Explore the vast range of titles available.

Infection by retroviruses as HIV-1 requires the stable integration of their genome into the host cells. This process needs the formation of integrase (IN)-viral DNA complexes, called intasomes, and their interaction with the target DNA wrapped around nucleosomes within cell chromatin. To provide new tools to analyze this association and select drugs, we applied the AlphaLISA technology to the complex formed between the prototype foamy virus (PFV) intasome and nucleosome reconstituted on 601 Widom sequence. This system allowed us to monitor the association between both partners and select small molecules that could modulate the intasome/nucleosome association. Using this approach, drugs acting either on the DNA topology within the nucleosome or on the IN/histone tail interactions have been selected. Within these compounds, doxorubicin and histone binders calixarenes were characterized using biochemical, in silico molecular simulations and cellular approaches. These drugs were shown to inhibit both PFV and HIV-1 integration in vitro . Treatment of HIV-1-infected PBMCs with the selected molecules induces a decrease in viral infectivity and blocks the integration process. Thus, in addition to providing new information about intasome-nucleosome interaction determinants, our work also paves the way for further unedited antiviral strategies that target the final step of intasome/chromatin anchoring. In this work, we report the first monitoring of retroviral intasome/nucleosome interaction by AlphaLISA. This is the first description of the AlphaLISA application for large nucleoprotein complexes (>200 kDa) proving that this technology is suitable for molecular characterization and bimolecular inhibitor screening assays using such large complexes. Using this system, we have identified new drugs disrupting or preventing the intasome/nucleosome complex and inhibiting HIV-1 integration both in vitro and in infected cells. This first monitoring of the retroviral/intasome complex should allow the development of multiple applications including the analyses of the influence of cellular partners, the study of additional retroviral intasomes, and the determination of specific interfaces. Our work also provides the technical bases for the screening of larger libraries of drugs targeting specifically these functional nucleoprotein complexes, or additional nucleosome-partner complexes, as well as for their characterization.

Background Stable insertion of the retroviral DNA genome into host chromatin requires the functional association between the intasome (integrase·viral DNA complex) and the nucleosome. The data from the literature suggest that direct protein–protein contacts between integrase and histones may be involved in anchoring the intasome to the nucleosome. Since histone tails are candidates for interactions with the incoming intasomes we have investigated whether they could participate in modulating the nucleosomal integration process. Results We show here that histone tails are required for an optimal association between HIV-1 integrase (IN) and the nucleosome for efficient integration. We also demonstrate direct interactions between IN and the amino-terminal tail of human histone H4 in vitro. Structure/function studies enabled us to identify amino acids in the carboxy-terminal domain of IN that are important for this interaction. Analysis of the nucleosome-binding properties of catalytically active mutated INs confirmed that their ability to engage the nucleosome for integration in vitro was affected. Pseudovirus particles bearing mutations that affect the IN/H4 association also showed impaired replication capacity due to altered integration and re-targeting of their insertion sites toward dynamic regions of the chromatin with lower nucleosome occupancy. Conclusions Collectively, our data support a functional association between HIV-1 IN and histone tails that promotes anchoring of the intasome to nucleosomes and optimal integration into chromatin.

The CCAAT-binding complex (CBC) is a conserved heterotrimeric transcription factor which, in fungi, requires additional regulatory subunits to act on transcription. In the pathogenic yeast Candida glabrata , CBC has a dual role. Together with the Hap4 regulatory subunit, it activates the expression of genes involved in respiration upon growth with non-fermentable carbon sources, while its association with the Yap5 regulatory subunit is required for the activation of iron tolerance genes in response to iron excess. In the present work, we investigated further the interplay between CBC, Hap4 and Yap5. We showed that Yap5 regulation requires a specific Yap Response Element in the promoter of its target gene GRX4 and that the presence of Yap5 considerably strengthens the binding of CBC to the promoters of iron tolerance genes. Chromatin immunoprecipitation (ChIP) and transcriptome experiments showed that Hap4 can also bind these promoters but has no impact on the expression of those genes when Yap5 is present. In the absence of Yap5 however, GRX4 is constitutively regulated by Hap4, similarly to the genes involved in respiration. Our results suggest that the distinction between the two types of CBC targets in C. glabrata is mainly due to the dependency of Yap5 for very specific DNA sequences and to the competition between Hap4 and Yap5 at the promoter of the iron tolerance genes.

The regulation of gene transcription is not simply dependent on the presence or absence of DNA-binding transcription factors that turn genes on or off, but also involves processes determining the ability of transcription factors to gain access to and bind their target DNA. Methylation of DNA cytosine bases leads to the inaccessibility of DNA regulatory elements to their transcription factors by a number of mechanisms. Our understanding of DNA methylation has advanced rapidly in recent years with the identification of an increasingly large number of novel proteins involved in this process. These include methylcytosine-binding proteins as well as additional members of the DNA methyltransferase family. The creation of mice with targeted deletions in a number of genes involved in DNA methylation has further elucidated the functions of many of these proteins. The characterization of complexes that contain proteins known to be involved in DNA methylation has led to the identification of additional proteins, especially those involved in histone deacetylation, indicating that DNA methylation and histone deacetylation very likely act in a synergistic fashion to regulate gene transcription. Finally, the implication of DNA methylation in tumorigenesis and the realization that some congenital diseases are caused by deficiency of proteins involved in DNA methylation has confirmed the importance of this process in regulating gene expression.

Circadian clock genes are regulated by a transcriptional-translational feedback loop. In Arabidopsis, LATE ELONGATED HYPOCOTYL (LHY) and CIRCADIAN CLOCK ASSOCIATED 1 (CCA1) transcripts are highly expressed in the morning. Translated LHY and CCA1 proteins repress the expression of TIMING OF CAB EXPRESSION 1 (TOC1), which peaks in the evening. TOC1 protein induces expression of LHY and CCA1, forming a negative feedback loop which is believed to constitute the oscillatory mechanism of the clock. The rhythmic oscillation of mouse clock genes mPERIOD 1 (mPER1) and mPER2 has been correlated with regular alteration of chromatin structure through histone acetylation/deacetylation. However, little is known about the relationship between the transcriptional activity of Arabidopsis clock genes and their chromatin status. Here, we report that histone H3 acetylation (H3Ac) and H3 lysine 4 tri-methylation (H3K4me3) levels at LHY, CCA1, and TOC1 are positively correlated with the rhythmic transcript levels of these genes, whereas H3K36me2 level shows a negative correlation. Thus, our study suggests rhythmic transcription of Arabidopsis clock genes might be regulated by rhythmic histone modification, and it provides a platform for future identification of clock-controlling histone modifiers.

The compaction level of arrays of nucleosomes may be understood in terms of the balance between the self-repulsion of DNA (principally linker DNA) and countering factors including the ionic strength and composition of the medium, the highly basic N termini of the core histones, and linker histones. However, the structural principles that come into play during the transition from a loose chain of nucleosomes to a compact 30-nm chromatin fiber have been difficult to establish, and the arrangement of nucleosomes and linker DNA in condensed chromatin fibers has never been fully resolved. Based on images of the solution conformation of native chromatin and fully defined chromatin arrays obtained by electron cryomicroscopy, we report a linker histone-dependent architectural motif beyond the level of the nucleosome core particle that takes the form of a stem-like organization of the entering and exiting linker DNA segments. DNA completes approximately 1.7 turns on the histone octamer in the presence and absence of linker histone. When linker histone is present, the two linker DNA segments become juxtaposed approximately 8 nm from the nucleosome center and remain apposed for 3-5 nm before diverging. We propose that this stem motif directs the arrangement of nucleosomes and linker DNA within the chromatin fiber, establishing a unique three-dimensional zigzag folding pattern that is conserved during compaction. Such an arrangement with peripherally arranged nucleosomes and internal linker DNA segments is fully consistent with observations in intact nuclei and also allows dramatic changes in compaction level to occur without a concomitant change in topology

Some genetic studies indicate that plant homologues of proteins involved in chromatin modification and remodeling in other organisms may regulate plant development. Previously, we described an Arabidopsis mutant with altered cold-responsive gene expression (acg1) displaying a late flowering phenotype, a null allele of fve. FVE is a homologue of the mammalian retinoblastoma-associated protein (RbAp), one component of a histone deacetylase (HDAC) complex involved in transcriptional repression, and has been shown to be involved in the deacetylation of the FLOWERING LOCUS C (FLC) chromatin encoding for a repressor of flowering. In an effort to gain insight into the biochemical functions of FVE, we overexpressed FVE tagged with the hemagglutinin (HA) and FLAG epitope at the N-terminus in acg1 mutants. The results of physiological and molecular analyses demonstrated that FVE overexpression in acg1 rescued the mutant phenotypes, including late flowering and alterations in floral pathway gene expression such as FLC, SUPPRESSOR OF OVER-EXPRESSION OF CO1 (SOC1), and FLOWERING LOCUS T (FT), and also super-induced cold-responsive reporter gene expression. The chromatin immunoprecipitation experiments revealed the amplification of specific DNA regions of FLC and COLD-REGULATED 15A (COR15A), indicating that FVE may bind to the FLC and COR15A chromatin. Gel-filtration chromatography and the immunoprecipitation of putative FVE complexes showed that FVE forms a protein complex of approximately 1.0 MDa. These results demonstrate that FVE may exist as a multiprotein complex, similar to the mammalian HDAC complex harboring RbAp, to regulate flowering time and cold response by associating with the FLC and COR chromatin.

Using a simple geometric model, we propose a general method for computing the linking number of the DNA embedded in chromatin fibers. The relevance of the method is reviewed through the single molecule experiments that have been performed in vitro with magnetic tweezers. We compute the linking number of the DNA in the manifold conformational states of the nucleosome which have been evidenced in these experiments and discuss the functional dynamics of chromosomes in the light of these manifold states.

For eukaryotes, fine tuning of gene expression is necessary to coordinate complex genetic information. Recent studies have shown that noncoding RNAs (ncRNAs) play central roles in this process. For example, ncRNAs participate in multiple diverse functions such as mRNA degradation, epigenetic regulation and alternative splicing. The findings regarding this new player in gene regulation suggest that the mechanism of gene regulation is much more complicated and subtle than previously thought. In this review, new findings concerning the role of ncRNAs in gene regulation are discussed.

Over the centuries, bio-sourced compounds isolated from plants, insects and microorganisms have been a potent source of drugs for the treatment of human diseases. In this review, we recapitulate the story of one of these compounds, 2-propylpentanoic acid, derived from the Valeriana officinalis flowering plant and its path to validation as a cancer treatment.