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A major goal of research in our laboratory is to understand, at a molecular level, how synthesis of eukaryotic messenger RNA by the multisubunit enzyme RNA polymerase II is regulated. In early work, our laboratory contributed to the identification, purification, and characterization of the mechanisms of action of the set of RNA polymerase II general initiation factors TFIIB, TFIID, TFIIE, TFIIF, and TFIIH, which are essential for transcription initiation by RNA polymerase II at most genes. In subsequent work, our laboratory has exploited this “minimal” enzyme system to investigate the mechanisms governing RNA polymerase II transcription. These efforts have led to the discovery of a collection of transcriptional regulatory proteins with roles in transcription initiation, elongation, and chromatin remodeling. In addition, an unanticipated outgrowth of these studies was the identification of a new family of E3 ubiquitin ligases, including the von Hippel-Lindau tumor suppressor complex as the founding member of this family. As discussed in more detail below, current research in our laboratory is focused on aspects of the mechanism and regulation of RNA polymerase II transcription, as well as investigations of the von Hippel-Lindau tumor suppressor complex and related ubiquitin ligases. Mechanism and Regulation of Transcription by RNA Polymerase II The Mediator The Mediator is a multisubunit transcription factor that performs a critical roles in both transcriptional activation and repression by promoting essential communication between DNA bound transcription factors and RNA polymerase II and the general initiation factors at the promoter. The Mediator was first identified in yeast and found to be composed of more than 20 distinct polypeptides. Our studies have focused on the Mediator complex from mammalian cells. In collaboration with Drs. Michael Washburn and Laurence Florens at the Stowers Institute, we have exploited a MudPIT-based proteomics approach to identify a collection of new mammalian Mediator subunits and to define a set of roughly 30 consensus mammalian Mediator subunits. These studies revealed that, although the overall architecture of the Mediator complex is strikingly conserved throughout evolution from yeast to man, the mammalian Mediator includes a set of additional, metazoan-specific subunits including an alternative form of the Cdk8/Cyclin C/Med12/Med13 kinase module consisting of a previously uncharacterized Cdk8-like kinase and paralogs of Med12 and Med13. Having in hand an apparently complete set of mammalian Mediator subunits is now allowing a more systematic investigation of the functions of individual Mediator subunits in vitro and in cells, with particular emphasis on the metazoan-specific Mediator proteins. A Family of Related Mammalian Chromatin Remodeling Enzymes In a line of investigation that began with our characterization of the mammalian TRRAP/Tip60 HAT complex, we identified and are now characterizing two new chromatin remodeling complexes, which we refer to as the SRCAP and Ino80-like complexes. These mammalian chromatin remodelers share some structural and functional similarities with the yeast SWR1 and Ino80 chromatin remodeling complexes. The TRRAP/Tip60, SRCAP, and Ino80-like complexes share a subset of subunits including the actin-related protein Arp4 and the Ruvb-like AAA+ ATPases Tip49a and Tip49b. The SRCAP complex includes the SNF2-related SRCAP ATPase, which was originally identified as a coactivator of CREB and some steroid receptors. Like the yeast SWR1 chromatin remodeling complex, the mammalian SRCAP complex catalyzes exchange of histone H2AZ/H2B dimers into nucleosomes. The Ino80-like complex includes a previously uncharacterized SNF2-related ATPase that resembles yeast Ino80. Like the yeast Ino80 chromatin remodeling complex, the mammalian Ino80-like complex is capable of catalyzing nucleosome sliding. Although the mammalian Ino80-like complex exhibits some similiarities in subunit composition with the yeast Ino80 complex, the Ino80-like complex includes a collection of subunits that appear to be metazoan-specific. Ongoing efforts in our laboratory are aimed at understanding the mechanisms of action of these mammalian chromatin remodeling complexes in vitro and in cells, with particular emphasis on the roles their metazoan-specific subunits play in cellular functions. RNA Polymerase II Elongation Factors Elongin and ELL In the past, we have exploited the minimal transcription system composed of RNA polymerase II and the general initiation factors as an assay to identify and purify additional factors that promote transcription elongation. These efforts led to the discovery of two such factors, Elongin and ELL, which both function to increase the overall rate of transcription elongation by RNA polymerase II by suppressing transient pausing by the enzyme at many sites along the DNA. Elongin is a 3-subunit complex composed of A, B, and C subunits of ~770, 118, and 112 amino acids. In initial studies, cloning of the genes encoding the Elongin subunits and reconstitution and characterization of the recombinant Elongin ABC complex revealed (i) that Elongin A is the transcriptionally active subunit of the Elongin complex and (ii) Elongins B and C form an isolable subcomplex that binds to an ~12 amino acid BC-box present in Elongin A and induces its transcriptional activity. Ongoing studies are aimed at understanding in greater detail the mechanism of Elongin action of regulation of RNA polymerase II activity in vitro and in cells. The human ELL gene was originally identified as a gene that undergoes frequent translocations with the MLL gene on chromosome 11q23 in a variety of leukemias. Two additional ELL family members, ELL2 and ELL3, have been identified and shown to function similarly to ELL to activate the rate of transcription elongation by RNA polymerase II. ELL family members function in cells as components of multiprotein complexes that include one of the related EAF1 or EAF2 proteins, which we recently showed are potent activators of ELL transcriptional activity. Ongoing studies are aimed at characterizing additional components of these ELL-containing complexes and understanding how ELL and its associated proteins promote RNA polymerase II transcription in vitro and in cells. Investigations of the VHL Tumor Suppressor Complex and Related E3 Ubiquitin Ligases In previous work, our laboratory contributed to the discovery that the von Hippel-Lindau (VHL) tumor suppressor protein is an integral component of an E3 ubiquitin ligase that negatively regulates expression of hypoxia-inducible genes by targeting hypoxia-inducible transcription factors ( HIFs ) for ubiquitination and degradation by the proteasome . The VHL gene on chromosome 3p25.5 is mutated in the majority of sporadic clear cell renal carcinomas and in VHL disease, an autosomal dominant familial cancer syndrome that predisposes affected individuals to a variety of tumors, including clear cell carcinoma, cerebellar hemangioblastomas and hemangiomas , retinal angiomas , and pheochromocytomas . These studies demonstrated that, like Elongin A, the VHL protein is a BC-box protein. Purification of the endogenous VHL tumor suppressor complex from rat liver in our laboratory led to the discovery that it is composed of VHL, Elongins B and C, Cullin family member Cul2, and a novel RING finger protein designated Rbx1 and, further, that it possesses intrinsic ubiquitin ligase activity driven by the Rbx1 RING finger. Subsequent studies in our laboratory and elsewhere have led to the identification of additional BC-box proteins (including the Suppressor of Cytokine Signaling (SOCS) proteins) and demonstrated that many are components of a larger family of Elongin BC- and Cul2/Cul5-containing ubiquitin ligases that include Elongins BC and Rbx1 or the related Rbx2 protein. In addition to our ongoing investigations of the mechanism of action of the VHL ubiquitin ligase complex in regulation of hypoxia-inducible transcription, we are currently attempting to develop MudPIT -based proteomics approaches to identify and characterize additional members of the family Elongin BC- and Cul2/Cul5-containing ubiquitin ligases and to identify their associated proteins and substrates. Academic Appointments: Professor, Department of Biochemistry & Molecular Biology, The University of Kansas School of Medicine Selected publications Cai
Y, Jin J, Florens L, Swanson SK, Kusch T, Li B, Workman JL, Washburn MP, Conaway RC, Conaway JW. The mammalian
YL1 protein is a shared subunit of the TRRAP/TIP60 histone acetyltransferase
and SRCAP complexes. J Biol Chem. 2005.
Abstract Sato
S, Tomomori-Sato C, Parmely TJ, Florens L, Zybailov B, Swanson SK, Banks CA,
Jin J, Cai Y, Washburn MP, Conaway JW,
Conaway RC. A set of consensus Mammalian mediator subunits identified by
multidimensional protein identification technology. Mol Cell. 2004;14:685-691.
Abstract Tomomori-Sato
C, Sato S, Parmely TJ, Banks CA, Sorokina I, Florens L, Zybailov B, Washburn
MP, Brower CS, Conaway RC, Conaway JW.
A mammalian mediator subunit that shares properties with Saccharomyces
cerevisiae mediator subunit Cse2. J Biol Chem. 2004. Abstract. Cai
Y, Jin J, Tomomori-Sato C, Sato S, Sorokina I, Parmely TJ, Conaway RC, Conaway JW. Identification of new subunits of the
multiprotein mammalian TRRAP/TIP60-containing histone acetyltransferase
complex. J Biol Chem. 2003;278:42733-42736. Abstract Conaway RC, Kong SE, Conaway JW. TFIIS and GreB: two
like-minded transcription elongation factors with sticky fingers. Cell.
2003;114:272-274. Abstract Dvir
A, Conaway JW, Conaway RC. Assays
for investigating the mechanism of promoter escape by RNA polymerase II. Methods
Enzymol. 2003;370:733-740. Abstract Kong
S, Shilatifard A, Conaway RC, Conaway JW.
Preparation and assay of RNA polymerase II elongation factors elongin and ELL. Methods
Enzymol. 2003;371:276-283. Abstract Kuznetsova
AV, Meller J, Schnell PO, Nash JA, Ignacak ML, Sanchez Y, Conaway JW, Conaway RC, Czyzyk-Krzeska MF. von Hippel-Lindau
protein binds hyperphosphorylated large subunit of RNA polymerase II through a
proline hydroxylation motif and targets it for ubiquitination. Proc Natl
Acad Sci U S A. 2003;100:2706-2711. Abstract. Maynard
MA, Qi H, Chung J, Lee EH, Kondo Y, Hara S, Conaway RC, Conaway JW, Ohh M. Multiple splice variants of the
human HIF-3 alpha locus are targets of the von Hippel-Lindau E3 ubiquitin
ligase complex. J Biol Chem. 2003;278:11032-11040. Abstract Sato
S, Tomomori-Sato C, Parmely TJ, Florens L, Zybailov B, Swanson SK, Banks CA,
Jin J, Cai Y, Washburn MP, Conaway JW, Conaway RC. A set of consensus Mammalian
mediator subunits identified by multidimensional protein identification
technology. Mol Cell. 2004;14:685-691. Abstract Sato
S, Tomomori-Sato C, Banks CA, Parmely TJ, Sorokina I, Brower CS, Conaway RC, Conaway JW. A mammalian
homolog of Drosophila melanogaster transcriptional coactivator intersex Is a
subunit of the mammalian mediator complex. J Biol Chem. 2003;278:49671-49674.
Abstract. Sato
S, Tomomori-Sato C, Banks CA, Sorokina I, Parmely TJ, Kong SE, Jin J, Cai Y,
Lane WS, Brower CS, Conaway RC, Conaway
JW. Identification of mammalian Mediator subunits with similarities to
yeast Mediator subunits Srb5, Srb6, Med11, and Rox3. J Biol Chem. 2003;278:15123-15127.
Abstract Shilatifard
A, Conaway RC, Conaway JW. The RNA
polymerase II elongation complex. Annu Rev Biochem. 2003;72:693-715.
Abstract Brower
CS, Sato S, Tomomori-Sato C, Kamura T, Pause A, Stearman R, Klausner RD, Malik
S, Lane WS, Sorokina I, Roeder RG, Conaway
JW, Conaway RC. Mammalian mediator subunit mMED8 is an Elongin
BC-interacting protein that can assemble with Cul2 and Rbx1 to reconstitute a
ubiquitin ligase. Proc Natl Acad Sci U S A. 2002;99:10353-10358. Abstract Conaway JW, Conaway RC. RNA Polymerases.
In: HH Kazazian, and John Wiley & Sons., eds. Wiley Eencyclopedia of
Molecular Medicine. Conaway RC, Brower CS, Conaway JW. Emerging roles of ubiquitin
in transcription regulation. Science. 2002;296:1254-1258. Abstract Conaway RC, Conaway JW. The von
Hippel-Lindau tumor suppressor complex and regulation of hypoxia-inducible
transcription. Adv Cancer Res. 2002;85:1-12. Abstract Ivan
M, Haberberger T, Gervasi DC, Michelson KS, Gunzler V, Kondo K, Yang H,
Sorokina I, Conaway RC, Conaway JW, Kaelin WG, Jr. Biochemical
purification and pharmacological inhibition of a mammalian prolyl hydroxylase
acting on hypoxia-inducible factor. Proc Natl Acad Sci U S A. 2002;99:13459-13464.
Abstract Kamura
T, Conaway JW, Conaway RC. Roles of
SCF and VHL ubiquitin ligases in regulation of cell growth. Prog Mol Subcell
Biol. 2002;29:1-15. Abstract Kamura
T, Brower CS, Conaway RC, Conaway JW.
A molecular basis for stabilization of the von Hippel-Lindau (VHL) tumor
suppressor protein by components of the VHL ubiquitin ligase. J Biol Chem.
2002;277:30388-30393. Abstract Zheng
N, Schulman BA, Song L, Miller JJ, Jeffrey PD, Wang P, Chu C, Koepp DM, Elledge
SJ, Pagano M, Conaway RC, Conaway JW,
Harper JW, Pavletich NP. Structure of the Cul1-Rbx1-Skp1-F boxSkp2 SCF
ubiquitin ligase complex. Nature. 2002;416:703-709. Abstract Querido E, Blanchette P, Yan Q, Kamura T, Morrison M, Boivin
D, Kaelin WG, Conaway RC, Conaway JW,
Branton PE. Degradation of p53 by adenovirus E4orf6 and E1B55K proteins occurs
via a novel mechanism involving a Cullin-containing complex. Genes Dev.
2001;15:3104-3117. Abstract Elmendorf BJ, Shilatifard A, Yan Q, Conaway RC, Conaway
JW. Transcription factors TFIIF, ELL, and Elongin negatively regulate
SII-induced nascent transcript cleavage by non-arrested RNA polymerase II
elongation intermediates. J Biol Chem. 2001;276:23109-23114. Abstract.
Kamura T, Burian D, Yan Q, Schmidt SL, Lane WS, Querido E,
Branton PE, Shilatifard A, Conaway RC, Conaway JW. Muf1, a novel Elongin
BC-interacting leucine-rich repeat protein that can assemble with Cul5 and Rbx1
to reconstitute a ubiquitin ligase. J Biol Chem. 2001;276:29748-29753. Abstract.
Kamura T, Sato S, Iwai K, Czyzk-Kryzeska M, Conaway RC,
Conaway JW. Activation of HIF1alpha ubiquitination by a reconstituted von
Hippel-Lindau (VHL) tumor suppressor complex. Proc Natl Acad Sci Kamura T, Conrad MN, Yan Q, Conaway RC, Conaway JW. The
Rbx1 subunit of SCF and VHL E3 ubiquitin ligase activates Rub1 modifications of
cullins Cdc53 and Cul2. Genes Dev. 1999;13:2928-2933. Abstract.
Kamura T, Koepp DM, Conrad MN, Skowyra D, Moreland RJ,
Iliopoulos O, Lane WS, Kaelin WG Jr, Elledge S, Harper W, Conaway RC,
Conaway JW. Rbx1, a Component of the VHL Tumor Suppressor Complex and SCF
Ubiquitin Ligase. Science. 1999;284:657-661. Abstract. Skowyra D, Koepp DM, Kamura T, Conrad M, Conaway RC,
Conaway JW, Elledge SJ, Harper JW. Reconstitution of G1 cyclin
ubiquitination with complexes containing SCFGrr1 and Rbx1. Science.
1999;284:662-665. Abstract. Kamura T, Haque D, Liu L, Kaelin WG, Conaway RC, Conaway
JW. The Elongin BC complex interacts with the SOCS-box motif present in
members of the suppressors of cytokine signalling (SOCS), Ras, WD-40 repeat,
and ankyrin repeat families. Genes Dev. 1998;12:3872-3881. Abstract.
Dvir A, Conaway RC, Conaway JW. A role for TFIIH in
controlling the activity of early RNA polymerase II elongation complexes. Proc
Natl Acad Sci Shilatifard A, Lane WS, Jackson KW, Conaway RC, Conaway
JW. An RNA polymerase II elongation factor encoded by the human ELL gene. Science.
1996;271:1873-1876. Abstract.
Duan DR , Pause A, Burgess WH, Aso T, Chen DYT, Garrett KP,
Conaway RC, Conaway JW, Linehan WM, Klausner RD. Inhibition of
transcription elongation by the von Hippel-Lindau tumor suppressor protein. Science.
1995;269:1402-1406. Abstract.
Aso T, Lane WS, Conaway RC, Conaway JW. Elongin
(SIII): A multisubunit regulator of elongation by RNA polymerase II. Science.
1995;269:1439-1443. Abstract.
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Conaway
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