Structural and DNA binding properties of ARID domains present in hSWI/SNF chromatin remodeling complex subunits

Abstract

The SWI/SNF complexes are multisubunit-containing protein complexes that are involved in chromatin-remodeling processes in the eukaryotic cells. In higher eukaryotes, SWI/SNF complexes contain one or more mutually exclusive AT-rich interaction domain (ARID)-containing proteins as one of the subunit. The SWI/SNF complexes can be further classified into sub complexes. Association of ARID-containing proteins BAF250a (also known as ARID1a) and BAF250b (also known as ARID1b) with the SWI/SNF complex results in BAF-A and BAF-B complexes respectively, whereas the association of ARID-containing BAF200 (also known as ARID2) results in PBAF complex. BAF250a, BAF250b and BAF200 subunits have an AT-rich interaction domain named as ARID. It has been proposed that BAF250a/b and BAF200 subunits likely recruit SWI/SNF complex through ARID domain to heterochromatin that allows transcriptional activation of normally silenced chromatin, thereby regulating the specific gene expression. The ARID is a conserved, all helical DNA binding domain found in several eukaryotic proteins. ARIDs in proteins such as human modulator recognition factor 2 (Mrf2), Drosophila Dead Ringer (Dri), and murine protein Bright were shown to recognize specific AT-rich DNA sequences. The evidences so far suggest that ARIDs of human SWI/SNF complexes interact with DNA without any sequence preference, therefore questioning the recruitment of SWI/SNF complexes by BAF250a to the target genes via its interaction with specific DNA sequences. The structure and functional annotation of BAF250a/b (> 2200 residues long proteins) remains poorly understood. The folded regions and domain boundaries of these lengthy proteins have not been clearly defined. Likewise, their DNA-binding specificities have not been studied systematically. With this background, we proposed to study the structure and DNA binding specificities of ARID domains in BAF250a, BAF250b, and BAF200 in this thesis. First, we have defined the domain architecture of BAF250a/ reveals the ARID and ARM-repeat domains with implication in function and assembly of the BAF remodeling complex. Next, systematically we have defined the domain boundary and DNA binding specificities of BAF250a ARID. Using NMR spectroscopy and ITC methods, our results showed that BAF250a ARID binds to AT rich DNA in a ‘specific’ manner with thermodynamic signatures for a specific DNA binding. NMR CSP driven model of BAF250a ARID – DNA complexes were generated and validated using mutagenesis approach. We have also completed the NMR chemical shift assignment of ARID domains of BAF250b and BAF200. We have generated a CS-Rosetta model structure of BAF200 ARID and currently we are in the process of refining the structure. DNA binding properties of BAF250b ARID have been studied using NMR and ITC methods. NMR CSP driven HADDOCK models of BAF250b ARID/DNA complexes suggest plausible mode of specific complex formation via DNA major groove recognition by helix H5 and minor groove interactions by loop L1. A temperature independent DNA binding thermodynamics was observed for BAF250b ARID unlike BAF250a ARID though these two proteins share > than 80% identity in their sequences. The reason of such differences may be due to the small and subtle deviations in loop L1 region. In case of BAF200 ARID, we have some preliminary results on its DNA binding properties obtained from NMR and SPR studies using AT-12 and GC-12 DNA.