Publications by authors named "Steven D Hanes"

26 Publications

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Structure analysis suggests Ess1 isomerizes the carboxy-terminal domain of RNA polymerase II via a bivalent anchoring mechanism.

Commun Biol 2021 Mar 25;4(1):398. Epub 2021 Mar 25.

Department of Biochemistry and Molecular Biology, SUNY-Upstate Medical University, Syracuse, NY, USA.

Accurate gene transcription in eukaryotes depends on isomerization of serine-proline bonds within the carboxy-terminal domain (CTD) of RNA polymerase II. Isomerization is part of the "CTD code" that regulates recruitment of proteins required for transcription and co-transcriptional RNA processing. Saccharomyces cerevisiae Ess1 and its human ortholog, Pin1, are prolyl isomerases that engage the long heptad repeat (YSPTSPS) of the CTD by an unknown mechanism. Here, we used an integrative structural approach to decipher Ess1 interactions with the CTD. Ess1 has a rigid linker between its WW and catalytic domains that enforces a distance constraint for bivalent interaction with the ends of long CTD substrates (≥4-5 heptad repeats). Our binding results suggest that the Ess1 WW domain anchors the proximal end of the CTD substrate during isomerization, and that linker divergence may underlie evolution of substrate specificity.
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http://dx.doi.org/10.1038/s42003-021-01906-8DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7994582PMC
March 2021

Prolyl isomerases in gene transcription.

Authors:
Steven D Hanes

Biochim Biophys Acta 2015 Oct 31;1850(10):2017-34. Epub 2014 Oct 31.

Department of Biochemistry and Molecular Biology, SUNY Upstate Medical University, 750 E Adams St., Syracuse, NY 13210 USA. Electronic address:

Background: Peptidyl-prolyl isomerases (PPIases) are enzymes that assist in the folding of newly-synthesized proteins and regulate the stability, localization, and activity of mature proteins. They do so by catalyzing reversible (cis-trans) rotation about the peptide bond that precedes proline, inducing conformational changes in target proteins.

Scope Of Review: This review will discuss how PPIases regulate gene transcription by controlling the activity of (1) DNA-binding transcription regulatory proteins, (2) RNA polymerase II, and (3) chromatin and histone modifying enzymes.

Major Conclusions: Members of each family of PPIase (cyclophilins, FKBPs, and parvulins) regulate gene transcription at multiple levels. In all but a few cases, the exact mechanisms remain elusive. Structure studies, development of specific inhibitors, and new methodologies for studying cis/trans isomerization in vivo represent some of the challenges in this new frontier that merges two important fields.

General Significance: Prolyl isomerases have been found to play key regulatory roles in all phases of the transcription process. Moreover, PPIases control upstream signaling pathways that regulate gene-specific transcription during development, hormone response and environmental stress. Although transcription is often rate-limiting in the production of enzymes and structural proteins, post-transcriptional modifications are also critical, and PPIases play key roles here as well (see other reviews in this issue). This article is part of a Special Issue entitled Proline-directed Foldases: Cell Signaling Catalysts and Drug Targets.
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http://dx.doi.org/10.1016/j.bbagen.2014.10.028DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4417086PMC
October 2015

Rpb4/7 facilitates RNA polymerase II CTD dephosphorylation.

Nucleic Acids Res 2014 Dec;42(22):13674-88

The Rpb4 and Rpb7 subunits of eukaryotic RNA polymerase II (RNAPII) participate in a variety of processes from transcription, DNA repair, mRNA export and decay, to translation regulation and stress response. However, their mechanism(s) of action remains unclear. Here, we show that the Rpb4/7 heterodimer in Saccharomyces cerevisiae plays a key role in controlling phosphorylation of the carboxy terminal domain (CTD) of the Rpb1 subunit of RNAPII. Proper phosphorylation of the CTD is critical for the synthesis and processing of RNAPII transcripts. Deletion of RPB4, and mutations that disrupt the integrity of Rpb4/7 or its recruitment to the RNAPII complex, increased phosphorylation of Ser2, Ser5, Ser7 and Thr4 within the CTD. RPB4 interacted genetically with genes encoding CTD phosphatases (SSU72, FCP1), CTD kinases (KIN28, CTK1, SRB10) and a prolyl isomerase that targets the CTD (ESS1). We show that Rpb4 is important for Ssu72 and Fcp1 phosphatases association, recruitment and/or accessibility to the CTD, and that this correlates strongly with Ser5P and Ser2P levels, respectively. Our data also suggest that Fcp1 is the Thr4P phosphatase in yeast. Based on these and other results, we suggest a model in which Rpb4/7 helps recruit and potentially stimulate the activity of CTD-modifying enzymes, a role that is central to RNAPII function.
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http://dx.doi.org/10.1093/nar/gku1227DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4267648PMC
December 2014

The Ess1 prolyl isomerase: traffic cop of the RNA polymerase II transcription cycle.

Authors:
Steven D Hanes

Biochim Biophys Acta 2014 12;1839(4):316-33. Epub 2014 Feb 12.

Department of Biochemistry and Molecular Biology, SUNY Upstate Medical University, 750 E Adams St., Syracuse, NY 13210, USA. Electronic address:

Ess1 is a prolyl isomerase that regulates the structure and function of eukaryotic RNA polymerase II. Ess1 works by catalyzing the cis/trans conversion of pSer5-Pro6 bonds, and to a lesser extent pSer2-Pro3 bonds, within the carboxy-terminal domain (CTD) of Rpb1, the largest subunit of RNA pol II. Ess1 is conserved in organisms ranging from yeast to humans. In budding yeast, Ess1 is essential for growth and is required for efficient transcription initiation and termination, RNA processing, and suppression of cryptic transcription. In mammals, Ess1 (called Pin1) functions in a variety of pathways, including transcription, but it is not essential. Recent work has shown that Ess1 coordinates the binding and release of CTD-binding proteins that function as co-factors in the RNA pol II complex. In this way, Ess1 plays an integral role in writing (and reading) the so-called CTD code to promote production of mature RNA pol II transcripts including non-coding RNAs and mRNAs.
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http://dx.doi.org/10.1016/j.bbagrm.2014.02.001DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4049342PMC
June 2014

The yeast Ess1 prolyl isomerase controls Swi6 and Whi5 nuclear localization.

G3 (Bethesda) 2014 Mar 20;4(3):523-37. Epub 2014 Mar 20.

Department of Biochemistry and Molecular Biology, SUNY Upstate Medical University, Syracuse, New York 13210.

The Ess1 prolyl isomerase from Saccharomyces cerevisiae and its human ortholog, Pin1, play critical roles in transcription by regulating RNA polymerase II. In human cells, Pin1 also regulates a variety of signaling proteins, and Pin1 misexpression is linked to several human diseases. To gain insight into Ess1/Pin1 function, we carried out a synthetic genetic array screen to identify novel targets of Ess1 in yeast. We identified potential targets of Ess1 in transcription, stress, and cell-cycle pathways. We focused on the cell-cycle regulators Swi6 and Whi5, both of which show highly regulated nucleocytoplasmic shuttling during the cell cycle. Surprisingly, Ess1 did not control their transcription but instead was necessary for their nuclear localization. Ess1 associated with Swi6 and Whi5 in vivo and bound directly to peptides corresponding to their nuclear localization sequences in vitro. Binding by Ess1 was significant only if the Swi6 and Whi5 peptides were phosphorylated at Ser-Pro motifs, the target sites of cyclin-dependent kinases. On the basis of these results, we propose a model in which Ess1 induces a conformational switch (cis-trans isomerization) at phospho-Ser-Pro sites within the nuclear targeting sequences of Swi6 and Whi5. This switch would promote nuclear entry and/or retention during late M and G1 phases and might work by stimulating dephosphorylation at these sites by the Cdc14 phosphatase. This is the first study to identify targets of Ess1 in yeast other than RNA polymerase II.
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http://dx.doi.org/10.1534/g3.113.008763DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3962490PMC
March 2014

Role of Ess1 in growth, morphogenetic switching, and RNA polymerase II transcription in Candida albicans.

PLoS One 2013 14;8(3):e59094. Epub 2013 Mar 14.

Department of Biomedical Sciences, School of Public Health, State University of New York, Albany, New York, United States of America.

Candida albicans is a fungal pathogen that causes potentially fatal infections among immune-compromised individuals. The emergence of drug resistant C. albicans strains makes it important to identify new antifungal drug targets. Among potential targets are enzymes known as peptidyl-prolyl cis/trans isomerases (PPIases) that catalyze isomerization of peptide bonds preceding proline. We are investigating a PPIase called Ess1, which is conserved in all major human pathogenic fungi. Previously, we reported that C. albicans Ess1 is essential for growth and morphogenetic switching. In the present study, we re-evaluated these findings using more rigorous genetic analyses, including the use of additional CaESS1 mutant alleles, distinct marker genes, and the engineering of suitably-matched isogenic control strains. The results confirm that CaEss1 is essential for growth in C. albicans, but show that reduction of CaESS1 gene dosage by half (δ/+) does not interfere with morphogenetic switching. However, further reduction of CaEss1 levels using a conditional allele does reduce morphogenetic switching. We also examine the role of the linker α-helix that distinguishes C. albicans Ess1 from the human Pin1 enzyme, and present results of a genome-wide transcriptome analysis. The latter analysis indicates that CaEss1 has a conserved role in regulation of RNA polymerase II function, and is required for efficient termination of small nucleolar RNAs and repression of cryptic transcription in C. albicans.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0059094PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3597612PMC
November 2013

Multiple roles for the Ess1 prolyl isomerase in the RNA polymerase II transcription cycle.

Mol Cell Biol 2012 Sep 9;32(17):3594-607. Epub 2012 Jul 9.

Division of Infectious Disease, Wadsworth Center, New York State Department of Health, Albany, New York, USA.

The Ess1 prolyl isomerase in Saccharomyces cerevisiae regulates RNA polymerase II (pol II) by isomerizing peptide bonds within the pol II carboxy-terminal domain (CTD) heptapeptide repeat (YSPTSPS). Ess1 preferentially targets the Ser5-Pro6 bond when Ser5 is phosphorylated. Conformational changes in the CTD induced by Ess1 control the recruitment of essential cofactors to the pol II complex and may facilitate the ordered transition between initiation, elongation, termination, and RNA processing. Here, we show that Ess1 associates with the phospho-Ser5 form of polymerase in vivo, is present along the entire length of coding genes, and is critical for regulating the phosphorylation of Ser7 within the CTD. In addition, Ess1 represses the initiation of cryptic unstable transcripts (CUTs) and is required for efficient termination of mRNA transcription. Analysis using strains lacking nonsense-mediated decay suggests that as many as half of all yeast genes depend on Ess1 for efficient termination. Finally, we show that Ess1 is required for trimethylation of histone H3 lysine 4 (H3K4). Thus, Ess1 has direct effects on RNA polymerase transcription by controlling cofactor binding via conformationally induced changes in the CTD and indirect effects by influencing chromatin modification.
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http://dx.doi.org/10.1128/MCB.00672-12DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3422015PMC
September 2012

The Bin3 RNA methyltransferase targets 7SK RNA to control transcription and translation.

Wiley Interdiscip Rev RNA 2012 Sep-Oct;3(5):633-47. Epub 2012 Jun 27.

Department of Biochemistry and Molecular Biology, SUNY-Upstate Medical University, Syracuse, NY, USA.

Bicoid-interacting protein 3 (Bin3) is a conserved RNA methyltransferase found in eukaryotes ranging from fission yeast to humans. It was originally discovered as a Bicoid (Bcd)-interacting protein in Drosophila, where it is required for anterior-posterior and dorso-ventral axis determination in the early embryo. The mammalian ortholog of Bin3 (BCDIN3), also known as methyl phosphate capping enzyme (MePCE), plays a key role in repressing transcription. In transcription, MePCE binds the non-coding 7SK RNA, which forms a scaffold for an RNA-protein complex that inhibits positive-acting transcription elongation factor b, an RNA polymerase II elongation factor. MePCE uses S-adenosyl methionine to transfer a methyl group onto the γ-phosphate of the 5' guanosine of 7SK RNA generating an unusual cap structure that protects 7SK RNA from degradation. Bin3/MePCE also has a role in translation regulation. Initial studies in Drosophila indicate that Bin3 targets 7SK RNA and stabilizes a distinct RNA-protein complex that assembles on the 3'-untranslated region of caudal mRNAs to prevent translation initiation. Much remains to be learned about Bin3/MeCPE function, including how it recognizes 7SK RNA, what other RNA substrates it might target, and how widespread a role it plays in gene regulation and embryonic development.
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http://dx.doi.org/10.1002/wrna.1123DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3629726PMC
January 2013

A universal RNA polymerase II CTD cycle is orchestrated by complex interplays between kinase, phosphatase, and isomerase enzymes along genes.

Mol Cell 2012 Jan;45(2):158-70

Institut de recherches cliniques de Montréal, Montréal, QC H2W 1R7, Canada.

Transcription by RNA polymerase II (RNAPII) is coupled to mRNA processing and chromatin modifications via the C-terminal domain (CTD) of its largest subunit, consisting of multiple repeats of the heptapeptide YSPTSPS. Pioneering studies showed that CTD serines are differentially phosphorylated along genes in a prescribed pattern during the transcription cycle. Genome-wide analyses challenged this idea, suggesting that this cycle is not uniform among different genes. Moreover, the respective role of enzymes responsible for CTD modifications remains controversial. Here, we systematically profiled the location of the RNAPII phosphoisoforms in wild-type cells and mutants for most CTD modifying enzymes. Together with results of in vitro assays, these data reveal a complex interplay between the modifying enzymes, and provide evidence that the CTD cycle is uniform across genes. We also identify Ssu72 as the Ser7 phosphatase and show that proline isomerization is a key regulator of CTD dephosphorylation at the end of genes.
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http://dx.doi.org/10.1016/j.molcel.2011.11.024DOI Listing
January 2012

Milestones in Candida albicans gene manipulation.

Fungal Genet Biol 2011 Sep 14;48(9):858-65. Epub 2011 Apr 14.

Department of Biomedical Sciences, School of Public Health, State University of New York, Albany, NY 12208, USA.

In the United States, candidemia is one of the most common hospital-acquired infections and is estimated to cause 10,000 deaths per year. The species Candida albicans is responsible for the majority of these cases. As C. albicans is capable of developing resistance against the currently available drugs, understanding the molecular basis of drug resistance, finding new cellular targets, and further understanding the overall mechanism of C. albicans pathogenesis are important goals. To study this pathogen it is advantageous to manipulate its genome. Numerous strategies of C. albicans gene manipulation have been introduced. This review evaluates a majority of these strategies and should be a helpful guide for researchers to identify gene targeting strategies to suit their requirements.
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http://dx.doi.org/10.1016/j.fgb.2011.04.003DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3143290PMC
September 2011

The Bin3 RNA methyltransferase is required for repression of caudal translation in the Drosophila embryo.

Dev Biol 2011 Apr 22;352(1):104-15. Epub 2011 Jan 22.

Wadsworth Center, New York State Department of Health, Albany, NY, USA.

Bin3 was first identified as a Bicoid-interacting protein in a yeast two-hybrid screen. In human cells, a Bin3 ortholog (BCDIN3) methylates the 5' end of 7SK RNA, but its role in vivo is unknown. Here, we show that in Drosophila, Bin3 is important for dorso-ventral patterning in oogenesis and for anterior-posterior pattern formation during embryogenesis. Embryos that lack Bin3 fail to repress the translation of caudal mRNA and exhibit head involution defects. bin3 mutants also show (1) a severe reduction in the level of 7SK RNA, (2) reduced binding of Bicoid to the caudal 3' UTR, and (3) genetic interactions with bicoid, and with genes encoding eIF4E, Larp1, polyA binding protein (PABP), and Ago2. 7SK RNA coimmunoprecipitated with Bin3 and is present in Bicoid complexes. These data suggest a model in which Bicoid recruits Bin3 to the caudal 3' UTR. Bin3's role is to bind and stabilize 7SK RNA, thereby promoting formation of a repressive RNA-protein complex that includes the RNA-binding proteins Larp1, PABP, and Ago2. This complex would prevent translation by blocking eIF4E interactions required for initiation. Our results, together with prior network analysis in human cells, suggest that Bin3 interacts with multiple partner proteins, methylates small non-coding RNAs, and plays diverse roles in development.
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http://dx.doi.org/10.1016/j.ydbio.2011.01.017DOI Listing
April 2011

Restricted domain mobility in the Candida albicans Ess1 prolyl isomerase.

Biochim Biophys Acta 2010 Jul 18;1804(7):1537-41. Epub 2010 Mar 18.

Wadsworth Center, New York State Department of Health, School of Public Health, University at Albany, Empire State Plaza, Albany, NY 12201, USA.

Ess1 is a peptidyl prolyl cis/trans isomerase that is required for virulence of the pathogenic fungi Candida albicans and Cryptococcus neoformans. The enzyme isomerizes the phospho-Ser-Pro linkages in the C-terminal domain of RNA polymerase II. Its human homolog, Pin1, has been implicated in a wide range of human diseases, including cancer and Alzheimer's disease. Crystallographic and NMR studies have demonstrated that the sequence linking the catalytic isomerase domain and the substrate binding WW domain of Pin1 is unstructured and that the two domains are only loosely associated in the absence of the substrate. In contrast, the crystal structure of C. albicans Ess1 revealed a highly ordered linker that contains a three turn alpha-helix and extensive association between the two tightly juxtaposed domains. In part to address the concern that the marked differences in the domain interactions for the human and fungal structures might reflect crystal lattice effects, NMR chemical shift analysis and 15N relaxation measurements have been employed to confirm that the linker of the fungal protein is highly ordered in solution. With the exception of two loops within the active site of the isomerase domain, the local backbone geometry observed in the crystal structure appears to be well preserved throughout the protein chain. The marked differences in interdomain interactions and linker flexibility between the human and fungal enzymes provide a structural basis for therapeutic targeting of the fungal enzymes.
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http://dx.doi.org/10.1016/j.bbapap.2010.03.005DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2951753PMC
July 2010

The Ess1 prolyl isomerase is required for transcription termination of small noncoding RNAs via the Nrd1 pathway.

Mol Cell 2009 Oct;36(2):255-66

Wadsworth Center, New York State Department of Health, 120 New Scotland Avenue, Albany, NY 12208, USA.

Genome-wide studies have identified abundant small, noncoding RNAs, including small nuclear RNAs, small nucleolar RNAs (snoRNAs), cryptic unstable transcripts (CUTs), and upstream regulatory RNAs (uRNAs), that are transcribed by RNA polymerase II (pol II) and terminated by an Nrd1-dependent pathway. Here, we show that the prolyl isomerase Ess1 is required for Nrd1-dependent termination of noncoding RNAs. Ess1 binds the carboxy-terminal domain (CTD) of pol II and is thought to regulate transcription by conformational isomerization of Ser-Pro bonds within the CTD. In ess1 mutants, expression of approximately 10% of the genome was altered, due primarily to defects in termination of snoRNAs, CUTs, stable unannotated transcripts, and uRNAs. Ess1 promoted dephosphorylation of Ser5 (but not Ser2) within the CTD, most likely by the Ssu72 phosphatase. We also provide evidence for a competition between Nrd1 and Pcf11 for CTD binding that is regulated by Ess1. These data indicate that a prolyl isomerase is required for specifying the "CTD code."
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http://dx.doi.org/10.1016/j.molcel.2009.08.018DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2770246PMC
October 2009

SAP18 promotes Krüppel-dependent transcriptional repression by enhancer-specific histone deacetylation.

J Biol Chem 2009 Jan 1;284(5):3012-3020. Epub 2008 Dec 1.

Abteilung Molekulare Entwicklungsbiologie, Max-Planck-Institut für biophysikalische Chemie, Am Fassberg 11, 37077 Göttingen, Germany. Electronic address:

Body pattern formation during early embryogenesis of Drosophila melanogaster relies on a zygotic cascade of spatially restricted transcription factor activities. The gap gene Krüppel ranks at the top level of this cascade. It encodes a C2H2 zinc finger protein that interacts directly with cis-acting stripe enhancer elements of pair rule genes, such as even skipped and hairy, at the next level of the gene hierarchy. Krüppel mediates their transcriptional repression by direct association with the corepressor Drosophila C terminus-binding protein (dCtBP). However, for some Krüppel target genes, deletion of the dCtBP-binding sites does not abolish repression, implying a dCtBP-independent mode of repression. We identified Krüppel-binding proteins by mass spectrometry and found that SAP18 can both associate with Krüppel and support Krüppel-dependent repression. Genetic interaction studies combined with pharmacological and biochemical approaches suggest a site-specific mechanism of Krüppel-dependent gene silencing. The results suggest that Krüppel tethers the SAP18 bound histone deacetylase complex 1 at distinct enhancer elements, which causes repression via histone H3 deacetylation.
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http://dx.doi.org/10.1074/jbc.M806163200DOI Listing
January 2009

The Drosophila calcipressin sarah is required for several aspects of egg activation.

Curr Biol 2006 Jul;16(14):1441-6

Department of Molecular Biology and Genetics, Cornell University, Ithaca, New York 14853, USA.

Activation of mature oocytes initiates development by releasing the prior arrest of female meiosis, degrading certain maternal mRNAs while initiating the translation of others, and modifying egg coverings. In vertebrates and marine invertebrates, the fertilizing sperm triggers activation events through a rise in free calcium within the egg. In insects, egg activation occurs independently of sperm and is instead triggered by passage of the egg through the female reproductive tract ; it is unknown whether calcium signaling is involved. We report here that mutations in sarah, which encodes an inhibitor of the calcium-dependent phosphatase calcineurin, disrupt several aspects of egg activation in Drosophila. Eggs laid by sarah mutant females arrest in anaphase of meiosis I and fail to fully polyadenylate and translate bicoid mRNA. Furthermore, sarah mutant eggs show elevated cyclin B levels, indicating a failure to inactivate M-phase promoting factor (MPF). Taken together, these results demonstrate that calcium signaling is involved in Drosophila egg activation and suggest a molecular mechanism for the sarah phenotype. We also find the conversion of the sperm nucleus into a functional male pronucleus is compromised in sarah mutant eggs, indicating that the Drosophila egg's competence to support male pronuclear maturation is acquired during activation.
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http://dx.doi.org/10.1016/j.cub.2006.06.024DOI Listing
July 2006

Bicoid cooperative DNA binding is critical for embryonic patterning in Drosophila.

Proc Natl Acad Sci U S A 2005 Sep 6;102(37):13176-81. Epub 2005 Sep 6.

Wadsworth Center, New York State Department of Health, 120 New Scotland Avenue, Albany, NY 12208, USA.

Cooperative interactions by DNA-binding proteins have been implicated in cell-fate decisions in a variety of organisms. To date, however, there are few examples in which the importance of such interactions has been explicitly tested in vivo. Here, we tested the importance of cooperative DNA binding by the Bicoid protein in establishing a pattern along the anterior-posterior axis of the early Drosophila embryo. We found that bicoid mutants specifically defective in cooperative DNA binding fail to direct proper development of the head and thorax, leading to embryonic lethality. The mutants did not faithfully stimulate transcription of downstream target genes such as hunchback (hb), giant, and Krüppel. Quantitative analysis of gene expression in vivo indicated that bcd cooperativity mutants were unable to accurately direct the extent to which hb is expressed along the anterior-posterior axis and displayed a reduced ability to generate sharp on/off transitions for hb gene expression. These failures in precise transcriptional control demonstrate the importance of cooperative DNA binding for embryonic patterning in vivo.
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http://dx.doi.org/10.1073/pnas.0506462102DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1201621PMC
September 2005

dSAP18 and dHDAC1 contribute to the functional regulation of the Drosophila Fab-7 element.

Nucleic Acids Res 2005 30;33(15):4857-64. Epub 2005 Aug 30.

Departament de Biologia Molecular i Cel.lular, Institut de Biologia Molecular de Barcelona, CSIC Parc Científic de Barcelona, Josep Samitier, 1-5 08028 Barcelona, Spain.

It was described earlier that the Drosophila GAGA factor [Trithorax-like (Trl)] interacts with dSAP18, which, in mammals, was reported to be a component of the Sin3-HDAC co-repressor complex. GAGA-dSAP18 interaction was proposed to contribute to the functional regulation of the bithorax complex (BX-C). Here, we show that mutant alleles of Trl, dsap18 and drpd3/hdac1 enhance A6-to-A5 transformation indicating a contribution to the regulation of Abd-B expression at A6. In A6, expression of Abd-B is driven by the iab-6 enhancer, which is insulated from iab-7 by the Fab-7 element. Here, we report that GAGA, dSAP18 and dRPD3/HDAC1 co-localize to ectopic Fab-7 sites in polytene chromosomes and that mutant Trl, dsap18 and drpd3/hdac1 alleles affect Fab-7-dependent silencing. Consistent with these findings, chromatin immunoprecipitation analysis shows that, in Drosophila embryos, the endogenous Fab-7 element is hypoacetylated at histones H3 and H4. These results indicate a contribution of GAGA, dSAP18 and dRPD3/HDAC1 to the regulation of Fab-7 function.
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http://dx.doi.org/10.1093/nar/gki776DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1196206PMC
September 2005

The Ess1 prolyl isomerase is dispensable for growth but required for virulence in Cryptococcus neoformans.

Microbiology (Reading) 2005 May;151(Pt 5):1593-1605

Department of Biomedical Sciences, School of Public Health, State University of New York, Albany, NY 12208, USA.

Cryptococcus neoformans is an important human fungal pathogen that also serves as a model for studies of fungal pathogenesis. C. neoformans contains several genes encoding peptidyl-prolyl cis/trans isomerases (PPIases), enzymes that catalyse changes in the folding and conformation of target proteins. Three distinct classes of PPIases have been identified: cyclophilins, FK506-binding proteins (FKBPs) and parvulins. This paper reports the cloning and characterization of ESS1, which is believed to be the first (and probably only) parvulin-class PPIase in C. neoformans. It is shown that ESS1 from C. neoformans is structurally and functionally homologous to ESS1 from Saccharomyces cerevisiae, which encodes an essential PPIase that interacts with RNA polymerase II and plays a role in transcription. In C. neoformans, ESS1 was found to be dispensable for growth, haploid fruiting and capsule formation. However, ESS1 was required for virulence in a murine model of cryptococcosis. Loss of virulence might have been due to the defects in melanin and urease production observed in ess1 mutants, or to defects in transcription of as-yet-unidentified virulence genes. The fact that Ess1 is not essential in C. neoformans suggests that, in this organism, some of its functions might be subsumed by other prolyl isomerases, in particular, cyclophilins Cpa1 or Cpa2. This is supported by the finding that ess1 mutants were hypersensitive to cyclosporin A. C. neoformans might therefore be a useful organism in which to investigate crosstalk among different families of prolyl isomerases.
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http://dx.doi.org/10.1099/mic.0.27786-0DOI Listing
May 2005

The structure of the Candida albicans Ess1 prolyl isomerase reveals a well-ordered linker that restricts domain mobility.

Biochemistry 2005 Apr;44(16):6180-9

Wadsworth Center, New York State Department of Health, School of Public Health, State University of New York, Albany, New York 12201-0509, USA.

Ess1 is a peptidyl-prolyl cis/trans isomerase (PPIase) that binds to the carboxy-terminal domain (CTD) of RNA polymerase II. Ess1 is thought to function by inducing conformational changes in the CTD that control the assembly of cofactor complexes on the transcription unit. Ess1 (also called Pin1) is highly conserved throughout the eukaryotic kingdom and is required for growth in some species, including the human fungal pathogen Candida albicans. Here we report the crystal structure of the C. albicansEss1 protein, determined at 1.6 A resolution. The structure reveals two domains, the WW and the isomerase domain, that have conformations essentially identical to those of human Pin1. However, the linker region that joins the two domains is quite different. In human Pin1, this linker is short and flexible, and part of it is unstructured. In contrast, the fungal Ess1 linker is highly ordered and contains a long alpha-helix. This structure results in a rigid juxtaposition of the WW and isomerase domains, in an orientation that is distinct from that observed in Pin1, and that eliminates a hydrophobic pocket between the domains that was implicated as the main substrate recognition site. These differences suggest distinct modes of interaction with long substrate molecules, such as the CTD of RNA polymerase II. We also show that C. albicans ess1(-)() mutants are attenuated for in vivo survival in mice. Together, these results suggest that CaEss1 might constitute a useful antifungal drug target, and that structural differences between the fungal and human enzymes could be exploited for drug design.
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http://dx.doi.org/10.1021/bi050115lDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4773908PMC
April 2005

Vanishingly low levels of Ess1 prolyl-isomerase activity are sufficient for growth in Saccharomyces cerevisiae.

J Biol Chem 2005 Apr 23;280(16):15510-7. Epub 2005 Feb 23.

Wadsworth Center, New York State Department of Health, Albany, New York 12208, USA.

Ess1 is an essential peptidylprolyl-cis/trans-isomerase in the yeast Saccharomyces cerevisiae. Ess1 and its human homolog, Pin1, bind to phospho-Ser-Pro sites within proteins, including the carboxyl-terminal domain (CTD) of Rpb1, the largest subunit of RNA polymerase II (pol II). Ess1 and Pin1 are thought to control mRNA synthesis by catalyzing conformational changes in Rpb1 that affect interaction of cofactors with the pol II transcription complex. Here we have characterized wild-type and mutant Ess1 proteins in vitro and in vivo. We found that Ess1 preferentially binds and isomerizes CTD heptad-repeat (YSPTSPS) peptides that are phosphorylated on Ser5. Binding by the mutant proteins in vitro was essentially normal, and the proteins were largely stable in vivo. However, their catalytic activities were reduced >1,000-fold. These data along with results of in vivo titration experiments indicate that Ess1 isomerase activity is required for growth, but only at vanishingly low levels. We found that although wild-type cells contain about approximately 200,000 molecules of Ess1, a level of fewer than 400 molecules per cell is sufficient for growth. In contrast, higher levels of Ess1 were required for growth in the presence of certain metabolic inhibitors, suggesting that Ess1 is important for tolerance to environmental challenge.
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http://dx.doi.org/10.1074/jbc.M412172200DOI Listing
April 2005

Sap18 is required for the maternal gene bicoid to direct anterior patterning in Drosophila melanogaster.

Dev Biol 2005 Feb;278(1):242-54

Molecular Genetics Program, Wadsworth Center, New York State Department of Health, 120 New Scotland Avenue, Albany, NY 12208, USA.

Development of the insect head is a complex process that in Drosophila requires the anterior determinant, Bicoid. Bicoid is present in an anterior-to-posterior concentration gradient, and binds DNA and stimulates transcription of head-specific genes. Many of these genes, including the gap-gene hunchback, are initially activated in a broad domain across the head primordium, but later retract so that their expression is cleared from the anterior-most segmented regions. Here, we show that retraction requires a Bicoid-interacting protein, Sap18, which is part of the Sin3/Rpd3 histone deacetylase complex. In sensitized-mutant backgrounds (e.g., bcdE1/+, removal of maternal sap18 results in embryos that are missing labrally derived parts of the cephalopharyngeal skeleton. These sap18 mutant embryos fail to repress hb expression, and show reduced anterior cap expression of the labral determinant cap 'n' collar. These phenotypes are enhanced by lowering the dose of rpd3, which encodes the catalytic subunit of the deacetylase complex. The results suggest a model where, in labral regions of the head, Bicoid is converted from an activator into a repressor by recruitment of a co-repressor to Bicoid-dependent promoters. Bicoid's activity, therefore, depends not only on its concentration gradient, but also on its interactions with modifier proteins within spatially restricted domains.
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http://dx.doi.org/10.1016/j.ydbio.2004.11.011DOI Listing
February 2005

Prolyl isomerases in yeast.

Front Biosci 2004 Sep 1;9:2420-46. Epub 2004 Sep 1.

Department of Molecular Genetics and Microbiology, Howard Hughes Medical Institute, Duke University Medical Center, Durham, NC 27710, USA.

Prolyl isomerases are enzymes that catalyze cis-trans isomerization of peptidyl-prolyl bonds and span three structurally unrelated protein families: the cyclophilins, FKBPs, and parvulins. The genome of the budding yeast Saccharomyces cerevisiae encodes eight different cyclophilins (Cpr1 to Cpr8), four FKBPs (Fpr1 to Fpr4), and a single parvulin (Ess1). Remarkably, two of these proteins, cyclophilin A and FKBP12, are conserved from yeast to humans and mediate virtually all of the intracellular actions of the immunosuppressive antifungal drugs cyclosporin A, FK506, and rapamycin. The study of prolyl isomerases in S. cerevisiae has proven invaluable to understand the elusive functions of these proteins, and continues to provide new insights into their diverse cellular roles. Here we review the current state of knowledge about prolyl-isomerases in this model organism.
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http://dx.doi.org/10.2741/1405DOI Listing
September 2004

Genetic interactions with C-terminal domain (CTD) kinases and the CTD of RNA Pol II suggest a role for ESS1 in transcription initiation and elongation in Saccharomyces cerevisiae.

Genetics 2004 May;167(1):93-105

Molecular Genetics Program, Wadsworth Center, New York State Department of Health, Albany, New York 12208, USA.

Ess1 is an essential prolyl isomerase that binds the C-terminal domain (CTD) of Rpb1, the large subunit of RNA polymerase II. Ess1 is proposed to control transcription by isomerizing phospho-Ser-Pro peptide bonds within the CTD repeat. To determine which step(s) in the transcription cycle might require Ess1, we examined genetic interactions between ESS1 and genes encoding the known CTD kinases (KIN28, CTK1, BUR1, and SRB10). Although genetic interactions were identified between ESS1 and all four kinases, the clearest interactions were with CTK1 and SRB10. Reduced dosage of CTK1 rescued the growth defect of ess1(ts) mutants, while overexpression of CTK1 enhanced the growth defects of ess1(ts) mutants. Deletion of SRB10 suppressed ess1(ts) and ess1Delta mutants. The interactions suggest that Ess1 opposes the functions of these kinases, which are thought to function in preinitiation and elongation. Using a series of CTD substitution alleles, we also identified Ser5-Pro6 as a potential target for Ess1 isomerization within the first "half" of the CTD repeats. On the basis of the results, we suggest a model in which Ess1-directed conformational changes promote dephosphorylation of Ser5 to stimulate preinitiation complex formation and, later, to inhibit elongation.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1470855PMC
http://dx.doi.org/10.1534/genetics.167.1.93DOI Listing
May 2004

Unexpected foraminiferal diversity revealed by small-subunit rDNA analysis of Antarctic sediment.

J Eukaryot Microbiol 2004 Mar-Apr;51(2):173-9

Wadsworth Center, P.O. Box 509, Albany, New York 12201, USA.

Studies of benthic Foraminifera typically rely on the morphological identification of dried specimens. This approach can introduce sampling bias against small, delicate, or morphologically ambiguous forms. To overcome this limitation, we extracted total DNA from sediment followed by PCR using group- and species-specific primers. Phylogenetic analyses revealed that approximately ninety percent of the PCR products represented previously undescribed sequence types that group with undersampled members of the allogromiid Foraminifera. We also used a modification of this technique to track individual species in sediment fractions too fine for normal morphological identification, and to confirm species placement of morphologically ambiguous foraminiferans. We were able to identify the DNA of several large foraminiferal species in fine fractions in a seasonally-dependent manner, indicating that in some seasons the majority of the standing stock of these species exists as gametes/juveniles. The approach outlined here represents a powerful strategy for exploring the total diversity of benthic foraminiferal communities.
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http://dx.doi.org/10.1111/j.1550-7408.2004.tb00542.xDOI Listing
August 2004

The ESS1 prolyl isomerase and its suppressor BYE1 interact with RNA pol II to inhibit transcription elongation in Saccharomyces cerevisiae.

Genetics 2003 Dec;165(4):1687-702

Molecular Genetics Program, Wadsworth Center, New York State Department of Health, Albany, NY 12208, USA.

Transcription by RNA polymerase II (pol II) requires the ordered binding of distinct protein complexes to catalyze initiation, elongation, termination, and coupled mRNA processing events. One or more proteins from each complex are known to bind pol II via the carboxy-terminal domain (CTD) of the largest subunit, Rpb1. How binding is coordinated is not known, but it might involve conformational changes in the CTD induced by the Ess1 peptidyl-prolyl cis/trans isomerase. Here, we examined the role of ESS1 in transcription by studying one of its multicopy suppressors, BYE1. We found that Bye1 is a negative regulator of transcription elongation. This led to the finding that Ess1 also inhibits elongation; Ess1 opposes elongation factors Dst1 and Spt4/5, and overexpression of ESS1 makes cells more sensitive to the elongation inhibitor 6-AU. In reporter gene assays, ess1 mutations reduce the ability of elongation-arrest sites to stall polymerase. We also show that Ess1 acts positively in transcription termination, independent of its role in elongation. We propose that Ess1-induced conformational changes attenuate pol II elongation and help coordinate the ordered assembly of protein complexes on the CTD. In this way, Ess1 might regulate the transition between multiple steps of transcription.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1462908PMC
December 2003

The Ess1 prolyl isomerase is required for growth and morphogenetic switching in Candida albicans.

Genetics 2002 Jan;160(1):37-48

Molecular Genetics Program, Wadsworth Center, New York State Department of Health, Albany, New York 12208, USA.

Prolyl-isomerases (PPIases) are found in all organisms and are important for the folding and activity of many proteins. Of the 13 PPIases in Saccharomyces cerevisiae only Ess1, a parvulin-class PPIase, is essential for growth. Ess1 is required to complete mitosis, and Ess1 and its mammalian homolog, Pin1, interact directly with RNA polymerase II. Here, we isolate the ESS1 gene from the pathogenic fungus Candida albicans and show that it is functionally homologous to the S. cerevisiae ESS1. We generate conditional-lethal (ts) alleles of C. albicans ESS1 and use these mutations to demonstrate that ESS1 is essential for growth in C. albicans. We also show that reducing the dosage or activity of ESS1 blocks morphogenetic switching from the yeast to the hyphal and pseudohyphal forms under certain conditions. Analysis of double mutants of ESS1 and TUP1 or CPH1, two genes known to be involved in morphogenetic switching, suggests that ESS1 functions in the same pathway as CPH1 and upstream of or in parallel to TUP1. Given that switching is important for virulence of C. albicans, inhibitors of Ess1 might be useful as antifungal agents.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1461953PMC
January 2002