Ssl2/TFIIH function in transcription start site scanning by RNA Polymerase II in Saccharomyces cerevisiae

In Saccharomyces cerevisiae, RNA Polymerase II (Pol II) selects transcription start sites (TSS) by a unidirectional scanning process. During scanning, a preinitiation complex (PIC) assembled at an upstream core promoter initiates at select positions within a window ~40-120 basepairs downstream. Several lines of evidence indicate that Ssl2, the yeast homolog of XPB and an essential and conserved subunit of the general transcription factor (GTF) TFIIH, drives scanning through its DNA-dependent ATPase activity, therefore potentially controlling both scanning rate and scanning extent (processivity). To address questions of how Ssl2 functions in promoter scanning and interacts with other initiation activities, we leveraged distinct initiation-sensitive reporters to identify novel ssl2 alleles. These ssl2 alleles, many of which alter residues conserved from yeast to human, confer either upstream or downstream TSS shifts at the model promoter ADH1 and genome-wide. Specifically, tested ssl2 alleles alter TSS selection by increasing or narrowing the distribution of TSSs used at individual promoters. Genetic interactions of ssl2 alleles with other initiation factors are consistent with ssl2 allele classes functioning through increasing or decreasing scanning processivity but not necessarily scanning rate. These alleles underpin a residue interaction network that likely modulates Ssl2 activity and TFIIH function in promoter scanning. We propose that the outcome of promoter scanning is determined by two functional networks, the first being Pol II activity and factors that modulate it to determine initiation efficiency within a scanning window, and the second being Ssl2/TFIIH and factors that modulate scanning processivity to determine the width of the scanning widow.

Download Full-text

Pausing sites of RNA polymerase II on actively transcribed genes are enriched in DNA double-stranded breaks

Journal of Biological Chemistry ◽

10.1074/jbc.ra119.011665 ◽

2020 ◽

Vol 295 (12) ◽

pp. 3990-4000 ◽

Cited By ~ 3

Author(s):

Sandeep Singh ◽

Karol Szlachta ◽

Arkadi Manukyan ◽

Heather M. Raimer ◽

Manikarna Dinda ◽

...

Keyword(s):

Transcriptional Regulation ◽

Rna Polymerase ◽

Rna Polymerase Ii ◽

Transcriptional Activation ◽

Topoisomerase I ◽

Cell Types ◽

Dna Breaks ◽

Transcription Start ◽

Pol Ii ◽

Transcription Start Sites

DNA double-stranded breaks (DSBs) are strongly associated with active transcription, and promoter-proximal pausing of RNA polymerase II (Pol II) is a critical step in transcriptional regulation. Mapping the distribution of DSBs along actively expressed genes and identifying the location of DSBs relative to pausing sites can provide mechanistic insights into transcriptional regulation. Using genome-wide DNA break mapping/sequencing techniques at single-nucleotide resolution in human cells, we found that DSBs are preferentially located around transcription start sites of highly transcribed and paused genes and that Pol II promoter-proximal pausing sites are enriched in DSBs. We observed that DSB frequency at pausing sites increases as the strength of pausing increases, regardless of whether the pausing sites are near or far from annotated transcription start sites. Inhibition of topoisomerase I and II by camptothecin and etoposide treatment, respectively, increased DSBs at the pausing sites as the concentrations of drugs increased, demonstrating the involvement of topoisomerases in DSB generation at the pausing sites. DNA breaks generated by topoisomerases are short-lived because of the religation activity of these enzymes, which these drugs inhibit; therefore, the observation of increased DSBs with increasing drug doses at pausing sites indicated active recruitment of topoisomerases to these sites. Furthermore, the enrichment and locations of DSBs at pausing sites were shared among different cell types, suggesting that Pol II promoter-proximal pausing is a common regulatory mechanism. Our findings support a model in which topoisomerases participate in Pol II promoter-proximal pausing and indicated that DSBs at pausing sites contribute to transcriptional activation.

Download Full-text

Quantitative analysis of transcription start site selection in Saccharomyces cerevisiae determines contributions of DNA sequence and RNA Polymerase II activity

10.1101/2021.11.09.467992 ◽

2021 ◽

Author(s):

Yunye Zhu ◽

Irina O. Vvedenskaya ◽

Bryce E. Nickels ◽

Craig D. Kaplan

Keyword(s):

Saccharomyces Cerevisiae ◽

Quantitative Analysis ◽

Rna Polymerase ◽

Rna Polymerase Ii ◽

Dna Sequence ◽

Transcription Initiation ◽

Transcription Start ◽

Pol Ii ◽

Wide Range

DNA sequence at Transcription Start Sites (TSSs) is a key determinant of initiation by RNA Polymerase II (Pol II). To function as a TSS, an initiation compatible sequence must be specified by a promoter in an appropriate chromatin context. We report the development of a method for quantitative analysis of transcription initiation by Pol II that involves construction of DNA libraries of barcoded promoter variants, production of RNA transcripts, and analysis of transcript 5' ends and transcript yields (Pol II MAssively Systematic Transcript End Readout, "Pol II MASTER"). Using Pol II MASTER, we measure the efficiency of transcription initiation during promoter scanning by Saccharomyces cerevisiae Pol II for ~1 million unique TSS sequences. Furthermore, we employ Pol II MASTER to determine how Pol II activity, known to widely alter TSS selection in vivo, alters TSS efficiencies across our promoter variants. Pol II MASTER recapitulates known critical qualities of Saccharomyces cerevisiae TSS -8, -1, and +1 positions while demonstrating that surrounding sequences modulate initiation efficiency over a wide range. We discover functional interactions between neighboring sequence positions, indicating that adjacent positions likely function together. We demonstrate that initiation efficiencies are altered for +1 A TSSs relative to +1 G TSSs when Pol II activity is perturbed through genetic means. Pol II MASTER provides data for predictive models of TSS initiation efficiency at genomic promoters.

Download Full-text

Computational identification and experimental characterization of preferred downstream positions in human core promoters

10.1101/2021.02.02.429413 ◽

2021 ◽

Author(s):

René Dreos ◽

Nati Malachi ◽

Anna Sloutskin ◽

Philipp Bucher ◽

Tamar Juven-Gershon

Keyword(s):

Rna Polymerase ◽

Rna Polymerase Ii ◽

Motif Discovery ◽

Core Promoter ◽

Sequence Motifs ◽

Promoter Element ◽

Pol Ii ◽

Core Promoters ◽

The Core

AbstractMetazoan core promoters, which direct the initiation of transcription by RNA polymerase II (Pol II), may contain short sequence motifs termed core promoter elements/motifs (e.g. the TATA box, initiator (Inr) and downstream core promoter element (DPE)), which recruit Pol II via the general transcription machinery. The DPE was discovered and extensively characterized in Drosophila, where it is strictly dependent on both the presence of an Inr and the precise spacing from it. Since the Drosophila DPE is recognized by the human transcription machinery, it is most likely that some human promoters contain a downstream element that is similar, though not necessarily identical, to the Drosophila DPE. However, only a couple of human promoters were shown to contain a functional DPE, and attempts to computationally detect human DPE-containing promoters have mostly been unsuccessful. Using a newly-designed motif discovery strategy based on Expectation-Maximization probabilistic partitioning algorithms, we discovered preferred downstream positions (PDP) in human promoters that resemble the Drosophila DPE. Available chromatin accessibility footprints revealed that Drosophila and human Inr+DPE promoter classes are not only highly structured, but also similar to each other, particularly in the proximal downstream region. Clustering of the corresponding sequence motifs using a neighbor-joining algorithm strongly suggests that canonical Inr+DPE promoters could be common to metazoan species. Using reporter assays we demonstrate the contribution of the identified downstream positions to the function of multiple human promoters. Furthermore, we show that alteration of the spacing between the Inr and PDP by two nucleotides results in reduced promoter activity, suggesting a strict spacing dependency of the newly discovered human PDP on the Inr. Taken together, our strategy identified novel functional downstream positions within human core promoters, supporting the existence of DPE-like motifs in human promoters.Author summaryTranscription of genes by the RNA polymerase II enzyme initiates at a genomic region termed the core promoter. The core promoter is a regulatory region that may contain diverse short DNA sequence motifs/elements that confer specific properties to it. Interestingly, core promoter motifs can be located both upstream and downstream of the transcription start site. Variable compositions of core promoter elements have been identified. The initiator (Inr) motif and the downstream core promoter element (DPE) is a combination of elements that has been identified and extensively characterized in fruit flies. Although a few Inr+DPE - containing human promoters have been identified, the presence of transcriptionally important downstream core promoter positions within human promoters has been a matter of controversy in the literature. Here, using a newly-designed motif discovery strategy, we discovered preferred downstream positions in human promoters that resemble fruit fly DPE. Clustering of the corresponding sequence motifs in eight additional species indicated that such promoters could be common to multicellular non-plant organisms. Importantly, functional characterization of the newly discovered preferred downstream positions supports the existence of Inr+DPE-containing promoters in human genes.

Download Full-text

JMJD5 couples with CDK9 to release the paused RNA polymerase II

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.2005745117 ◽

2020 ◽

Vol 117 (33) ◽

pp. 19888-19895

Author(s):

Haolin Liu ◽

Srinivas Ramachandran ◽

Nova Fong ◽

Tzu Phang ◽

Schuyler Lee ◽

...

Keyword(s):

Rna Polymerase ◽

Rna Polymerase Ii ◽

Elongation Factor ◽

Pol Ii ◽

Transcription Start Sites ◽

Terminal Domain ◽

Carboxyl Terminal Domain ◽

Pol Ii Pausing ◽

Carboxyl Terminal ◽

Heptad Repeats

More than 30% of genes in higher eukaryotes are regulated by RNA polymerase II (Pol II) promoter proximal pausing. Pausing is released by the positive transcription elongation factor complex (P-TEFb). However, the exact mechanism by which this occurs and whether phosphorylation of the carboxyl-terminal domain of Pol II is involved in the process remains unknown. We previously reported that JMJD5 could generate tailless nucleosomes at position +1 from transcription start sites (TSS), thus perhaps enable progression of Pol II. Here we find that knockout of JMJD5 leads to accumulation of nucleosomes at position +1. Absence of JMJD5 also results in loss of or lowered transcription of a large number of genes. Interestingly, we found that phosphorylation, by CDK9, of Ser2 within two neighboring heptad repeats in the carboxyl-terminal domain of Pol II, together with phosphorylation of Ser5 within the second repeat, HR-Ser2p (1, 2)-Ser5p (2) for short, allows Pol II to bind JMJD5 via engagement of the N-terminal domain of JMJD5. We suggest that these events bring JMJD5 near the nucleosome at position +1, thus allowing JMJD5 to clip histones on this nucleosome, a phenomenon that may contribute to release of Pol II pausing.

Download Full-text

The Chd1 chromatin remodeler shifts hexasomes unidirectionally

eLife ◽

10.7554/elife.21356 ◽

2016 ◽

Vol 5 ◽

Cited By ~ 32

Author(s):

Robert F Levendosky ◽

Anton Sabantsev ◽

Sebastian Deindl ◽

Gregory D Bowman

Keyword(s):

Saccharomyces Cerevisiae ◽

Rna Polymerase ◽

Rna Polymerase Ii ◽

Histone Variants ◽

Chromatin Remodeler ◽

Post Translational Modifications ◽

Pol Ii ◽

Specific Orientation ◽

Chromatin Reorganization

Despite their canonical two-fold symmetry, nucleosomes in biological contexts are often asymmetric: functionalized with post-translational modifications (PTMs), substituted with histone variants, and even lacking H2A/H2B dimers. Here we show that the Widom 601 nucleosome positioning sequence can produce hexasomes in a specific orientation on DNA, providing a useful tool for interrogating chromatin enzymes and allowing for the generation of nucleosomes with precisely defined asymmetry. Using this methodology, we demonstrate that the Chd1 chromatin remodeler from Saccharomyces cerevisiae requires H2A/H2B on the entry side for sliding, and thus, unlike the back-and-forth sliding observed for nucleosomes, Chd1 shifts hexasomes unidirectionally. Chd1 takes part in chromatin reorganization surrounding transcribing RNA polymerase II (Pol II), and using asymmetric nucleosomes we show that ubiquitin-conjugated H2B on the entry side stimulates nucleosome sliding by Chd1. We speculate that biased nucleosome and hexasome sliding due to asymmetry contributes to the packing of arrays observed in vivo.

Download Full-text

RNA polymerase II-independent recruitment of SPT6L at transcription start sites in Arabidopsis

Nucleic Acids Research ◽

10.1093/nar/gkz465 ◽

2019 ◽

Vol 47 (13) ◽

pp. 6714-6725 ◽

Cited By ~ 3

Author(s):

Chen Chen ◽

Jie Shu ◽

Chenlong Li ◽

Raj K Thapa ◽

Vi Nguyen ◽

...

Keyword(s):

Rna Polymerase ◽

Rna Polymerase Ii ◽

Transcription Initiation ◽

Elongation Factor ◽

Proper Function ◽

Gene Body ◽

Transcription Start ◽

Transcription Start Sites ◽

Genome Wide ◽

Yeast Mutants

Abstract SPT6 is a conserved elongation factor that is associated with phosphorylated RNA polymerase II (RNAPII) during transcription. Recent transcriptome analysis in yeast mutants revealed its potential role in the control of transcription initiation at genic promoters. However, the mechanism by which this is achieved and how this is linked to elongation remains to be elucidated. Here, we present the genome-wide occupancy of Arabidopsis SPT6-like (SPT6L) and demonstrate its conserved role in facilitating RNAPII occupancy across transcribed genes. We also further demonstrate that SPT6L enrichment is unexpectedly shifted, from gene body to transcription start site (TSS), when its association with RNAPII is disrupted. Protein domains, required for proper function and enrichment of SPT6L on chromatin, are subsequently identified. Finally, our results suggest that recruitment of SPT6L at TSS is indispensable for its spreading along the gene body during transcription. These findings provide new insights into the mechanisms underlying SPT6L recruitment in transcription and shed light on the coordination between transcription initiation and elongation.

Download Full-text

Three Key Subregions Contribute to the Function of the Downstream RNA Polymerase II Core Promoter

Molecular and Cellular Biology ◽

10.1128/mcb.00053-10 ◽

2010 ◽

Vol 30 (14) ◽

pp. 3471-3479 ◽

Cited By ~ 34

Author(s):

Joshua W. M. Theisen ◽

Chin Yan Lim ◽

James T. Kadonaga

Keyword(s):

Rna Polymerase ◽

Rna Polymerase Ii ◽

Mutational Analysis ◽

Core Promoter ◽

Regulatory Element ◽

Consensus Sequence ◽

Transcription Start ◽

The Core ◽

Close Proximity ◽

Core Promoter Activity

ABSTRACT The RNA polymerase II core promoter is a diverse and complex regulatory element. To gain a better understanding of the core promoter, we examined the motif 10 element (MTE), which is located downstream of the transcription start site and acts in conjunction with the initiator (Inr). We found that the MTE promotes the binding of purified TFIID to the core promoter and that the TAF6 and TAF9 subunits of TFIID appear to be in close proximity to the MTE. To identify the specific nucleotides that contribute to MTE activity, we performed a detailed mutational analysis and determined a functional MTE consensus sequence. These studies identified favored as well as disfavored nucleotides and demonstrated the previously unrecognized importance of nucleotides in the subregion of nucleotides 27 to 29 (+27 to + 29 relative to A+1 in the Inr consensus) for MTE function. Further analysis led to the identification of three downstream subregions (nucleotides 18 to 22, 27 to 29, and 30 to 33) that contribute to core promoter activity. The three binary combinations of these subregions lead to the MTE (nucleotides 18 to 22 and 27 to 29), a downstream core promoter element (nucleotides 27 to 29 and 30 to 33), and a novel “bridge” core promoter motif (nucleotides 18 to 22 and 30 to 33). These studies have thus revealed a tripartite organization of key subregions in the downstream core promoter.

Download Full-text

CUT&RUN detects distinct DNA footprints of RNA polymerase II near the transcription start sites

Chromosome Research ◽

10.1007/s10577-020-09643-0 ◽

2020 ◽

Vol 28 (3-4) ◽

pp. 381-393

Author(s):

Michi Miura ◽

Honglin Chen

Keyword(s):

Rna Polymerase ◽

Rna Polymerase Ii ◽

Chromatin Immunoprecipitation ◽

Biological Significance ◽

Micrococcal Nuclease ◽

Dna Fragments ◽

Transcription Start ◽

Transcription Start Sites ◽

Human Lung Carcinoma Cell

AbstractCUT&RUN is a powerful tool to study protein-DNA interactions in vivo. DNA fragments cleaved by the targeted micrococcal nuclease identify the footprints of DNA-binding proteins on the chromatin. We performed CUT&RUN on human lung carcinoma cell line A549 maintained in a multi-well cell culture plate to profile RNA polymerase II. Long (> 270 bp) DNA fragments released by CUT&RUN corresponded to the bimodal peak around the transcription start sites, as previously seen with chromatin immunoprecipitation. However, we found that short (< 120 bp) fragments identify a well-defined peak localised at the transcription start sites. This distinct DNA footprint of short fragments, which constituted only about 5% of the total reads, suggests the transient positioning of RNA polymerase II before promoter-proximal pausing, which has not been detected in the physiological settings by standard chromatin immunoprecipitation. We showed that the positioning of the large-size-class DNA footprints around the short-fragment peak was associated with the directionality of transcription, demonstrating the biological significance of distinct CUT&RUN footprints of RNA polymerase II.

Download Full-text

Requirement of ELC1 for RNA Polymerase II Polyubiquitylation and Degradation in Response to DNA Damage in Saccharomyces cerevisiae

Molecular and Cellular Biology ◽

10.1128/mcb.00293-06 ◽

2006 ◽

Vol 26 (11) ◽

pp. 3999-4005 ◽

Cited By ~ 40

Author(s):

Balazs Ribar ◽

Louise Prakash ◽

Satya Prakash

Keyword(s):

Saccharomyces Cerevisiae ◽

Rna Polymerase ◽

Rna Polymerase Ii ◽

Ubiquitin Ligase ◽

Excision Repair ◽

Uv Light ◽

Yeast Cells ◽

Pol Ii ◽

Dna Damaging Agents ◽

Nucleotide Excision

ABSTRACT Treatment of Saccharomyces cerevisiae and human cells with DNA-damaging agents such as UV light or 4-nitroquinoline-1-oxide induces polyubiquitylation of the largest RNA polymerase II (Pol II) subunit, Rpb1, which results in rapid Pol II degradation by the proteasome. Here we identify a novel role for the yeast Elc1 protein in mediating Pol II polyubiquitylation and degradation in DNA-damaged yeast cells and propose the involvement of a ubiquitin ligase, of which Elc1 is a component, in this process. In addition, we present genetic evidence for a possible involvement of Elc1 in Rad7-Rad16-dependent nucleotide excision repair (NER) of lesions from the nontranscribed regions of the genome and suggest a role for Elc1 in increasing the proficiency of repair of nontranscribed DNA, where as a component of the Rad7-Rad16-Elc1 ubiquitin ligase, it would promote the efficient turnover of the NER ensemble from the lesion site in a Rad23-19S proteasomal complex-dependent reaction.

Download Full-text

Properties of an Intergenic Terminator and Start Site Switch That Regulate IMD2 Transcription in Yeast

Molecular and Cellular Biology ◽

10.1128/mcb.00380-08 ◽

2008 ◽

Vol 28 (12) ◽

pp. 3883-3893 ◽

Cited By ~ 60

Author(s):

M. Harley Jenks ◽

Thomas W. O'Rourke ◽

Daniel Reines

Keyword(s):

Rna Polymerase ◽

Rna Polymerase Ii ◽

Initiation Site ◽

Start Codon ◽

Start Site ◽

Transcription Start ◽

Transcription Start Sites ◽

Short Transcript ◽

Alternative Transcription ◽

Polyadenylation Sites

ABSTRACT The IMD2 gene in Saccharomyces cerevisiae is regulated by intracellular guanine nucleotides. Regulation is exerted through the choice of alternative transcription start sites that results in synthesis of either an unstable short transcript terminating upstream of the start codon or a full-length productive IMD2 mRNA. Start site selection is dictated by the intracellular guanine nucleotide levels. Here we have mapped the polyadenylation sites of the upstream, unstable short transcripts that form a heterogeneous family of RNAs of ≈200 nucleotides. The switch from the upstream to downstream start sites required the Rpb9 subunit of RNA polymerase II. The enzyme's ability to locate the downstream initiation site decreased exponentially as the start was moved downstream from the TATA box. This suggests that RNA polymerase II's pincer grip is important as it slides on DNA in search of a start site. Exosome degradation of the upstream transcripts was highly dependent upon the distance between the terminator and promoter. Similarly, termination was dependent upon the Sen1 helicase when close to the promoter. These findings extend the emerging concept that distinct modes of termination by RNA polymerase II exist and that the distance of the terminator from the promoter, as well as its sequence, is important for the pathway chosen.

Download Full-text