtf1, Cdc73, Leo1, and Ctr9. Previous efforts identified clear Drosophila homologs of Paf1, Rtf1, and Cdc73 based on sequence similarity. Further work showed that these proteins help to regulate gene expression and are required for viability. To date, a Drosophila homolog of Ctr9 has remained uncharacterized. Here, we show that the gene CG2469 encodes a functional Drosophila Ctr9 homolog. Both human and Drosophila Ctr9 localize to the nuclei of Drosophila cells and appear enriched in histone locus bodies. RNAi knockdown of Drosophila Ctr9 results in a germline stem cell loss phenotype marked by defects in the morphology of germ cell nuclei. A molecular null mutation of Drosophila Ctr9 results in lethality and a human cDNA CTR9 transgene rescues this phenotype. Clonal analysis in the ovary using this null allele reveals that loss of Drosophila Ctr9 results in a reduction of global levels of histone H3 trimethylation of lysine 4 (H3K4me3), but does not compromise the maintenance of stem cells in ovaries. Given the differences between the null mutant and RNAi knockdown phenotypes, the germ cell defects caused by RNAi likely result from the combined loss of Drosophila Ctr9 and other unidentified genes. These data provide further evidence that the function of this Paf1 complex component is conserved across species.

During infection by human adenovirus (HAdV), the proteins encoded by the early region 1A (E1A) gene bind and appropriate components of the cellular transcriptional machinery to activate the transcription of viral early genes. Previously, we identified roles for the human Bre1 (hBre1) and hPaf1 complexes in E1A-mediated transcriptional activation of HAdV early genes. Here we show that E1A binds hBre1 directly and that this complex targets the hPaf1 complex via the Rtf1 subunit. Depletion of hPaf1 reduces E1A-dependent activation of transcription from the E2e, E3, and E4 viral transcription units, and this does not result from a reduced ability of RNA polymerase II to be recruited to the promoter-proximal regions of these genes. In contrast, depletion of hPaf1 reduces the occupancy of RNA polymerase II across these transcription units. This is accompanied by reductions in the level of H3K36 trimethylation, a posttranslational histone modification associated with efficient transcriptional elongation, and the number of full-length transcripts from these genes. Together, these results indicate that E1A uses hBre1 to recruit the hPaf1 complex in order to optimally activate viral early transcription by enhancing transcriptional elongation.