Development of a mouse embryonic stem cell model for investigating the functions of the linker histone H1-4.

The linker histone H1 C-terminal domain (CTD) plays a pivotal role in chromatin condensation. De novo frameshift mutations within the CTD coding region of H1.4 have recently been reported to be associated with Rahman syndrome, a neurological disease that causes intellectual disability and overgrowth. To investigate the mechanisms and pathogenesis of Rahman syndrome, we developed a cellular model using murine embryonic stem cells (mESCs) and CRISPR/Cas9 genome engineering. Our engineered mES cells facilitate detailed investigations, such as H1-4 dynamics, immunoprecipitation, and nuclear localization; in addition, we tagged the mutant H1-4 with a photoactivatable GFP (PA-GFP) and an HA tag to facilitate pulldown assays. We anticipate that these engineered cells could also be used for the development of a mouse model to study the in vivo role of the H1-4 protein.

The CENP-A nucleosome: where and when it happens during the inner kinetochore's assembly.

CENP-A is an essential histone variant that replaces the canonical H3 at the centromeres and marks these regions epigenetically. The CENP-A nucleosome is the specific building block of centromeric chromatin, and it is recognized by CENP-C and CENP-N, two components of the constitutive centromere-associated network (CCAN), the first protein layer of the kinetochore. Recent proposals of the yeast and human (h)CCAN structures position the assembly on exposed DNA, suggesting an elusive spatiotemporal recognition. We summarize the data on the structural organization of the CENP-A nucleosome and the binding of CENP-C and CENP-N. The latter posits an apparent contradiction in engaging the CENP-A nucleosome versus the CCAN. We propose a reconciliatory model for the assembly of CCAN on centromeric chromatin.

Capturing Protein-Nucleic Acid Interactions by High-Intensity Laser-Induced Covalent Cross-Linking†.

Interactions of DNA with structural proteins such as histones, regulatory proteins and enzymes play a crucial role in major cellular processes such as transcription, replication and repair. The in vivo mapping and characterization of the binding sites of the involved biomolecules are of primary importance for a better understanding of genomic deployment that is implicated in tissue and developmental stage-specific gene expression regulation. The most powerful and commonly used approach to date is immunoprecipitation of chemically cross-linked chromatin (XChIP) coupled with sequencing analysis (ChIP-seq). While the resolution and the sensitivity of the high-throughput sequencing techniques have been constantly improved, little progress has been achieved in the cross-linking step. Because of its low efficiency, the use of the conventional UVC lamps remains very limited while the formaldehyde method was established as the "gold standard" cross-linking agent. Efficient biphotonic cross-linking of directly interacting nucleic acid-protein complexes by a single short UV laser pulse has been introduced as an innovative technique for overcoming limitations of conventionally used chemical and photochemical approaches. In this survey, the main available methods including the laser approach are critically reviewed for their ability to generate DNA-protein cross-links in vitro model systems and cells.

Interstrand Crosslinking Involving Guanine: A New Major UVC Laser-Induced Biphotonic Oxidatively Generated DNA Damage.

Several classes of oxidatively generated DNA damage including oxidized purine and pyrimidine bases, interstrand base crosslinks and DNA-protein crosslinks have been previously shown to be generated in both isolated DNA and cellular DNA upon exposure to either 266-nm laser irradiation or one-electron oxidants. In this study, we provide evidence that biphotonic ionization of guanine bases by UVC laser irradiation of double-stranded deoxyoligonucleotides in aerated aqueous solutions induces the formation of interstrand crosslinks (ICLs). This is supported by various experiments including sequencing gel analyses of formed photoproducts and effects of UVC laser intensity on their formation. This constitutes a novel example of the diversity of reactions of guanine radical cation that can be generated by various one-electron oxidants including UVC laser biphotonic ionization, direct effect of ionization radiation and type I photosensitizers. However, the exact structure of the interstrand base adducts that is a challenging analytical issue remains to be further established. Examples of relevant biochemical/structural applications of biphotonic induction of ICLs in DNA samples by high-intensity UVC laser pulses are provided.

The Role of Histone Variants in the Epithelial-To-Mesenchymal Transition.

The epithelial-to-mesenchymal transition (EMT) is a physiological process activated during early embryogenesis, which continues to shape tissues and organs later on. It is also hijacked by tumor cells during metastasis. The regulation of EMT has been the focus of many research groups culminating in the last few years and resulting in an elaborate transcriptional network buildup. However, the implication of epigenetic factors in the control of EMT is still in its infancy. Recent discoveries pointed out that histone variants, which are key epigenetic players, appear to be involved in EMT control. This review summarizes the available data on histone variants' function in EMT that would contribute to a better understanding of EMT itself and EMT-related diseases.

Structure and Dynamics of a 197 bp Nucleosome in Complex with Linker Histone H1.

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 bp nucleosome bearing symmetric 25 bp 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.

FACT Assists Base Excision Repair by Boosting the Remodeling Activity of RSC.

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.

The thyroid hormone nuclear receptor TRα1 controls the Notch signaling pathway and cell fate in murine intestine.

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.

Binding of NF-κB to nucleosomes: effect of translational positioning, nucleosome remodeling and linker histone H1.

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.

Evident stabilization of the clinical profile in HIV/AIDS as evaluated in an open label clinical trial using a polyherbal formulation.

BACKGROUND & OBJECTIVES: The complementary and alternative medicines (CAM) have not been systematically evaluated for the management of HIV/AIDS patients. In a prospective, single-site, open-label, non-randomized, controlled, pilot trial, we evaluated a polyherbal formulation (PHF) for its safety and efficacy in treating subjects with HIV-AIDS. METHODS: A total of 32 and 31 subjects were enrolled under the PHF and highly active antiretroviral treatment (HAART) arms, respectively, and followed up for a period of 24 months. Plasma viral RNA, CD4 cell count and blood chemistry were monitored at 3-month intervals. Following mid-term safety evaluation, 12 subjects from the PHF arm were shifted to HAART and were followed separately as PHF-to-HAART arm, for the rest of the period. RESULTS: The HAART arm was characterized by significant improvements in CD4 cell count (154.4 cells/µl/year, P<0.001) and reduction in plasma viral load within 3 to 6 months (-0.431+ 0.004 log 10 IU/month, P<0.001). In contrast, the PHF arm showed a profile of CD4 cell loss at remarkably slower kinetics (14.3 cells/µl/year, P=0.021) and insignificant reduction in the viral load. The PHF and HAART arms did not differ significantly in the occurrence of AIDS-related illnesses over the study period of 24 months. In the PHF-to-HAART arm, the rates of CD4 count and reduction in viral load were significant and comparable to that of the HAART group. In the PHF arm, at 1 month, a significant increase in CD4 cell count and a concomitant decrease in viral load were seen. INTERPRETATION & CONCLUSIONS: The PHF appears to have provided protection by delaying the kinetics of CD4 cell reduction. Given the several study limitations, drawing assertive inferences from the data is challenging. Future studies with a stringent study design are warranted to confirm these findings.

Multi-level interactions between the nuclear receptor TRα1 and the WNT effectors ß-catenin/Tcf4 in the intestinal epithelium.

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 ß-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 ß-catenin/Tcf4. Moreover, TRα1 physically interacted with ß-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 ß-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.

Extensive characterization of NF-κB binding uncovers non-canonical motifs and advances the interpretation of genetic functional traits.

BACKGROUND: Genetic 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. RESULTS: We 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. CONCLUSIONS: NF-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.