The Wittenberg Laboratory

Research

Control of Cell Cycle Initiation. 
Progress through the eukaryotic cell cycle is driven by sequential waves of accumulation of regulatory proteins that determine the sequence of cell cycle events (Wittenberg and Reed, 2005). As a consequence, periodic transcription regulates the sequence and timing of cell cycle events and it is, in turn, regulated by those events.  Our laboratory studies the regulation of G1/S transcription (sometimes called G1-specific transcription), the wave of expression associated with commitment to a new cell cycle. The G1/S gene family in the budding yeast is comprised of about three hundred genes that are co-expressed as cells initiate a new cycle during G1 phase. Those genes are expressed under the control of two distinct transcription factors known as SBF and MBF (Fig. 1). We have identified two transcriptional repressors that regulate the G1/S transcription factors. Whi5 specifically associates with and represses SBF-dependent transcription during early G1 phase (de Bruin et al., 2004). It is inactivated as a consequence of phosphorylation by the G1 cyclin/CDK complex, Cln3/Cdc28 leading to SBF activation. The second repressor, Nrm1, acts as a corepressor together with MBF to repress transcription as cells exit G1 phase (de Bruin et al., 2006). Nrm1 is encoded by an MBF target and, thereby, participates in a negative feedback loop that limits G1-specific transcription.  Furthermore, Nrm1 is antagonized by the Rad53 checkpoint kinase in cells responding to DNA replication stress (Travesa et al, 2012). Whi5 and Nrm1 share a small domain called the GTB motif that mediates their repressive interaction with their cognate transcription factor (Travesa et al, 2013).  Together, they impose the transcriptional repression that is central to the architecture of G1/S regulatory circuitry. A similar wave of gene expression encoding many orthologous proteins occurs during G1 phase in metazoans, including humans, and is controlled by the E2F family of transcription factors. Deregulation of the Rb/E2F transcriptional circuit is tightly associated with disease including cancer.


Role of the SCF^Grr1 E3 Ubiquitin Ligase in Nutrient Uptake and Cell Proliferation.  The role of ubiquitylation is pervasive in biological regulatory mechanisms and defects in that process are associated with numerous human diseases.  Ubiquitin-mediated proteolysis, as well as non-proteolytic processes involving ubiquitylation, is mediated by an enzymatic cascade that, like ubiquitin itself, is highly conserved throughout eukaryotes. One of the primary determinants of specificity of ubiquitylation occurs at the level of substrate selection.  That process is mediated, in most cases, by the E3 component of the cascade, the ubiquitin ligase. Because of the diversity of proteins regulated by ubiquitylation the family of ubiquitin ligases is large and diverse.#160; Among E3 ligases, the SCF (Skp1-Cullin-F-box protein) family is the largest. The diversity is primarily a consequence of the diversity of F-box proteins. Our laboratory has studied the SCF^Grr1 E3 ubiquitin ligase, a critical element of both nutritional regulation of cell growth (Spielewoy et al, 2004; 2010) and cell cycle control (Lanker et al, 1995; Hsiung et al, 2001) targeting both transcriptional regulators and cyclin proteins for ubiquitylation and degradation. We have undertaken a detailed investigation of the nature of substrate selection and discrimination by the SCF^Grr1 E3 ubiquitin ligase (Lanker et al, 1995; Hsiung et al, 2001; Berset et al, 2002; Spielewoy et al, 2004). The F-box Grr1 mediates the interaction with its substrates, in part, via its leucine-rich repeat (LRR; Fig. 2). Because of its central role in pathways critical for the regulation of cell growth and the diversity of targets that it recognizes, establishing the basis for specificity of Grr1 is expected to be particularly useful in developing an understanding of the manner in which F-box proteins participate in biological regulatory mechanisms.  Elucidation of the mechanisms by which E3 ubiquitin ligases, in general, and LRR-containing F-box proteins, specifically, recognize and regulate protein stability is of critical importance for the development of treatments via drug targeting and other approaches. 

Ribosome Biogenesis in Yeast  (In collaboration with Jamie Williamson, TSRI).  The eukaryotic ribosome biogenesis pathway involves the coordinated high level expression ~100 ribosomal protein (RP) genes, hundreds of ribosome biogenesis factor (RiBi) genes, as well as the multicopy ribosomal RNA (rRNA) genes. While a great deal is known about the inventory of RiBis, and the role of several of these in specific steps in biogenesis, there is a tremendous amount that remains to be discovered about the mechanism of binding of ribosomal proteins, and about the specific roles of RiBis. We have recently initiated studies in collaboration with the laboratory of Jamie Williamson at TSRI to provide a better understanding of pathways for ribosomal protein association during biogenesis and the connection of those pathways to cell growth and proliferation.  One of our main approaches is to utilize stable isotope pulse labeling and quantitative mass spectrometry (QMS) to probe the dynamics of RPs and intermediates in ribosome biogenesis. QMS provides the opportunity to identify the protein composition in each of the intermediate pools, and the rate of synthesis can be derived from the rate of labeling (Fig.3).  Information about the role of RPs and RiBis can be obtained by perturbing the pathway, and observing the resulting changes in the metabolic pools.  Cell growth is intimately related to the biogenesis of the protein synthetic machinery. The impact of defects in ribosome biogenesis factors on cell growth and cell cycle is well documented. However, a systematic approach to understanding these defects has been lacking. We propose to exploit our knowledge of the pathways for ribosome biogenesis to interrogate the relationship between that process, cell growth and cell cycle. 

Selected Publications:

Travesa, A., Kalashnikova, T.I., de Bruin, R.A.M., Cass, S.R., Chahwan, C., Lee, D.E., Lowndes, N., and Wittenberg, C. (2013).  Repression of G1/S transcription is mediated via interaction of the GTB motif of Nrm1 and Whi5 with Swi6. Mol. Cell. Biol. In press.

Travesa, A., Kuo, D., de Bruin, R.A.M., Kalashnikova, T.I., Guaderrama, M., Thai, K., Aslanian, A., Smolka, M.B., Yates, J.R., Ideker, T., and Wittenberg, C. (2012).  DNA replication stress differentially regulates G1/S genes via Rad53-dependent inactivation of Nrm1. EMBO J 31, 1811–1822.

Spielewoy, N., Guaderrama, M., Wohlschlegel, J.A., Ashe, M., Yates, J.R., and C. Wittenberg. (2010).  Npr2, yeast homolog of the human tumor suppressor NPRL2, is a target of Grr1 required for adaptation to growth under suboptimal nutrient conditions. Eukaryotic Cell 9, 592–601.

de Bruin, R., Kalashnikova, T.I., Aslanian, A., Wohlschlegel, J.A., Chahwan, C., Yates, J., 3rd, Russell, P., and Wittenberg, C. (2008).  DNA replication checkpoint promotes G1/S-specific transcription via Cds1-dependent phosphorylation of the MBF-bound repressor, Nrm1. Proc. Natl. Acad. Sci. USA 105, 11230-11235.

de Bruin, R., Kalashnikova, T.I., Chahwan, C., McDonald, W.H., Wohlschlegel, J.A., Yates, J., 3rd, Russell, P., and Wittenberg, C. (2006).  Constraining G1-specific transcription to late G1-phase:  The MBF-associated corepressor Nrm1 acts via negative feedback. Molecular Cell 23, 483-496.

Wittenberg, C., and Reed, S.I. (2005).  Cell cycle-dependent transcription in yeast: promoters, transcription factors, and transcriptomes. Oncogene 24, 2746–2755.

Spielewoy, N., Flick, K., Kalashnikova, T.I., Walker, J.R., and Wittenberg, C.  (2004).  Regulation and recognition of SCF^Grr1 targets in the glucose and amino acid signaling pathways. Mol. Cell. Biol. 24, 8994–9005.

de Bruin, R., McDonald, W.H., Kalashnikova, T.I., Yates, J., 3rd, and Wittenberg, C. (2004).  Cln3 activates G1-specific Transcription via phosphorylation of the SBF-Bound Repressor, Whi5. Cell 117, 887-898.

Berset, C., Griac, P., Tempel, R., La Rue, J., Wittenberg, C., and Lanker, S. (2002).  Transferable domain in the G(1) cyclin Cln2 sufficient to switch degradation of Sic1 from the E3 ubiquitin ligase SCF(Cdc4) to SCF(Grr1). Mol Cell Biol 22, 4463–4476.

Hsiung, Y.G., Chang, H.C., Pellequer, J.L., La Valle, R., Lanker, S., and Wittenberg, C. (2001).  F-box protein Grr1 interacts with phosphorylated targets via the cationic surface of its leucine-rich repeat. Mol Cell Biol 21, 2506–2520.

Lanker, S., Valdivieso, M.H., and Wittenberg, C. (1996).  Rapid Degradation of the G1 Cyclin Cln2 Induced by CDK-Dependent Phosphorylation. Science 271, 1597–1601.