No abstract

SUMMARY Linker histones associate with nucleosomes to promote the formation of higher-order chromatin structure, but the underlying molecular details are unclear. We investigated the structure of a 197 base-pair nucleosome bearing symmetric 25 base-pair linker DNA arms in complex with vertebrate linker histone H1. We determined electron cryo-microscopy (cryo-EM) and crystal structures of unbound and H1-bound nucleosomes and validated these structures by site-directed protein cross-linking and hydroxyl radical footprinting experiments. Histone H1 shifts the conformational landscape of the nucleosome by drawing the two linkers together and reducing their flexibility. The H1 C-terminal domain (CTD) localizes primarily to a single linker, while the H1 globular domain contacts the nucleosome dyad and both linkers, associating more closely with the CTD-distal linker. These findings reveal that H1 imparts a strong degree of asymmetry to the nucleosome, which is likely to influence the assembly and architecture of higher-order structures.

BackgroundGenetic studies have provided ample evidence of the influence of non-coding DNA polymorphisms on trait variance, particularly those occurring within transcription factor binding sites. Protein binding microarrays and other platforms that can map these sites with great precision have enhanced our understanding of how a single nucleotide polymorphism can alter binding potential within an in vitro setting, allowing for greater predictive capability of its effect on a transcription factor binding site.ResultsWe have used protein binding microarrays and electrophoretic mobility shift assay-sequencing (EMSA-Seq), a deep sequencing based method we developed to analyze nine distinct human NF-κB dimers. This family of transcription factors is one of the most extensively studied, but our understanding of its DNA binding preferences has been limited to the originally described consensus motif, GGRRNNYYCC. We highlight differences between NF-κB family members and also put under the spotlight non-canonical motifs that have so far received little attention. We utilize our data to interpret the binding of transcription factors between individuals across 1,405 genomic regions laden with single nucleotide polymorphisms. We also associated binding correlations made using our data with risk alleles of disease and demonstrate its utility as a tool for functional studies of single nucleotide polymorphisms in regulatory regions.ConclusionsNF-κB dimers bind specifically to non-canonical motifs and these can be found within genomic regions in which a canonical motif is not evident. Binding affinity data generated with these different motifs can be used in conjunction with data from chromatin immunoprecipitation-sequencing (ChIP-Seq) to enable allele-specific analyses of expression and transcription factor-DNA interactions on a genome-wide scale.

CENP-A is a histone variant, which replaces histone H3 at centromeres and confers unique properties to centromeric chromatin. The crystal structure of CENP-A nucleosome suggests flexible nucleosomal DNA ends, but their dynamics in solution remains elusive and their implication in centromere function is unknown. Using electron cryo-microscopy, we determined the dynamic solution properties of the CENP-A nucleosome. Our biochemical, proteomic, and genetic data reveal that higher flexibility of DNA ends impairs histone H1 binding to the CENP-A nucleosome. Substituting the 2-turn αN-helix of CENP-A with the 3-turn αN-helix of H3 results in compact particles with rigidified DNA ends, able to bind histone H1. In vivo replacement of CENP-A with H3-CENP-A hybrid nucleosomes leads to H1 recruitment, delocalization of kinetochore proteins, and significant mitotic and cytokinesis defects. Our data reveal that the evolutionarily conserved flexible ends of the CENP-A nucleosomes are essential to ensure the fidelity of the mitotic pathway.

NF-κB is a key transcription factor regulating the expression of inflammatory responsive genes. How NF-κB binds to naked DNA templates is well documented, but how it interacts with chromatin is far from being clear. Here we used a combination of UV laser footprinting, hydroxyl footprinting and electrophoretic mobility shift assay to investigate the binding of NF-κB to nucleosomal templates. We show that NF-κB p50 homodimer is able to bind to its recognition sequence, when it is localized at the edge of the core particle, but not when the recognition sequence is at the interior of the nucleosome. Remodeling of the nucleosome by the chromatin remodeling machine RSC was not sufficient to allow binding of NF-κB to its recognition sequence located in the vicinity of the nucleosome dyad, but RSC-induced histone octamer sliding allowed clearly detectable binding of NF-κB with the slid particle. Importantly, nucleosome dilution-driven removal of H2A–H2B dimer led to complete accessibility of the site located close to the dyad to NF-κB. Finally, we found that NF-κB was able to displace histone H1 and prevent its binding to nucleosome. These data provide important insight on the role of chromatin structure in the regulation of transcription of NF-κB dependent genes.

FACT, in addition to its role in transcription, is likely implicated in both transcription-coupled nucleotide excision repair and DNA double strand break repair. Here, we present evidence that FACT could be directly involved in Base Excision Repair and elucidate the chromatin remodeling mechanisms of FACT during BER. We found that, upon oxidative stress, FACT is released from transcription related protein complexes to get associated with repair proteins and chromatin remodelers from the SWI/SNF family. We also showed the rapid recruitment of FACT to the site of damage, coincident with the glycosylase OGG1, upon the local generation of oxidized DNA. Interestingly, FACT facilitates uracil-DNA glycosylase in the removal of uracil from nucleosomal DNA thanks to an enhancement in the remodeling activity of RSC. This discloses a novel property of FACT wherein it has a co-remodeling activity and strongly enhances the remodeling capacity of the chromatin remodelers. Altogether, our data suggest that FACT may acts in concert with RSC to facilitate excision of DNA lesions during the initial step of BER.

Thyroid hormones control various aspects of gut development and homeostasis. The best-known example is in gastrointestinal tract remodeling during amphibian metamorphosis. It is well documented that these hormones act via the TR nuclear receptors, which are hormone-modulated transcription factors. Several studies have shown that thyroid hormones regulate the expression of several genes in the Notch signaling pathway, indicating a possible means by which they participate in the control of gut physiology. However, the mechanisms and biological significance of this control have remained unexplored. Using multiple in vivo and in vitro approaches, we show that thyroid hormones positively regulate Notch activity through the TRα1 receptor. From a molecular point of view, TRα1 indirectly controls Notch1, Dll1, Dll4 and Hes1 expression but acts as a direct transcriptional regulator of the Jag1 gene by binding to a responsive element in the Jag1 promoter. Our findings show that the TRα1 nuclear receptor plays a key role in intestinal crypt progenitor/stem cell biology by controlling the Notch pathway and hence the balance between cell proliferation and cell differentiation.

Intestinal homeostasis results from complex cross-regulation of signaling pathways; their alteration induces intestinal tumorigenesis. Previously, we found that the thyroid hormone nuclear receptor TRα1 activates and synergizes with the WNT pathway, inducing crypt cell proliferation and promoting tumorigenesis. Here, we investigated the mechanisms and implications of the cross-regulation between these two pathways in gut tumorigenesis in vivo and in vitro . We analyzed TRα1 and WNT target gene expression in healthy mucosae and tumors from mice overexpressing TRα1 in the intestinal epithelium in a WNT-activated genetic background ( vil -TRα1/Apc mice). Interestingly, increased levels of β-catenin/Tcf4 complex in tumors from vil -TRα1/Apc mice blocked TRα1 transcriptional activity. This observation was confirmed in Caco2 cells, in which TRα1 functionality on a luciferase reporter-assay was reduced by the overexpression of β-catenin/Tcf4. Moreover, TRα1 physically interacted with β-catenin/Tcf4 in the nuclei of these cells. Using molecular approaches, we demonstrated that the binding of TRα1 to its DNA target sequences within the tumors was impaired, while it was newly recruited to WNT target genes. In conclusion, our observations strongly suggest that increased β-catenin/Tcf4 levels i) correlated with reduced TRα1 transcriptional activity on its target genes and, ii) were likely responsible for the shift of TRα1 binding on WNT targets. Together, these data suggest a novel mechanism for the tumor-promoting activity of the TRα1 nuclear receptor.