Research Projects

Rationale: Post translationally modified eukaryotic chromatin gets leads to context dependent gene expression program(s). Such covalent modification are carried out by ‘Chromatin writers’ giving rise to different chromatin states. Inhibitor of kB Kinase 1 (IKK1 or IKKalpha), a pleotropic Ser/Thr kinase, is one such 'chromatin writer' that phosphorylate Histone H3. IKK1 is essential for the activation of NF-kB. Furthermore, IKK1 plays pivotal roles in organismal well-being in both NF-kB dependent and independent manners. It can exert its decisive roles both in the cytosol and the nucleus, with or without employing its kinase activity. Point mutations in and away from the kinase domain in the CHUK gene encoding IKK1 are known to cause severe immunological abnormalities in humans. Skewed distribution of IKK1’s in the cytosol or nucleus results in opposite pathophysiological outcomes. While the cytosolic roles of IKK1 have been studied in extraordinary details, nuclear roles of this kinase remain poorly understood. Most prominent nuclear function of this kinase has been ascribed to its catalytic activity to phosphorylate Ser10 on H3 (H3 S10). However, in instances like terminal differentiation of keratinocytes catalytic activity of IKK1 is not required though its presence is essential. It is not clear how IKK1 performs this function without exercising the catalytic prowess. This project aims to understand the underlying mechanism(s) of very specific aspects of nuclear functions of IKK1 employing multidisciplinary experimental approaches. Scientific Objectives: The following scientific objectives are set to duly address intriguing nuclear functions of IKK1 1) Deciphering the de novo nucleosome assembly promoting function of IKK1 2) Elucidating the structural basis of nucleosome recognition and assembly by IKK1 3) Assessing the effect of IKK1 on chromatin organization Hypothesis/model to be tested: Nuclear IKK1 interacts with the nucleosome and phosphorylates histone H3 in the chromatin context to function as a chromatin writer. In this proposal we would like to investigate the mechanistic basis of IKK1’s nuclear function as and beyond a chromatin writer at highest possible resolution. Based on our preliminary data, pose the possibility that IKK1 is not just a chromatin writer – it also acts as a nucleosome assembly factor that promotes de novo nucleosome assembly. We would like to decipher the biochemical and structural basis of nucleosome recognition and de novo nucleosome assembly by IKK1, and its impact on chromatin organization. In vitro and cellular assays using mouse and human cells will be used to test our hypothesis. Main experiments: Broad outline of the main experiments are furnished below • Biochemical and biophysical investigation of de novo nucleosome assembly from constituent histones and DNA in presence of IKK1 purified from Sf9 cells. • CryoEM analyses of IKK1’s complex with H2A-H2B, H3-H4 and mononucleosome. • NGS-based ATAC-seq and transcriptomic analyses to assess IKK1’s role in chromatin organization using WT and knockout MEFs or knocking down IKK1 by shRNA in human cells. • Similar NGS-based analyses of IKK1’s role in chromatin organization in response to known stimuli like LTbeta. Significance: It is known that IKK1 is a chromatin writer that phosphorylate H3 S10, like many other kinases. However, no report ever pointed or indicated that it may also act as a de novo nucleosome assembly factor. In fact, no other protein kinase has ever been shown to possess this activity. Our discovery is the first report of such a fundamental function of any eukaryotic kinase ever studied. Establishment of this phenomenon with detailed analyses will entice the community to think differently not only about IKK1 but also about other nuclear kinases. It is a breakthrough discovery with anticipated far reaching consequences in diverse developmental, immunologial and malignant pathologies where IKK1 is implicated.