The yeast histone deacetylase Rpd3 can be recruited to promoters to repress transcription initiation. Biochemical, genetic, and gene-expression analyses show that Rpd3 exists in two distinct complexes. The smaller complex, Rpd3C(S), shares Sin3 and Ume1 with Rpd3C(L) but contains the unique subunits Rco1 and Eaf3. Rpd3C(S) mutants exhibit phenotypes remarkably similar to those of Set2, a histone methyltransferase associated with elongating RNA polymerase II. Chromatin immunoprecipitation and biochemical experiments indicate that the chromodomain of Eaf3 recruits Rpd3C(S) to nucleosomes methylated by Set2 on histone H3 lysine 36, leading to deacetylation of transcribed regions. This pathway apparently acts to negatively regulate transcription because deleting the genes for Set2 or Rpd3C(S) bypasses the requirement for the positive elongation factor Bur1/Bur2.

To physically characterize the web of interactions connecting the Saccharomyces cerevisiae proteins suspected to be RNA polymerase II (RNAPII) elongation factors, subunits of Spt4/Spt5 and Spt16/Pob3 (corresponding to human DSIF and FACT), Spt6, TFIIF (Tfg1, -2, and -3), TFIIS, Rtf1, and Elongator (Elp1, -2, -3, -4, -5, and -6) were affinity purified under conditions designed to minimize loss of associated polypeptides and then identified by mass spectrometry. Spt16/Pob3 was discovered to associate with three distinct complexes: histones; Chd1/casein kinase II (CKII); and Rtf1, Paf1, Ctr9, Cdc73, and a previously uncharacterized protein, Leo1. Rtf1 and Chd1 have previously been implicated in the control of elongation, and the sensitivity to 6-azauracil of strains lacking Paf1, Cdc73, or Leo1 suggested that these proteins are involved in elongation by RNAPII as well. Confirmation came from chromatin immunoprecipitation (ChIP) assays demonstrating that all components of this complex, including Leo1, cross-linked to the promoter, coding region, and 3 end of the ADH1 gene. In contrast, the three subunits of TFIIF cross-linked only to the promoter-containing fragment of ADH1. Spt6 interacted with the uncharacterized, essential protein Iws1 (interacts with Spt6), and Spt5 interacted either with Spt4 or with a truncated form of Spt6. ChIP on Spt6 and the novel protein Iws1 resulted in the cross-linking of both proteins to all three regions of the ADH1 gene, suggesting that Iws1 is likely an Spt6-interacting elongation factor. Spt5, Spt6, and Iws1 are phosphorylated on consensus CKII sites in vivo, conceivably by the Chd1/CKII associated with Spt16/Pob3. All the elongation factors but Elongator copurified with RNAPII.The synthesis of functional mRNA precursors by eukaryotic RNA polymerase II (RNAPII) is a multistage process consisting of three major steps: initiation, elongation, and termination. Although much has been learned about steps involved in initiation, including formation of the initiation complex, initiation of the RNA chain, and promoter clearance, much less is known about later stages in transcription, especially chain elongation. It is known, however, that the transition from transcriptional initiation to elongation is associated with a change in the factors that are associated with RNAPII.Whereas general transcription factors are required for promoter-directed transcription initiation, and the multisubunit Srb/Mediator complex is associated with the RNAPII holoenzyme that binds to promoters in the yeast Saccharomyces cerevisiae, RNAPII can elongate the transcript unaided. However, several factors have been discovered that stimulate elongation on nonchromatin, "naked" DNA templates (e.g., TFIIS and TFIIF) (32,47). Recently, other factors have been identified that are thought to affect RNAPII transcript elongation via effects on chromatin. These include the yeast factors Spt4 and Spt5 (corresponding to human 5,6-dichloro-1-␤-D-ribofuranosylbenzimidazole [DRB] sensitivity-inducing factor [DSIF], which is req...

The largest subunit of RNA polymerase II contains a unique C-terminal domain important for coupling of transcription and mRNA processing. This domain consists of a repeated heptameric sequence (YSPTSPS) phosphorylated at serines 2 and 5. Serine 5 is phosphorylated during initiation and recruits capping enzyme. Serine 2 is phosphorylated during elongation by the Ctk1 kinase, a protein similar to mammalian Cdk9/P-TEFb. Chromatin immunoprecipitation was used to map positions of transcription elongation and mRNA processing factors in strains lacking Ctk1. Ctk1 is not required for association of elongation factors with transcribing polymerase. However, in ctk1Delta strains, the recruitment of polyadenylation factors to 3' regions of genes is disrupted and changes in 3' ends are seen. Therefore, Serine 2 phosphorylation by Ctk1 recruits factors for cotranscriptional 3' end processing in vivo.

To understand the factor interactions of transcribing RNA polymerase II (RNApII) in vivo, chromatin immunoprecipitations were used to map the crosslinking patterns of multiple elongation and polyadenylation factors across transcribed genes. Transcription through the polyadenylation site leads to a reduction in the levels of the Ctk1 kinase and its associated phosphorylation of the RNApII C-terminal domain. One group of elongation factors (Spt4/5, Spt6/Iws1, and Spt16/Pob3), thought to mediate transcription through chromatin, shows patterns matching that of RNApII. In contrast, the Paf and TREX/THO complexes partially overlap RNApII, but do not crosslink to transcribed regions downstream of polyadenylation sites. In a complementary pattern, polyadenylation factors crosslink strongly at the 3 0 ends of genes. Mutation of the 3 0 polyadenylation sequences or the Rna14 protein causes loss of polyadenylation factor crosslinking and readthrough of termination sequences. Therefore, transcription termination and polyadenylation involve transitions at the 3 0 end of genes that may include an exchange of elongation and polyadenylation/termination factors.

We previously reported that apicidin arrested human cancer cell growth through selective induction of p21 WAF1/Cip1 . In this study, the apoptotic potential of apicidin and its mechanism in HL60 cells was investigated. Treatment of HL60 cells with apicidin caused a decrease in viable cell number in a dose-dependent manner and an increase in DNA fragmentation, nuclear morphological change, and apoptotic body formation, concomitant with progressive accumulation of hyperacetylated histone H4. In addition, apicidin converted the procaspase-3 form to catalytically active effector protease, resulting in subsequent cleavages of poly-(ADP-ribose) polymerase and p21 WAF1/Cip1 . Incubation of HL60 cells with z-DEVD-fmk, a caspase-3 inhibitor, almost completely abrogated apicidin-induced activation of caspase-3, DNA fragmentation, and cleavages of poly-(ADP-ribose) polymerase and p21 WAF1/Cip1 . Moreover, these effects were preceded by an increase in translocation of Bax into the mitochondria, resulting in the release of cytochrome c and cleavage of procaspase-9. The addition of cycloheximide greatly inhibited activation of caspase-3 by apicidin by interfering with cleavage of procaspase-3 and DNA fragmentation, suggesting that apicidin-induced apoptosis was dependent on de novo protein synthesis. Consistent with these results, apicidin transiently increased the expressions of both Fas and Fas ligand. Preincubation with NOK-1 monoclonal antibody, which prevents the Fas-Fas ligand interaction and is inhibitory to Fas signaling, interfered with apicidin-induced translocation of Bax, cytochrome c release, cleavage of procaspase-3, and DNA fragmentation. Taken together, the results suggest that apicidin might induce apoptosis through selective induction of Fas/Fas ligand, resulting in the release of cytochrome c from the mitochondria to the cytosol and subsequent activation of caspase-9 and caspase-3.