Biomedical subjects

Timothy R Hughes

Publications and source records attributed to Timothy R Hughes.

At least 19 recordsLinked to original sources

PubMed2026

An expanded codebook of human transcription factor DNA-binding specificity.

Gene expression is regulated by transcription factors (TFs), which recognize specific DNA sequence motifs. Several hundred putative human TFs, identified mainly by an apparent DNA-binding domain, lack known binding motifs1. Furthermore, even for well-characterized TFs, it remains controversial the degree to which motifs accurately reflect binding sites in living cells2. Here we describe a systematic effort ('Codebook') to determine the sequence specificity of 332 putative and poorly characterized human TFs. More than 4,000 independent experiments, encompassing multiple in vitro and in vivo assays, produced motifs for just over half (177; 53%) of the TFs, of which most are associated with only a single protein. These results extend the vocabulary of sequence recognition encoded by human TFs by around 130 distinct motifs. Moreover, binding motifs identified in vitro are strongly enriched in cellular binding sites. Collectively, the data reveal tens of thousands of previously unknown, conserved and direct TF-binding sites across the human genome. These sites are concentrated in promoter regions and are predictive of gene expression. In summary, this new codebook provides an important step forward in decoding the human genome.

Humans↗

PubMed2026

GHT-SELEX demonstrates unexpectedly high intrinsic sequence specificity and complex DNA binding of many human transcription factors.

There is ongoing debate regarding the degree to which transcription factors (TFs) independently specify genomic binding: TF binding motifs are typically short and degenerate, yielding many more binding site predictions than observed in cells. Here we present genomic high-throughput SELEX (GHT-SELEX)-a scalable method that surveys intrinsic binding of purified TFs to the fragmented, naked and unmodified genome. GHT-SELEX peaks for 179 diverse human TFs display surprisingly high overlap with chromatin immunoprecipitation sequencing peaks for the same TF. Comparable overlap can be obtained from motifs using appropriate analytical approaches. For C2H2 zinc finger (zf) proteins-the largest class of human TFs-GHT-SELEX shows that modular, alternative engagement of C2H2-zf domains is the norm, enabling several types of distinct target sites, and frequently involving internal duplication and divergence within the C2H2-zf array. Altogether, it is common for TFs to delineate a large fraction of in vivo genomic binding sites independently of other cellular factors.

Humans↗

PubMed2024

GHT-SELEX demonstrates unexpectedly high intrinsic sequence specificity and complex DNA binding of many human transcription factors.

A long-standing challenge in human regulatory genomics is that transcription factor (TF) DNA-binding motifs are short and degenerate, while the genome is large. Motif scans therefore produce many false-positive binding site predictions. By surveying 179 TFs across 25 families using >1,500 cyclic in vitro selection experiments with fragmented, naked, and unmodified genomic DNA - a method we term GHT-SELEX (Genomic HT-SELEX) - we find that many human TFs possess much higher sequence specificity than anticipated. Moreover, genomic binding regions from GHT-SELEX are often surprisingly similar to those obtained in vivo (i.e. ChIP-seq peaks). We find that comparable specificity can also be obtained from motif scans, but performance is highly dependent on derivation and use of the motifs, including accounting for multiple local matches in the scans. We also observe alternative engagement of multiple DNA-binding domains within the same protein: long C2H2 zinc finger proteins often utilize modular DNA recognition, engaging different subsets of their DNA binding domain (DBD) arrays to recognize multiple types of distinct target sites, frequently evolving via internal duplication and divergence of one or more DBDs. Thus, contrary to conventional wisdom, it is common for TFs to possess sufficient intrinsic specificity to independently delineate cellular targets.

C2H2↗

PubMed2004

Regulation of chromosome stability by the histone H2A variant Htz1, the Swr1 chromatin remodeling complex, and the histone acetyltransferase NuA4.

NuA4, the only essential histone acetyltransferase complex in Saccharomyces cerevisiae, acetylates the N-terminal tails of histones H4 and H2A. Affinity purification of NuA4 revealed the presence of three previously undescribed subunits, Vid21/Eaf1/Ydr359c, Swc4/Eaf2/Ygr002c, and Eaf7/Ynl136w. Experimental analyses revealed at least two functionally distinct sets of polypeptides in NuA4: (i) Vid21 and Yng2, and (ii) Eaf5 and Eaf7. Vid21 and Yng2 are required for bulk histone H4 acetylation and are functionally linked to the histone H2A variant Htz1 and the Swr1 ATPase complex (SWR-C) that assembles Htz1 into chromatin, whereas Eaf5 and Eaf7 have a different, as yet undefined, role. Mutations in Htz1, the SWR-C, and NuA4 cause defects in chromosome segregation that are consistent with genetic interactions we have observed between the genes encoding these proteins and genes encoding kinetochore components. Because SWR-C-dependent recruitment of Htz1 occurs in both transcribed and centromeric regions, a NuA4/SWR-C/Htz1 pathway may regulate both transcription and centromere function in S. cerevisiae.

Acetyltransferases↗

PubMed2004

Exploration of essential gene functions via titratable promoter alleles.

Nearly 20% of yeast genes are required for viability, hindering genetic analysis with knockouts. We created promoter-shutoff strains for over two-thirds of all essential yeast genes and subjected them to morphological analysis, size profiling, drug sensitivity screening, and microarray expression profiling. We then used this compendium of data to ask which phenotypic features characterized different functional classes and used these to infer potential functions for uncharacterized genes. We identified genes involved in ribosome biogenesis (HAS1, URB1, and URB2), protein secretion (SEC39), mitochondrial import (MIM1), and tRNA charging (GSN1). In addition, apparent negative feedback transcriptional regulation of both ribosome biogenesis and the proteasome was observed. We furthermore show that these strains are compatible with automated genetic analysis. This study underscores the importance of analyzing mutant phenotypes and provides a resource to complement the yeast knockout collection.

Alleles↗

PubMed2004

ESF1 is required for 18S rRNA synthesis in Saccharomyces cerevisiae.

We report that Esf1p (Ydr365cp), an essential, evolutionarily conserved nucleolar protein, is required for the biogenesis of 18S rRNA in Saccharomyces cerevisiae. Depletion of Esf1p resulted in delayed processing of 35S precursor and a striking loss of 18S rRNA. Esf1p physically associated with ribosomal proteins and proteins involved in 18S rRNA biogenesis. Consistent with its role in 18S rRNA biogenesis, Esf1p also physically associated with U3 and U14 snoRNAs, but did not appear to be a core component of the SSU processome. These data indicate that Esf1p plays a direct role in early pre-rRNA processing.

Amino Acid Sequence↗

PubMed2004

The specificities of four yeast dihydrouridine synthases for cytoplasmic tRNAs.

Dihydrouridine is a highly abundant modified nucleoside found widely in tRNAs of eubacteria, eukaryotes, and some archaea. In cytoplasmic tRNA of Saccharomyces cerevisiae, dihydrouridine occurs exclusively at positions 16, 17, 20, 20A, 20B, and 47. Here we show that the known dihydrouridine synthases Dus1p and Dus2p and two previously uncharacterized homologs, Dus3p (encoded by YLR401c) and Dus4p (YLR405w), are required for all of the dihydrouridine modification of cytoplasmic tRNAs in S. cerevisiae. We have mapped the in vivo position specificity of the four Dus proteins, by three complementary approaches: determination of the molar ratio of dihydrouridine in purified tRNAs from different dus mutants; microarray analysis of a large number of tRNAs based on differential hybridization of uridine and dihydrouridine-containing tRNAs to the complementary oligonucleotides; and the development and use of a novel dihydrouridine mapping technique, employing primer extension. We show that each of the four Dus proteins has a distinct position specificity: Dus1p for U(16) and U(17), Dus2p for U(20), Dus3p for U(47), and Dus4p for U(20a) and U(20b).

Chromatography, High Pressure Liquid↗

PubMed2004

High-definition macromolecular composition of yeast RNA-processing complexes.

A remarkably large collection of evolutionarily conserved proteins has been implicated in processing of noncoding RNAs and biogenesis of ribonucleoproteins. To better define the physical and functional relationships among these proteins and their cognate RNAs, we performed 165 highly stringent affinity purifications of known or predicted RNA-related proteins from Saccharomyces cerevisiae. We systematically identified and estimated the relative abundance of stably associated polypeptides and RNA species using a combination of gel densitometry, protein mass spectrometry, and oligonucleotide microarray hybridization. Ninety-two discrete proteins or protein complexes were identified comprising 489 different polypeptides, many associated with one or more specific RNA molecules. Some of the pre-rRNA-processing complexes that were obtained are discrete sub-complexes of those previously described. Among these, we identified the IPI complex required for proper processing of the ITS2 region of the ribosomal RNA primary transcript. This study provides a high-resolution overview of the modular topology of noncoding RNA-processing machinery.

Amino Acid Sequence↗

PubMed2004

Genome-wide analysis of mRNA stability using transcription inhibitors and microarrays reveals posttranscriptional control of ribosome biogenesis factors.

Using DNA microarrays, we compared global transcript stability profiles following chemical inhibition of transcription to rpb1-1 (a temperature-sensitive allele of yeast RNA polymerase II). Among the five inhibitors tested, the effects of thiolutin and 1,10-phenanthroline were most similar to rpb1-1. A comparison to various microarray data already in the literature revealed similarity between mRNA stability profiles and the transcriptional response to stresses such as heat shock, consistent with the fact that the general stress response includes a transient shutoff of general mRNA transcription. Genes encoding factors involved in rRNA synthesis and ribosome assembly, which are often observed to be coordinately down-regulated in yeast microarray data, were among the least stable transcripts. We examined the effects of deletions of genes encoding deadenylase components Ccr4p and Pan2p and putative RNA-binding proteins Pub1p and Puf4p on the genome-wide pattern of mRNA stability after inhibition of transcription by chemicals and/or heat stress. This examination showed that Ccr4p, the major yeast mRNA deadenylase, contributes to the degradation of transcripts encoding both ribosomal proteins and rRNA synthesis and ribosome assembly factors and mediates a large part of the transcriptional response to heat stress. Pan2p and Puf4p also contributed to the degradation rate of these mRNAs following transcriptional shutoff, while Pub1p preferentially stabilized transcripts encoding ribosomal proteins. Our results indicate that the abundance of ribosome biogenesis factors is controlled at the level of mRNA stability.

Gene Expression Profiling↗

PubMed2004

RPAP1, a novel human RNA polymerase II-associated protein affinity purified with recombinant wild-type and mutated polymerase subunits.

We have programmed human cells to express physiological levels of recombinant RNA polymerase II (RNAPII) subunits carrying tandem affinity purification (TAP) tags. Double-affinity chromatography allowed for the simple and efficient isolation of a complex containing all 12 RNAPII subunits, the general transcription factors TFIIB and TFIIF, the RNAPII phosphatase Fcp1, and a novel 153-kDa polypeptide of unknown function that we named RNAPII-associated protein 1 (RPAP1). The TAP-tagged RNAPII complex is functionally active both in vitro and in vivo. A role for RPAP1 in RNAPII transcription was established by shutting off the synthesis of Ydr527wp, a Saccharomyces cerevisiae protein homologous to RPAP1, and demonstrating that changes in global gene expression were similar to those caused by the loss of the yeast RNAPII subunit Rpb11. We also used TAP-tagged Rpb2 with mutations in fork loop 1 and switch 3, two structural elements located strategically within the active center, to start addressing the roles of these elements in the interaction of the enzyme with the template DNA during the transcription reaction.

Animals↗

PubMed2003

Integration of chemical-genetic and genetic interaction data links bioactive compounds to cellular target pathways.

Bioactive compounds can be valuable research tools and drug leads, but it is often difficult to identify their mechanism of action or cellular target. Here we investigate the potential for integration of chemical-genetic and genetic interaction data to reveal information about the pathways and targets of inhibitory compounds. Taking advantage of the existing complete set of yeast haploid deletion mutants, we generated drug-hypersensitivity (chemical-genetic) profiles for 12 compounds. In addition to a set of compound-specific interactions, the chemical-genetic profiles identified a large group of genes required for multidrug resistance. In particular, yeast mutants lacking a functional vacuolar H(+)-ATPase show multidrug sensitivity, a phenomenon that may be conserved in mammalian cells. By filtering chemical-genetic profiles for the multidrug-resistant genes and then clustering the compound-specific profiles with a compendium of large-scale genetic interaction profiles, we were able to identify target pathways or proteins. This method thus provides a powerful means for inferring mechanism of action.

Biotechnology↗

PubMed2003

A panoramic view of yeast noncoding RNA processing.

Predictive analysis using publicly available yeast functional genomics and proteomics data suggests that many more proteins may be involved in biogenesis of ribonucleoproteins than are currently known. Using a microarray that monitors abundance and processing of noncoding RNAs, we analyzed 468 yeast strains carrying mutations in protein-coding genes, most of which have not previously been associated with RNA or RNP synthesis. Many strains mutated in uncharacterized genes displayed aberrant noncoding RNA profiles. Ten factors involved in noncoding RNA biogenesis were verified by further experimentation, including a protein required for 20S pre-rRNA processing (Tsr2p), a protein associated with the nuclear exosome (Lrp1p), and a factor required for box C/D snoRNA accumulation (Bcd1p). These data present a global view of yeast noncoding RNA processing and confirm that many currently uncharacterized yeast proteins are involved in biogenesis of noncoding RNA.

Cells, Cultured↗

PubMed2003

RAM: a conserved signaling network that regulates Ace2p transcriptional activity and polarized morphogenesis.

In Saccharomyces cerevisiae, polarized morphogenesis is critical for bud site selection, bud development, and cell separation. The latter is mediated by Ace2p transcription factor, which controls the daughter cell-specific expression of cell separation genes. Recently, a set of proteins that include Cbk1p kinase, its binding partner Mob2p, Tao3p (Pag1p), and Hym1p were shown to regulate both Ace2p activity and cellular morphogenesis. These proteins seem to form a signaling network, which we designate RAM for regulation of Ace2p activity and cellular morphogenesis. To find additional RAM components, we conducted genetic screens for bilateral mating and cell separation mutants and identified alleles of the PAK-related kinase Kic1p in addition to Cbk1p, Mob2p, Tao3p, and Hym1p. Deletion of each RAM gene resulted in a loss of Ace2p function and caused cell polarity defects that were distinct from formin or polarisome mutants. Two-hybrid and coimmunoprecipitation experiments reveal a complex network of interactions among the RAM proteins, including Cbk1p-Cbk1p, Cbk1p-Kic1p, Kic1p-Tao3p, and Kic1p-Hym1p interactions, in addition to the previously documented Cbk1p-Mob2p and Cbk1p-Tao3p interactions. We also identified a novel leucine-rich repeat-containing protein Sog2p that interacts with Hym1p and Kic1p. Cells lacking Sog2p exhibited the characteristic cell separation and cell morphology defects associated with perturbation in RAM signaling. Each RAM protein localized to cortical sites of growth during both budding and mating pheromone response. Hym1p was Kic1p- and Sog2p-dependent and Sog2p and Kic1p were interdependent for localization, indicating a close functional relationship between these proteins. Only Mob2p and Cbk1p were detectable in the daughter cell nucleus at the end of mitosis. The nuclear localization and kinase activity of the Mob2p-Cbk1p complex were dependent on all other RAM proteins, suggesting that Mob2p-Cbk1p functions late in the RAM network. Our data suggest that the functional architecture of RAM signaling is similar to the S. cerevisiae mitotic exit network and Schizosaccharomyces pombe septation initiation network and is likely conserved among eukaryotes.

Cell Cycle Proteins↗

PubMed2003

Interferon-gamma stimulates the expression of the inducible cAMP early repressor in macrophages through the activation of casein kinase 2. A potentially novel pathway for interferon-gamma-mediated inhibition of gene transcription.

Interferon-gamma (IFN-gamma) is a pleiotropic cytokine that modulates the immune function, cell proliferation, apoptosis, macrophage activation, and numerous other cellular responses. These biological actions of IFN-gamma are characterized by both the activation and the inhibition of gene transcription. Unfortunately, in contrast to gene activation, the mechanisms through which the cytokine suppresses gene transcription remain largely unclear. We show here for the first time that exposure of macrophages to IFN-gamma leads to a dramatic induction in the expression of the inducible cAMP early repressor (ICER), a potent inhibitor of gene transcription. In addition, a synergistic action of IFN-gamma and calcium in the activation of ICER expression was identified. The IFN-gamma-mediated activation of ICER expression was not blocked by H89, bisindoylmaleimide, SB202190, PD98059, W7, and AG490, which inhibit protein kinase A, protein kinase C, p38 mitogen-activated protein kinase, extracellular signal-regulated kinase, calcium-calmodulin-dependent protein kinase, and Janus kinase-2, respectively. In contrast, apigenin, a selective casein kinase 2 (CK2) inhibitor, was found to inhibit response. Consistent with this finding, IFN-gamma stimulated CK2 activity and the level of phosphorylated cAMP response element-binding protein, which is known to induce ICER gene transcription, and this response was inhibited in the presence of apigenin. These studies, therefore, identify a previously uncharacterized pathway, involving the IFN-gamma-mediated stimulation of CK2 activity, activation of cAMP response element-binding protein, and increased production of ICER, which may then play an important role in the inhibition of macrophage gene transcription by this cytokine.

8-Bromo Cyclic Adenosine Monophosphate↗

PubMed2003

A Snf2 family ATPase complex required for recruitment of the histone H2A variant Htz1.

Deletions of three yeast genes, SET2, CDC73, and DST1, involved in transcriptional elongation and/or chromatin metabolism were used in conjunction with genetic array technology to screen approximately 4700 yeast deletions and identify double deletion mutants that produce synthetic growth defects. Of the five deletions interacting genetically with all three starting mutations, one encoded the histone H2A variant Htz1 and three encoded components of a novel 13 protein complex, SWR-C, containing the Snf2 family ATPase, Swr1. The SWR-C also copurified with Htz1 and Bdf1, a TFIID-interacting protein that recognizes acetylated histone tails. Deletions of the genes encoding Htz1 and seven nonessential SWR-C components caused a similar spectrum of synthetic growth defects when combined with deletions of 384 genes involved in transcription, suggesting that Htz1 and SWR-C belong to the same pathway. We show that recruitment of Htz1 to chromatin requires the SWR-C. Moreover, like Htz1 and Bdf1, the SWR-C promotes gene expression near silent heterochromatin.

Adenosine Triphosphatases↗

PubMed2003

Yeast genomics and proteomics in drug discovery and target validation.

Small, cell permeable, and target-specific chemical ligands are highly valuable, not only as therapeutics but also as research tools. The synthesis, identification and characterization of these compounds is often a difficult task. The straightforward genetics of the budding yeast Saccharomyces cerevisiae, and the high degree of conservation of basic cellular processes between yeast and higher organisms makes yeast an excellent tool for drug development studies, particularly in regards to anticancer and anti-fungal drug discovery. Recent advances in yeast functional genomics and proteomics studies are changing the field of yeast research. Many of these new technologies are readily applicable to drug target identification and other aspects of drug discovery. This review will focus on current genetic, genomic, and proteomic methodologies in S. cerevisiae that have the potential to be useful in drug discovery and target validation.

Animals↗

PubMed2002

FunSpec: a web-based cluster interpreter for yeast.

BACKGROUND: For effective exposition of biological information, especially with regard to analysis of large-scale data types, researchers need immediate access to multiple categorical knowledge bases and need summary information presented to them on collections of genes, as opposed to the typical one gene at a time. RESULTS: We present here a web-based tool (FunSpec) for statistical evaluation of groups of genes and proteins (e.g. co-regulated genes, protein complexes, genetic interactors) with respect to existing annotations (e.g. functional roles, biochemical properties, localization). FunSpec is available online at http://funspec.med.utoronto.ca CONCLUSION: FunSpec is helpful for interpretation of any data type that generates groups of related genes and proteins, such as gene expression clustering and protein complexes, and is useful for predictive methods employing "guilt-by-association."

Cluster Analysis↗

PubMed2002

Large-scale prediction of Saccharomyces cerevisiae gene function using overlapping transcriptional clusters.

Genome sequencing has led to the discovery of tens of thousands of potential new genes. Six years after the sequencing of the well-studied yeast Saccharomyces cerevisiae and the discovery that its genome encodes approximately 6,000 predicted proteins, more than 2,000 have not yet been characterized experimentally, and determining their functions seems far from a trivial task. One crucial constraint is the generation of useful hypotheses about protein function. Using a new approach to interpret microarray data, we assign likely cellular functions with confidence values to these new yeast proteins. We perform extensive genome-wide validations of our predictions and offer visualization methods for exploration of the large numbers of functional predictions. We identify potential new members of many existing functional categories including 285 candidate proteins involved in transcription, processing and transport of non-coding RNA molecules. We present experimental validation confirming the involvement of several of these proteins in ribosomal RNA processing. Our methodology can be applied to a variety of genomics data types and organisms.

Algorithms↗