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Kevin Struhl

Publications and source records attributed to Kevin Struhl.

At least 37 records · Page 2Linked to original sources
PubMed2004

The transcription factor Ifh1 is a key regulator of yeast ribosomal protein genes.

Ribosomal protein (RP) genes in eukaryotes are coordinately regulated in response to growth stimuli and environmental stress, thereby permitting cells to adjust ribosome number and overall protein synthetic capacity to physiological conditions. Approximately 50% of RNA polymerase II transcription is devoted to RP genes. The transcriptional regulator Rap1 binds most yeast RP promoters, and Rap1 sites are important for coordinate regulation of RP genes. However, Rap1 is not the specific regulator that controls RP transcription because it also functions as a repressor, and many Rap1-activated promoters are not coordinately regulated with RP promoters. Here we show that the transcription factors Fhl1 and Ifh1 associate almost exclusively with RP promoters; association depends on Rap1 and (to a lesser extent) a DNA element at many RP promoters. Ifh1 is recruited to promoters via the forkhead-associated (FHA) domain of Fhl1; the level of Ifh1 associated with RP promoters determines the level of transcription; and environmental stress causes a marked reduction in the association of Ifh1, but not Fhl1 or Rap1. Thus, Ifh1 association with promoters is the key regulatory step for coordinate expression of RP genes.

Base Sequence↗
PubMed2004

Association of RNA polymerase with transcribed regions in Escherichia coli.

We examine the association of the beta-, alpha-, and sigma(70)-subunits of Escherichia coli RNA polymerase (RNAP) and the NusA elongation factor with transcribed regions in vivo by using chromatin immunoprecipitation. RNAP preferentially associates with the promoter-proximal region of several operons, and this preference is particularly pronounced at the lexA-dinF promoter. When cells are grown in exponential phase, little or no sigma(70) is associated with RNAP during early elongation. However, during stationary phase, sigma(70) is retained in a fraction of elongating RNAP complexes throughout the melAB operon. In contrast, sigma(70) is not observed in elongating RNAP complexes at the lacZYA operon during stationary phase. At both operons, NusA associates with RNAP during early elongation, and this association is greatly reduced during stationary phase. These observations suggest that in vivo association of sigma(70) and NusA with elongating RNAP is regulated by growth conditions.

Chromatin Immunoprecipitation↗
PubMed2004

Quantitative sequential chromatin immunoprecipitation, a method for analyzing co-occupancy of proteins at genomic regions in vivo.

Sequential chromatin immunoprecipitation (SeqChIP) is a procedure in which formaldehyde-crosslinked, protein-DNA complexes from living cells are subjected to two sequential immunoprecipitations with antibodies of different specificity. SeqChIP has been used to address, in a qualitative manner, whether two proteins can simultaneously co-occupy a stretch of DNA in vivo. Here, we expand on our earlier work and describe theoretical and practical considerations for performing and interpreting SeqChIP experiments in a quantitative manner. We provide a detailed experimental procedure for designing and performing SeqChIP experiments as well as experimental examples of the three possible outcomes: full co-occupancy, no co-occupancy and partial co-occupancy. In some cases of partial co-occupancy, the order of immunoprecipitations in SeqChIP can strongly influence the outcome. We experimentally confirm a quantitative parameter that provides a measure of co-occupancy of two proteins on a given region of DNA and provide information on how to interpret the results of SeqChIP experiments. Our quantitative treatment of SeqChIP data substantially expands the usefulness of the technique for elucidating molecular mechanisms in vivo.

Chromatin↗
PubMed2004

MAP kinase-mediated stress relief that precedes and regulates the timing of transcriptional induction.

In yeast, hyperosmotic stress causes an immediate dissociation of most proteins from chromatin, presumably because cells are unprepared for, and initially unresponsive to, increased ion concentrations in the nucleus. Osmotic stress activates Hog1 MAP kinase, which phosphorylates at least two proteins located at the plasma membrane, the Nha1 Na+/H+ antiporter and the Tok1 potassium channel. Hog1 phosphorylation stimulates Nha1 activity, and this is crucial for the rapid reassociation of proteins with their target sites in chromatin. This initial response to hyperosmolarity precedes and temporally regulates the activation of stress-response genes that depends on Hog1 phosphorylation of transcription factors in the nucleus. Thus, a single MAP kinase coordinates temporally, spatially, and mechanistically distinct responses to stress, thereby providing very rapid stress relief that facilitates subsequent changes in gene expression that permit long-term adaptation to harsh environmental conditions.

Cation Transport Proteins↗
PubMed2004

Cellular stress alters the transcriptional properties of promoter-bound Mot1-TBP complexes.

Mot1 associates with transcriptionally active promoters, and it directly affects transcriptional activity in a positive or negative manner, depending on the gene. As determined by sequential chromatin immunoprecipitation, Mot1 co-occupies promoters with TBP, but not with TFIIB, TFIIA, or Pol II when cells are grown in normal conditions. This strongly suggests that the Mot1-TBP complex is transcriptionally inactive, and hence is in dynamic equilibrium with transcriptionally active forms of TBP. Surprisingly, in response to heat shock and other forms of environmental stress, Mot1 co-occupies promoters with TFIIB and elongation-competent Pol II, but not with TFIIA. This suggests that functional preinitiation complexes can contain Mot1 instead of TFIIA in vivo. Thus, Mot1-TBP complexes can exist in active and inactive forms that are regulated by environmental stress.

Adenosine Triphosphatases↗
PubMed2004

Unbiased mapping of transcription factor binding sites along human chromosomes 21 and 22 points to widespread regulation of noncoding RNAs.

Using high-density oligonucleotide arrays representing essentially all nonrepetitive sequences on human chromosomes 21 and 22, we map the binding sites in vivo for three DNA binding transcription factors, Sp1, cMyc, and p53, in an unbiased manner. This mapping reveals an unexpectedly large number of transcription factor binding site (TFBS) regions, with a minimal estimate of 12,000 for Sp1, 25,000 for cMyc, and 1600 for p53 when extrapolated to the full genome. Only 22% of these TFBS regions are located at the 5' termini of protein-coding genes while 36% lie within or immediately 3' to well-characterized genes and are significantly correlated with noncoding RNAs. A significant number of these noncoding RNAs are regulated in response to retinoic acid, and overlapping pairs of protein-coding and noncoding RNAs are often coregulated. Thus, the human genome contains roughly comparable numbers of protein-coding and noncoding genes that are bound by common transcription factors and regulated by common environmental signals.

Amino Acid Motifs↗
PubMed2004

Genomic studies with Escherichia coli MelR protein: applications of chromatin immunoprecipitation and microarrays.

Escherichia coli MelR protein is a transcription activator that is essential for melibiose-dependent expression of the melAB genes. We have used chromatin immunoprecipitation to study the binding of MelR and RNA polymerase to the melAB promoter in vivo. Our results show that MelR is associated with promoter DNA, both in the absence and presence of the inducer melibiose. In contrast, RNA polymerase is recruited to the melAB promoter only in the presence of inducer. The MelR DK261 positive control mutant binds to the melAB promoter but cannot recruit RNA polymerase. Further analysis of immunoprecipitated DNA, by using an Affymetrix GeneChip array, showed that the melAB promoter is the major, if not the sole, target in E. coli for MelR. This was confirmed by a transcriptomics experiment to analyze RNA in cells either with or without melR.

Chromatin↗
PubMed2004

Genome-wide occupancy profile of the RNA polymerase III machinery in Saccharomyces cerevisiae reveals loci with incomplete transcription complexes.

We used chromatin immunoprecipitation, followed by microarray hybridization, to determine the genome-wide distribution of the RNA polymerase (Pol) III transcription apparatus in the yeast Saccharomyces cerevisiae. The Pol III transcriptome includes all tRNA genes, previously identified non-tRNA Pol III genes, and SNR52, which encodes a small nucleolar RNA. Unexpectedly, we identify eight ETC loci that are occupied by TFIIIC but not by other components of the Pol III machinery. Some ETC loci contain stretches of DNA that are highly conserved among closely related yeast species, suggesting that they may encode functional RNAs. ETC6 is located upstream of the gene encoding the tau 91 subunit of TFIIIC, suggesting the possibility of Pol III-regulated expression of a critical Pol III factor. We also identify the ZOD1 locus, which is bound by all components of the Pol III machinery and yet does not appear to express an RNA conserved among closely related yeast species. The B block motifs and several flanking nucleotides of the ZOD1 and ETC loci are very similar to each other and are highly conserved across the yeast species. Furthermore, the unusual profile of Pol III factor association with ZOD1 and the ETC loci is perfectly preserved in a different Saccharomyces species, indicating that these loci represent novel functional entities.

Base Sequence↗
PubMed2004

Eaf3 regulates the global pattern of histone acetylation in Saccharomyces cerevisiae.

Saccharomyces cerevisiae has a global pattern of histone acetylation in which histone H3 and H4 acetylation levels are lower at protein-coding sequences than at promoter regions. The loss of Eaf3, a subunit of the NuA4 histone acetylase and Rpd3 histone deacetylase complexes, greatly alters the genomic profile of histone acetylation, with the effects on H4 appearing to be more pronounced than those on H3. Specifically, the loss of Eaf3 causes increases in H3 and H4 acetylation at coding sequences and decreases at promoters, such that histone acetylation levels become evenly distributed across the genome. Eaf3 does not affect the overall level of H4 acetylation, the recruitment of the NuA4 catalytic subunit Esa1 to target promoters, or the level of transcription of the genes analyzed for histone acetylation. Whole-genome transcriptional profiling indicates that Eaf3 plays a positive, but quantitatively modest, role in the transcription of a small subset of genes, whereas it has a negative effect on very few genes. We suggest that Eaf3 regulates the genomic profile of histone H3 and H4 acetylation in a manner that does not involve targeted recruitment and is independent of transcriptional activity.

Acetylation↗
PubMed2004

Evidence for eviction and rapid deposition of histones upon transcriptional elongation by RNA polymerase II.

Biochemical experiments indicate that transcriptional elongation by RNA polymerase II (Pol II) is inhibited by nucleosomes and hence requires chromatin-modifying activities. Here, we examine the fate of histones upon passage of elongating Pol II in vivo. Histone density throughout the entire Saccharomyces cerevisiae GAL10 coding region is inversely correlated with Pol II association and transcriptional activity, suggesting that the elongating Pol II machinery efficiently evicts core histones from the DNA. Furthermore, new histones appear to be deposited onto DNA less than 1 min after passage of Pol II. Transcription-dependent deposition of histones requires the FACT complex that travels with elongating Pol II. Our results suggest that Pol II transcription generates a highly dynamic equilibrium of histone eviction and histone deposition and that there is significant histone exchange throughout most of the yeast genome within a single cell cycle.

Chromatin↗
PubMed2003

The Rtf1 component of the Paf1 transcriptional elongation complex is required for ubiquitination of histone H2B.

In yeast cells, the Rtf1 and Paf1 components of the Paf1 transcriptional elongation complex are important for recruitment of Set1, the histone H3-lysine 4 (H3-Lys4) methylase, to a highly localized domain at the 5' portion of active mRNA coding regions. Here, we show that Rtf1 is essential for global methylation of H3-Lys4 and H3-Lys79, but not H3-Lys36. This role of Rtf1 resembles that of Rad6, which mediates ubiquitination of histone H2B at lysine 123. Indeed, Rtf1 is required for H2B ubiquitination, suggesting that its effects on H3-Lys4 and H3-Lys79 methylation are an indirect consequence of its effect on H2B ubiquitination. Rtf1 is important for telomeric silencing, with loss of H3-Lys4 and H3-Lys79 methylation synergistically reducing Sir2 association with telomeric DNA. Dot1, the H3-Lys79 methylase, associates with transcriptionally active genes, but unlike the association of Set1 and Set2 (the H3-Lys36 methylase), this association is largely independent of Rtf1. We suggest that Rtf1 affects genome-wide ubiquitination of H2B by a mechanism that is distinct from its function as a transcriptional elongation factor.

Blotting, Western↗
PubMed2003

Lysine-79 of histone H3 is hypomethylated at silenced loci in yeast and mammalian cells: a potential mechanism for position-effect variegation.

Methylation of lysine-79 (K79) within the globular domain of histone H3 by Dot1 methylase is important for transcriptional silencing and for association of the Sir silencing proteins in yeast. Here, we show that the level of H3-K79 methylation is low at all Sir-dependent silenced loci but not at other transcriptionally repressed regions. Hypomethylation of H3-K79 at the telomeric and silent mating-type loci, but not the ribosomal DNA, requires the Sir proteins. Overexpression of Sir3 concomitantly extends the domain of Sir protein association and H3-K79 hypomethylation at telomeres. In mammalian cells, H3-K79 methylation is found at loci that are active for V(D)J recombination, but not at recombinationally inactive loci that are heterochromatic. These results suggest that H3-K79 methylation is an evolutionarily conserved marker of active chromatin regions, and that silencing proteins block the ability of Dot1 to methylate histone H3. Further, they suggest that Sir proteins preferentially bind chromatin with hypomethylated H3-K79 and then block H3-K79 methylation. This positive feedback loop, and the reverse loop in which H3-K79 methylation weakens Sir protein association and leads to further methylation, suggests a model for position-effect variegation.

Acetylation↗
PubMed2003

Targeted recruitment of Set1 histone methylase by elongating Pol II provides a localized mark and memory of recent transcriptional activity.

Set1, the yeast histone H3-lysine 4 (H3-K4) methylase, is recruited by the Pol II elongation machinery to a highly localized domain at the 5' portion of active mRNA coding regions. Set1 association depends upon the TFIIH-associated kinase that phosphorylates the Pol II C-terminal domain (CTD) and mediates the transition between initiation and elongation, and Set1 interacts with the form of Pol II whose CTD is phosphorylated at serine 5 but not serine 2. The Rtf1 and Paf1 components of the Pol II-associated Paf1 complex are also important for Set1 recruitment. Although the level of dimethylated H3-K4 is fairly uniform throughout the genome, the pattern of trimethylated H3-K4 strongly correlates with Set1 occupancy. Hypermethylated H3-K4 within the mRNA coding region persists for considerable time after transcriptional inactivation and Set1 dissociation from the chromatin, indicating that H3-K4 hypermethylation provides a molecular memory of recent transcriptional activity.

Blotting, Western↗
PubMed2003

The FACT complex travels with elongating RNA polymerase II and is important for the fidelity of transcriptional initiation in vivo.

The FACT complex facilitates transcription on chromatin templates in vitro, and it has been functionally linked to nucleosomes and putative RNA polymerase II (Pol II) elongation factors. In Saccharomyces cerevisiae cells, FACT specifically associates with active Pol II genes in a TFIIH-dependent manner and travels across the gene with elongating Pol II. Conditional inactivation of the FACT subunit Spt16 results in increased Pol II density, transcription, and TATA-binding protein (TBP) occupancy in the 3' portion of certain coding regions, indicating that FACT suppresses inappropriate initiation from cryptic promoters within coding regions. Conversely, loss of Spt16 activity reduces the association of TBP, TFIIB, and Pol II with normal promoters. Thus, FACT is required for wild-type cells to restrict initiation to normal promoters, thereby ensuring that only appropriate mRNAs are synthesized. We suggest that FACT contributes to the fidelity of Pol II transcription by linking the processes of initiation and elongation.

Base Sequence↗
PubMed2002

The VP16 activation domain interacts with multiple transcriptional components as determined by protein-protein cross-linking in vivo.

Transcriptional activator proteins recruit the RNA polymerase II machinery and chromatin-modifying activities to promoters. Biochemical experiments indicate that activator proteins can associate with a large number of proteins, and many such proteins have been proposed to be direct targets of activators. However, there is great uncertainty about which biochemical interactions are physiologically relevant. Here, we develop a formaldehyde-based cross-linking procedure to identify protein-protein interactions that occur under physiological conditions. We show that the VP16 activation domain directly interacts with TATA-binding protein (TBP), TFIIB, and the SAGA histone acetylase complex in vivo.

Acetyltransferases↗
PubMed2002

Ubiquitination of histone H2B by Rad6 is required for efficient Dot1-mediated methylation of histone H3 lysine 79.

Dot1 is a non-SET domain protein that methylates histone H3 at lysine 79, a surface-exposed residue that lies within the globular domain. In the context of a nucleosome, H3 lysine 79 is located in close proximity with lysine 123 of histone H2B, a major site for ubiquitination by Rad6. Here we show that Rad6-mediated ubiquitination of H2B lysine 123 is important for efficient methylation of lysine 79, but not lysine 36, of histone H3. In contrast, lysine 79 methylation of H3 is not required for ubiquitination of H2B. Our study provides a new example of trans-histone regulation between modifications on different histones. In addition, it suggests that Rad6 affects telomeric silencing, at least in part, by influencing methylation of histone H3.

Histone-Lysine N-Methyltransferase↗
PubMed2002

Methylation of H3-lysine 79 is mediated by a new family of HMTases without a SET domain.

The N-terminal tails of core histones are subjected to multiple covalent modifications, including acetylation, methylation, and phosphorylation. Similar to acetylation, histone methylation has emerged as an important player in regulating chromatin dynamics and gene activity. Histone methylation occurs on arginine and lysine residues and is catalyzed by two families of proteins, the protein arginine methyltransferase family and the SET-domain-containing methyltransferase family. Here, we report that lysine 79 (K79) of H3, located in the globular domain, can be methylated. K79 methylation occurs in a variety of organisms ranging from yeast to human. In budding yeast, K79 methylation is mediated by the silencing protein DOT1. Consistent with conservation of K79 methylation, DOT1 homologs can be found in a variety of eukaryotic organisms. We identified a human DOT1-like (DOT1L) protein and demonstrated that this protein possesses intrinsic H3-K79-specific histone methyltransferase (HMTase) activity in vitro and in vivo. Furthermore, we found that K79 methylation level is regulated throughout the cell cycle. Thus, our studies reveal a new methylation site and define a novel family of histone lysine methyltransferase.

Amino Acid Sequence↗