The Spt5 C-Terminal Region Recruits Yeast 3′ RNA Cleavage Factor I

Spt6 is a multifunctional histone chaperone involved in the maintenance of chromatin structure during elongation by RNA polymerase II (Pol II). Spt6 has a tandem SH2 (tSH2) domain within its C terminus that recognizes Pol II C-terminal domain (CTD) peptides phosphorylated on Ser2, Ser5, or Try1 in vitro. Deleting the tSH2 domain, however, only has a partial effect on Spt6 occupancy in vivo, suggesting that more complex mechanisms are involved in the Spt6 recruitment. Our results show that the Ser2 kinases Bur1 and Ctk1, but not the Ser5 kinase Kin28, cooperate in recruiting Spt6, genomewide. Interestingly, the Ser2 kinases promote the association of Spt6 in early transcribed regions and not toward the 3= ends of genes, where phosphorylated Ser2 reaches its maximum level. In addition, our results uncover an unexpected role for histone deacetylases (Rpd3 and Hos2) in promoting Spt6 interaction with elongating Pol II. Finally, our data suggest that phosphorylation of the Pol II CTD on Tyr1 promotes the association of Spt6 with the 3= ends of transcribed genes, independently of Ser2 phosphorylation. Collectively, our results show that a complex network of interactions, involving the Spt6 tSH2 domain, CTD phosphorylation, and histone deacetylases, coordinate the recruitment of Spt6 to transcribed genes in vivo. D ynamic reorganization of chromatin structure is important for the regulation of transcription by RNA polymerase II (Pol II). Several ATP-dependent chromatin remodelers, histone-modifying enzymes, and histone chaperones promote the disruption of chromatin structure to allow transcription by Pol II and to restore chromatin structure in the wake of transcription (1, 2). Spt6 is a highly conserved and multifunctional protein shown to regulate multiple steps of transcription, including initiation (3, 4) and elongation (5, 6). In addition, it is important for other DNAdependent processes such as recombination (7,8), mRNA export (9), and viral replication (10).Spt6 interacts with histones H3 and H4 (11) and functions as a histone chaperone to regulate cotranscriptional nucleosome reassembly and to modulate chromatin structure, including histone modifications (11-15). Depletion of Spt6 in yeast (Saccharomyces cerevisiae) causes a widespread reduction in histone H3 primarily from the 5= ends of transcribed genes, indicating the importance of Spt6 in maintaining histone occupancy (16). One of the effects of losing Spt6 function is altered gene expression and activation of intragenic cryptic transcription (17-19). Aberrant transcription is also associated with histone deacetylase (HDAC) mutants. Impairing the function of the HDAC Rpd3-small (Rpd3S) and Hos2-Set3 complexes leads to cryptic and antisense transcription genome-wide (20, 21). It is not clear whether Spt6 and HDACs collaborate to suppress spurious transcription.Spt6 localization to transcribed regions strongly correlates with Pol II occupancy (15,22). It possesses a tandem Src homology 2 domain (tSH2) that interacts with phosphorylated Pol II C-terminal doma...

Section: Discussionsupporting

confidence: 52%

Histone Deacetylases and Phosphorylated Polymerase II C-Terminal Domain Recruit Spt6 for Cotranscriptional Histone Reassembly

Burugula

Jeronimo

Pathak

et al. 2014

“…In vitro, Spt5 binds and activates the human capping enzyme (37), and its C-terminal repeat region (CTR) binds S. pombe triphosphatase and guanylyltransferase (38,40). To test whether Spt5 is involved in recruiting capping enzymes in vivo, we carried out ChIP analysis in S. cerevisiae strains expressing only Spt5 lacking the CTR (50,51). We detected a decrease in the occupancy of Cet1, Ceg1, and Abd1 to about 65%, 50%, and 35% of wild-type levels, respectively, at the 5= region of the ADH1 gene, whereas Pol II occupancy was relatively unaffected (Fig.…”

Section: Resultsmentioning

confidence: 99%

“…We carried out a correlation analysis with genome-wide occupancy profiles reported here and elsewhere (21,23,51). Pearson correlation coefficients between profiles were provided as a similarity metric to calculate a correlation-based two-dimensional network using the GraphViz's Neato algorithm (49).…”

Section: Figmentioning

confidence: 99%

Cap Completion and C-Terminal Repeat Domain Kinase Recruitment Underlie the Initiation-Elongation Transition of RNA Polymerase II

Lidschreiber

Leike

Cramer

2013

Self Cite

After transcription initiation, RNA polymerase (Pol) II escapes from the promoter and recruits elongation factors. The molecular basis for the initiation-elongation factor exchange during this transition remains poorly understood. Here, we used chromatin immunoprecipitation (ChIP) to elucidate the initiation-elongation transition of Pol II in the budding yeast Saccharomyces cerevisiae. We show that the early Pol II elongation factor Spt5 contributes to stable recruitment of the mRNA capping enzymes Cet1, Ceg1, and Abd1. Genome-wide occupancy for Cet1 and Ceg1 is restricted to the transcription start site (TSS), whereas occupancy for Abd1 peaks at ϳ110 nucleotides downstream, and occupancy for the cap-binding complex (CBC) rises subsequently. Abd1 and CBC are important for recruitment of the kinases Ctk1 and Bur1, which promote elongation and capping enzyme release. These results suggest that cap completion stimulates productive Pol II elongation.T ranscription of protein-coding genes by RNA polymerase (Pol) II begins with assembly of an initiation complex at the promoter. As the RNA grows, initiation factors dissociate from Pol II, and elongation factors are recruited (1-3). The initiationelongation transition begins when the nascent RNA grows to about 12 nucleotides (nt), which releases initiation factor TFIIB (4, 5). Release of initiation factors frees the Pol II clamp domain, which binds the conserved elongation factor Spt5 (6, 7). Here, we refer to the exchange of initiation by elongation factors as the initiation-elongation transition. The transition starts with the dissociation of initiation factors and ends with completed recruitment of elongation factors.During initiation, the C-terminal repeat domain (CTD) of Pol II gets phosphorylated at the serine 5 (S5) and S7 residues (8-10). CTD S5 phosphorylation is important for recruitment of RNA 5= capping enzymes (11-13). Capping starts with removal of the terminal ␥-phosphate from the 5= triphosphate end of the nascent RNA, which is catalyzed by the RNA triphosphatase (14-16). The truncated RNA 5= end is then linked to an inverted guanylyl group by the guanylyltransferase. Finally, the cap methyltransferase methylates position N7 of the newly added terminal guanine. In Saccharomyces cerevisiae the three catalytic activities are encoded by Cet1, Ceg1, and Abd1, respectively. In metazoans the first two steps of the capping reaction are catalyzed by a single capping enzyme consisting of an N-terminal triphosphatase and a C-terminal guanylyltransferase domain. The resulting 7-methylguanosine (m7G) cap protects the transcript from degradation and promotes translation initiation of the mRNA (17, 18). The complete cap associates with the cap-binding complex (CBC) that functions in pre-mRNA splicing and mRNA export (19,20).We have previously obtained high-resolution genome-wide occupancy profiles for Pol II initiation and elongation factors by chromatin immunoprecipitation (ChIP) in S. cerevisiae. These studies showed that initiation factor occupancy peaked around 50...

“…Despite well-supported genetic and functional studies that implicate Spt4-Spt5 in transcription-coupled processes ranging from chromatin modification and RNA processing (5) to 3=-end formation (74)(75)(76), not much is known about how Spt4-Spt5 interacts with the RNAP ternary complex to function in these nuclear processes. Mechanistic understanding is limited to an NA encirclement model based on the binding of the Spt5 NGN domain to the RNAP Clamp module (26,27,34) and a general understanding of the Spt5 KOW domains' involvement in binding RNAP (11,17,59).…”

Section: Discussionmentioning

confidence: 99%

Structures and Functions of the Multiple KOW Domains of Transcription Elongation Factor Spt5

Meyer

Zhang

et al. 2015

The eukaryotic Spt4-Spt5 heterodimer forms a higher-order complex with RNA polymerase II (and I) to regulate transcription elongation. Extensive genetic and functional data have revealed diverse roles of Spt4-Spt5 in coupling elongation with chromatin modification and RNA-processing pathways. A mechanistic understanding of the diverse functions of Spt4-Spt5 is hampered by challenges in resolving the distribution of functions among its structural domains, including the five KOW domains in Spt5, and a lack of their high-resolution structures. We present high-resolution crystallographic results demonstrating that distinct structures are formed by the first through third KOW domains (KOW1-Linker1 [K1L1] and KOW2-KOW3) of Saccharomyces cerevisiae Spt5. The structure reveals that K1L1 displays a positively charged patch (PCP) on its surface, which binds nucleic acids in vitro, as shown in biochemical assays, and is important for in vivo function, as shown in growth assays. Furthermore, assays in yeast have shown that the PCP has a function that partially overlaps that of Spt4. Synthesis of our results with previous evidence suggests a model in which Spt4 and the K1L1 domain of Spt5 form functionally overlapping interactions with nucleic acids upstream of the transcription bubble, and this mechanism may confer robustness on processes associated with transcription elongation.T ranscription by RNA polymerase (RNAP) II is a regulated process that requires over 60 different accessory factors that interact dynamically with the polymerase as it proceeds through the three main stages of transcription: initiation, elongation, and termination (1). Each RNAP II complex is structurally and biochemically tuned to cope with a set of conditions (e.g., the chromatin state) and to accomplish specific functions (e.g., promoter-dependent initiation) associated with a particular stage of transcription. In eukaryotes, the processes of RNAP II elongation and termination interact closely with pathways of pre-mRNA processing, 3=-end formation, and RNA export (2-4) and thus form temporally and spatially coupled activities that together determine transcriptional responses to intrinsic and extrinsic signaling and regulate RNA metabolism. The heterodimeric complex of Spt4-Spt5 is one of several protein factors that participate in all of the steps that follow transcription initiation. Saccharomyces cerevisiae Spt4-Spt5 is known to coordinate transcription elongation with chromatin remodeling and histone modification; it functions as a general elongation factor for both RNAP II and RNAP I and coordinates with 5= capping, splicing, and 3=-end processing of transcripts (reviewed by Hartzog and Fu [5]). The metazoan homolog of Spt4-Spt5, DSIF, partners with NELF and additional RNAP IIassociated complexes (e.g., P-TEFb and Mediator) to impart a pause-and-release mechanism that regulates RNAP II activity during the initiation-to-elongation transition near promoterproximal regions (6-8). While facilitative for transcription elongation, Spt4-Spt5 also pl...