FACT Prevents the Accumulation of Free Histones Evicted from Transcribed Chromatin and a Subsequent Cell Cycle Delay in G1

The FACT complex participates in chromatin assembly and disassembly during transcription elongation. The yeast mutants affected in the SPT16 gene, which encodes one of the FACT subunits, alter the expression of G1 cyclins and exhibit defects in the G1/S transition. Here we show that the dysfunction of chromatin reassembly factors, like FACT or Spt6, down-regulates the expression of the gene encoding the cyclin that modulates the G1 length (CLN3) in START by specifically triggering the repression of its promoter. The G1 delay undergone by spt16 mutants is not mediated by the DNA–damage checkpoint, although the mutation of RAD53, which is otherwise involved in histone degradation, enhances the cell-cycle defects of spt16-197. We reveal how FACT dysfunction triggers an accumulation of free histones evicted from transcribed chromatin. This accumulation is enhanced in a rad53 background and leads to a delay in G1. Consistently, we show that the overexpression of histones in wild-type cells down-regulates CLN3 in START and causes a delay in G1. Our work shows that chromatin reassembly factors are essential players in controlling the free histones potentially released from transcribed chromatin and describes a new cell cycle phenomenon that allows cells to respond to excess histones before starting DNA replication

Nuclear inclusion of nontargeted and chromatin-targeted polystyrene beads and plasmid DNA containing nanoparticles

The nuclear membrane is one of the major cellular barriers in the delivery of plasmid DNA (pDNA). Cell division has a positive influence on the expression efficiency since, at the end of mitosis, pDNA or pDNA containing complexes near the chromatin are probably included by a random process in the nuclei of the daughter cells. However, very little is known about the nuclear inclusion of nanoparticles during cell division. Using the Xenopus nuclear envelope reassembly (XNER) assay, we found that the nuclear enclosure of nanoparticles was dependent on size (with 100 and 200 nm particles being better included than the 500 nm ones) and charge (with positively charged particles being better included than negatively charged cr polyethyleneglycolated (PEGylated) ones) of the beads. Also, coupling chromatin-targeting peptides to the polystyrene beads or pDNA complexes improved their inclusion by 2- to 3-fold. Upon microinjection in living HeLa cells, however, nanoparticles were never observed in the nuclei of cells postdivision but accumulated in a specific perinuclear region, which was identified as the lysosomal compartment. This indicates that nanoparticles can end up in the lysosomes even when they were not delivered through endocytosis. To elucidate if the chromatin binding peptides also have potential in living cells, this additional barrier first has to be tackled, since it prevents free particles from being present near the chromatin at the moment of cell division

A Genome-wide gene-expression analysis and database in transgenic mice during development of amyloid or tau pathology

We provide microarray data comparing genome-wide differential expression and pathology throughout life in four lines of "amyloid" transgenic mice (mutant human APP, PSEN1, or APP/PSEN1) and "TAU" transgenic mice (mutant human MAPT gene). Microarray data were validated by qPCR and by comparison to human studies, including genome-wide association study (GWAS) hits. Immune gene expression correlated tightly with plaques whereas synaptic genes correlated negatively with neurofibrillary tangles. Network analysis of immune gene modules revealed six hub genes in hippocampus of amyloid mice, four in common with cortex. The hippocampal network in TAU mice was similar except that Trem2 had hub status only in amyloid mice. The cortical network of TAU mice was entirely different with more hub genes and few in common with the other networks, suggesting reasons for specificity of cortical dysfunction in FTDP17. This Resource opens up many areas for investigation. All data are available and searchable at http://www.mouseac.org

The histone chaperones Nap1 and Vps75 bind histones H3 and H4 in a tetrameric conformation

Histone chaperones physically interact with histones to direct proper assembly and disassembly of nucleosomes regulating diverse nuclear processes such as DNA replication, promoter remodeling, transcription elongation, DNA damage, and histone variant exchange. Currently, the best-characterized chaperone-histone interaction is that between the ubiquitous chaperone Asf1 and a dimer of H3 and H4. Nucleosome assembly proteins (Nap proteins) represent a distinct class of histone chaperone. Using pulsed electron double resonance (PELDOR) measurements and protein crosslinking, we show that two members of this class, Nap1 and Vps75, bind histones in the tetrameric conformation also observed when they are sequestered within the nucleosome. Furthermore, H3 and H4 trapped in their tetrameric state can be used as substrates in nucleosome assembly and chaperone-mediated lysine acetylation. This alternate mode of histone interaction provides a potential means of maintaining the integrity of the histone tetramer during cycles of nucleosome reassembly

Tousled kinase TLK1B mediates chromatin assembly in conjunction with Asf1 regardless of its kinase activity

<p>Abstract</p> <p>Background</p> <p>The Tousled Like Kinases (TLKs) are involved in chromatin dynamics, including DNA replication and repair, transcription, and chromosome segregation. Indeed, the first two TLK1 substrates were identified as the histone H3 and Asf1 (a histone H3/H4 chaperone), which immediately suggested a function in chromatin remodeling. However, despite the straightforward assumption that TLK1 acts simply by phosphorylating its substrates and hence modifying their activity, TLK1 also acts as a chaperone. In fact, a kinase-dead (KD) mutant of TLK1B is functional in stimulating chromatin assembly in vitro. However, subtle effects of Asf1 phosphorylation are more difficult to probe in chromatin assembly assays. Not until very recently was the Asf1 site phosphorylated by TLK1 identified. This has allowed for probing directly the functionality of a site-directed mutant of Asf1 in chromatin assembly assays.</p> <p>Findings</p> <p>Addition of either wt or non-phosphorylatable mutant Asf1 to nuclear extract stimulates chromatin assembly on a plasmid. Similarly, TLK1B-KD stimulates chromatin assembly and it synergizes in reactions with supplemental Asf1 (wt or non-phosphorylatable mutant).</p> <p>Conclusions</p> <p>Although the actual function of TLKs as mediators of Asf1 activity cannot be easily studied in vivo, particularly since in mammalian cells there are two TLK genes and two Asf1 genes, we were able to study specifically the stimulation of chromatin assembly in vitro. In such assays, clearly the TLK1 kinase activity was not critical, as neither a non-phosphorylatable Asf1 nor use of the TLK1B-KD impaired the stimulation of nucleosome formation.</p

A Microarray study of Carpet-Shell Clam (Ruditapes decussatus) shows common and organ-specific growth-related gene expression Differences in gills and digestive gland

Growth rate is one of the most important traits from the point of view of individual fitness and commercial production in mollusks, but its molecular and physiological basis is poorly known. We have studied differential gene expression related to differences in growth rate in adult individuals of the commercial marine clam Ruditapes decussatus. Gene expression in the gills and the digestive gland was analyzed in 5 fast-growing and five slow-growing animals by means of an oligonucleotide microarray containing 14,003 probes. A total of 356 differentially expressed genes (DEG) were found. We tested the hypothesis that differential expression might be concentrated at the growth control gene core (GCGC), i. e., the set of genes that underlie the molecular mechanisms of genetic control of tissue and organ growth and body size, as demonstrated in model organisms. The GCGC includes the genes coding for enzymes of the insulin/ insulin-like growth factor signaling pathway (IIS), enzymes of four additional signaling pathways (Raf/ Ras/ Mapk, Jnk, TOR, and Hippo), and transcription factors acting at the end of those pathways. Only two out of 97 GCGC genes present in themicroarray showed differential expression, indicating a very little contribution of GCGC genes to growth-related differential gene expression. Forty eight DEGs were shared by both organs, with gene ontology (GO) annotations corresponding to transcription regulation, RNA splicing, sugar metabolism, protein catabolism, immunity, defense against pathogens, and fatty acid biosynthesis. GO termenrichment tests indicated that genes related to growth regulation, development and morphogenesis, extracellular matrix proteins, and proteolysis were overrepresented in the gills. In the digestive gland overrepresented GO terms referred to gene expression control through chromatin rearrangement, RAS-related small GTPases, glucolysis, and energy metabolism. These analyses suggest a relevant role of, among others, some genes related to the IIS, such as the ParaHox gene Xlox, CCAR and the CCN family of secreted proteins, in the regulation of growth in bivalves.Direccion General de Investigacion Cientifica y Tecnica of the Spanish Government [AGL2010-16743, AGL2013-49144-C3-3-R]; COMPETE Program; Portuguese National Funds [PEst-255 C/MAR/LA0015/2011]; Portuguese FCT [UID/Multi/04326/2013]; Generalitat Valenciana; Ministry of Education, Culture, and Sports of the Spanish Government; Association of European Marine Biology Laboratoriesinfo:eu-repo/semantics/publishedVersio

Interplay between Transcription Factors and the Epigenome: Insight from the Role of RUNX1 in Leukemia

The genome has the ability to respond in a precise and co-ordinated manner to cellular signals. It achieves this through the concerted actions of transcription factors and the chromatin platform, which are targets of the signalling pathways. Our understanding of the molecular mechanisms through which transcription factors and the chromatin landscape each control gene activity has expanded dramatically over recent years, and attention has now turned to understanding the complex, multifaceted interplay between these regulatory layers in normal and disease states. It has become apparent that transcription factors as well as the components and modifiers of the epigenetic machinery are frequent targets of genomic alterations in cancer cells. Through the study of these factors, we can gain unique insight into the dynamic interplay between transcription factors and the epigenome, and how their dysregulation leads to aberrant gene expression programs in cancer. Here we will highlight how these factors normally co-operate to establish and maintain the transcriptional and epigenetic landscape of cells, and how this is reprogrammed in cancer, focusing on the RUNX1 transcription factor and oncogenic derivative RUNX1-ETO in leukemia as paradigms of transcriptional and epigenetic reprogramming

ASF1 Proteins are Involved in UV-induced DNA Damage Repair and are Cell Cycle Regulated by E2F Transcription Factors in Arabidopsis thaliana

ASF1 is a key histone H3/H4 chaperone that participates in a variety of DNA and chromatin-related processes, including DNA repair, where chromatin assembly and disassembly is of primaryrelevance. Information concerning the role of ASF1 proteins in post-UV response in higher plants is currently limited. In Arabidopsis thaliana, an initial analysis of in vivo localization of ASF1A andASF1B indicates that both proteins are mainly expressed in proliferative tissues. In silico promoteranalysis identified ASF1A and ASF1B as potential targets of E2F transcription factors. Theseobservations were experimentally validated, both in vitro by electrophoretic mobility shift assays, and in vivo by chromatin immunoprecipitation assays and expression analysis using transgenic plants with altered levels of different E2F transcription factors. These data suggest that ASF1A and ASF1B are regulated during cell cycle progression through E2F transcription factors. In addition, we found that ASF1A and ASF1B are associated with the UV-B induced DNA damage response in A. thaliana. Transcript levels of ASF1A and ASF1B were increased following a UV-B-treatment. Consistent with a potential role in ultraviolet-B (UV-B) response, RNAi silenced plants of both genes showed increased sensitivity to UV-B compared to wild type plants. Finally, by coimmunoprecipitation analysis, we found that ASF1 physically interacts with N-terminal acetylated histones H3 and H4, and with acetyltransferases of the HAM subfamily, which are known to be involved in cell cycle control and DNA repair, among other functions. Together, here we provide evidence that ASF1A and ASF1B are regulated by cell cycle progression and are involved in DNA repair after UV-B irradiation.Fil: Lario, Luciana Daniela. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Rosario. Centro de Estudios Fotosintéticos y Bioquímicos (i); ArgentinaFil: Gutierrez, Crisanto. Universidad Autónoma de Madrid; EspañaFil: Ramirez Parra, Elena. Universidad Politecnica de Madrid; EspañaFil: Spampinato, Claudia Patricia. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Rosario. Centro de Estudios Fotosintéticos y Bioquímicos (i); ArgentinaFil: Casati, Paula. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Rosario. Centro de Estudios Fotosintéticos y Bioquímicos (i); Argentin

Flexible model-based clustering of mixed binary and continuous data: application to genetic regulation and cancer

Clustering is used widely in ‘omics’ studies and is often tackled with standard methods, e.g. hierarchical clustering. However, the increasing need for integration of multiple data sets leads to a requirement for clustering methods applicable to mixed data types, where the straightforward application of standard methods is not necessarily the best approach. A particularly common problem involves clustering entities characterized by a mixture of binary data (e.g. presence/absence of mutations, binding, motifs and epigenetic marks) and continuous data (e.g. gene expression, protein abundance, metabolite levels). Here we present a generic method based on a probabilistic model for clustering this type of data, and illustrate its application to genetic regulation and the clustering of cancer samples. We show that the resulting clusters lead to useful hypotheses: in the case of genetic regulation these concern regulation of groups of genes by specific sets of transcription factors and in the case of cancer samples combinations of gene mutations are related to patterns of gene expression. The clusters have potential mechanistic significance and in the latter case are significantly linked to survival. The method is available as a stand-alone software package (GNU General Public Licence) from https://github.com/BioToolsLeeds/FlexiCoClusteringPackage.git