Baughman, H. E. R. | Search bioRxiv

bioRxiv2024

The Interaction of NF-κB Transcription Factor with Centromeric Chromatin

Centromeric chromatin is a subset of chromatin structure and governs chromosome segregation. The centromere is composed of both CENP-A nucleosomes (CENP-Anuc) and H3 nucleosomes (H3nuc) and is enriched with alpha-satellite (-sat) DNA repeats. These CENP-Anuc have a different structure than H3nuc, decreasing the base pairs (bp) of wrapped DNA from 147 bp for H3nuc to 121 bp for CENP-Anuc. All these factors can contribute to centromere function. We investigated the interaction of H3nuc and CENP-Anuc with NF-{kappa}B, a crucial transcription factor in regulating immune response and inflammation. We utilized Atomic Force Microscopy (AFM) to characterize complexes of both types of nucleosomes with NF-{kappa}B. We found that NF-{kappa}B unravels H3nuc, removing more than 20 bp of DNA, and that NF-{kappa}B binds to the nucleosomal core. Similar results were obtained for the truncated variant of NF-{kappa}B comprised only of the Rel Homology domain and missing the transcription activation domain (TAD), suggesting the RelA TAD is not critical in unraveling H3nuc. By contrast, NF-{kappa}B did not bind to or unravel CENP- Anuc. These findings with different affinities for two types of nucleosomes to NF-{kappa}B may have implications for understanding the mechanisms of gene expression in bulk and centromere chromatin.

biophysics↗

bioRxiv2022

An intrinsically disordered transcription activation domain alters the DNA binding affinity and specificity of NFκB p50/RelA

Many transcription factors contain intrinsically disordered transcription activation domains (TADs), which mediate interactions with co-activators to activate transcription. Historically, DNA-binding domains and TADs have been considered as modular units, but recent studies have shown that TADs can influence DNA binding. We biophysically characterized the NF{kappa}B p50/RelA heterodimer including the RelA TAD and investigated the TADs influence on NF{kappa}B-DNA interactions. In solution the RelA TAD is disordered but compact, with helical tendency in two regions that interact with co-activators. The presence of the TAD increased the stoichiometry of NF{kappa}B-DNA complexes containing promoter DNA sequences with tandem {kappa}B recognition motifs by promoting the binding of NF{kappa}B dimers in excess of the number of {kappa}B sites. We measured the binding affinity of p50/RelA for DNA containing tandem {kappa}B sites and single {kappa}B sites. While the presence of the TAD enhanced the binding affinity of p50/RelA for all {kappa}B sequences tested, it increased the affinity for non-specific DNA sequences by over 10-fold, leading to an overall decrease in specificity for {kappa}B DNA sequences. Our results reveal a novel function of the RelA TAD in promoting binding to non-consensus DNA previously observed by in vivo studies of NF{kappa}B-DNA binding in response to strong inflammatory signals.

biochemistry↗