Loss of the Rtf1 subunit of the yeast Paf1 complex has the greatest effect on histone H2B monoubiquitination and H3K4 methylation. To investigate the conservation of Rtf1's role in histone methylation and regulation of gene expression in a higher eukaryote, the role of subunit Rtf1 in histone methylation and transcriptional regulation was characterized in Drosophila. CG10955 is the Drosophila structural homologue of yeast Rtf1, dRtf1. dRtf1 mRNA levels were visualized by Northern blot during eight developmental stages. A transcript of ~3 kb was detected by using dRtf1-radiolabeled antisense RNA probe. Much higher levels of transcript were observed in embryos than in any other developmental stage. In this respect, dRtf1 expression is similar to elongation factor dELL, but it contrasted with elongation factor dEloA expression, which is most abundant in late larvae and during pupariation (Tenney, 2006).
RNAi was used to lower Rtf1 mRNA and protein levels, to determine whether dRtf1 is required for Drosophila viability. RNAi knockdown of dRTF1 was accomplished by using P element-mediated transformation of the SympUAST vector with a 600-bp insertion of dRtf1 cDNA sequence. The SympUAST vector contains two convergent Gal4-inducible UAS promoters that can be used to transcribe double-stranded RNA from the dRtf1 fragment, activating the Drosophila RNAi machinery SympUASTdRtf1 flies were crossed to the actin-Gal4 driver line to activate RNAi. Rtf1 RNAi is expressed only in actin-Gal4 driven dRtf1 progeny, and not control siblings. When one line, 10A, 235 CyO, was used, y+/SympUASTdRtf1 adults were recovered with no actin-Gal4 driven dRtf1 progeny observed. When a second independent line, 17C, 310 CyO, was used, y+/SympUASTdRtf1 adults were recovered with no actin-Gal4 driven dRtf1 progeny observed. A large number of dead pupae were observed in both crosses, suggesting that the Rtf1 RNAi flies were dying during the pupal stage. Because lethality was observed by using two independent SympUASTdRtf1 insertion lines, lethality was unlikely to be due to ectopic gene activation at the SympUASTdRtf1 insertion site (Tenney, 2006).
In actin-Gal4 driven dRtf1 RNAi third instar larvae, Rtf1 protein levels on polytene chromosomes were reduced compared with those in their control siblings. Although Northern blot analysis showed that dRtf1 transcripts decreased by ~20%, this level of reduction seems to be sufficient to cause 100% lethality. The data indicate that, upon expression of Rtf1 RNAi in actin-Gal4 driven dRtf1 progeny, the overall level of chromosomal Rtf1 was substantially reduced, suggesting that the small RNAs generated by Dicer from the long double-stranded Rtf1 RNA precursors may act as microRNAs (miRNAs) and may inhibit Rtf1 translation as well (Tenney, 2006).
Knockdown dRtf1 by RNAi enhances Nnd-1: The Notch signaling pathway is required for regulation of cell fate decisions throughout metazoan development. Although Notch signaling is known to activate transcription of numerous target genes, little is known regarding the molecular details of this process. Recently, Bray (2005) reported that the Drosophila homologue of yeast Bre1 is required for Notch target gene expression in Drosophila. Because Rtf1 is also required for regulation of Rad6/Bre1 in yeast, whether dRtf1 is required for proper Notch signaling was tested (Tenney, 2006).
The hypomorphic Notch allele Nnd-1 causes limited nicking in the margin of the adult wing. Mutations in factors required for Notch signaling enhance this wing nicking; strong enhancement results in a reduced wing width. Mild, nonlethal activation of two independent dRtf1 RNAi knockdown transgenes, dRtf110A and dRtf117C, by an Hsp70Gal4 driver at 27°C decreased the width of Nnd-1 wings relative to a no-RNAi control (y w) and relative to RNAi directed against dEloA (dEloA18D). Both dRtf1 RNAi insertions gave significant reductions in wing width relative to the dEloA and y w controls. There was no significant difference in wing width between y w and dEloA RNAi, demonstrating that the effect on Nnd-1 was not due to ectopic Gal4 expression (Tenney, 2006).
Reference names in red indicate recommended papers.
Search PubMed for articles about Drosophila Rtf1
Adelman, K., Wei, W., Ardehali, M. B., Werner, J., Zhu, B. Reinberg, D. and Lis, J. T. (2006). Drosophila Paf1 modulates chromatin structure at actively transcribed genes. Mol. Cell. Biol. 26(1): 250-60. Medline abstract: 16354696
Bray, S., Musisi, H. and Bienz, M. (2005). Bre1 is required for Notch signaling and histone modification. Dev. Cell 8(2): 279-86. Medline abstract: 15691768
Costa, P. J., and Arndt, K. M. (2000). Synthetic lethal interactions suggest a role for the Saccharomyces cerevisiae Rtf1 protein in transcription elongation. Genetics 156: 535-547. Medline abstract: 11014804
Dover, J., Schneider, J., Tawiah-Boateng, M. A., Wood, A., Dean, K., Johnston, M. & Shilatifard, A. (2002). Methylation of histone H3 by COMPASS requires ubiquitination of histone H2B by Rad6. J. Biol. Chem. 277: 28368-28371. Medline abstract: 12070136
Kaplan, C. D., Holland, M. J. and Winston, F. (2005). Interaction between transcription elongation factors and mRNA 3'-end formation at the Saccharomyces cerevisiae GAL10-GAL7 locus. J. Biol. Chem. 280: 913-922. Medline abstract: 15531585
Krogan, N. J., et al. (2003a). The Paf1 complex is required for histone H3 methylation by COMPASS and Dot1p: linking transcriptional elongation to histone methylation. Mol. Cell 11: 721-729. Medline abstract: 12667454
Krogan, N. J., et al. (2003b). Methylation of histone H3 by Set2 in Saccharomyces cerevisiae is linked to transcriptional elongation by RNA polymerase II. Mol. Cell. Biol. 23(12): 4207-18. Medline abstract: 12773564
Mueller, C. L., et al. (2004). The Paf1 complex has functions independent of actively transcribing RNA polymerase II. Mol. Cell 14: 447-456. Medline abstract: 15149594
Ng, H. H., Dole, S. and Struhl, K. (2003). The Rtf1 component of the Paf1 transcriptional elongation complex is required for ubiquitination of histone H2B. J. Biol. Chem. 278: 33625-33628. Medline abstract: 12876293
Porter, S. E., Penheiter, K. L. and Jaehning, J. A. (2005). Separation of the Saccharomyces cerevisiae Paf1 complex from RNA polymerase II results in changes in its subnuclear localization. Eukaryot. Cell 4: 209-220. Medline abstract: 15643076
Rozenblatt-Rosen, O., et al. (2005). The parafibromin tumor suppressor protein is part of a human Paf1 complex. Mol. Cell. Biol. 25: 612-620. Medline abstract: 15632063
Shi, X., et al. (1996). Paf1p, an RNA polymerase II-associated factor in Saccharomyces cerevisiae, may have both positive and negative roles in transcription. Mol. Cell. Biol. 16: 669-676. Medline abstract: 8552095
Shi, X., et al. (1997). Cdc73p and Paf1p are found in a novel RNA polymerase II-containing complex distinct from the Srbp-containing holoenzyme. Mol. Cell. Biol. 17: 1160-1169. Medline abstract: 9032243
Simic, R., et al. (2003). Chromatin remodeling protein Chd1 interacts with transcription elongation factors and localizes to transcribed genes. EMBO J. 22: 1846-1856. Medline abstract: 12682017
Smith, S. T., et al. (2004). Modulation of heat shock gene expression by the TAC1 chromatin-modifying complex. Nat. Cell Biol. 6: 162-167. Medline abstract: 14730313
Sun, Z. W., and Allis, C. D. (2002). Ubiquitination of histone H2B regulates H3 methylation and gene silencing in yeast. Nature 418: 104-108. Medline abstract: 12077605
Tenney, K., et al. (2006). Drosophila Rtf1 functions in histone methylation, gene expression, and Notch signaling. Proc. Natl. Acad. Sci. 103(32): 11970-4. Medline abstract: 16882721
Wood, A., Schneider, J., Dover, J., Johnston, M. and Shilatifard, A. (2003). The Paf1 complex is essential for histone monoubiquitination by the Rad6-Bre1 complex, which signals for histone methylation by COMPASS and Dot1p J. Biol. Chem. 278: 34739-34742. Medline abstract: 12876294
Yart, A., et al. (2005). The HRPT2 tumor suppressor gene product parafibromin associates with human PAF1 and RNA polymerase II. Mol. Cell. Biol. 25: 5052-5060. Medline abstract: 15923622
Zhu, B., Mandal, S. S., Pham, A. D., Zheng, Y., Erdjument-Bromage, H., Batra, S. K., Tempst, P. and Reinberg, D. (2005). The human PAF complex coordinates transcription with events downstream of RNA synthesis. Genes Dev. 19: 1668-1673. Medline abstract: 16024656
date revised: 9 April 2007
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