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San1 ubiquitin ligase is involved in nuclear protein quality control via its interaction with intrinsically disordered proteins for ubiquitylation and proteasomal degradation. Since several transcription/chromatin regulatory factors contain intrinsically disordered domains and can be inhibitory to transcription when in excess, San1 might be involved in transcription regulation. To address this, we analyzed the role of San1 in the genome-wide association of TATA box binding protein (TBP; which nucleates preinitiation complex [PIC] formation for transcription initiation) and RNA polymerase II (Pol II). Our results reveal the roles of San1 in regulating TBP recruitment to the promoters and Pol II association with the coding sequences and, hence, PIC formation and coordination of elongating Pol II, respectively. Consistently, transcription is altered in the absence of San1. Such transcriptional alteration is associated with impaired ubiquitylation and proteasomal degradation of Spt16 and gene association of Paf1 but not the incorporation of centromeric histone, Cse4, into the active genes in the Δsan1 strain. Collectively, our results demonstrate distinct functions of a nuclear protein quality control factor in regulating the genome-wide PIC formation and elongating Pol II (and hence transcription), thus unraveling new gene regulatory mechanisms. [ABSTRACT FROM AUTHOR]

San1 ubiquitin ligase is involved in nuclear protein quality control via its interaction with intrinsically disordered proteins for ubiquitylation and proteasomal degradation. Since several transcription/chromatin regulatory factors contain intrinsically disordered domains and can be inhibitory to transcription when in excess, San1 might be involved in transcription regulation. To address this, we analyzed the role of San1 in the genome-wide association of TATA box binding protein (TBP; which nucleates preinitiation complex [PIC] formation for transcription initiation) and RNA polymerase II (Pol II). Our results reveal the roles of San1 in regulating TBP recruitment to the promoters and Pol II association with the coding sequences and, hence, PIC formation and coordination of elongating Pol II, respectively. Consistently, transcription is altered in the absence of San1. Such transcriptional alteration is associated with impaired ubiquitylation and proteasomal degradation of Spt16 and gene association of Paf1 but not the incorporation of centromeric histone, Cse4, into the active genes in the Δsan1 strain. Collectively, our results demonstrate distinct functions of a nuclear protein quality control factor in regulating the genome-wide PIC formation and elongating Pol II (and hence transcription), thus unraveling new gene regulatory mechanisms. [ABSTRACT FROM AUTHOR]

FACT (facilitates chromatin transcription), an essential and evolutionarily conserved heterodimer from yeast to humans, controls transcription and is found to be upregulated in various cancers. However, the basis for such upregulation is not clearly understood. Our recent results deciphering a new ubiquitin-proteasome system regulation of the FACT subunit SPT16 in orchestrating transcription in yeast hint at the involvement of the proteasome in controlling FACT in humans, with a link to cancer. To test this, we carried out experiments in human embryonic kidney (HEK293) cells, which revealed that human SPT16 undergoes ubiquitylation and that its abundance is increased following inhibition of the proteolytic activity of the proteasome, thus implying proteasomal regulation of human SPT16. Furthermore, we find that the increased abundance/expression of SPT16 in HEK293 cells alters the transcription of genes, including ones associated with cancer, and that the proteasomal degradation of SPT16 is impaired in kidney cancer (Caki-2) cells to upregulate SPT16. Like human SPT16, murine SPT16 in C2C12 cells also undergoes ubiquitylation and proteasomal degradation to regulate transcription. Collectively, our results reveal a proteasomal regulation of mammalian SPT16, with physiological relevance in controlling transcription, and implicate such proteasomal control in the upregulation of SPT16 in cancer. [ABSTRACT FROM AUTHOR]

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 co-transcriptionally via its interaction with Mdm30. Further, it is unclear whether impaired UPS regulation of Sub2 in 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 shut-down of Mdm30 enhances Sub2's abundance and impairs mRNA 40 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 transcription-dependent manner. Consistently, chromatin-bound Sub2 level is increased in absence of Mdm30. Further, we find that Mdm30 does not facilitate splicing of export factors. Moreover, Mdm30 does not have dramatic effect on mitochondrial respiration/function, and mRNA export occurs in absence of Fzo1 that is required for mitochondrial dynamics/respiration. Collective results reveal that Mdm30 interacts with Sub2 for proteasomal degradation in transcription-dependent manner to promote mRNA export independently of splicing or mitochondrial function, thus advancing our understanding of mRNA export. [ABSTRACT FROM AUTHOR]

Cap-binding complex (CBC) associates co-transcriptionally 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 mRNA export adaptor, Yra1 (that forms transcription-export or TREX complex with THO and Sub2), to the active genes to enhance mRNA export in Saccharomyces cerevisiae. Likewise, recruitment of Npl3 (a heteronuclear ribonuclear protein involved in mRNA export via formation of export competent ribonuclear protein complex or RNP) to the active genes is facilitated by CBC. Thus, CBC enhances targeting of the export factors to promote 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 TREX-2 or Transcription-export 2). However, CBC promotes splicing of SUS1 mRNA, and consequently, Sus1 protein level and mRNA export via TREX-2. Collectively, our results support that CBC promotes recruitment of Yra1 and Npl3 to the active genes, independently of THO, Sub2 or cleavage and polyadenylation factors, to enhance mRNA export via TREX and RNP, respectively, in addition to its role in facilitating SUS1 mRNA splicing to increase mRNA export through TREX-2, thus revealing distinct stimulatory functions of CBC in mRNA export. [ABSTRACT FROM AUTHOR]

Non-coding antisense transcripts arise from the strand opposite the sense strand. Over 70% of the human genome generates non-coding antisense transcripts while less than 2% of the genome codes for proteins. Antisense transcripts and/or the act of antisense transcription regulate gene expression and genome integrity by interfering with sense transcription and modulating histone modifications or DNA methylation. Hence, they have significant pathological and physiological relevance. Indeed, antisense transcripts were found to be associated with various diseases including cancer, diabetes, cardiac and neurodegenerative disorders, and, thus, have promising potentials for prognostic and diagnostic markers and therapeutic development. However, it is not clearly understood how antisense transcription is initiated and epigenetically regulated. Such knowledge would provide new insights into the regulation of antisense transcription, and hence disease pathogenesis with therapeutic development. The recent studies on antisense transcription initiation and its epigenetic regulation, which are limited, are discussed here. Furthermore, we concisely describe how antisense transcription/transcripts regulate gene expression and genome integrity with implications in disease pathogenesis and therapeutic development. [ABSTRACT FROM AUTHOR]

TOR (target of rapamycin) has been previously implicated in transcriptional stimulation of the ribosomal protein (RP) genes via enhanced recruitment of NuA4 (nucleosome acetyltransferase of H4) to the promoters. However, it is not clearly understood how TOR enhances NuA4 recruitment to the promoters of the RP genes. Here we show that TOR facilitates the recruitment of the 19S proteasome subcomplex to the activator to enhance the targeting of NuA4 to the promoters of the RP genes. NuA4, in turn, promotes the recruitment of TFIID (transcription factor IID, composed of TATA box-binding protein [TBP] and a set of TBP-associated factors [TAFs]) and RNA polymerase II to the promoters of the RP genes to enhance transcriptional initiation. Therefore, our results demonstrate that TOR facilitates the recruitment of the 19S proteasome subcomplex to the promoters of the RP genes to promote the targeting of NuA4 for enhanced preinitiation complex (PIC) formation and consequently transcriptional initiation, hence illuminating TOR regulation of RP gene activation. Further, our results reveal that TOR differentially regulates PIC formation (and hence transcription) at the non-RP genes, thus demonstrating a complex regulation of gene activation by TOR. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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 Δeaf1 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. [ABSTRACT FROM AUTHOR]

Rad26p is a SWI/SNF-like ATPase in yeast, and is conserved among eukaryotes. Both Rad26p and its human homolog CSB (Cockayne syndrome group B) are involved in regulation of chromatin structure, transcription and DNA repair.  Thus, mutations or malfunctions of these proteins have significant effects on cellular functions. Mutations in CSB are associated with Cockayne syndrome (CS) that is characterized by heterogeneous pathologies such as mental and physical retardation, sun sensitivity, premature aging, muscular and skeletal abnormalities, and progressive decline in neurological and cognitive functions. Therefore, many research groups focused their studies to understand the mechanisms of Rad26p/CSB functions to illuminate the molecular bases of CS. These studies have provided significant functional and mechanistic insights of Rad26p/CSB in regulation of gene expression and genome integrity as described here. [ABSTRACT FROM PUBLISHER]

The authors discuss aspects of the study regarding the implication of both repressive functions and transcriptional stimulatory of histone H2B ubiquitin ligase at the same of different genes. The study hydrolization of ubiquitylated-histone H2B to histone H2B by an ubiquitin protease, Ubp8, which is the main component of the Spt-Ada-Gcn5-acetyltransferase (SAGA) complex. Results showed that Brel RING domain has a stimulatory function in histone H2B ubiquitylation to improve transcription.

In spite of the important regulatory functions of antisense transcripts in gene expression, it remains unknown how antisense transcription is initiated. Recent studies implicated RNA polymerase II in initiation of antisense transcription. However, how RNA polymerase II is targeted to initiate antisense transcription has not been elucidated. Here, we have analyzed the association of RNA polymerase II with the antisense initiation site at the 3' end of the GAL10 coding sequence in dextrose-containing growth medium that induces antisense transcription. We find that RNA polymerase II is targeted to the antisense initiation site at GAL10 by Reb1p activator as well as general transcription factors (e.g., TFIID, TFIIB, and Mediator) for antisense transcription initiation. Intriguingly, while GAL10 antisense transcription is dependent on TFIID, its sense transcription does not require TFIID. Further, the Gal4p activator that promotes GAL10 sense transcription is dispensable for antisense transcription. Moreover, the proteasome that facilitates GAL10 sense transcription does not control its antisense transcription. Taken together, our results reveal that GAL10 sense and antisense transcriptions are regulated differently and shed much light on the mechanisms of antisense transcription initiation. [ABSTRACT FROM AUTHOR]

The fourth lysine of histone H3 is post-translationally modified by a methyl group via the action of histone methyltransferase, and such a covalent modification is associated with transcriptionally active and/or repressed chromatin states. Thus, histone H3 lysine 4 methylation has a crucial role in maintaining normal cellular functions. In fact, misregulation of this covalent modification has been implicated in various types of cancer and other diseases. Therefore, a large number of studies over recent years have been directed towards histone H3 lysine 4 methylation and the enzymes involved in this covalent modification in eukaryotes ranging from yeast to human. These studies revealed a set of histone H3 lysine 4 methyltransferases with important cellular functions in different eukaryotes, as discussed here. [ABSTRACT FROM AUTHOR]

The life of any protein within a cell begins with transcriptional activation, and ends with proteolytic degradation. Intriguingly, the 26S proteasome complex, a non-lysosomal protein degradation machine comprising the 20S proteolytic core and 19S regulatory particles, has been implicated in intimate regulation of eukaryotic transcriptional activation through diverse mechanisms in a proteolysis-dependent as well as independent manner. Here, we discuss the intricate mechanisms of such proteasomal regulation of eukaryotic gene activation via multiple pathways. [ABSTRACT FROM AUTHOR]

Covalent modifications of histones are central to the regulation of chromatin dynamics, and, therefore, many biological processes involving chromatin, such as replication, repair, transcription and genome stability, are regulated by chromatin and its modifications. In this review, we discuss the biochemical, molecular and genetic properties of the enzymatic machinery involved in four different types of histone modification: acetylation, ubiquitination, phosphorylation and methylation. We also discuss how perturbation of the activity of this enzymatic machinery can cause developmental defects and disease. [ABSTRACT FROM AUTHOR]

Histone H2B ubiquitylation is involved in regulation of transcription and other DNA transacting processes. It is performed by a RING finger-containing ubiquitin ligase, Bre1, which is conserved from yeast to humans. Recent studies have implicated both transcriptional stimulatory and repressive functions of histone H2B ubiquitin ligase at the same or different genes as discussed here. [ABSTRACT FROM AUTHOR]

A comparative global proteomic screen identified factors required for COMPASS (complex of proteins associated with Set1)-mediated mono-, di-, and trimethylation of the fourth lysine of histone H3 (H3K4), which included components of a cyclin-dependent protein kinase (Ctk complex) that phosphorylates the C-terminal domain of the largest subunit of RNA polymerase II (Pol II). Our results indicate that histone H3K4 methylation levels are regulated by the Ctk1, Ctk2, and Ctk3 components of the Ctk complex. We show that loss of Ctk1 kinase activity results in reduced histone H3K4 monomethylation levels, followed by a global increase in histone H3K4 trimethylation levels on chromatin. Ctk1 loss does not appear to have a substantial effect on histone H2B monoubiquitination levels or COMPASS and Paf1 complex phosphorylation. Our chromatin immunoprecipitation studies demonstrate that histone H3 eviction during active transcription is decelerated in a CTK1 deletion strain in response to reduced levels of Pol II recruitment. Our in vitro studies show that the onset of monomethylation on an unmethylated histone H3 by COMPASS is virtually immediate, while the onset of trimethylation occurs upon extended time of association between the histone tail and COMPASS. Our study suggests a role for the Ctk complex in the regulation of the pattern of H3K4 mono-, di-, and trimethylation via COMPASS. [ABSTRACT FROM AUTHOR]

Despite recent advances in characterizing the regulation of histone H3 lysine 4 (H3-K4) methylation at the GAL1 gene by the H2B-K123-specific deubiquitinase activity of Saccharomyces cerevisiae SAGA (Spt-Ada-Gcn5-acetyltransferase)-associated Ubp8p, our knowledge on the general role of Ubp8p at the SAGA-dependent genes is lacking. For this study, using a formaldehyde-based in vivo cross-linking and chromatin immunoprecipitation (ChIP) assay, we have analyzed the role of Ubp8p in the regulation of H3-K4 methylation at three other SAGA-dependent yeast genes, namely, PHO84, ADH1, and CUP1. Like that at GAL1, H3-K4 methylation is increased at the PHO84 core promoter in the UBP8 deletion mutant. We also show that H3-K4 methylation remains invariant at the PHO84 open reading frame in the Δ ubp8 mutant, demonstrating a highly localized role of Upb8p in regulation of H3-K4 methylation at the promoter in vivo. However, unlike that at PHO84, H3-K4 methylation at the two other SAGA-dependent genes is not controlled by Ubp8p. Interestingly, Ubp8p and H3-K4 methylation are dispensable for preinitiation complex assembly at the core promoters of these genes. Our ChIP assay further demonstrates that the association of Ubp8p with SAGA is mediated by Sgf11p, consistent with recent biochemical data. Collectively, the data show that Ubp8p differentially controls H3-K4 methylation at the SAGA-dependent promoters, revealing a complex regulatory network of histone methylation in vivo. [ABSTRACT FROM AUTHOR]