Frank C. P. Holstege

Dissecting the Regulatory Circuitry of a Eukaryotic Genome

Genome-wide expression analysis was used to identify genes whose expression depends on the functions of key components of the transcription initiation machinery in yeast. Components of the RNA polymerase II holoenzyme, the general transcription factor TFIID, and the SAGA chromatin modification complex were found to have roles in expression of distinct sets of genes. The results reveal an unanticipated level of regulation which is superimposed on that due to gene-specific transcription factors, a novel mechanism for coordinate regulation of specific sets of genes when cells encounter limiting nutrients, and evidence that the ultimate targets of signal transduction pathways can be identified within the initiation apparatus.

Toward a Comprehensive Atlas of the Physical Interactome of Saccharomyces cerevisiae

Defining protein complexes is critical to virtually all aspects of cell biology. Two recent affinity purification/mass spectrometry studies in Saccharomyces cerevisiae have vastly increased the available protein interaction data. The practical utility of such high throughput interaction sets, however, is substantially decreased by the presence of false positives. Here we created a novel probabilistic metric that takes advantage of the high density of these data, including both the presence and absence of individual associations, to provide a measure of the relative confidence of each potential protein-protein interaction. This analysis largely overcomes the noise inherent in high throughput immunoprecipitation experiments. For example, of the 12,122 binary interactions in the general repository of interaction data (BioGRID) derived from these two studies, we marked 7504 as being of substantially lower confidence. Additionally, applying our metric and a stringent cutoff we identified a set of 9074 interactions (including 4456 that were not among the 12,122 interactions) with accuracy comparable to that of conventional small scale methodologies. Finally we organized proteins into coherent multisubunit complexes using hierarchical clustering. This work thus provides a highly accurate physical interaction map of yeast in a format that is readily accessible to the biological community.

Specific inhibition of gene expression using a stably integrated, inducible small-interfering-RNA vector

We have designed a doxycycline‐regulated form of the H1 promoter of RNA polymerase III that allows the inducible knockdown of gene expression by small interfering RNAs (siRNAs). As a proof‐of‐principle, we have targeted β‐catenin in colorectal cancer (CRC) cells. T‐cell factor (TCF) target‐gene expression is induced by accumulated β‐catenin, and is the main transforming event in these cells. We have shown previously that the disruption of β‐catenin/TCF4 activity in CRC cells by the overexpression of dominant‐negative TCF induces rapid G1 arrest and differentiation. Stable integration of our inducible siRNA vector allowed the rapid production of siRNAs on doxycycline induction, followed by specific downregulation of β‐catenin. In these CRC cells, TCF reporter‐gene activity was inhibited, and G1 arrest and differentiation occurred. The inhibition of two other genes using this vector system shows that it should be useful for the inducible knockdown of gene expression.

An expression profile for diagnosis of lymph node metastases from primary head and neck squamous cell carcinomas

Metastasis is the process by which cancers spread to distinct sites in the body. It is the principal cause of death in individuals suffering from cancer. For some types of cancer, early detection of metastasis at lymph nodes close to the site of the primary tumor is pivotal for appropriate treatment. Because it can be difficult to detect lymph node metastases reliably, many individuals currently receive inappropriate treatment. We show here that DNA microarray gene-expression profiling can detect lymph node metastases for primary head and neck squamous cell carcinomas that arise in the oral cavity and oropharynx. The predictor, established with an 82-tumor training set, outperforms current clinical diagnosis when independently validated. The 102 predictor genes offer unique insights into the processes underlying metastasis. The results show that the metastatic state can be deciphered from the primary tumor gene-expression pattern and that treatment can be substantially improved.

Redundant roles for the TFIID and SAGA complexes in global transcription

The transcription factors TFIID and SAGA are multi-subunit complexes involved in transcription by RNA polymerase II. TFIID and SAGA contain common TATA-binding protein (TBP)-associated factor (TAFII) subunits and each complex contains a subunit with histone acetyltransferase activity. These observations have raised questions about whether the functions of the two complexes in vivo are unique or overlapping. Here we use genome-wide expression analysis to investigate how expression of the yeast genome depends on both shared and unique subunits of these two complexes. We find that expression of most genes requires one or more of the common TAF II subunits, indicating that the functions of TFIID and SAGA are widely required for gene expression. Among the subunits shared by TFIID and SAGA are three histone-like TAFIIs, which have been proposed to form a sub-complex and mediate a common function in global transcription. Unexpectedly, we find that the histone-like TAFIIs have distinct roles in expression of the yeast genome. Most importantly, we show that the histone acetylase components of TFIID and SAGA (TAFII145 and Gcn5) are functionally redundant, indicating that expression of a large fraction of yeast genes can be regulated through the action of either complex.

Functional Organization of the S. cerevisiae Phosphorylation Network

Reversible protein phosphorylation is a signaling mechanism involved in all cellular processes. To create a systems view of the signaling apparatus in budding yeast, we generated an epistatic miniarray profile (E-MAP) comprised of 100,000 pairwise, quantitative genetic interactions, including virtually all protein and small-molecule kinases and phosphatases as well as key cellular regulators. Quantitative genetic interaction mapping reveals factors working in compensatory pathways (negative genetic interactions) or those operating in linear pathways (positive genetic interactions). We found an enrichment of positive genetic interactions between kinases, phosphatases, and their substrates. In addition, we assembled a higher-order map from sets of three genes that display strong interactions with one another: triplets enriched for functional connectivity. The resulting network view provides insights into signaling pathway regulation and reveals a link between the cell-cycle kinase, Cak1, the Fus3 MAP kinase, and a pathway that regulates chromatin integrity during transcription by RNA polymerase II.

Opening of an RNA polymerase II promoter occurs in two distinct steps and requires the basal transcription factors IIE and IIH.

We have studied promoter opening in assays reconstituted with purified RNA polymerase II and basal transcription factors. We found that creating a region of heteroduplex DNA around the start site of the adenovirus major late (AdML) promoter circumvents the requirement for TFIIE and TFIIH in transcription. The critical size and position of the heteroduplex region that alleviates the requirement for TFIIE and TFIIH is six nucleotides, from −4 to +2. Promoter opening was investigated directly with potassium permanganate (KMnO4), a chemical probe specific for single‐stranded thymidines. We found that KMnO4‐detectable opening of the AdML promoter requires the presence of the complete pre‐initiation complex, DBpolFEH, and that opening occurs in two discrete steps. First, dependent on ATP but prior to initiation, the −9 to +1 region becomes single‐stranded. Second, formation of the first phosphodiester bond results in expansion of the open region to the +8 position. Our results lead to a model in which the critical function of the TFIIH‐associated DNA helicases is to create a single‐stranded region. This gives RNA polymerase II access to the nucleotides of the template strand and allows expansion of the open region upon formation of the first phosphodiester bond.

Protein Interaction Verification and Functional Annotation by Integrated Analysis of Genome-Scale Data

Assays capable of determining the properties of thousands of genes in parallel present challenges with regard to accurate data processing and functional annotation. Collections of microarray expression data are applied here to assess the quality of different high-throughput protein interaction data sets. Significant differences are found. Confidence in 973 out of 5342 putative two-hybrid interactions from S. cerevisiae is increased. Besides verification, integration of expression and interaction data is employed to provide functional annotation for over 300 previously uncharacterized genes. The robustness of these approaches is demonstrated by experiments that test the in silico predictions made. This study shows how integration improves the utility of different types of functional genomic data and how well this contributes to functional annotation.

An Unusual Eukaryotic Protein Phosphatase Required for Transcription by RNA Polymerase II and CTD Dephosphorylation in S. cerevisiae

The carboxy-terminal domain (CTD) of the largest subunit of RNA polymerase II is phosphorylated soon after transcriptional initiation. We show here that the essential FCP1 gene of S. cerevisiae is linked genetically to RNA polymerase II and encodes a CTD phosphatase essential for dephosphorylation of RNA polymerase II in vivo. Fcp1p contains a phosphatase motif, psi psi psi DXDX(T/V)psi psi, which is novel for eukaryotic protein phosphatases and essential for Fcp1p to function in vivo. This motif is also required for recombinant Fcp1p to dephosphorylate the RNA polymerase II CTD or the artificial substrate p-nitrophenylphosphate in vitro. The effects of fcp1 mutations in global run-on and genome-wide expression studies show that transcription by RNA polymerase II in S. cerevisiae generally requires CTD phosphatase.

Monitoring global messenger RNA changes in externally controlled microarray experiments

Expression profiling is a universal tool, with a range of applications that benefit from the accurate determination of differential gene expression. To allow normalization using endogenous transcript levels, current microarray analyses assume that relatively few transcripts vary, or that any changes that occur are balanced. When normalization using endogenous genes is carried out, changes in expression levels are calculated relative to the behaviour of most of the transcripts. This does not reflect absolute changes if global shifts in messenger RNA populations occur. Using external RNA controls, we have set up microarray experiments to monitor global changes. The levels of most mRNAs were found to change during yeast stationary phase and human heat shock when external controls were included. Even small global changes had a significant effect on the number of genes reported as being differentially expressed. This suggests that global mRNA changes occur more frequently than is assumed at present, and shows that monitoring such effects may be important for the accurate determination of changes in gene expression.