Abstract
Phosphorylation of the large RNA Polymerase II subunit C-terminal domain (CTD) is believed to be important in promoter clearance and for recruiting protein factors that function in messenger RNA synthesis and processing. P-TEFb is a protein kinase that targets the (CTD). The goal of this study was to identify chromatin modifications and associations that require P-TEFb activity in vivo. We knocked down the catalytic subunit of P-TEFb, Cdk9, in Drosophila melanogaster using RNA interference. Cdk9 knockdown flies die during metamorphosis. Phosphorylation at serine 2 and serine 5 of the CTD heptad repeat were both dramatically reduced in knockdown larvae. Hsp 70 mRNA induction by heat shock was attenuated in Cdk9 knockdown larvae. Both mono- and trimethylation of histone H3 at lysine 4 were dramatically reduced, suggesting a link between CTD phosphorylation and histone methylation in transcribed chromatin in vivo. Levels of the chromo helicase protein CHD1 were reduced in Cdk9 knockdown chromosomes, suggesting that CHD1 is targeted to chromosomes through P-TEFb-dependent histone methylation. Dimethylation of histone H3 at lysine 36 was significantly reduced in knockdown larvae, implicating CTD phosphorylation in the regulation of this chromatin modification. Binding of the RNA Polymerase II elongation factor ELL was reduced in knockdown chromosomes, suggesting that ELL is recruited to active polymerase via CTD phosphorylation.
| Original language | English (US) |
|---|---|
| Pages (from-to) | 101-114 |
| Number of pages | 14 |
| Journal | Molecular Genetics and Genomics |
| Volume | 277 |
| Issue number | 2 |
| DOIs | |
| State | Published - Feb 2007 |
Funding
Acknowledgments We are grateful to Aharon Rosenbloom for performing several of the western blots. We thank Dr. J. T. Lis for anti-Cdk9, anti-Pol II and anti-Spt5 antiserum, Dr. R. P. Perry for CHD1 antiserum and Dr. A. Shearn for Ash1 antiserum. We are grateful to Dr. Dale Dorsett for the use of his Xuorescence microscope. We thank Adam Wood for a critical reading of the manuscript and helpful discussion. This work was supported by NSF grant MCB 0131414 (JCE) and by grants from the National Institutes of Health (2R01CA089455 and 1R01GM069905) and the American Cancer Society (AS). A. Shilatifard is a Scholar of the Leukemia and Lymphoma Society.
Keywords
- CHD1
- CTD phosphorylation
- Cdk9
- Elongation factor
- Histone methylation
- P-TEFb
- RNA Polymerase II
ASJC Scopus subject areas
- Genetics
- Molecular Biology