The attachment of the small protein, ubiquitin, to target proteins regulates a vast array of biological processes. We are interested in the role of ubiquitination in regulating transcription and the DNA damage response. We study the protein complexes that attach, recognize and remove ubiquitin modifications, as well as the way in which cross-talk between ubiquitination and other post-translational modifications regulates chromatin activity. We use x-ray crystallography, enzymology, cell-based assays and a variety of biophysical tools to gain insights into the mechanisms underlying these essential cellular processes.
Below is a summary of ongoing research projects:
SAGA: Ubiquitination and acetylation in gene activation
The 1.9 MDa yeast SAGA complex has been a paradigm for understanding the connection between histone modifications and gene activation as well as the cross-talk between different types of modifications. SAGA functions in a cascade of posttranslational modifications that begins with monoubiquitination of histone H2B-K123m which triggers methylation of histone H3-K4. The SAGA complex is recruited, where it deubiquitinates H2B, recognizes the H3K4 trimethyl mark and acetylates histone H3. The 19 SAGA subunits are conserved from yeast to humans and are organized into subcomplexes including the four-protein SAGA deubiquitinating module (DUBm) and the four-protein SAGA histone acetyltransferase (HAT) module. Our structural and biochemical studies are aimed at uncovering how SAGA recognizes its nucleosomal substrates and how the multiple histone modifying activities of SAGA are coordinated during the process of gene activation.
Morgan, Haj-Yahya, Ringel, Bandi, Brik, Wolberger (2016) Structural basis for histone H2B deubiquitination by the SAGA DUB module. Science 351: 725-8. PubMed
Ringel, Cieniewicz, Taverna, Wolberger (2015) Nucleosome competition reveals processive acetylation by the SAGA HAT module. PNAS 6;112(40):E5461-70. PubMed
Samara, Ringel, Wolberger (2012) A role for intersubunit interactions in maintaining SAGA deubiquitinating module structure and activity. Structure 20:1414-24. PubMed
Samara, Datta et al. (2010) Structural insights into the assembly and function of the SAGA deubiquitinating module. Science 328: 1025-9. PubMed
Ubiquitin Signaling in the DNA damage response
Attachment of ubiquitin or the ubiquitin-like protein, SUMO, plays a critical role in the response to DNA damage. Monoubiquitin, K63-linked polyubiquitin, K48-linked polyubiquitin and mixed SUMO-ubiquitin chains play distinct roles in signaling DNA damage and recruiting the necessary enzymes needed for DNA repair. We are interested in the enzymes that specifically assemble and disassemble ubiquitin and SUMO signals at damage sites, as well as the protein that recognize different types of chains as distinct signals. One area of study is the OTUB1 deubiquitinating enzyme, which forms complexes with E2 ubiquitin conjugating enzymes that can both regulate polyubiquitin chain elongation as well as hydrolysis of K48-linked polyubiquitin chains. We are also interested in the role of hybrid SUMO-ubiquitin chains in signaling DNA double strand breaks.
Wiener, DiBello, Lombardi, Guzzo, Zhang, Matunis, Wolberger (2013) E2 ubiquitin-conjugating enzymes regulate the deubiquitinating activity of OTUB1. NSMB 20:1033-9. PubMed
Guzzo, Berndsen, Zhu, Gupta, Datta, Greenberg, Wolberger, Matunis (2012) RNF4-dependent hybrid SUMO-ubiquitin chains are signals for RAP80 and thereby mediate the recruitment of BRCA1 to sites of DNA damage. Sci Signal. 5(253):ra88. PubMed
Wiener, Zhang, Wang, Wolberger (2012) The mechanism of OTUB1-mediated inhibition of ubiquitination. Nature 483:618-22. PubMed
The Sir2 Family of NAD+-dependent Deacetylases
Sirtuins comprise an unusual class of enzymes that deacetylate lysine side chains in a reaction that consumes NAD+. Enzymes similar to Sir2 are found in nearly all organisms, where they regulate processes including transcriptional silencing, DNA repair, chromosome stability, neuronal degeneration and fat mobilization. In addition to their principal activity in deacetylating substrates, can also remove other types of acyl modification from residues such as propionyl-, succinyl- and myristoyl-lysine. Understanding the novel chemistry of these enzymes and how it is exploited to regulate sirtuin activity in the cell has been an ongoing focus of our research.
Bheda, Jing, Wolberger, Lin (2016) The Substrate Specificity of Sirtuins. Annu Rev Biochem. 85:405-29.
Ringel, Roman, Wolberger (2014) Alternate deacylating specificities of the archaeal sirtuins Sir2Af1 and Sir2Af2. Protein Sci. 23(12):1686-97. Article
Bheda, Swatkoski, Fiedler, Boeke, Cotter, Wolberger (2012) Biotinylation of lysine method identifies acetylated histone H3 lysine 79 in Saccharomyces cerevisiae as a substrate for Sir2. PNAS 109(16):E916-25. Article
Bheda, Wang, Escalante-Semerena, Wolberger (2011) Structure of Sir2Tm bound to a propionylated peptide. Protein Sci. 20(1):131-9. PubMed