Transcription Factor NF-κB Unravels Nucleosomes

Stormberg, Tommy; Filliaux, Shaun; Baughman, Hannah E.R.; Komives, Elizabeth A.; Lyubchenko, Yuri L.

doi:10.1101/2021.03.18.436050

Cited by 4 publications

(8 citation statements)

References 23 publications

(34 reference statements)

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“…Although only weak ChIP-Seq peaks are generally detected within the first 10-15 minutes following inflammatory stimulation, widespread nucleosome repositioning has been observed during this time period prior to the appearance of robust ChIP-Seq peaks at 30 minutes (33,34). Consistent with this in vivo result, we recently found that the p50/RelARHD is capable of binding nucleosomes and unraveling nucleosomal DNA in vitro through interactions with both specific κB sequences and nonspecific regions (59). We predict that p50/RelAFL would be even more effective in opening nucleosomes, given its enhanced ability to bind nonspecific DNA sequences.…”

Section: Discussionsupporting

confidence: 76%

“…The p50/RelA RHD , p50/RelA FL , and RelA TAD constructs were lysed by sonication and purified by Ni 2+ -NTA chromatography as described previously for p50/RelA RHD (59). Following overnight dialysis, protein was aliquoted and stored at -80°C.…”

Section: Methodsmentioning

confidence: 99%

“…Prior to experiments, aliquots were thawed and further purified. p50/RelA RHD and p50/RelA FL were purified by cation exchange chromatography (MonoS; GE healthcare) to remove bound nucleic acids, as described previously (59). Protein was further purified by size-exclusion chromatography using a Superdex 200 column (GE healthcare) in SEC buffer (25 mM Tris, 150 mM NaCl, 0.5 mM EDTA, 1 mM DTT, adjusted to pH 7.5 at room temperature).…”

Section: Methodsmentioning

confidence: 99%

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

Baughman

Narang

Chen

et al. 2022

Preprint

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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 NFκB p50/RelA heterodimer including the RelA TAD and investigated the influence of the TAD on NFκB-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 NFκB-DNA complexes containing promoter DNA sequences with tandem κB recognition motifs by promoting the binding of NFκB dimers in excess of the number of κB sites. We measured the binding affinity of p50/RelA for DNA containing tandem κB sites and single κB sites. While the presence of the TAD enhanced the binding affinity of p50/RelA for all κB sequences tested, it increased the affinity for non-specific DNA sequences by over 10-fold, leading to an overall decrease in specificity for κB 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 NFκB-DNA binding in response to strong inflammatory signals.

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Section: Discussionsupporting

confidence: 76%

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

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

Baughman

Narang

Chen

et al. 2022

Preprint

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“…Other pioneer transcription factors, such as Rap1 [55] and NF-κB [56] have also been shown to induce or stabilize open conformations of chromatin fibers or mononucleosomes configurations. Rap1 also uses intrinsic nucleosome and chromatin dynamics to facilitate binding [55].…”

Section: Discussionmentioning

confidence: 99%

OCT4 interprets and enhances nucleosome flexibility

Huertas

Ortmeier

et al. 2021

Preprint

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Pioneer transcription factors induce cellular identity transitions by binding to sites on inaccessible DNA wrapped in nuclear chromatin. They contribute to chromatin opening and recruit other factors to regulatory DNA sites. The structural basis of their interaction with the chromatin structural unit, the nucleosome, is still unresolved. From a combination of experiments and molecular simulations we reveal here how Oct4, the master regulator and inducer of cellular pluripotency, interprets and enhances nucleosome structural flexibility. The magnitude of Oct4 impact on nucleosome dynamics depends on the binding site position and the mobility of unstructured tails of nucleosomal histone proteins. Oct4 propagates and stabilizes open nucleosome conformations by specific sequence recognition and unspecific DNA exploration. Our findings provide a structural basis for the versatility of transcription factors in engaging with nucleosomes and have implications for understanding how pioneer factors induce chromatin dynamics.

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“…The goal of this study was to reveal nanoscale structural characteristics of nucleosomes assembled on the α-satellite sequence. We utilized Atomic Force Microscopy (AFM) with the capability to characterize nucleosomes on the single molecule level with nanometer resolution to address this issue (22)(23)(24)(25)(26). We assembled CENP-A nucleosomes on three different substrates: a substrate containing an α-satellite motif, a substrate containing non-specific DNA and nucleosomes assembled on the Widom 601 positioning motif to determine whether or not CENP-A has distinguishing structural characteristics between α-satellite and other DNA sequences.…”

Section: Introductionmentioning

confidence: 99%

Sequence Dependent Nanoscale Structure of CENP-A Nucleosomes

Stormberg¹,

Lyubchenko²

2022

Preprint

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CENP-A is a histone variant found in high abundance at the centromere. The centromere chromatin similar to the bulk chromatin consist of nucleosomes, but H3 histone of the bulk chromatin nucleosomes (H3 nucleosomes) is replaced with CENP-A histone. Additionally, the centromere comprises tandem repeats of α-satellite DNA which CENP-A nucleosomes assemble upon. However, the effect of the DNA sequence on the nucleosome assembly and centromere formation remains poorly understood. Here we investigated the structure of nucleosomes assembled with the CENP-A variant using Atomic Force Microscopy. We assembled both CENP-A nucleosomes and H3 nucleosomes on a DNA substrate containing an α-satellite motif and characterized their positioning and wrapping efficiency. We also studied CENP-A nucleosomes on the 601-positioning motif and non-specific DNA to compare their relative positioning and stability. CENP-A nucleosomes assembled on α-satellite DNA did not show any positional preference along the substrate, which is similar to H3 nucleosomes and CENP-A nucleosomes on non-specific DNA. The range of nucleosome wrapping efficiency was narrower on α-satellite DNA compared with non-specific DNA, suggesting a more stable complex. These findings indicate that DNA sequence and histone composition may be two of many factors required for accurate centromere assembly.

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Transcription Factor NF-κB Unravels Nucleosomes

Cited by 4 publications

References 23 publications

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

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

OCT4 interprets and enhances nucleosome flexibility

Sequence Dependent Nanoscale Structure of CENP-A Nucleosomes

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