Biomedical subjects

Selene K Swanson

Publications and source records attributed to Selene K Swanson.

At least 19 recordsLinked to original sources

PubMed2006

Ctk complex-mediated regulation of histone methylation by COMPASS.

A comparative global proteomic screen identified factors required for COMPASS (complex of proteins associated with Set1)-mediated mono-, di-, and trimethylation of the fourth lysine of histone H3 (H3K4), which included components of a cyclin-dependent protein kinase (Ctk complex) that phosphorylates the C-terminal domain of the largest subunit of RNA polymerase II (Pol II). Our results indicate that histone H3K4 methylation levels are regulated by the Ctk1, Ctk2, and Ctk3 components of the Ctk complex. We show that loss of Ctk1 kinase activity results in reduced histone H3K4 monomethylation levels, followed by a global increase in histone H3K4 trimethylation levels on chromatin. Ctk1 loss does not appear to have a substantial effect on histone H2B monoubiquitination levels or COMPASS and Paf1 complex phosphorylation. Our chromatin immunoprecipitation studies demonstrate that histone H3 eviction during active transcription is decelerated in a CTK1 deletion strain in response to reduced levels of Pol II recruitment. Our in vitro studies show that the onset of monomethylation on an unmethylated histone H3 by COMPASS is virtually immediate, while the onset of trimethylation occurs upon extended time of association between the histone tail and COMPASS. Our study suggests a role for the Ctk complex in the regulation of the pattern of H3K4 mono-, di-, and trimethylation via COMPASS.

Chromatin Immunoprecipitation↗

PubMed2006

Thermus thermophilus bacteriophage phiYS40 genome and proteomic characterization of virions.

We determined the sequence of the 152,372 bp genome of phiYS40, a lytic tailed bacteriophage of Thermus thermophilus. The genome contains 170 putative open reading frames and three tRNA genes. Functions for 25% of phiYS40 gene products were predicted on the basis of similarity to proteins of known function from diverse phages and bacteria. phiYS40 encodes a cluster of proteins involved in nucleotide salvage, such as flavin-dependent thymidylate synthase, thymidylate kinase, ribonucleotide reductase, and deoxycytidylate deaminase, and in DNA replication, such as DNA primase, helicase, type A DNA polymerase, and predicted terminal protein involved in initiation of DNA synthesis. The structural genes of phiYS40, most of which have no similarity to sequences in public databases, were identified by mass spectrometric analysis of purified virions. Various phiYS40 proteins have different phylogenetic neighbors, including myovirus, podovirus, and siphovirus gene products, bacterial genes and, in one case, a dUTPase from a eukaryotic virus. phiYS40 has apparently arisen through multiple acts of recombination between different phage genomes as well as through acquisition of bacterial genes.

Amino Acid Sequence↗

PubMed2006

Proteasome recruitment and activation of the Uch37 deubiquitinating enzyme by Adrm1.

Uch37 is one of the three principal deubiquitinating enzymes (DUBs), and the only ubiquitin carboxy-terminal hydrolase (UCH)-family protease, that is associated with mammalian proteasomes. We show that Uch37 is responsible for the ubiquitin isopeptidase activity in the PA700 (19S) proteasome regulatory complex. PA700 isopeptidase disassembles Lys 48-linked polyubiquitin specifically from the distal end of the chain, a property that may be used to clear poorly ubiquitinated or unproductively bound substrates from the proteasome. To better understand Uch37 function and the mechanism responsible for its specificity, we investigated how Uch37 is recruited to proteasomes. Uch37 binds through Adrm1, a previously unrecognized orthologue of Saccharomyces cerevisiae Rpn13p, which in turn is bound to the S1 (also known as Rpn2) subunit of the 19S complex. Adrm1 (human Rpn13, hRpn13) binds the carboxy-terminal tail of Uch37, a region that is distinct from the UCH catalytic domain, which we show inhibits Uch37 activity. Following binding, Adrm1 relieves Uch37 autoinhibition, accelerating the hydrolysis of ubiquitin-7-amido-4-methylcoumarin (ubiquitin-AMC). However, neither Uch37 alone nor the Uch37-Adrm1 or Uch37-Adrm1-S1 complexes can hydrolyse di-ubiquitin efficiently; rather, incorporation into the 19S complex is required to enable processing of polyubiquitin chains.

Amino Acid Sequence↗

PubMed2006

A role for Yin Yang-1 (YY1) in the assembly of snRNA transcription complexes.

The RNA polymerase (pol) II and III human small nuclear RNA (snRNA) genes have very similar promoters and recruit a number of common factors. In particular, both types of promoters utilize the small nuclear RNA activating protein complex (SNAP(c)) and the TATA box binding protein (TBP) for basal transcription, and are activated by Oct-1. We find that SNAP(c) purified from cell lines expressing tagged SNAP(c) subunits is associated with Yin Yang-1 (YY1), a factor implicated in both activation and repression of transcription. Recombinant YY1 accelerates the binding of SNAP(c) to the proximal sequence element, its target within snRNA promoters. Moreover, it enhances the formation of a complex on the pol III U6 snRNA promoter containing all the factors (SNAP(c), TBP, TFIIB-related factor 2 (Brf2), and B double prime 1 (Bdp1)) that are sufficient to direct in vitro U6 transcription when complemented with purified pol III, as well as that of a subcomplex containing TBP, Brf2, and Bdp1. YY1 is found on both the RNA polymerase II U1 and the RNA polymerase III U6 promoters as determined by chromatin immunoprecipitations. Thus, YY1 represents a new factor that participates in transcription complexes formed on both pol II and III promoters.

Animals↗

PubMed2006

Analyzing chromatin remodeling complexes using shotgun proteomics and normalized spectral abundance factors.

Mass spectrometry-based approaches are commonly used to identify proteins from multiprotein complexes, typically with the goal of identifying new complex members or identifying post-translational modifications. However, with the recent demonstration that spectral counting is a powerful quantitative proteomic approach, the analysis of multiprotein complexes by mass spectrometry can be reconsidered in certain cases. Using the chromatography-based approach named multidimensional protein identification technology, multiprotein complexes may be analyzed quantitatively using the normalized spectral abundance factor that allows comparison of multiple independent analyses of samples. This study describes an approach to visualize multiprotein complex datasets that provides structure function information that is superior to tabular lists of data. In this method review, we describe a reanalysis of the Rpd3/Sin3 small and large histone deacetylase complexes previously described in a tabular form to demonstrate the normalized spectral abundance factor approach.

Chromatin Assembly and Disassembly↗

PubMed2006

The essential gene wda encodes a WD40 repeat subunit of Drosophila SAGA required for histone H3 acetylation.

Histone acetylation provides a switch between transcriptionally repressive and permissive chromatin. By regulating the chromatin structure at specific promoters, histone acetyltransferases (HATs) carry out important functions during differentiation and development of higher eukaryotes. HAT complexes are present in organisms as diverse as Saccharomyces cerevisiae, humans, and flies. For example, the well-studied yeast SAGA is related to three mammalian complexes. We previously identified Drosophila melanogaster orthologues of yeast SAGA components Ada2, Ada3, Spt3, and Tra1 and demonstrated that they associate with dGcn5 in a high-molecular-weight complex. To better understand the function of Drosophila SAGA (dSAGA), we sought to affinity purify and characterize this complex in more detail. A proteomic approach led to the identification of an orthologue of the yeast protein Ada1 and the novel protein encoded by CG4448, referred to as WDA (will decrease acetylation). Embryos lacking both alleles of the wda gene exhibited reduced levels of histone H3 acetylation and could not develop into adult flies. Our results point to a critical function of dSAGA and histone acetylation during Drosophila development.

Acetylation↗

PubMed2006

Host cell factor and an uncharacterized SANT domain protein are stable components of ATAC, a novel dAda2A/dGcn5-containing histone acetyltransferase complex in Drosophila.

Gcn5 is a conserved histone acetyltransferase (HAT) found in a number of multisubunit complexes from Saccharomyces cerevisiae, mammals, and flies. We previously identified Drosophila melanogaster homologues of the yeast proteins Ada2, Ada3, Spt3, and Tra1 and showed that they associate with dGcn5 to form at least two distinct HAT complexes. There are two different Ada2 homologues in Drosophila named dAda2A and dAda2B. dAda2B functions within the Drosophila version of the SAGA complex (dSAGA). To gain insight into dAda2A function, we sought to identify novel components of the complex containing this protein, ATAC (Ada two A containing) complex. Affinity purification and mass spectrometry revealed that, in addition to dAda3 and dGcn5, host cell factor (dHCF) and a novel SANT domain protein, named Atac1 (ATAC component 1), copurify with this complex. Coimmunoprecipitation experiments confirmed that these proteins associate with dGcn5 and dAda2A, but not with dSAGA-specific components such as dAda2B and dSpt3. Biochemical fractionation revealed that ATAC has an apparent molecular mass of 700 kDa and contains dAda2A, dGcn5, dAda3, dHCF, and Atac1 as stable subunits. Thus, ATAC represents a novel histone acetyltransferase complex that is distinct from previously purified Gcn5/Pcaf-containing complexes from yeast and mammalian cells.

Amino Acid Sequence↗

PubMed2006

Mnd1/Hop2 facilitates Dmc1-dependent interhomolog crossover formation in meiosis of budding yeast.

During meiosis, each chromosome must pair with its homolog and undergo meiotic crossover recombination in order to segregate properly at the first meiotic division. Recombination in meiosis in Saccharomyces cerevisiae relies on two Escherichia coli recA homologs, Rad51 and Dmc1, as well as the more recently discovered heterodimer Mnd1/Hop2. Meiotic recombination in S. cerevisiae mnd1 and hop2 single mutants is initiated via double-strand breaks (DSBs) but does not progress beyond this stage; heteroduplex DNA, joint molecules, and crossovers are not detected. Whereas hop2 and mnd1 single mutants are profoundly recombination defective, we show that mnd1 rad51, hop2 rad51, and mnd1 rad17 double mutants are able to carry out crossover recombination. Interestingly, noncrossover recombination is absent, indicating a role for Mnd1/Hop2 in the designation of DSBs for noncrossover recombination. We demonstrate that in the rad51 mnd1 double mutant, recombination is more likely to occur between repetitive sequences on nonhomologous chromosomes. Our results support a model in which Mnd1/Hop2 is required for DNA-DNA interactions that help ensure Dmc1-mediated stable strand invasion between homologous chromosomes, thereby preserving genomic integrity.

Adenosine Triphosphatases↗

PubMed2005

Histone H3 methylation by Set2 directs deacetylation of coding regions by Rpd3S to suppress spurious intragenic transcription.

Yeast Rpd3 histone deacetylase plays an important role at actively transcribed genes. We characterized two distinct Rpd3 complexes, Rpd3L and Rpd3S, by MudPIT analysis. Both complexes shared a three subunit core and Rpd3L contains unique subunits consistent with being a promoter targeted corepressor. Rco1 and Eaf3 were subunits specific to Rpd3S. Mutants of RCO1 and EAF3 exhibited increased acetylation in the FLO8 and STE11 open reading frames (ORFs) and the appearance of aberrant transcripts initiating within the body of these ORFs. Mutants in the RNA polymerase II-associated SET2 histone methyltransferase also displayed these defects. Set2 functioned upstream of Rpd3S and the Eaf3 methyl-histone binding chromodomain was important for recruitment of Rpd3S and for deacetylation within the STE11 ORF. These data indicate that Pol II-associated Set2 methylates H3 providing a transcriptional memory which signals for deacetylation of ORFs by Rpd3S. This erases transcription elongation-associated acetylation to suppress intragenic transcription initiation.

Acetylation↗

PubMed2005

The HIR corepressor complex binds to nucleosomes generating a distinct protein/DNA complex resistant to remodeling by SWI/SNF.

The histone regulatory (HIR) and histone promoter control (HPC) repressor proteins regulate three of the four histone gene loci during the Saccharomyces cerevisiae cell cycle. Here, we demonstrate that Hir1, Hir2, Hir3, and Hpc2 proteins form a stable HIR repressor complex. The HIR complex promotes histone deposition onto DNA in vitro and constitutes a novel nucleosome assembly complex. The HIR complex stably binds to DNA and nucleosomes. Furthermore, HIR complex binding to nucleosomes forms a distinct protein/DNA complex resistant to remodeling by SWI/SNF. Thus, the HIR complex is a novel nucleosome assembly complex which functions with SWI/SNF to regulate transcription.

DNA, Fungal↗

PubMed2005

Stable incorporation of sequence specific repressors Ash1 and Ume6 into the Rpd3L complex.

Histone deacetylation by Saccharomyces cerevisiae Rpd3 represses genes regulated by the Ash1 and Ume6 DNA-binding proteins. Rpd3 exists in a small 0.6 MDa (Rpd3S) and large 1.2 MDa (Rpd3L) corepressor complex. In this report, we identify by mass spectrometry and MudPIT the subunits of the Rpd3L complex. These included Rpd3, Sds3, Pho23, Dep1, Rxt2, Sin3, Ash1, Ume1, Sap30, Cti6, Rxt3 and Ume6. Dep1 and Sds3, unique components of Rpd3L, were required for Rpd3L integrity and HDAC activity. Similar to RPD3, deletion of DEP1 enhanced telomeric silencing and derepressed INO1. Two sequence-specific repressors, Ash1 and Ume6, were stably associated with Rpd3L. While both of these proteins localized to the INO1 and HO promoters, the repression of these genes were dependent only on Ume6 and Ash1, respectively. Thus, the Rpd3L complex is directly recruited to specific promoters through multiple integral DNA-binding proteins.

Acetylation↗

PubMed2005

A mammalian chromatin remodeling complex with similarities to the yeast INO80 complex.

The mammalian Tip49a and Tip49b proteins belong to an evolutionarily conserved family of AAA+ ATPases. In Saccharomyces cerevisiae, orthologs of Tip49a and Tip49b, called Rvb1 and Rvb2, respectively, are subunits of two distinct ATP-dependent chromatin remodeling complexes, SWR1 and INO80. We recently demonstrated that the mammalian Tip49a and Tip49b proteins are integral subunits of a chromatin remodeling complex bearing striking similarities to the S. cerevisiae SWR1 complex (Cai, Y., Jin, J., Florens, L., Swanson, S. K., Kusch, T., Li, B., Workman, J. L., Washburn, M. P., Conaway, R. C., and Conaway, J. W. (2005) J. Biol. Chem. 280, 13665-13670). In this report, we identify a new mammalian Tip49a- and Tip49b-containing ATP-dependent chromatin remodeling complex, which includes orthologs of 8 of the 15 subunits of the S. cerevisiae INO80 chromatin remodeling complex as well as at least five additional subunits unique to the human INO80 (hINO80) complex. Finally, we demonstrate that, similar to the yeast INO80 complex, the hINO80 complex exhibits DNA- and nucleosome-activated ATPase activity and catalyzes ATP-dependent nucleosome sliding.

ATPases Associated with Diverse Cellular Activitie↗

PubMed2005

Molecular regulation of histone H3 trimethylation by COMPASS and the regulation of gene expression.

The Set1-containing complex COMPASS, which is the yeast homolog of the human MLL complex, is required for mono-, di-, and trimethylation of lysine 4 of histone H3. We have performed a comparative global proteomic screen to better define the role of COMPASS in histone trimethylation. We report that both Cps60 and Cps40 components of COMPASS are required for proper histone H3 trimethylation, but not for proper regulation of telomere-associated gene silencing. Purified COMPASS lacking Cps60 can mono- and dimethylate but is not capable of trimethylating H3(K4). Chromatin immunoprecipitation (ChIP) studies indicate that the loss subunits of COMPASS required for histone trimethylation do not affect the localization of Set1 to chromatin for the genes tested. Collectively, our results suggest a molecular requirement for several components of COMPASS for proper histone H3 trimethylation and regulation of telomere-associated gene expression, indicating multiple roles for different forms of histone methylation by COMPASS.

DNA-Binding Proteins↗

PubMed2005

The continuing evolution of shotgun proteomics.

Shotgun proteomics has emerged as a powerful approach for the analysis of complex protein mixtures, including biofluids, tissues, cells, organelles or protein complexes. Having evolved from the integration of chromatography and mass spectrometry, innovations in sample preparation, multidimensional chromatography, mass spectrometry and proteomic informatics continually facilitate, enable and challenge shotgun proteomics. As a result, shotgun proteomics continues to evolve and enable new areas of biological research, and is beginning to impact human disease diagnosis and therapeutic intervention.

Chromatography, High Pressure Liquid↗

PubMed2005

The mammalian Mediator complex.

The multiprotein Mediator (Med) complex is an evolutionarily conserved transcriptional regulator that plays important roles in activation and repression of RNA polymerase II transcription. Prior studies identified a set of more than twenty distinct polypeptides that compose the Saccharomyces cerevisiae Mediator. Here we discuss efforts to characterize the subunit composition and associated activities of the mammalian Med complex.

Animals↗

PubMed2005

Characterization of the yeast trimeric-SAS acetyltransferase complex.

The yeast SAS2 (Something About Silencing 2) gene encodes a member of the MYST protein family of histone acetyltransferases (HATs) and is involved in transcriptional silencing at all silent loci (HML, HMR, telomeres, and rDNA) in Saccharomyces cerevisiae. Sas2 is the catalytic subunit of a yeast histone acetyltransferase complex termed SAS complex. The enzymatic activity of SAS complex on free histones has been reported, but nucleosomal HAT activity has not yet been documented. Here we show that the native yeast SAS complex is a small trimeric protein complex composed solely of Sas2, Sas4, and Sas5 with a molecular mass of about 125 kDa. It is capable of acetylating both free histones and nucleosomes, although the nucleosomal HAT activity of SAS complex is very weak when compared with that of NuA4, the other member of MYST HAT complex. We also demonstrate that the putative acetyl CoA binding motif in Sas2 is essential for both the in vivo silencing function and the enzymatic activity of SAS complex. Unlike NuA4, which acetylates all four available lysines at the N-terminal tail of histone H4, SAS complex exclusively acetylates lysine 16 of histone H4 in vitro and is required for the bulk of H4 lysine 16 acetylation in vivo. This specific lysine preference corresponds to the role of SAS complex in antagonizing the spreading of Sir proteins at silent loci in S. cerevisiae.

Acetyl Coenzyme A↗

PubMed2005

The mammalian YL1 protein is a shared subunit of the TRRAP/TIP60 histone acetyltransferase and SRCAP complexes.

The multiprotein mammalian TRRAP/TIP60-containing histone acetyltransferase (HAT) complex performs critical functions in a variety of cellular processes including transcriptional activation, double strand DNA break repair, and apoptosis. We previously isolated the TRRAP/TIP60 complex from HeLa cells (Cai, Y., Jin, J., Tomomori-Sato, C., Sato, S., Sorokina, I., Parmely, T. J., Conaway, R. C., and Conaway, J. W. (2003) J. Biol. Chem. 278, 42733-42736). Analysis of proteins present in preparations of the TRRAP/TIP60 complex led to the identification of several new subunits, as well as several potential subunits including the YL1 protein. Here we present evidence that the YL1 protein is a previously unrecognized subunit of the TRRAP/TIP60 HAT complex. In addition, we present evidence that YL1 is also a component of a novel mammalian multiprotein complex that includes the SNF2-related helicase SRCAP and resembles the recently described Saccharomyces cerevisiae SWR1 chromatin remodeling complex. Taken together, our findings identify the YL1 protein as a new subunit of the TRRAP/TIP60 HAT complex, and they suggest that YL1 plays multiple roles in chromatin modification and remodeling in cells.

Acetyltransferases↗

PubMed2005

The deubiquitylation activity of Ubp8 is dependent upon Sgf11 and its association with the SAGA complex.

Covalent modifications of the histone tails and the cross talk between these modifications are hallmark features of gene regulation. The SAGA histone acetyltransferase complex is one of the most well-characterized complexes involved in these covalent modifications. The recent finding that the removal of the ubiquitin group from H2B is performed by a component of SAGA, Ubp8, is intriguing as it assigns two posttranslation modification processes to one complex. In this work, we characterize the association of Ubp8 with SAGA and the effect that acetylation and deubiquitylation have on one another in vitro and in vivo. We found not only that Ubp8 is a part of the SAGA complex, but also that its deubiquitylation activity requires Ubp8's association with SAGA. Furthermore, we found that the Ubp8 association with SAGA requires Sgf11 and that this requirement is reciprocal. We also found that the acetylation and deubiquitylation activities of SAGA are independent of one another. However, we found that preacetylating histone H2B inhibited subsequent deubiquitylation. Additionally, we found that increasing the ubiquitylation state of H2B inhibited the expression of the ARG1 gene, whose repression was previously shown to require the RAD6 ubiquitin ligase. Taken together, these data indicate that the expression of some genes, including ARG1, is regulated by a balance of histone H2B ubiquitylation in the cell.

Acetylation↗