The Mediator complex is central to transcription by RNA polymerase II (Pol II) in eukaryotes. In ... more The Mediator complex is central to transcription by RNA polymerase II (Pol II) in eukaryotes. In yeast, Mediator is recruited by activators via its tail module and then facilitates assembly of the pre-initiation complex (PIC), including Pol II, setting the stage for productive transcription. Mediator occupies proximal promoter regions only transiently prior to Pol II escape; interruption of the transcription cycle by inactivation or depletion of Kin28 inhibits Pol II escape and stabilizes Mediator occupancy at promoters. However, whether Mediator occupancy and dynamics differ for gene cohorts induced by stress or alternative growth conditions has not been examined on a genome-wide scale. Here we investigate Mediator occupancy following heat shock or CdCl2 induction, with or without depletion of Kin28. We find that Pol II occupancy exhibits similar dependence on Mediator under normal and heat shock conditions; however, Mediator occupancy does not increase upon Kin28 depletion at most...
Previous investigations of topoisomer distributions of simian virus 40 (SV40) DNA from monkey cel... more Previous investigations of topoisomer distributions of simian virus 40 (SV40) DNA from monkey cells have revealed that these circular mini-chromosomes, like relaxed, naked, closed circular DNA, exist as a Gaussian distribution of topoisomers. I have extended this comparison by measuring topoisomer distributions for a variety of plasmid episomes that are stably propagated in cells of the yeast Saccharomyces cerevisiae. The breadth of the topoisomer distributions for plasmid chromatin, including SV40, is approximately constant when normalized for DNA length, as is the breadth of distribution for naked DNA. However, the distributions for plasmid chromatin are substantially broader than those for the corresponding relaxed, naked DNAs. The breath is constant for plasmids differing in transcriptional activity, and varies only slightly between synchronized and unsynchronized populations of yeast cells, suggesting that variation in plasmid linking number with transcription or replication does not account for the observed heterogeneity in linking number. Topoisomer heterogeneity for plasmid chromatin in vivo may be due to heterogeneity in the number of nucleosomes on each plasmid, which could reflect either the nature of the assembly process or the dynamics of nucleosomes within the cell.
Transcriptional activators function in vivo via binding sites that may be packaged into chromatin... more Transcriptional activators function in vivo via binding sites that may be packaged into chromatin. Here we show that whereas the transcriptional activator GAL4 is strongly able to perturb chromatin structure via a nucleosomal binding site in yeast, GCN4 does so poorly. Correspondingly, GCN4 requires assistance from an accessory protein, RAP1, for activation of theHIS4promoter, whereas GAL4 does not. The requirement for RAP1 for GCN4-mediatedHIS4activation is dictated by the DNA-binding domain of GCN4 and not the activation domain, suggesting that RAP1 assists GCN4 in gaining access to its binding site. Consistent with this, overexpression of GCN4 partially alleviates the requirement for RAP1, whereasHIS4activation via a weak GAL4 binding site requires RAP1. RAP1 is extremely effective at interfering with positioning of a nucleosome containing its binding site, consistent with a role in opening chromatin at theHIS4promoter. Furthermore, increasing the spacing between binding sites fo...
Thermal untwisting of DNA is suppressed in vitro in nucleosomes formed with chicken or monkey his... more Thermal untwisting of DNA is suppressed in vitro in nucleosomes formed with chicken or monkey histones. In contrast, results obtained for the 2 micron plasmid in Saccharomyces cerevisiae are consistent with only 30% of the DNA being constrained from thermal untwisting in vivo. In this paper, we examine thermal untwisting of several plasmids in yeast cells, nuclei, and nuclear extracts. All show the same quantitative degree of thermal untwisting, indicating that this phenomenon is independent of DNA sequence. Highly purified yeast plasmid chromatin also shows a large degree of thermal untwisting, whereas circular chromatin reconstituted using chicken histones is restrained from thermal untwisting in yeast nuclear extracts. Thus, the difference in thermal untwisting between yeast chromatin and that assembled with chicken histones is most likely due to differences in the constituent histone proteins.
ABSTRACT DNA in eukaryotes is packaged into chromatin by association with the four histone protei... more ABSTRACT DNA in eukaryotes is packaged into chromatin by association with the four histone proteins, H2A, H2B, H3, and H4, to form nucleosomes. Transcription factors must recognize particular DNA sequences to activate genes and so must compete with the histones for occupancy of their cognate binding sites. Beyond this repressive role, the histones have also adopted regulatory roles, both positive and negative, which are largely mediated by amino terminal “tails” that do not contribute to the basic structure of the nucleosome. Here, we describe some of our recent progress in understanding how transcription factors compete with histones for binding site occupancy and also discuss recent results on the global role of the histone H3 amino terminus in regulating gene expression in the budding yeast Saccharomyces cerevisiae.
Previous studies have described a transcriptional "memory effect", whereby transcript l... more Previous studies have described a transcriptional "memory effect", whereby transcript levels of many Abf1-regulated genes in the budding yeast Saccharomyces cerevisiae are undiminished even after Abf1 has dissociated from its regulatory sites. Here we provide additional support for this effect and investigate its molecular basis. We show that the effect is observed in a distinct abf1 ts mutant from that used in earlier studies, demonstrating that it is robust, and use chromatin immunoprecipitation (ChIP) to show that Abf1 association is decreased similarly from memory effect and transcriptionally responsive genes at the restrictive temperature. We also demonstrate that the association of TBP and Pol II decreases following loss of Abf1 binding for transcriptionally responsive genes but not for memory effect genes. Examination of genome-wide nucleosome occupancy data reveals that while transcriptionally responsive genes exhibit increased nucleosome occupancy in abf1 ts yeast...
The tail module subunits of Mediator complex are targets of activators both in yeast and metazoan... more The tail module subunits of Mediator complex are targets of activators both in yeast and metazoans. Here we discuss recent evidence from studies in yeast for tail module specificity for SAGA-dependent, TATA-containing genes including highly regulated stress response genes, and for independent recruitment and function of the tail module.
Saccharomyces cerevisiae adapts to hypoxia by expressing a large group of "anaerobic" g... more Saccharomyces cerevisiae adapts to hypoxia by expressing a large group of "anaerobic" genes. Among these, the eight DAN/TIR genes are regulated by the repressors Rox1 and Mot3 and the activator Upc2/Mox4. In attempting to identify factors recruited by the DNA binding repressor Mot3 to enhance repression of the DAN/TIR genes, we found that the histone deacetylase and global repressor complex, Rpd3-Sin3-Sap30, was not required for repression. Strikingly, the complex was instead required for activation. In addition, the histone H3 and H4 amino termini, which are targets of Rpd3, were also required for DAN1 expression. Epistasis tests demonstrated that the Rpd3 complex is not required in the absence of the repressor Mot3. Furthermore, the Rpd3 complex was required for normal function and stable binding of the activator Upc2 at the DAN1 promoter. Moreover, the Swi/Snf chromatin remodeling complex was strongly required for activation of DAN1, and chromatin immunoprecipitation an...
Methods in molecular biology (Clifton, N.J.), 1999
Topology of closed circular DNA is affected by its packaging into nucleosomes and potentially by ... more Topology of closed circular DNA is affected by its packaging into nucleosomes and potentially by alteration of nucleosome structure. Changes in topology that reflect alterations in chromatin structure can be measured and quantified using closed circular plasmids from living yeast. Here we describe detailed protocols for measuring DNA topology in yeast chromatin.
The yeast transcriptional activator Gal4p can bind to sites in nucleosomal DNA in vivo which it i... more The yeast transcriptional activator Gal4p can bind to sites in nucleosomal DNA in vivo which it is unable to access in vitro. One event which could allow proteins to bind to otherwise inaccessible sites in chromatin in living cells is DNA replication. To determine whether replication is required for Gal4p to bind to nucleosomal sites in yeast, we have used previously characterized chromatin reporters in which Gal4p binding sites are incorporated into nucleosomes. We find that Gal4p is able to perturb nucleosome positioning via nucleosomal binding sites in yeast arrested either in G1, with alpha-factor, or in G2/M, with nocodazole. Similar results were obtained whether Gal4p synthesis was induced from the endogenous promoter by growth in galactose medium or by an artificial, hormone-inducible system. We also examined binding of the Drosophila transcriptional activator Bicoid, which belongs to the homeodomain class of transcription factors. We show that Bicoid, like Gal4p, can bind to...
The SWI-SNF complex in yeast and related complexes in higher eukaryotes have been implicated in a... more The SWI-SNF complex in yeast and related complexes in higher eukaryotes have been implicated in assisting gene activation by overcoming the repressive effects of chromatin. We show that the ability of the transcriptional activator GAL4 to bind to a site in a positioned nucleosome is not appreciably impaired in swi mutant yeast cells. However, chromatin remodeling that depends on a transcriptional activation domain shows a considerable, although not complete, SWI-SNF dependence, suggesting that the SWI-SNF complex exerts its major effect at a step subsequent to activator binding. We tested this idea further by comparing the SWI-SNF dependence of a reporter gene based on the GAL10 promoter, which has an accessible upstream activating sequence and a nucleosomal TATA element, with that of a CYC1-lacZ reporter, which has a relatively accessible TATA element. We found that the GAL10-based reporter gene showed a much stronger SWI-SNF dependence than did the CYC1-lacZ reporter with several ...
We examine the generality of transcription factor-mediated chromatin remodeling by monitoring cha... more We examine the generality of transcription factor-mediated chromatin remodeling by monitoring changes in chromatin structure in a yeast (Saccharomyces cerevisiae) episome outside of the context of a natural promoter. The episome has a well defined chromatin structure and a binding site for the transcription factor GAL4 but lacks a nearby functional TATA element or transcription start site, so that changes in chromatin structure are unlikely to be caused by transcription. To separate changes caused by binding and by activation domains, we use both GAL4 and a chimeric, hormone-dependent activator consisting of the GAL4 DNA-binding domain, an estrogen receptor (ER) hormone-binding domain, and a VP16 activation domain (Louvion, J.-F., Havaux-Copf, B. and Picard, D. (1993) Gene (Amst.) 131, 129-134). Both GAL4 and GAL4.ER.VP16 show very little perturbation of chromatin structure in their nonactivating configurations. Substantial additional perturbation occurs upon activation. This additi...
To examine the effect of nucleosomes on in vitro transcription, purified chicken erythrocyte core... more To examine the effect of nucleosomes on in vitro transcription, purified chicken erythrocyte core histones and plasmid DNA bearing the Xenopus 5S RNA gene were assembled into nucleosomes and used as templates for transcription in a Xenopus oocyte nuclear extract. Plasmids having a nucleosome incorporating a specific region of the gene were selected by treating the reconstituted molecules with restriction endonucleases. In this way, it was shown that a nucleosome on or close to the internal control region of the 5S RNA gene inhibits transcription. Furthermore, experiments with 5S maxigenes showed that RNA polymerase III, in contrast to SP6 RNA polymerase, will not transcribe through a nucleosome in vitro.
We have defined protein-DNA interactions associated with the transcription complex of a Xenopus s... more We have defined protein-DNA interactions associated with the transcription complex of a Xenopus somatic 5 S RNA gene under efficient in vitro transcription conditions. Transcription factor IIIA, the internal control region of the 5 S RNA gene and specific DNA sequences 5' and 3' of the internal control region are all involved in the formation of a multiprotein complex. Specific protein-DNA interactions outside of the 5 S RNA gene itself, revealed by DNase I footprinting, have no apparent role in the transcription process. Sequences within the 5 S RNA gene, 5' of the internal control region, are not essential for transcription by RNA polymerase III in vitro, but do contribute to its efficiency.
Coding regions of transcribed and non-transcribed genes typically differ in chromatin structure. ... more Coding regions of transcribed and non-transcribed genes typically differ in chromatin structure. However, it is not known what kind of alterations in nucleosome or chromatin structure these differences reflect. To determine whether changes in nucleosome topology accompany transcription, we introduced into yeast a multicopy plasmid bearing the gene coding for the heat shock protein HSP26. The plasmid-borne gene is assembled into chromatin, and is induced by heat shock in the same manner as the endogenous HSP26 gene. A small change in linking number in the HSP26 plasmid accompanies heat shock. This change is consistent with that previously reported for thermal untwisting of DNA in yeast chromatin, and is equivalent in magnitude to that observed in control plasmids which lack heat-shock response elements. These data indicate that no stable alteration in nucleosome topology accompanies transcription of the heat shock gene. Moreover, the kinetics of the observed changes in linking number...
Incorporation into a positioned nucleosome of a cis-acting element essential for replication in S... more Incorporation into a positioned nucleosome of a cis-acting element essential for replication in Saccharomyces cerevisiae disrupts the function of the element in vivo [R. T. Simpson, Nature (London) 343:387-389, 1990]. Furthermore, nucleosome positioning has been implicated in repression of transcription by RNA polymerase II in yeast cells. We have now asked whether the function of cis-acting elements essential for transcription of a gene transcribed by RNA polymerase III can be similarly affected. A tRNA gene was fused to either of two nucleosome positioning signals such that the predicted nucleosome would incorporate near its center the tRNA start site and essential A-box element. These constructs were then introduced into yeast cells on stably maintained, multicopy plasmids. Competent tRNA genes were transcribed in vivo and were not incorporated into positioned nucleosomes. Mutated, inactive tRNA genes were incorporated into nucleosomes whose positions were as predicted. This find...
Mediator is a large, multisubunit complex that is required for essentially all mRNA transcription... more Mediator is a large, multisubunit complex that is required for essentially all mRNA transcription in eukaryotes. In spite of the importance of Mediator, the range of its targets and how it is recruited to these is not well understood. Previous work showed that in Saccharomyces cerevisiae, Mediator contributes to transcriptional activation by two distinct mechanisms, one depending on the tail module triad and favoring SAGA-regulated genes, and the second occurring independently of the tail module and favoring TFIID-regulated genes. Here, we use chromatin immunoprecipitation sequencing (ChIP-seq) to show that dependence on tail module subunits for Mediator recruitment and polymerase II (Pol II) association occurs preferentially at SAGA-regulated over TFIID-regulated genes on a genome-wide scale. We also show that recruitment of tail module subunits to active gene promoters continues genome-wide when Mediator integrity is compromised in med17 temperature-sensitive (ts) yeast, demonstra...
The Mediator complex is central to transcription by RNA polymerase II (Pol II) in eukaryotes. In ... more The Mediator complex is central to transcription by RNA polymerase II (Pol II) in eukaryotes. In yeast, Mediator is recruited by activators via its tail module and then facilitates assembly of the pre-initiation complex (PIC), including Pol II, setting the stage for productive transcription. Mediator occupies proximal promoter regions only transiently prior to Pol II escape; interruption of the transcription cycle by inactivation or depletion of Kin28 inhibits Pol II escape and stabilizes Mediator occupancy at promoters. However, whether Mediator occupancy and dynamics differ for gene cohorts induced by stress or alternative growth conditions has not been examined on a genome-wide scale. Here we investigate Mediator occupancy following heat shock or CdCl2 induction, with or without depletion of Kin28. We find that Pol II occupancy exhibits similar dependence on Mediator under normal and heat shock conditions; however, Mediator occupancy does not increase upon Kin28 depletion at most...
Previous investigations of topoisomer distributions of simian virus 40 (SV40) DNA from monkey cel... more Previous investigations of topoisomer distributions of simian virus 40 (SV40) DNA from monkey cells have revealed that these circular mini-chromosomes, like relaxed, naked, closed circular DNA, exist as a Gaussian distribution of topoisomers. I have extended this comparison by measuring topoisomer distributions for a variety of plasmid episomes that are stably propagated in cells of the yeast Saccharomyces cerevisiae. The breadth of the topoisomer distributions for plasmid chromatin, including SV40, is approximately constant when normalized for DNA length, as is the breadth of distribution for naked DNA. However, the distributions for plasmid chromatin are substantially broader than those for the corresponding relaxed, naked DNAs. The breath is constant for plasmids differing in transcriptional activity, and varies only slightly between synchronized and unsynchronized populations of yeast cells, suggesting that variation in plasmid linking number with transcription or replication does not account for the observed heterogeneity in linking number. Topoisomer heterogeneity for plasmid chromatin in vivo may be due to heterogeneity in the number of nucleosomes on each plasmid, which could reflect either the nature of the assembly process or the dynamics of nucleosomes within the cell.
Transcriptional activators function in vivo via binding sites that may be packaged into chromatin... more Transcriptional activators function in vivo via binding sites that may be packaged into chromatin. Here we show that whereas the transcriptional activator GAL4 is strongly able to perturb chromatin structure via a nucleosomal binding site in yeast, GCN4 does so poorly. Correspondingly, GCN4 requires assistance from an accessory protein, RAP1, for activation of theHIS4promoter, whereas GAL4 does not. The requirement for RAP1 for GCN4-mediatedHIS4activation is dictated by the DNA-binding domain of GCN4 and not the activation domain, suggesting that RAP1 assists GCN4 in gaining access to its binding site. Consistent with this, overexpression of GCN4 partially alleviates the requirement for RAP1, whereasHIS4activation via a weak GAL4 binding site requires RAP1. RAP1 is extremely effective at interfering with positioning of a nucleosome containing its binding site, consistent with a role in opening chromatin at theHIS4promoter. Furthermore, increasing the spacing between binding sites fo...
Thermal untwisting of DNA is suppressed in vitro in nucleosomes formed with chicken or monkey his... more Thermal untwisting of DNA is suppressed in vitro in nucleosomes formed with chicken or monkey histones. In contrast, results obtained for the 2 micron plasmid in Saccharomyces cerevisiae are consistent with only 30% of the DNA being constrained from thermal untwisting in vivo. In this paper, we examine thermal untwisting of several plasmids in yeast cells, nuclei, and nuclear extracts. All show the same quantitative degree of thermal untwisting, indicating that this phenomenon is independent of DNA sequence. Highly purified yeast plasmid chromatin also shows a large degree of thermal untwisting, whereas circular chromatin reconstituted using chicken histones is restrained from thermal untwisting in yeast nuclear extracts. Thus, the difference in thermal untwisting between yeast chromatin and that assembled with chicken histones is most likely due to differences in the constituent histone proteins.
ABSTRACT DNA in eukaryotes is packaged into chromatin by association with the four histone protei... more ABSTRACT DNA in eukaryotes is packaged into chromatin by association with the four histone proteins, H2A, H2B, H3, and H4, to form nucleosomes. Transcription factors must recognize particular DNA sequences to activate genes and so must compete with the histones for occupancy of their cognate binding sites. Beyond this repressive role, the histones have also adopted regulatory roles, both positive and negative, which are largely mediated by amino terminal “tails” that do not contribute to the basic structure of the nucleosome. Here, we describe some of our recent progress in understanding how transcription factors compete with histones for binding site occupancy and also discuss recent results on the global role of the histone H3 amino terminus in regulating gene expression in the budding yeast Saccharomyces cerevisiae.
Previous studies have described a transcriptional "memory effect", whereby transcript l... more Previous studies have described a transcriptional "memory effect", whereby transcript levels of many Abf1-regulated genes in the budding yeast Saccharomyces cerevisiae are undiminished even after Abf1 has dissociated from its regulatory sites. Here we provide additional support for this effect and investigate its molecular basis. We show that the effect is observed in a distinct abf1 ts mutant from that used in earlier studies, demonstrating that it is robust, and use chromatin immunoprecipitation (ChIP) to show that Abf1 association is decreased similarly from memory effect and transcriptionally responsive genes at the restrictive temperature. We also demonstrate that the association of TBP and Pol II decreases following loss of Abf1 binding for transcriptionally responsive genes but not for memory effect genes. Examination of genome-wide nucleosome occupancy data reveals that while transcriptionally responsive genes exhibit increased nucleosome occupancy in abf1 ts yeast...
The tail module subunits of Mediator complex are targets of activators both in yeast and metazoan... more The tail module subunits of Mediator complex are targets of activators both in yeast and metazoans. Here we discuss recent evidence from studies in yeast for tail module specificity for SAGA-dependent, TATA-containing genes including highly regulated stress response genes, and for independent recruitment and function of the tail module.
Saccharomyces cerevisiae adapts to hypoxia by expressing a large group of "anaerobic" g... more Saccharomyces cerevisiae adapts to hypoxia by expressing a large group of "anaerobic" genes. Among these, the eight DAN/TIR genes are regulated by the repressors Rox1 and Mot3 and the activator Upc2/Mox4. In attempting to identify factors recruited by the DNA binding repressor Mot3 to enhance repression of the DAN/TIR genes, we found that the histone deacetylase and global repressor complex, Rpd3-Sin3-Sap30, was not required for repression. Strikingly, the complex was instead required for activation. In addition, the histone H3 and H4 amino termini, which are targets of Rpd3, were also required for DAN1 expression. Epistasis tests demonstrated that the Rpd3 complex is not required in the absence of the repressor Mot3. Furthermore, the Rpd3 complex was required for normal function and stable binding of the activator Upc2 at the DAN1 promoter. Moreover, the Swi/Snf chromatin remodeling complex was strongly required for activation of DAN1, and chromatin immunoprecipitation an...
Methods in molecular biology (Clifton, N.J.), 1999
Topology of closed circular DNA is affected by its packaging into nucleosomes and potentially by ... more Topology of closed circular DNA is affected by its packaging into nucleosomes and potentially by alteration of nucleosome structure. Changes in topology that reflect alterations in chromatin structure can be measured and quantified using closed circular plasmids from living yeast. Here we describe detailed protocols for measuring DNA topology in yeast chromatin.
The yeast transcriptional activator Gal4p can bind to sites in nucleosomal DNA in vivo which it i... more The yeast transcriptional activator Gal4p can bind to sites in nucleosomal DNA in vivo which it is unable to access in vitro. One event which could allow proteins to bind to otherwise inaccessible sites in chromatin in living cells is DNA replication. To determine whether replication is required for Gal4p to bind to nucleosomal sites in yeast, we have used previously characterized chromatin reporters in which Gal4p binding sites are incorporated into nucleosomes. We find that Gal4p is able to perturb nucleosome positioning via nucleosomal binding sites in yeast arrested either in G1, with alpha-factor, or in G2/M, with nocodazole. Similar results were obtained whether Gal4p synthesis was induced from the endogenous promoter by growth in galactose medium or by an artificial, hormone-inducible system. We also examined binding of the Drosophila transcriptional activator Bicoid, which belongs to the homeodomain class of transcription factors. We show that Bicoid, like Gal4p, can bind to...
The SWI-SNF complex in yeast and related complexes in higher eukaryotes have been implicated in a... more The SWI-SNF complex in yeast and related complexes in higher eukaryotes have been implicated in assisting gene activation by overcoming the repressive effects of chromatin. We show that the ability of the transcriptional activator GAL4 to bind to a site in a positioned nucleosome is not appreciably impaired in swi mutant yeast cells. However, chromatin remodeling that depends on a transcriptional activation domain shows a considerable, although not complete, SWI-SNF dependence, suggesting that the SWI-SNF complex exerts its major effect at a step subsequent to activator binding. We tested this idea further by comparing the SWI-SNF dependence of a reporter gene based on the GAL10 promoter, which has an accessible upstream activating sequence and a nucleosomal TATA element, with that of a CYC1-lacZ reporter, which has a relatively accessible TATA element. We found that the GAL10-based reporter gene showed a much stronger SWI-SNF dependence than did the CYC1-lacZ reporter with several ...
We examine the generality of transcription factor-mediated chromatin remodeling by monitoring cha... more We examine the generality of transcription factor-mediated chromatin remodeling by monitoring changes in chromatin structure in a yeast (Saccharomyces cerevisiae) episome outside of the context of a natural promoter. The episome has a well defined chromatin structure and a binding site for the transcription factor GAL4 but lacks a nearby functional TATA element or transcription start site, so that changes in chromatin structure are unlikely to be caused by transcription. To separate changes caused by binding and by activation domains, we use both GAL4 and a chimeric, hormone-dependent activator consisting of the GAL4 DNA-binding domain, an estrogen receptor (ER) hormone-binding domain, and a VP16 activation domain (Louvion, J.-F., Havaux-Copf, B. and Picard, D. (1993) Gene (Amst.) 131, 129-134). Both GAL4 and GAL4.ER.VP16 show very little perturbation of chromatin structure in their nonactivating configurations. Substantial additional perturbation occurs upon activation. This additi...
To examine the effect of nucleosomes on in vitro transcription, purified chicken erythrocyte core... more To examine the effect of nucleosomes on in vitro transcription, purified chicken erythrocyte core histones and plasmid DNA bearing the Xenopus 5S RNA gene were assembled into nucleosomes and used as templates for transcription in a Xenopus oocyte nuclear extract. Plasmids having a nucleosome incorporating a specific region of the gene were selected by treating the reconstituted molecules with restriction endonucleases. In this way, it was shown that a nucleosome on or close to the internal control region of the 5S RNA gene inhibits transcription. Furthermore, experiments with 5S maxigenes showed that RNA polymerase III, in contrast to SP6 RNA polymerase, will not transcribe through a nucleosome in vitro.
We have defined protein-DNA interactions associated with the transcription complex of a Xenopus s... more We have defined protein-DNA interactions associated with the transcription complex of a Xenopus somatic 5 S RNA gene under efficient in vitro transcription conditions. Transcription factor IIIA, the internal control region of the 5 S RNA gene and specific DNA sequences 5' and 3' of the internal control region are all involved in the formation of a multiprotein complex. Specific protein-DNA interactions outside of the 5 S RNA gene itself, revealed by DNase I footprinting, have no apparent role in the transcription process. Sequences within the 5 S RNA gene, 5' of the internal control region, are not essential for transcription by RNA polymerase III in vitro, but do contribute to its efficiency.
Coding regions of transcribed and non-transcribed genes typically differ in chromatin structure. ... more Coding regions of transcribed and non-transcribed genes typically differ in chromatin structure. However, it is not known what kind of alterations in nucleosome or chromatin structure these differences reflect. To determine whether changes in nucleosome topology accompany transcription, we introduced into yeast a multicopy plasmid bearing the gene coding for the heat shock protein HSP26. The plasmid-borne gene is assembled into chromatin, and is induced by heat shock in the same manner as the endogenous HSP26 gene. A small change in linking number in the HSP26 plasmid accompanies heat shock. This change is consistent with that previously reported for thermal untwisting of DNA in yeast chromatin, and is equivalent in magnitude to that observed in control plasmids which lack heat-shock response elements. These data indicate that no stable alteration in nucleosome topology accompanies transcription of the heat shock gene. Moreover, the kinetics of the observed changes in linking number...
Incorporation into a positioned nucleosome of a cis-acting element essential for replication in S... more Incorporation into a positioned nucleosome of a cis-acting element essential for replication in Saccharomyces cerevisiae disrupts the function of the element in vivo [R. T. Simpson, Nature (London) 343:387-389, 1990]. Furthermore, nucleosome positioning has been implicated in repression of transcription by RNA polymerase II in yeast cells. We have now asked whether the function of cis-acting elements essential for transcription of a gene transcribed by RNA polymerase III can be similarly affected. A tRNA gene was fused to either of two nucleosome positioning signals such that the predicted nucleosome would incorporate near its center the tRNA start site and essential A-box element. These constructs were then introduced into yeast cells on stably maintained, multicopy plasmids. Competent tRNA genes were transcribed in vivo and were not incorporated into positioned nucleosomes. Mutated, inactive tRNA genes were incorporated into nucleosomes whose positions were as predicted. This find...
Mediator is a large, multisubunit complex that is required for essentially all mRNA transcription... more Mediator is a large, multisubunit complex that is required for essentially all mRNA transcription in eukaryotes. In spite of the importance of Mediator, the range of its targets and how it is recruited to these is not well understood. Previous work showed that in Saccharomyces cerevisiae, Mediator contributes to transcriptional activation by two distinct mechanisms, one depending on the tail module triad and favoring SAGA-regulated genes, and the second occurring independently of the tail module and favoring TFIID-regulated genes. Here, we use chromatin immunoprecipitation sequencing (ChIP-seq) to show that dependence on tail module subunits for Mediator recruitment and polymerase II (Pol II) association occurs preferentially at SAGA-regulated over TFIID-regulated genes on a genome-wide scale. We also show that recruitment of tail module subunits to active gene promoters continues genome-wide when Mediator integrity is compromised in med17 temperature-sensitive (ts) yeast, demonstra...
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