Drosophila Rtf1 functions in histone methylation, gene expression, and Notch signaling

Abstract

The Rtf1 subunit of the Paf1 complex is required for proper monoubiquitination of histone H2B and methylation of histone H3 on lysines 4 (H3K4) and 79 in yeast Saccharomyces cerevisiae. Using RNAi, we examined the role of Rtf1 in histone methylation and gene expression in Drosophila melanogaster. We show that Drosophila Rtf1 (dRtf1) is required for proper gene expression and development. Furthermore, we show that RNAi-mediated reduction of dRtf1 results in a reduction in histone H3K4 trimethylation levels on bulk histones and chromosomes in vivo, indicating that the histone modification pathway via Rtf1 is conserved among yeast, Drosophila, and human. Recently, it was demonstrated that histone H3K4 methylation mediated via the E3 ligase Bre1 is critical for transcription of Notch target genes in Drosophila. Here we demonstrate that the dRtf1 component of the Paf1 complex functions in Notch signaling.chromatin ͉ elongation ͉ RNA polymerase II ͉ transcription ͉ monoubiquitination I nterplay between transcriptional activators and repressors regulates gene expression by RNA polymerase II (RNA Pol II). In several cases, chromatin structure is implicated in transcriptional activation and repression. Posttranslational methylation of lysines on the N-terminal tails of histones is thought to modulate higher-order chromatin folding and can activate or repress transcription, depending on the residue being methylated, the regulatory protein recruited by the methyl mark, and whether the lysine is mono-, di-, or trimethylated (1).Histone modifications can be interdependent, such that one modification requires another preexisting modification (1). Histone H3 methylation at lysines 4 and 79 is catalyzed by the complex of proteins associated with Set1 (COMPASS) and Dot1p, respectively in Saccharomyces cerevisiae (1-8). Methylation at these residues requires monoubiquitination of histone H2B at lysine 123 by Rad6͞Bre1 (9-11). The Paf1 complex indirectly regulates histone methylation through its regulation of H2B monoubiquitination and interaction of COMPASS with RNA Pol II (12-14).The Paf1 complex in yeast is composed of five subunits, Paf1, Rtf1, Cdc73, Ctr9, and Leo1, and is associated with the elongating form of RNA Pol II (1, 15-18). The Rtf1 component of Paf1 is required for H2B ubiquitination by and for the recruitment of Set1͞COMPASS to elongating RNA Pol II (12, 13). Because Rtf1 is essential for histone monoubiquitination, methylation, and transcriptional control in yeast, we sought the Drosophila homologue dRtf1 to characterize its role in higher eukaryotes. Here we use RNAi to reduce dRtf1 expression levels and examine the in vivo effect of dRtf1 reduction on transcription and development in the fly. We show that RNAi knockdown of dRtf1 causes pupal lethality. To demonstrate a role for dRtf1 in gene expression, we tested the effect of dRtf1 RNAi knockdown on heat shock gene expression and found that Rtf1 knockdown results in a reduction in heat shock (Hsp70) gene expression. Recently, Bray et al. (19) demonstrated th...

Small region of Rtf1 protein can substitute for complete Paf1 complex in facilitating global histone H2B ubiquitylation in yeast

“…Paf1C associates with RNA pol II from the 5′ end of a gene to the poly(A) site (22, 23); interacts functionally and physically with the transcription elongation factors Spt4-Spt5/DSIF, Spt16-Pob3/FACT, and TFIIS (8,(24)(25)(26)(27); regulates the phosphorylation state of RNA pol II (28, 29); and is required for proper 3′ end formation of certain transcripts (30-32). Important to this study, deletion of RTF1 from yeast cells causes dramatic reductions in H2B K123 ubiquitylation and H3 K4 and K79 methylation (33-36), and this role in histone modification, like other Paf1C functions, is conserved in higher eukaryotes (37)(38)(39)(40).The mechanisms by which Paf1C promotes histone modifications have yet to be elucidated. Deletion of RTF1, CTR9, or PAF1 reduces the occupancies of Rad6 and Set1/COMPASS on coding regions (33,35,41), and purified Paf1C interacts with Bre1 in vitro (42,43), suggesting that Paf1C serves as a platform for recruiting histone-modifying enzymes to RNA pol II during elongation.…”

“…Paf1C associates with RNA pol II from the 5′ end of a gene to the poly(A) site (22, 23); interacts functionally and physically with the transcription elongation factors Spt4-Spt5/DSIF, Spt16-Pob3/FACT, and TFIIS (8,(24)(25)(26)(27); regulates the phosphorylation state of RNA pol II (28, 29); and is required for proper 3′ end formation of certain transcripts (30-32). Important to this study, deletion of RTF1 from yeast cells causes dramatic reductions in H2B K123 ubiquitylation and H3 K4 and K79 methylation (33-36), and this role in histone modification, like other Paf1C functions, is conserved in higher eukaryotes (37)(38)(39)(40).…”

Flickin’ the ubiquitin switch

“…26 It is also of interest to note that knockdown of the histone chaperones ASF1 and NAP1 in insect cells results in derepression of Notch-target genes, and both chaperones physically interact with the H3K4me2/3 demethylase LID/KDM5. 27,28 Depletion of either histone chaperone results in an increase in H3K4me3 at the promoters of Notch target genes, while global levels of this methylated histone were unaffected (Fig.…”

Genetic mechanisms regulating proliferation and cell specification in the Drosophila embryonic CNS

“…Moreover, Drosophila Rtf1 (Restores TBP function 1) was identified as an essential protein during development and found to be involved in the histone modification for H3K4 and H2b, suggesting that its function is conserved from yeast to human. The same study showed that dRtf1 affects the transcription level of Notch target genes [235]. Also, dpaf-1 is an essential gene and involves in methylation of H3K4 [246].…”

“…For instance, several studies showed that Paf1C promote histone trimethylation of H3K4 and H3K36, and ubiquitination of H2BK123; Paf1C is involved in chromatin remodeling, transcription elongation and it mediates transcriptional termination for RNA polymerase II [224,230]. Inanition, several studies have suggested relations between Wnt or Notch signaling and different components of Paf1C [233][234][235][236]. Further, some of the Paf1C components e.g., Ctr9 are identified as a tumor suppressor factor in various organisms especially in human [232,[237][238][239][240][241].…”