Previous studies have implicated SAGA (Spt-Ada-Gcn5-acetyltransferase) and TFIID (Transcription factor-IID)-dependent mechanisms of transcriptional activation in yeast. SAGA-dependent transcriptional activation is further regulated by the 19S proteasome subcomplex. However, the role of the 19S proteasome subcomplex in transcriptional activation of the TFIID-dependent genes has not been elucidated. Therefore, we have performed a series of chromatin immunoprecipitation, mutational and transcriptional analyses at the TFIID-dependent ribosomal protein genes such as RPS5, RPL2B and RPS11B. We find that the 19S proteasome subcomplex is recruited to the promoters of these ribosomal protein genes, and promotes the association of NuA4 (Nucleosome acetyltransferase of histone H4) co-activator, but not activator Rap1p (repressor-activator protein 1). These observations support that the 19S proteasome subcomplex enhances the targeting of co-activator at the TFIID-dependent promoter. Such an enhanced targeting of NuA4 HAT (histone acetyltransferase) promotes the recruitment of the TFIID complex for transcriptional initiation. Collectively, our data demonstrate that the 19S proteasome subcomplex enhances the targeting of NuA4 HAT to activator Rap1p at the promoters of ribosomal protein genes to facilitate the recruitment of TFIID for transcriptional stimulation, hence providing a new role of the 19S proteasome subcomplex in establishing a specific regulatory network at the TFIID-dependent promoter for productive transcriptional initiation in vivo.

Recently, we have demonstrated a predominant association of Rad26p with the coding sequences but not promoters of several GAL genes following transcriptional induction. Here, we show that the occupancy of histone H2A–H2B dimer at the coding sequences of these genes is not altered following transcriptional induction in the absence of Rad26p. A histone H2A–H2B dimer-enriched chromatin in Δrad26 is correlated to decreased association of RNA polymerase II with the active coding sequences (and hence transcription). However, the reduced association of RNA polymerase II with the active coding sequence in the absence of Rad26p is not due to the defect in formation of transcription complex at the promoter. Thus, Rad26p regulates the occupancy of histone H2A–H2B dimer, which is correlated to the association of elongating RNA polymerase II with active GAL genes. Similar results are also found at other inducible non-GAL genes. Collectively, our results define a new role of Rad26p in orchestrating chromatin structure and hence transcription in vivo.

NuA4 (nucleosome acetyltransferase of H4) promotes transcriptional initiation of TFIID (a complex of TBP and TBP-associated factors [TAFs])-dependent ribosomal protein genes involved in ribosome biogenesis. However, it is not clearly understood how NuA4 regulates the transcription of ribosomal protein genes. Here, we show that NuA4 is recruited to the promoters of ribosomal protein genes, such as RPS5, RPL2B, and RPS11B, for TFIID recruitment to initiate transcription, and the recruitment of NuA4 to these promoters is impaired in the absence of its Eaf1p component. Intriguingly, impaired NuA4 recruitment in a ⌬eaf1 strain depletes recruitment of TFIID (a TAF-dependent form of TBP) but not the TAF-independent form of TBP to the promoters of ribosomal protein genes. However, in the absence of NuA4, SAGA (Spt-Ada-Gcn5-acetyltransferase) is involved in targeting the TAF-independent form of TBP to the promoters of ribosomal protein genes for transcriptional initiation. Thus, NuA4 plays an important role in targeting TFIID to the promoters of ribosomal protein genes for transcriptional initiation in vivo. Such a function is mediated via its targeted histone acetyltransferase activity. In the absence of NuA4, ribosomal protein genes lose TFIID dependency and become SAGA dependent for transcriptional initiation. Collectively, these results provide significant insights into the regulation of ribosomal protein gene expression and, hence, ribosome biogenesis and functions.H istone H4 acetylation plays important roles in the regulation of eukaryotic transcription and other biological processes (1-3). In Saccharomyces cerevisiae, NuA4 (nucleosome acetyltransferase of H4) acetylates histone H4. In addition, NuA4 is involved in acetylation of histones H2A and H2A.Z (4-7). NuA4 is a multisubunit protein complex and is conserved from yeast to humans (Tip60 is the human homologue of yeast NuA4) (8). Like other histone lysine (K) acetyltransferases (KATs), NuA4 is involved in various cellular events, such as transcription, DNA repair, and cell cycle progression (9-27). In addition, NuA4 is proposed to regulate cellular aging and autophagy via acetylation of nonhistone proteins (28-30). Likewise, Tip60 has numerous nonhistone targets involved in various cellular activities (31, 32). In addition, Tip60 has been found to be involved in performing critical functions in DNA repair and stem cell regulation (33-36). Therefore, NuA4 and its human homologue are multifunctional in maintaining normal cellular functions.Esa1p is the catalytic subunit of NuA4 with KAT activity (37, 38). In addition, NuA4 has 12 other subunits (39, 40). These subunits include Tra1p (ATM-related factor), Epl1p (enhancer of polycomb homologue), Arp4p (actin-related protein), Yaf9p (leukemogenic factor ENL/AF9 homologue), Act1p, and 7 Esa1p-associated factors, Eaf1p to Eaf7p. Eaf2p and Eaf4p are also known as Swc4p and Yng2p, respectively. Although Esa1p is the catalytic subunit of NuA4, it cannot acetylate nucleosomal histones on its own but can acetylate naked/f...

NuA4 histone lysine (K) acetyltransferase (KAT) promotes transcriptional initiation of TATA-binding protein (TBP)-associated factor (TAF)-dependent ribosomal protein genes. TAFs have also been recently found to enhance antisense transcription from the 3= end of the GAL10 coding sequence. However, it remains unknown whether, like sense transcription of the ribosomal protein genes, TAF-dependent antisense transcription of GAL10 also requires NuA4 KAT. Here, we show that NuA4 KAT associates with the GAL10 antisense transcription initiation site at the 3= end of the coding sequence. Such association of NuA4 KAT depends on the Reb1p-binding site that recruits Reb1p activator to the GAL10 antisense transcription initiation site. Targeted recruitment of NuA4 KAT to the GAL10 antisense transcription initiation site promotes GAL10 antisense transcription. Like NuA4 KAT, histone H3 K4/36 methyltransferases and histone H2B ubiquitin conjugase facilitate GAL10 antisense transcription, while the Swi/Snf and SAGA chromatin remodeling/modification factors are dispensable for antisense, but not sense, transcription of GAL10. Taken together, our results demonstrate for the first time the roles of NuA4 KAT and other chromatin regulatory factors in controlling antisense transcription, thus illuminating chromatin regulation of antisense transcription. Noncoding RNAs have been implicated in various cellular processes such as X-chromosome inactivation, genomic imprinting, dosage compensation, heterochromatin formation, metabolism, development, and differentiation (1-5). There are several classes of noncoding RNAs, which include microRNAs, small nuclear RNAs, small interfering RNAs, Piwi-interacting RNAs, and natural antisense transcripts (6). About 72% of genes in human and mouse are associated with antisense transcription (7,8). Antisense transcripts arise from the strand opposite to the sense strand and play regulatory functions in interfering with the stability of sense transcripts, and hence gene expression. Therefore, a number of studies have been focused on the use of antisense oligonucleotides in regulation of gene expression and treatment of diseases without permanently altering the genes. In fact, antisense oligonucleotides are in various clinical trials for treatment of diseases such as cancers, hypertension, respiratory illness, and HIV infection (9-13).Despite great potentials of antisense transcripts/transcription in disease pathogenesis and treatment, it is not clearly understood how antisense transcription is initiated. Recently, we have demonstrated that, like in sense transcription, RNA polymerase II is targeted to the 3= end of the GAL10 coding sequence by an activator Reb1p or Reb1p-binding site and general transcription factors (GTFs) such as transcription factor IID (TFIID) (which is composed of TATA-binding protein [TBP] and a set of TBP-associated factors [TAFs]), TFIIB, and Mediator to initiate antisense transcription (14). Further, we have shown that the Gal4p activator and proteasome that facilitate GAL10 s...

Yeast mRNA 59-triphosphatase, Cet1p, recognizes phosphorylated-RNA polymerase II as a component of capping machinery via Ceg1p for cotranscriptional formation of mRNA cap structure that recruits cap-binding complex (CBC) and protects mRNA from exonucleases. Here, we show that the accumulation of RNA polymerase II at the promoter proximal site of ADH1 is significantly enhanced in the absence of Cet1p. Similar results are also found at other genes. Cet1p is recruited to the 59 end of the coding sequence, and its absence impairs mRNA capping, and hence CBC recruitment. However, such an impaired recruitment of CBC does not enhance promoter proximal accumulation of RNA polymerase II. Thus, Cet1p specifically lowers the accumulation of RNA polymerase II at the promoter proximal site independently of mRNA cap structure or CBC. Further, we show that Cet1p's N-terminal domain, which is not involved in mRNA capping, decreases promoter proximal accumulation of RNA polymerase II. An accumulation of RNA polymerase II at the promoter proximal site in the absence of Cet1p's N-terminal domain is correlated with reduced transcription. Collectively, our results demonstrate a novel role of Cet1p in regulation of promoter proximal accumulation of RNA polymerase II independently of mRNA capping activity, and hence transcription in vivo.