Spt6 directly interacts with Cdc73 and is required for Paf1 complex occupancy at active genes in <i>Saccharomyces cerevisiae</i>

Ellison, Mitchell A.; Namjilsuren, Sanchirmaa; Shirra, Margaret K.; Blacksmith, Matthew; Schusteff, Rachel A; Kerr, Eleanor M.; Fang, Fei; Xiang, Yufei; Shi, Yi; Arndt, Karen M.

doi:10.1093/nar/gkad180

Cited by 14 publications

(7 citation statements)

References 111 publications

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“…For example, by phosphorylating the linker region between the polymerase core and the CTD, P-TEFb also promotes the binding of the histone chaperone SPT6 to RNAP II at this region [213,250]. SPT6 interacts with PAF1c and helps stabilize its presence on chromatin [251]. This recent finding aligns with the implicated role of P-TEFb activity in remodeling the chromatin barriers encountered on gene bodies by the elongation machinery during transcription.…”

Section: Chromatin Modification During Elongation By Tat and P-tefbsupporting

confidence: 56%

The cell biology of HIV-1 latency and rebound

Mbonye,

Karn

2024

Retrovirology

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Transcriptionally latent forms of replication-competent proviruses, present primarily in a small subset of memory CD4+ T cells, pose the primary barrier to a cure for HIV-1 infection because they are the source of the viral rebound that almost inevitably follows the interruption of antiretroviral therapy. Over the last 30 years, many of the factors essential for initiating HIV-1 transcription have been identified in studies performed using transformed cell lines, such as the Jurkat T-cell model. However, as highlighted in this review, several poorly understood mechanisms still need to be elucidated, including the molecular basis for promoter-proximal pausing of the transcribing complex and the detailed mechanism of the delivery of P-TEFb from 7SK snRNP. Furthermore, the central paradox of HIV-1 transcription remains unsolved: how are the initial rounds of transcription achieved in the absence of Tat? A critical limitation of the transformed cell models is that they do not recapitulate the transitions between active effector cells and quiescent memory T cells. Therefore, investigation of the molecular mechanisms of HIV-1 latency reversal and LRA efficacy in a proper physiological context requires the utilization of primary cell models. Recent mechanistic studies of HIV-1 transcription using latently infected cells recovered from donors and ex vivo cellular models of viral latency have demonstrated that the primary blocks to HIV-1 transcription in memory CD4+ T cells are restrictive epigenetic features at the proviral promoter, the cytoplasmic sequestration of key transcription initiation factors such as NFAT and NF-κB, and the vanishingly low expression of the cellular transcription elongation factor P-TEFb. One of the foremost schemes to eliminate the residual reservoir is to deliberately reactivate latent HIV-1 proviruses to enable clearance of persisting latently infected cells—the “Shock and Kill” strategy. For “Shock and Kill” to become efficient, effective, non-toxic latency-reversing agents (LRAs) must be discovered. Since multiple restrictions limit viral reactivation in primary cells, understanding the T-cell signaling mechanisms that are essential for stimulating P-TEFb biogenesis, initiation factor activation, and reversing the proviral epigenetic restrictions have become a prerequisite for the development of more effective LRAs.

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Section: Chromatin Modification During Elongation By Tat and P-tefbsupporting

confidence: 56%

The cell biology of HIV-1 latency and rebound

Mbonye,

Karn

2024

Retrovirology

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“…As in other systems, RNAPII occupancy fluctuates over gene bodies in budding yeast. 26,[65][66][67] If RNAPII processivity is high, changing occupancy suggests that transcription elongation rates evolve over gene bodies as RNAPII escapes the promoter, proceeds to productive elongation, approaches the cleavage and polyadenylation site, and prepares for termination. Therefore, our model defines variable zones of transcriptional behavior that smoothly transition through user-defined elongation parameters along a simulated gene body (Figure 4B).…”

Section: Defining a Flexible Model Of Transcription Elongation Dynamicsmentioning

confidence: 99%

“…25 Importantly, Cdc73, Rtf1, and Leo1 have all been implicated in coupling Paf1C to the activated elongation complex through their direct interactions with Spt6 and RNAPII, the phosphorylated Spt5 CTR, and RNA, respectively. 10,26,27 As core structural subunits, Paf1 and Ctr9 are presumed to be necessary for the proper execution of all Paf1C-related functions, as described above. 28 Generally, paf1Δ and ctr9Δ alleles phenocopy one another and confer the strongest mutant phenotypes in yeast.…”

Section: Introductionmentioning

confidence: 99%

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Multiple direct and indirect roles of Paf1C in elongation, splicing, and histone post-translational modifications

Francette,

Arndt

2024

Preprint

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Paf1C is a highly conserved protein complex with critical functions during eukaryotic transcription. Previous studies have shown that Paf1C is multi-functional, controlling specific aspects of transcription, ranging from RNAPII processivity to histone modifications. However, it is unclear how specific Paf1C subunits directly impact transcription and coupled processes. We have compared conditional depletion to steady-state deletion for each Paf1C subunit to determine the direct and indirect contributions to gene expression in Saccharomyces cerevisiae. Using nascent transcript sequencing, RNAPII profiling, and modeling of transcription elongation dynamics, we have demonstrated direct effects of Paf1C subunits on RNAPII processivity and elongation rate and indirect effects on transcript splicing and repression of antisense transcripts. Further, our results suggest that the direct transcriptional effects of Paf1C cannot be readily assigned to any particular histone modification. This work comprehensively analyzes both the immediate and extended roles of each Paf1C subunit in transcription elongation and transcript regulation.

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“…Unlike most classical genetic approaches, essential genes or synthetic lethal interactions can be studied using the AID system. We and others have used the AID system to achieve specific degradation of chromatin readers, chromatin remodeling factors, transcription factors, and transcriptional coactivators in S. cerevisiae (9,(12)(13)(14)(15).…”

Section: Introductionmentioning

confidence: 99%

An end-to-end workflow to study newly synthesized mRNA following rapid protein depletion inSaccharomyces cerevisiae

Ridenour,

Donczew

2024

Preprint

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Accurate regulation of gene transcription by RNA polymerase II is essential for the growth and development of eukaryotic cells. Although significant progress has been made in understanding the mechanisms that regulate transcription, many questions remain unanswered. Defining the direct effects of transcriptional regulators is critically important to answering these questions. An effective approach for identifying the direct targets of transcriptional regulators is combining rapid protein depletion and quantification of newly transcribed RNA. The auxin-inducible degron (AID) system and thiol (SH)-linked alkylation for the metabolic sequencing of RNA (SLAM-seq) are powerful methods to rapidly degrade a target protein and directly quantify newly transcribed RNA, respectively. Both methods have been widely applied to study transcriptional regulation. To address unresolved questions in transcription, we engineered an end-to-end workflow in Saccharomyces cerevisiae to deplete proteins of interest using the AID system and measure newly transcribed RNA using SLAM-seq. We provide a step-by-step protocol to support rapid implementation and demonstrate that the workflow can help define the direct effects of transcriptional regulators using the BET proteins Bdf1 and Bdf2 as a test case. This workflow will help address outstanding questions underlying the molecular basis of transcription and other biological processes in S. cerevisiae and other systems.

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Spt6 directly interacts with Cdc73 and is required for Paf1 complex occupancy at active genes in Saccharomyces cerevisiae

Cited by 14 publications

References 111 publications

The cell biology of HIV-1 latency and rebound

The cell biology of HIV-1 latency and rebound

Multiple direct and indirect roles of Paf1C in elongation, splicing, and histone post-translational modifications

An end-to-end workflow to study newly synthesized mRNA following rapid protein depletion inSaccharomyces cerevisiae

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