Uncovering the Histone Interactome of Cat Eye Syndrome Critical Region 2 (CECR2) Bromodomain

Sahoo, Snehasudha Subhadarsini (2022) Uncovering the Histone Interactome of Cat Eye Syndrome Critical Region 2 (CECR2) Bromodomain. Masters thesis, Indian Institute of Science Education and Research Kolkata.

Text (MS dissertation of Snehasudha Subhadarsini Sahoo (17MS198)) 17MS198_Thesis_file.pdf - Submitted Version Restricted to Repository staff only Download (10MB)

Abstract

Cat Eye Syndrome Critical Region Protein 2 (CECR2) is a multi-domain transcription factor, that is part of the CERF chromatin remodelling complex. It has been linked to several key developmental processes and transcription regulation. Among its multiple domains, the bromodomain motif, which recognises acetylated lysine residues, is of particular interest as it has been reported to act as both a tumour suppressor and tumour repressor. However, the exact molecular mechanisms, i.e, the exact acetylated lysines recognised by the CECR2 bromodomain remain unelucidated. Structural analysis of the CECR2 bromodomain further suggests that the CECR2 BRD has the potential to recognise more acylated lysine residues than other bromodomain-containing proteins. However, the lack of extensive studies involving CECR2 BRD’s interactome has prevented us from making any conclusions as of yet. In light of the same, we have designed a multidisciplinary project, to uncover the histone interactome of CECR2 BRD, using both synthesized and endogenous histones as the test subjects. Potential acetylated histone marks recognized and bound by CECR2 BRD were first identified using a molecular docking approach and the validity of these binding interactions was then screened through high throughput fluorescence polarization assays. The binding affinity of these acetylated histone marks for CECR2 BRD was subsequently quantified using Isothermal Titration Calorimetry (ITC) experiments. Ni-NTA enrichment assays of endogenous histones with CECR2 BRD proteins were also undertaken to study if there is any discrimination between synthesized histone peptides and endogenous histones. Finally, pocket residues potentially interacting with the neutralised lysine residue were mutated to confirm if they were indeed crucial for acetyllysine recognition and binding. By the end of this project, we were able to identify novel acetyl lysine marks recognized by CECR2 BRD and were more interestingly, able to observe a trend in their binding affinity. CECR2 BRD shows strongest binding affinity for H4K5acK12ac, followed by H4K5ac, H4K8ac, H4K16ac, H4K12ac, H4Kac4 and weak affinity for H4K5acK8ac and acetylated lysine residues on other histone tails. Thus, this study provides us with key insights into potential factors influencing molecular interactions between CECR2 BRD and its binding histone marks, which might help us understand the divergent roles of CECR2 BRD in the context of cancer.

Actions (login required)

View Item