Chromatin binding dynamics Our analysis of transcription preinitiation complex assembly led us to consider fundamental questions about chromatin binding dynamics in cells. We seek to understand not only pathways for assembly and activity of transcription complexes in vivo, but the time scales on which these processes occur, how efficient they are in a cell population, and how stable complexes are once they assemble on chromatin. To address these questions, we have developed methods that allow us to measure chromatin binding kinetics at specific chromosomal sites in cells.
Transcriptional regulatory mechanisms involve a complex interplay of factors whose effects are mediated by physical interactions among them and diverse enzymatic activtites, all occurring in the context of the native chromatin fiber. To understand aspects of this regulation we apply approaches that span the full range from biochemistry, biophysics and structural biology, to molecular biology, cell biology, genetics and genomics.
For investigation of transcriptional regulatory mechanisms in yeast cells, we use genetic and molecular biological approaches to engineer strains with particular defects, and we characterize the effects of these mutations on transcription by measuring transcription factor interactions with chromatin and RNA output at particular genes as well as genome-wide.
The cartoon above shows a number of the key factors required for assembly of transcription preinitiation complexes (PICs) at promoters, many of which are themselves multisubunit complexes. There is a wealth of structural data that show how these factors actually interact with one another. The figure on the left shows an example; this is what a minimal version of the yeast PIC looks like during the initial steps of RNA synthesis, with TATA binding protein (TBP) shown in dark blue, TFIIB shown in orange, TFIIF in yellow, and RNA polymerase II (Pol II) in gray. The DNA (cyan) can be seen disappearing into the Pol II active site.
An essential and conserved regulator of PIC assembly and activity is an enzyme called Mot1. It interacts with TBP, which you can see in the figure above is a central component of the PIC. Mot1 has the remarkable property of using ATP hydrolysis to remove TBP from DNA. Mot1 is comprised of two functional domains. Its N-terminal domain (NTD) has an extended corkscrew-like shape that interacts with the saddle-shaped TBP molecule. The image on the home page shows the co-crystal structure of the NTD-TBP complex. The C-terminal region of Mot1 possesses an ATPase domain that is highly related to a large number of ATPases belonging to the Snf2/Swi2 family. These enzymes play critical roles in all aspects of DNA metabolism including transcription, replication and repair. In general, enzymes in this family use ATP hydrolysis to catalyze changes in protein-DNA interactions, and understanding how they do this on a molecular level has been the subject of intense interest by many labs. We apply biochemical, biophysical and structural biological approaches to better understand the Mot1 catalytic mechanism, and in so doing seek to better understand how enzymes in this important class function in general. Through our longstanding collaboration with friends in Munich, we now have high resolution models of how the Mot1 NTD interacts with the TBP-DNA-NC2 complex as well as the ring-like structure of full length Mot1 alone.
Our recent work on Mot1 and PIC dynamics showed that in general, the interactions between general transcription factors and chromatin are surprisingly short-lived in vivo. Biochemical studies, however, had established that a subset of PIC components can be stably bound to promoters in vitro, nicely explaining in principle how the activated state of a gene can be perpetuated. We wondered what the relationship of the rapid dynamics we observed could be to the normal process of gene expression and regulation. One possibility was that the biochemical studies did not accurately capture the dynamic processes occuring in vivo. Another possibility was that binding dynamics vary, potentially dramatically, at different promoters in vivo- an idea with interesting implications for gene regulation. Alternatively, stable complexes may form in vivo, but perhaps they do so inefficiently. In support of a fundamentally important role for chromatin binding dynamics, we found that Mot1-mediated TBP turnover is essential genome-wide for accurate and efficient transcription, and perturbing it can impact apparently all steps of the process of RNA synthesis- from initiation, to elongation, to termination. For this reason, a major focus of our current work is to develop and exploit methods for measuring chromatin binding dynamics at single copy genes in vivo. In a recently developed method which we call Crosslinking Kinetics (or CLK, pronounced "clock") Analysis we measure how the chromatin immunoprecipitation (ChIP) signal depends on the formaldehyde incubation time. By modeling this process we can obtain estimates of the on-rate, the off-rate and the fractional occupancy for chromatin binding, among other parameters. In parallel, we have also been applying an approach called competition ChIP, which allows us to extract kinetic parameters by measuring the rates of replacement of one form of a transcription factor at a chromatin site with an induced isoform distinguished by a different epitope tag. So far, we have seen that chromatin complexes span an enormous range of stability, with half-lives ranging from just a few seconds to an hour or more. How regulators influence this dynamic behavior is just beginning to be explored, but we have already observed that quantitative analyses of chromatin binding in vivo by our methods can challenge models derived from conventional ChIP studies and provide new insight into regulatory mechanisms operating at promoters.
Uchiyama, R., K. Kupkova, S.J. Shetty, A.S. Linford, M.G. Pray-Grant, L.E. Wagar, M.M. Davis, R. Haque, A. Gaultier, M.W. Mayo, P.A. Grant, W.A. Petri, Jr., S. Bekiranov, and D.T. Auble. Histone H3 Lysine 4 Methylation Signature Associated with Human Undernutrition. Proc. Nat. Acad Sci. USA, in press. <PNAS>
Butryn, A., S. Woike, S.J. Shetty, D.T. Auble, and K.-P. Hopfner. Crystal Structure of the Full Swi2/Snf2 Remodeler Mot1 in the Resting State. eLife 7:e37774, 2018. <PubMed>
Hoffman, E. A., Zaidi, H., Shetty, S. J., Bekiranov, S. and Auble, D. T. An Improved Method for Measuring Chromatin-binding Dynamics Using Time-dependent Formaldehyde Crosslinking. Bio-protocol 8(4): e2905, 2018. <Bio-protocol>
H. Zaidi, E.A. Hoffman, S.J. Shetty, S. Bekiranov, and D.T. Auble. Second-generation method for analysis of chromatin binding with formaldehyde crosslinking kinetics. J. Biol. Chem. 292:19338-19355, 2017. <PubMed>
X. Chen, K. Poorey, M.N. Wells, U. Müller, S. Bekiranov, D.T. Auble*, and D.A. Brow*. Transcriptomes of six mutants in the Sen1 pathway reveal combinatorial control of transcription termination across the Saccharomyces cerevisiae genome. PLoS Genetics 13: e1006863, 2017. <PubMed>
H.A. Zaidi, D.T. Auble, and S. Bekiranov. RNA Synthesis is Associated with Multiple TBP-Chromatin Binding Events. Scientific Reports 7:39631, 2017. <PubMed>
R. Viswanathan, J.D. True, and D.T. Auble. Molecular mechanism of Mot1, a TATA-binding protein (TBP)-DNA Dissociating Enzyme. J. Biol. Chem. 291:15714-15726, 2016. <PubMed>
J.D. True, J.J. Muldoon, M.N. Carver, K. Poorey, S.J. Shetty, S. Bekiranov, and D.T. Auble. The Modifier of Transcription 1 (Mot1) ATPase and Spt16 histone chaperone co-regulate transcription through preinitiation complex assembly and nucleosome organization. J. Biol. Chem. 291:15307-15319, 2016 <PubMed>
A. Butryn, J.M. Schuller, G. Stoehr, P. Runge-Wollmann, F. Forster, D.T. Auble and K.P. Hopfner. Structural Basis for recognition and remodeling of the TBP:DNA:NC2 complex by Mot1. eLife 4:e07432, 2015 <PubMed>
R. Viswanathan, E.A. Hoffman, S.J. Shetty, S. Bekiranov, and D.T. Auble. Analysis of chromatin binding dynamics using the crosslinking kinetics (CLK) method. Methods 70:97-107, 2014 <PubMed>
T. Ahmed, D. Auble, J.A. Berkley R. Black P.P. Ahern M. Hossain, A. Hsieh, S. Ireen, M. Arabi, J.I. Gordon. An evolving perspective about the origins of childhood undernutrition and nutritional interventions that includes the gut microbiome. Ann. N.Y. Acad. Sci. 1332:22-38, 2014. <PubMed>
K. Poorey, R. Viswanathan, M.N. Carver, T.S. Karpova, S.M. Cirimotich, J.G. McNally, S. Bekiranov, and D.T. Auble. Measuring Chromatin Interaction Dynamics on the Second Time Scale at Single-Copy Genes. Science 342:369-372, 2013. <PubMed>
G. Moyle-Heyrman, R. Viswanathan, J. Widom, and D.T. Auble. Two-step Mechanism for Modifier of Transcription (Mot1) Enzyme-catalyzed Displacement of TATA-binding Protein (TBP) from DNA. J. Biol. Chem. 287:9002-9012, 2012. <PubMed>
R. Viswanathan and D.T. Auble. One Small Step for Mot1; One Giant Leap for Other Swi2/Snf2 Enzymes? BBA Gene Reg. Mech. 1809:488-496, 2011. <PubMed>
P. Wollmann, S. Cui, R. Viswanathan, O. Berninghausen, M.N. Wells, M. Moldt, G. Witte, A. Butryn, P. Wendler, R. Beckmann, D.T. Auble* and K.-P. Hopfner*. Structure and mechanism of the Swi2/Snf2 remodeler Mot1 in complex with its substrate TBP. Nature 475:403-407, 2011. *co-corresponding authors <PubMed > <EcTBP structure (PDB 3OCI)> <EcMot1 NTD-TBP complex (PDB 3OC3)>
E. M. Hyland, H. Molina, K. Poorey, C. Jie, Z. Xie, J. Dai, J. Qian, S. Bekiranov, D.T. Auble, A. Pandey, and J.D. Boeke. An Evolutionarily "Young" Lysine Residue In Histone H3 Attenuates Transcriptional Output in Saccharomyces cerevisiae. Genes & Dev. 25:1306-1319, 2011. <PubMed> <Table S1> <Table S2> <Table S3> <Supplemental Figures>
K. Poorey, R.O. Sprouse, M.N. Wells, R. Viswanathan, S. Bekiranov, and D.T. Auble. RNA Synthesis Precision Is Regulated by Preinitiation Complex Turnover. Genome Res. 20:1679-1688, 2010. <PubMed> <Supplemental Material>
S.L. Wade and D.T. Auble. The Rad23 Ubiquitin Receptor, the Proteasome and Functional Specificity in Transcriptional Control. Transcription 1:22-26, 2010. <PubMed>
S.L. Wade, K. Poorey, S. Bekiranov, and D.T. Auble. The Snf1 kinase and proteasome-associated Rad23 regulate UV-responsive gene expression. EMBO J. 28:2919–2931, 2009. <PubMed> <Supplemental Results> <List of all genes with a significant UV effect in WT cells>
R.O. Sprouse, M.N. Wells, and D.T. Auble. TATA Binding Protein Variants that Bypass the Requirement for Mot1 In Vivo. J. Biol. Chem. 284:4525-4535, 2009. <PubMed> <Supplemental Results>
D. Basu, M. P. Walkiewicz, M. Frieman, R.S. Baric, D.T. Auble, and D.A. Engel. Novel influenza NS1 antagonists block replication and restore innate immune function. J. Virol. 83:1881-1891, 2009. <PubMed>
D.T. Auble. The Dynamic Personality of TATA-Binding Protein. Trends in Biochem. Sci. 34:49-52, 2009. <PubMed>
R.O. Sprouse, T.S. Karpova, F. Mueller, A. Dasgupta, J.G. McNally, and D.T. Auble. Regulation of TATA-Binding Protein Dynamics in Living Yeast Cells. Proc. Nat. Acad. Sci. USA 105:13304 –13308, 2008. <PubMed> <Supporting Information>
R.O. Sprouse, I. Shcherbakova, H. Cheng, E. Jamison, M. Brenowitz, and D.T. Auble. Functional and Structural Organization of Mot1 Bound to a Natural Target Promoter. J. Biol. Chem. 283:24935-24948, 2008. <PubMed> <Table S1> <Figure S1> (Figure S1 is a movie.)
A. Dasgupta, R.O. Sprouse, S. French, P. Aprikian, R. Hontz, S.A. Juedes, J.S. Smith, A.L. Beyer, and D.T. Auble. Regulation of rRNA Synthesis by TATA-Binding Protein-Associated Factor Mot1. Mol. Cell. Biol. 27:2886-2896, 2007. <PubMed>
R.O. Sprouse, M. Brenowitz, and D.T. Auble. Snf2/Swi2-Related ATPase Mot1 Drives Displacement of TATA-Binding Protein by Gripping DNA. EMBO J. 25: 1492-1504, 2006. <PubMed>
Supplemental data:<Figure S1> <Figure S2> <Figure S3> <Figure S4>
A. Dasgupta, S.A. Juedes, R.O. Sprouse, and D.T. Auble. Mot1-Mediated Control of Transcription Complex Assembly and Activity. EMBO J. 24: 1717-1729, 2005. <PubMed>
Supplemental data: <Figure S1> <Figure S2> <Figure S3> <Figure S4> <Table S1>
T. Miyake, J. Reese, C. Loch, D.T. Auble, and R. Li. Genome-Wide Analysis of ARS Binding Factor 1 (Abf1p)-Mediated Transcriptional Regulation in Saccharomyces cerevisiae. J. Biol. Chem., 279: 34865-34872, 2004. <PubMed>
K.L. Ramsey, J. J. Smith, A. Dasgupta, N. Maqani, Patrick Grant and D.T. Auble. The NEF4 complex regulates Rad4 levels and utilizes Snf2/Swi2-related ATPase activity for nucleotide excision repair. Mol. Cell. Biol., 24: 6362-6378, 2004. <PubMed> <Supplemental data>
A. Dasgupta, K.L. Ramsey, J.S. Smith and D.T. Auble. Sir Antagonist 1 (San1) is a Ubiquitin Ligase. J. Biol. Chem., 279: 26830-26838, 2004. <PubMed>
R. P. Darst, A. Dasgupta, C. Zhu, J.-Y. Hsu, A. Vroom, T. Muldrow, and D. T. Auble. Mot1 Regulates the DNA Binding Activity of Free TBP in an ATP-Dependent Manner. J. Biol. Chem, 278:13216-13226, 2003. <PubMed>
A. Dasgupta, R.P. Darst, K.J. Martin, C.A. Afshari, and D.T. Auble. Mot1 Activates and Represses Transcription by Direct, ATPase-Dependent Mechanisms. Proc. Nat. Acad. Sci. USA 99:2666-2671, 2002. <PubMed> <Supplemental data>
R.P. Darst, D. Wang, and D.T. Auble. Mot1-Catalyzed TBP-DNA Disruption: Uncoupling DNA Conformational Change and Role of Upstream DNA. EMBO J. 20:2028-2040, 2001. <PubMed>
Y. Cang, D.T. Auble, and G. Prelich. A New Regulatory Domain on the TATA-Binding Protein. EMBO J. 18:6662-6671, 1999. <PubMed>
T.A. Muldrow, A.M. Campbell, P.A. Weil, and D.T. Auble. Mot1 Can Activate Basal Transcription In Vitro by Regulating the Distribution of TBP Between Promoter and Nonpromoter Sites. Mol. Cell. Biol. 19:2835-2845, 1999. <PubMed>
D.T. Auble, and S.M. Steggerda. Testing for DNA Tracking by Mot1, a Snf2/Swi2 Protein Family Member. Mol. Cell. Biol. 19:412-423, 1999. <PubMed>
Chen et al. PLoS Genetics 13:e1006863, 2017.
RNA tiling array data are available from the NCBI Gene Expression Omnibus, accession number GSE100283
True et al. JBC 291:15307-15319, 2016.
ChIP-seq, MNase-seq and RNA tiling data are available from the NCBI Gene Expression Omnibus, SuperSeries assession number GSE80235
Hyland et al. Genes & Dev. 25:1306-1319, 2011.
RNA tiling array data are available from the NCBI Gene Expression Omnibus, accession number GSE13889
Poorey et al. Genome Res. 20:1679-1688, 2010.
RNA tiling and ChIP-on-chip data are available from the NCBI Gene Expression Omnibus, accession number GSE18283
Download processed RNA and ChIP-chip data files here.
Wade et al. EMBO J. 28:2919–2931, 2009.
all microarray gene expression data reported in this study are available from the NCBI Gene Expression Omnibus (GEO), accession number GSE16799
Sprouse et al. JBC 284:4525-4535, 2009.
bypass TBP microarray data are available from the NCBI Gene Expression
Omnibus, accession number GSE12371
Miyake et al. JBC 279: 34865-34872, 2004.
view list of Abf1-regulated genes with links to SGD
Dasgupta et al. JBC 279:26830-26838, 2004.
san1 microarray data are available from YFGdb
Dasgupta et al. PNAS 99:2666-2671, 2002.
browse or download mot1 microarray data
Microarray Data Parser: here
Affinity purification of Mot1
ChIP (standard assay)
FeBABE cleavage protocol
Gel shift assay
Immobilized template assay
In vitro NER assay
Northern blot protocol
pCORE modification protocol
Plasmid mini prep
Primers for yeast genome modification
Preparation of Py antibody-coupled beads
Quantitation of RNA by real time PCR
Yeast whole cell extract prep (galactose-induced cells)
Yeast genomic DNA prep
Yeast genomic DNA prep (10 min)
Yeast total RNA prep
Yeast transformation for integration (tagging, KO, pCORE, etc)
auble [at] virginia.edu
Department of Biochemistry and Molecular Genetics
Box 800733 Pinn Hall
University of Virginia Health System
Charlottesville, VA 22908
Department of Biochemistry and Molecular Genetics
Room 6213 Pinn Hall
1340 Jefferson Park Avenue
University of Virginia Health System
Charlottesville, VA 22908
Wednesdays at 9:00 a.m. in David's office