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department of molecular biosciences, evanston, il 60208

 
 
 
 
 

   
Research

The Radhakrishnan lab is interested in the molecular mechanisms of transcription regulation, specifically, how transcription factors bind DNA and recruit coactivators thereby nucleating protein-protein interaction networks that extend to the transcription machinery, and how chromatin-modifying complexes read, interpret, and modify the histone code. We are asking these questions in the context of (i) cAMP signaling mediated by CREB and its coactivators and (ii) a cohort of related, yet functionally distinct, histone deacetylase (HDAC)-associated chromatin-modifying complexes. Separately, we are also developing software for analyzing experimentally-determined structures of protein-protein/nucleic acid complexes with the goal of automating the process of deciphering the 'rules' of macromolecular associations.

Molecular Mechanisms of Recruitment and Assembly of Sin3/HDAC Corepressor Complexes in Transcriptional Repression

Multiprotein complexes containing chromatin-modifying enzymes play an important role in reading, interpreting, and altering the histone code thereby changing the transcriptional status of specific genetic loci. We are investigating how two functionally distinct forms of the Sin3/HDAC corepressor complex are recruited to specific sites on the genome, how these complexes are assembled, and what role(s) the constitutively-associated subunits of the complex play in effecting specific transcriptional outcomes. These questions bear significance, as not only have multiple components of these complexes been implicated in the genesis and progression of cancers, the key enzymatic components of this complex - the so-called HDACs - are proven targets of cancer therapy.

Phosphorylation-Independent Mechanism of Transcriptional Activation by CREB

The cyclic AMP responsive transcription factor CREB activates transcription via both phosphorylation-dependent and -independent mechanisms. Whereas the phosphorylation-dependent mechanism involving cyclic AMP dependent kinases relies on the recruitment of p300/CBP coactivators and is well characterized, phosphorylation-independent mechanisms of transcriptional activation are poorly understood. Since members of the CRTC/TORC family of coactivators have been recently implicated in the latter pathway, which regulates blood glucose levels during fasting and is relevant to the development of type 2 diabetes, we are seeking to clarify the molecular basis of these interactions with CREB and also with downstream effectors including coactivators and histone acetyltransferases. Since several CRTC/TORC family members are involved in chromosomal translocations that lead to certain cancers, we are also seeking to clarify the precise molecular roles of these aberrant proteins.

Methods for Automated Analysis of Macromolecular Complexes

We had previously developed a web application that detects stabilizing intermolecular interactions in macromolecular complexes from atomic coordinate data. The core software called MONSTER comprises a PERL wrapper that takes advantage of established software in the public domain to validate atomic coordinate files, identify interacting residues, and assign the nature of these interactions. The results are integrated and presented in an intuitive and interactive graphical format. Applications of MONSTER range from mining and validating experimentally-determined structures to guiding mutational analysis. We are presently expanding the scope of MONSTER to guide (i) automated sequence motif identification, (ii) molecular modeling of homologous complexes and (iii) developing a hierarchical classification scheme for macromolecular complexes. A version of the software is available at http://monster.northwestern.edu.

 
 

 

SELECTED PUBLICATIONS

Clark, M.D., Kumar, G.S., Marcum, R., Luo, Q., Zhang, Y., and Radhakrishnan, I. (2015). Molecular basis for the mechanism of constitutive CBP/p300 coactivator recruitment by CRTC1-MAML2 and its implications in cAMP signaling. Biochemistry 54, 5439-5446.

Clark, M.D., Marcum, R., Graveline, R., Chan, C.W., Xie, R. Chen, Z., Ding, Y., Zhang, Y., Mondragón, A., David, G., and Radhakrishnan, I. (2015). Structural insights into the assembly of the histone deacetylase-associated Sin3L/Rpd3L corepressor complex. Proc. Natl. Acad. Sci. USA 112, E3669-E3678.

Xie, T., Zmyslowski, M., Zhang, Y., and Radhakrishnan, I. (2015). Multi-specificity of MRG domains. Structure 23, 1049-1057.

Luo, Q., Viste, K., Zaa, J.C., Kumar, G.S., Tsai, W.W., Talai, A., Mayo, K.E., Montminy, M., and Radhakrishnan, I. (2012). Mechanism of CREB recognition and coactivation by the CREB-regulated transcriptional coactivator CRTC2. Proc. Natl. Acad. Sci. USA 109, 20865-20870.

Kumar, G.S., Chang, W., Xie, T., Patel, A., Zhang, Y., Wang, G.G., David, G., and Radhakrishnan, I. (2012). Sequence requirements for combinatorial recognition of histone H3 by the MRG15 and Pf1 subunits of the Rpd3S/Sin3S corepressor complex. J. Mol. Biol. 422, 519-531.

Xie, T., Graveline, R., Kumar, G.S., Zhang, Y., Krishnan, A., David, G. and Radhakrishnan, I. (2012). Structural basis for molecular interactions involving MRG domains: Implications in chromatin biology. Structure 20, 151-160.

Xie, T., He, Y., Korkeamaki, H., Zhang, Y., Imhoff, R., Lohi, O., and Radhakrishnan, I. (2011). Structure of the 30 kDa Sin3-associated protein (SAP30) in complex with the mammalian Sin3A corepressor and its role in nucleic acid binding. J. Biol. Chem. 286, 27814-27824.

Kumar, G.S., Xie, T., Zhang, Y., and Radhakrishnan, I. (2011). Solution structure of the mSin3A PAH2-Pf1 SID1 Complex: a Mad1/Mxd1-like interaction disrupted by MRG15 in the mammalian Rpd3S/Sin3S complex. J. Mol. Biol. 408, 987-1000.

He, Y., Imhoff, R., Sahu, A., and Radhakrishnan, I. (2009). Solution structure of a novel zinc finger motif in the SAP30 polypeptide of the Sin3 corepressor complex and its potential role in nucleic acid recognition. Nucleic Acids Res. 37, 2142-2152.

Sahu, S.C., Swanson, K.A., Kang, R.S., Huang, K., Brubaker, K., Ratcliff, K., & Radhakrishnan, I. (2008). Conserved themes in target recognition by the PAH1 and PAH2 domains of the Sin3 transcriptional corepressor. J. Mol. Biol. 375, 1444-1456.

Little, T.H., Zhang, Y., Matulis, C.K., Weck, J., Zhang, Z., Ramachandran, A., Mayo, K.E. and Radhakrishnan, I. (2005). Sequence-specific DNA recognition by Steroidogenic Factor 1: A helix at the carboxy-terminus of the DNA binding domain is necessary for complex stability. Mol. Endocrinol. 20, 831-843.

Swanson, K.A., Knoepfler, P.S., Huang, K., Kang, R.S., Cowley, S.M., Laherty, C.D., Eisenman, R.N., and Radhakrishnan, I. (2004). HBP1 and Mad1 repressors bind the Sin3 corepressor PAH2 domain with opposite helical orientations. Nat. Struct. Mol. Biol. 11, 738-746.

Swanson, K.A., Kang, R.S., Stamenova, S.D., Hicke, L., and Radhakrishnan, I. (2003). Solution structure of Vps27 UIM-ubiquitin complex important for endosomal sorting and receptor downregulation. EMBO J. 22, 4597-4606.

Kang, R.S., Daniels, C.M., Francis, S.A., Shih, S.C., Salerno, W.J., Hicke, L., and Radhakrishnan, I. (2003). Solution structure of a CUE-ubiquitin complex reveals a conserved mode of ubiquitin binding. Cell 113, 621-630.

Brubaker, K., Cowley, S.M., Huang, K., Loo, L., Yochum, G.S., Ayer, D.E., Eisenman, R.N., and Radhakrishnan, I. (2000). Solution structure of the interacting domains of the Mad-Sin3 complex: Implications for recruitment of a chromatin-modifying repression complex. Cell 103, 655-665.

Radhakrishnan, I., Pérez-Alvarado, G.C., Parker, D., Dyson, H.J., Montminy, M.R., and Wright, P.E. (1997). Solution structure of the KIX binding domain of CBP bound to the transactivation domain of CREB: A model for activator:coactivator interactions. Cell 91, 741-752.

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