FACT (facilitates chromatin transcription), an evolutionarily conserved histone chaperone involved in transcription and other DNA transactions, is upregulated in cancers, and its downregulation is associated with cellular death. However, it is not clearly understood how FACT is fine-tuned for normal cellular functions. Here, we show that the FACT subunit Spt16 is ubiquitylated by San1 (an E3 ubiquitin ligase) and degraded by the 26S proteasome. Enhanced abundance of Spt16 in the absence of San1 impairs transcriptional elongation. Likewise, decreased abundance of Spt16 also reduces transcription. Thus, an optimal level of Spt16 is required for efficient transcriptional elongation, which is maintained by San1 via ubiquitylation and proteasomal degradation. Consistently, San1 associates with the coding sequences of active genes to regulate Spt16's abundance. Further, we found that enhanced abundance of Spt16 in the absence of San1 impairs chromatin reassembly at the coding sequence, similarly to the results seen following inactivation of Spt16. Efficient chromatin reassembly enhances the fidelity of transcriptional elongation. Taken together, our results demonstrate for the first time a fine-tuning of FACT by a ubiquitin proteasome system in promoting chromatin reassembly in the wake of elongating RNA polymerase II and transcriptional elongation, thus revealing novel regulatory mechanisms of gene expression. In eukaryotes, DNA is packaged into nucleosomes to form chromatin. Each nucleosome within chromatin consists of ϳ147 bp of DNA wrapped around a histone octamer containing one histone H3-H4 tetramer and two histone H2A-H2B dimers (1). Thus, chromatin structure plays crucial functions in regulation of DNA transactions such as transcription, replication, and DNA repair (2-4). A variety of factors are involved in altering chromatin structure and, hence, DNA transactions. These factors are generally classified as ATP-independent histone modifying enzymes, ATP-dependent chromatin remodelers, and histone chaperones. Histone modifying enzymes function through addition or removal of specific chemical groups (e.g., acetyl, methyl, ubiquitin, phospho, and SUMO), while ATP-dependent chromatin remodelers alter DNA-histone interactions or the composition of the nucleosome in an ATP-dependent manner. On the other hand, histone chaperones function by binding with nucleosomes or histones to facilitate assembly and/or disassembly of nucleosomes in an ATP-independent fashion. The histone chaperone that was first found to alter chromatin structure during transcription is FACT (facilitates chromatin transcription), which is evolutionarily conserved among eukaryotes (5, 6). In budding yeast (Saccharomyces cerevisiae), FACT is composed of Spt16 (suppressor of Ty) and Pob3 and physically interacts with nucleosomes with the assistance of the HMG (high mobility group) protein Nhp6. Likewise, FACT is also a heterodimer of Spt16 and SSRP1 (structurespecific recognition protein 1) in humans. SSRP1 contains HMG domain, while HMG domain is p...

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...

Cap-binding complex (CBC) associates cotranscriptionally with the cap structure at the 5= end of nascent mRNA to protect it from exonucleolytic degradation. Here, we show that CBC promotes the targeting of an mRNA export adaptor, Yra1 (forming transcription export [TREX] complex with THO and Sub2), to the active genes and enhances mRNA export in Saccharomyces cerevisiae. Likewise, recruitment of Npl3 (an hnRNP involved in mRNA export via formation of export-competent ribonuclear protein complex [RNP]) to the active genes is facilitated by CBC. Thus, CBC enhances targeting of the export factors and promotes mRNA export. Such function of CBC is not mediated via THO and Sub2 of TREX, cleavage and polyadenylation factors, or Sus1 (that regulates mRNA export via transcription export 2 [TREX-2]). However, CBC promotes splicing of SUS1 mRNA and, consequently, Sus1 protein level and mRNA export via TREX-2. Collectively, our results support the hypothesis that CBC promotes recruitment of Yra1 and Npl3 to the active genes, independently of THO, Sub2, or cleavage and polyadenylation factors, and enhances mRNA export via TREX and RNP, respectively, in addition to its role in facilitating SUS1 mRNA splicing to increase mRNA export through TREX-2, revealing distinct stimulatory functions of CBC in mRNA export.

We have recently demonstrated that an mRNA capping enzyme, Cet1, impairs promoter-proximal accumulation/pausing of RNA polymerase II (Pol II) independently of its capping activity in Saccharomyces cerevisiae to control transcription. However, it is still unknown how Pol II pausing is regulated by Cet1. Here, we show that Cet1's N-terminal domain (NTD) promotes the recruitment of FACT (facilitates chromatin transcription that enhances the engagement of Pol II into transcriptional elongation) to the coding sequence of an active gene, ADH1, independently of mRNAcapping activity. Absence of Cet1's NTD decreases FACT targeting to ADH1 and consequently reduces the engagement of Pol II in transcriptional elongation, leading to promoter-proximal accumulation of Pol II. Similar results were also observed at other genes. Consistently, Cet1 interacts with FACT. Collectively, our results support the notion that Cet1's NTD promotes FACT targeting to the active gene independently of mRNA-capping activity in facilitating Pol II's engagement in transcriptional elongation, thus deciphering a novel regulatory pathway of gene expression. KEYWORDS Cet1, FACT, RNA polymerase II, transcription, mRNA capping R NA polymerase II (Pol II) pauses at the promoter-proximal regions in Drosophila and mammals to provide an additional layer of regulation of transcription. NELF (negative elongation factor) and DSIF (DRB sensitivity factor) play important roles in such pausing of Pol II (1, 2). DSIF alone does not pause Pol II (1, 2). Rather, it targets NELF to associate with Pol II for pausing (1, 2). DSIF is present in all eukaryotes and archaea and shares homology with a bacterial transcription factor, NusG (1, 3). However, NELF is conserved only in higher eukaryotes (1), and thus, the promoter-proximal pausing of Pol II is observed in higher eukaryotes. Such pausing of Pol II has emerged as an important regulatory step of transcription (1, 2). The dissociation of NELF releases paused Pol II for productive transcriptional elongation. P-TEFb, a kinase, triggers the dissociation of NELF via phosphorylation (1, 2). In addition, P-TEFb phosphorylates DSIF and serine-2 at the carboxy-terminal domain (CTD) of the Rpb1 subunit of Pol II (1, 2). Such phosphorylation of DSIF and Pol II has stimulatory effects on transcriptional elongation (1). Thus, P-TEFb performs a crucial function in releasing paused Pol II and enhances transcriptional elongation. The recruitment of P-TEFb to the gene may occur in several ways, such as interaction with DNA-binding proteins, like c-Myc and NF-B, or association with mediator or Brd4 (which is, in turn, bound to the acetylated tail of histone H4) (1, 2, 4-7). Further, the amount and availability of active P-TEFb are regulated via sequestering of P-TEFb into an inactive complex with 7SK RNA and HEXIM protein (1, 2, 8, 9). Thus, various factors, including signaling molecules and chromatin structure/modification, play crucial roles in controlling P-TEFb and, hence, the release

SAGA (Spt-Ada-Gcn5-Acetyltransferase) and TFIID (transcription factor IID) have been previously shown to facilitate the formation of the PIC (pre-initiation complex) at the promoters of two distinct sets of genes. Here, we demonstrate that TFIID and SAGA differentially participate in the stimulation of PIC formation (and hence transcriptional initiation) at the promoter of , a gene for the high-affinity inorganic phosphate (P) transporter for crucial cellular functions, in response to nutrient signaling. We show that transcriptional initiation of occurs predominantly in a TFIID-dependent manner in the absence of P in the growth medium. Such TFIID dependency is mediated via the NuA4 (nucleosome acetyltransferase of H4) histone acetyltransferase (HAT). Intriguingly, transcriptional initiation of also occurs in the presence of P in the growth medium, predominantly via the SAGA complex, but independently of NuA4 HAT. Thus, P in the growth medium switches transcriptional initiation of from NuA4-TFIID to SAGA dependency. Further, we find that both NuA4-TFIID- and SAGA-dependent transcriptional initiations of are facilitated by the 19S proteasome subcomplex or regulatory particle (RP) via enhanced recruitment of the coactivators SAGA and NuA4 HAT, which promote TFIID-independent and -dependent PIC formation for transcriptional initiation, respectively. NuA4 HAT does not regulate activator binding to , but rather facilitates PIC formation for transcriptional initiation in the absence of Pi in the growth medium. On the other hand, SAGA promotes activator recruitment to for transcriptional initiation in the growth medium containing Pi. Collectively, our results demonstrate two distinct stimulatory pathways for PIC formation (and hence transcriptional initiation) at by TFIID, SAGA, NuA4, and 19S RP in the presence and absence of an essential nutrient, P, in the growth media, thus providing new regulatory mechanisms of transcriptional initiation in response to nutrient signaling.

The evolutionarily conserved RNA polymerase II-associated factor 1 (Paf1) from yeast to humans regulates transcription and associated processes, and thus, malfunctions and/or misregulations of Paf1 are associated with cellular pathologies. Indeed, Paf1 (also known as PD2 or pancreatic differentiation 2) is found to be upregulated in poorly differentiated cancer cells, and such upregulation is involved in cellular transformation or oncogenesis. However, the basis for Paf1 upregulation in these cells remains largely unknown. In light of this, we have tested here the idea that the ubiquitin-proteasome system (UPS) regulates the cellular abundance of Paf1. In this direction, we analyzed the role of UPS in regulation of Paf1's abundance in yeast. We find that Paf1 undergoes ubiquitylation and is degraded by the 26S proteasome in yeast, thus deciphering UPS regulation of an evolutionarily conserved factor, Paf1, involved in various cellular processes at the crossroads of the cancer networks. Likewise, Paf1 undergoes proteasomal degradation in well-differentiated, but not poorly differentiated, pancreatic cancer cells, hence pointing to the UPS in upregulation of Paf1 in poorly differentiated cancers. Collectively, our results reveal UPS regulation of Paf1 and suggest downregulation of UPS in elevating Paf1's abundance in poorly differentiated cancers.

Although an F-box protein, Mdm30, is found to regulate ubiquitylation of the Sub2 component of TREX (transcription-export) complex for proteasomal degradation in stimulation of mRNA export, it remains unknown whether such ubiquitin-proteasome system (UPS) regulation of Sub2 occurs cotranscriptionally via its interaction with Mdm30. Further, it is unclear whether impaired UPS regulation of Sub2 in the absence of Mdm30 alters mRNA export via splicing defects of export factors and/or mitochondrial dynamics/function, since Sub2 controls mRNA splicing and Mdm30 regulates mitochondrial aggregation. Here, we show that Mdm30 interacts with Sub2, and temporary shutdown of Mdm30 enhances Sub2’s abundance and impairs mRNA export. Likewise, Sub2’s abundance is increased following transcriptional inhibition. These results support Mdm30’s direct role in regulation of Sub2’s cellular abundance in a transcription-dependent manner. Consistently, the chromatin-bound Sub2 level is increased in the absence of Mdm30. Further, we find that Mdm30 does not facilitate splicing of export factors. Moreover, Mdm30 does not have a dramatic effect on mitochondrial respiration/function, and mRNA export occurs in the absence of Fzo1, which is required for mitochondrial dynamics/respiration. Collective results reveal that Mdm30 interacts with Sub2 for proteasomal degradation in a transcription-dependent manner to promote mRNA export independently of splicing or mitochondrial function, thus advancing our understanding of mRNA export.

Ataxin-7 maintains the integrity of SAGA (Spt-Ada-Gcn5-Acetyltransferase), an evolutionarily conserved co-activator in stimulating pre-initiation complex (PIC) formation for transcription initiation, and thus, its up- and/or down-regulation is associated with various diseases. However, it remains unknown how ataxin-7 is regulated that could provide new insights into disease pathogenesis and therapeutic interventions. Here, we show that ataxin-7’s yeast homologue, Sgf73, undergoes ubiquitylation and proteasomal degradation. Impairment of such regulation increases Sgf73’s abundance, which enhances recruitment of TBP (that nucleates PIC formation) to the promoter, but impairs transcription elongation. However, decreased Sgf73 level reduces PIC formation and transcription. Thus, Sgf73 is fine-tuned by ubiquitin-proteasome system (UPS) in orchestrating transcription. Likewise, ataxin-7 undergoes ubiquitylation and proteasomal degradation, alteration of which changes ataxin-7’s abundance that is associated with altered transcription and cellular pathologies/diseases. Collectively, our results unveil a novel UPS regulation of Sgf73/ataxin-7 for normal cellular health, and implicate alteration of such regulation in diseases.