Nonshivering thermogenesis capacity associated to mitochondrial DNA haplotypes and gender in the greater white-toothed shrew, Crocidura russula.

A selection gradient was recently suggested as one possible cause for a clinal distribution of mitochondrial DNA (mtDNA) haplotypes along an altitudinal transect in the greater white-toothed shrew, Crocidura russula (Ehinger et al. 2002). One mtDNA haplotype (H1) rare in lowland, became widespread when approaching the altitudinal margin of the distribution. As H1 differs from the main lowland haplotype by several nonsynonymous mutations (including on ATP6), and as mitochondria play a crucial role in metabolism and thermogenesis, distribution patterns might stem from differences in the thermogenic capacity of different mtDNA haplotypes. In order to test this hypothesis, we measured the nonshivering thermogenesis (NST) associated with different mtDNA haplotypes. Sixty-two shrews, half of which had the H1 haplotype, were acclimated in November at semioutdoor conditions and measured for NST throughout winter. Our results showed the crucial role of NST for winter survival in C. russula. The individuals that survived winter displayed a higher significant increase in NST during acclimation, associated with a significant gain in body mass, presumably from brown fat accumulation. The NST capacity (ratio of NST to basal metabolic rate) was exceptionally high for such a small species. NST was significantly affected by a gender x haplotype interaction after winter-acclimation: females bearing the H1 haplotype displayed a better thermogenesis at the onset of the breeding season, while the reverse was true for males. Altogether, our results suggest a sexually antagonistic cyto-nuclear selection on thermogenesis.

The E3-Ubiquitin Ligase TRIM50 Interacts with HDAC6 and p62, and Promotes the Sequestration and Clearance of Ubiquitinated Proteins into the Aggresome.

In this study we report that, in response to proteasome inhibition, the E3-Ubiquitin ligase TRIM50 localizes to and promotes the recruitment and aggregation of polyubiquitinated proteins to the aggresome. Using Hdac6-deficient mouse embryo fibroblasts (MEF) we show that this localization is mediated by the histone deacetylase 6, HDAC6. Whereas Trim50-deficient MEFs allow pinpointing that the TRIM50 ubiquitin-ligase regulates the clearance of polyubiquitinated proteins localized to the aggresome. Finally we demonstrate that TRIM50 colocalizes, interacts with and increases the level of p62, a multifunctional adaptor protein implicated in various cellular processes including the autophagy clearance of polyubiquitinated protein aggregates. We speculate that when the proteasome activity is impaired, TRIM50 fails to drive its substrates to the proteasome-mediated degradation, and promotes their storage in the aggresome for successive clearance.

Identification and characterization of the members of the pho1 gene family in moss and rice

Inorganic phosphate (Pi) is one of the main nutrients limiting plant growth anddevelopment in many agro-ecosystems. In plants, phosphate is acquired from the soil by theroots, and is then transferred to the shoot via the xylem. In the model plant Arabidopsisthaliana, PHO1 was previously identified as being involved in loading Pi into the xylem ofroots. AtPHO1, belongs to a multigenic family composed of 10 additional members, namelyAtPHO1;H1 to AtPHO1;10. In this study, we aimed at further investigating the role of thePHO1 gene family in Pi homeostasis in plants, and to this end we isolated and characterizedthe PHO1 members of two main model plants, the moss Physcomitrella patens and the riceOryza sativa.In the bryophyte P. patens, bioinformatic analyses revealed the presence of seven AtPHO1homologues, highly similar to AtPHO1. The seven moss PHO1 genes, namely PpPHO1;1 toPpPHO1;7 appeared to be differentially regulated, both at the tissue level and in response toPi status. However only PpPHO1;1 and PpPHO1;7 were specifically up-regulated upon Pistarvation, suggesting a potential role in Pi homeostasis. We also characterized the responseof P. patens to Pi starvation, showing that higher and lower plants share some commonstrategies to adapt to Pi-deficiency.In the second part, focusing on the monocotyledon rice, we showed the existence of threePHO1 homologues OsPHO1;1 to OsPHO1;3, with the unique particularity of each havingNatural Antisense Transcripts (NATs). Molecular analyses revealed that both the sense andthe antisense OsPHO1;2 transcripts were by far the most abundantly expressed transcripts ofthe family, preferentially expressed in the roots. The stable expression of OsPHO1;2 in allconditions tested, in opposition with the highly induced antisense transcript upon Pistarvation, suggest a putative role for the antisense in regulating the sense transcript.Moreover, mutant analyses revealed that OsPHO1;2 plays a key role in Pi homeostasis, intransferring Pi from the root to the shoot. Finally, complementing the pho1 mutant inArabidopsis, characterized by low Pi in the shoot and reduced growth, with the riceOsPHO1;2 gene revealed a new role for PHO1 in Pi signaling. Indeed, the complementedplants showed normal growth, with however low Pi content.

Structural variation-associated expression changes are paralleled by chromatin architecture modifications.

Copy number variants (CNVs) influence the expression of genes that map not only within the rearrangement, but also to its flanks. To assess the possible mechanism(s) underlying this "neighboring effect", we compared intrachromosomal interactions and histone modifications in cell lines of patients affected by genomic disorders and control individuals. Using chromosome conformation capture (4C-seq), we observed that a set of genes flanking the Williams-Beuren Syndrome critical region (WBSCR) were often looping together. The newly identified interacting genes include AUTS2, mutations of which are associated with autism and intellectual disabilities. Deletion of the WBSCR disrupts the expression of this group of flanking genes, as well as long-range interactions between them and the rearranged interval. We also pinpointed concomitant changes in histone modifications between samples. We conclude that large genomic rearrangements can lead to chromatin conformation changes that extend far away from the structural variant, thereby possibly modulating expression globally and modifying the phenotype. GEO SERIES ACCESSION NUMBER: GSE33784, GSE33867.

Changes in ubiquitin immunoreactivity in developing rat brain: a putative role for ubiquitin and ubiquitin conjugates in dendrite outgrowth and differentiation.

The role of ubiquitin in development of the mammalian brain has been studied using a monoclonal antibody, RHUb1, specific for ubiquitin. Immunodevelopment of western blots of homogenate samples of the cerebral cortex, hippocampus and cerebellum prepared from animals of known postnatal age show marked developmental changes in conjugate level. Striking decreases in the level of a prominent conjugate of molecular weight 22,000, which is identified as ubiquitinated histone, are observed during the first postnatal week in the cerebral cortex and hippocampus, but not the cerebellum. A marked overall developmental decrease in the level of high-molecular-weight (> 40,000) ubiquitin conjugates which occurs predominantly during the third, but also the fourth, postnatal week is observed in all three regions. Immunocytochemical data obtained with the RHUb1 antibody show intense staining of neuronal perikarya, nuclei and dendrites in early postnatal cerebral cortex and hippocampus. Staining of pyramidal cell perikarya and dendrites is particularly prominent. The intensity of dendritic staining, particularly for the cerebral cortex, shows a striking decrease after postnatal day 14 and only faint dendritic staining is observed in the adult. In early postnatal cerebellum, immunoreactivity is predominantly nuclear, though some staining of the proximal regions of Purkinje cell dendrites is observed between postnatal days 4 and 19. As with the cerebral cortex and hippocampus, most of the ubiquitin reactivity is lost in adult animals. The loss of dendritic staining, particularly in the cerebral cortex, correlates with the decrease in the level of high-molecular-weight ubiquitin conjugates observed on the western blots. Immunodevelopment of western blots of a range of subcellular fractions prepared from developing rat forebrain shows that the developmental decrease in the level of high-molecular-weight ubiquitin conjugates is not uniform for all fractions. The decrease in conjugate level is most marked for the cell-soluble, mitochondrial and detergent-insoluble cytoskeletal fractions. Taken overall, the data suggest a role for ubiquitin in dendrite outgrowth and arborization, loss of dendritic ubiquitin immunoreactivity correlating with completion of these processes.

PPARβ Interprets a Chromatin Signature of Pluripotency to Promote Embryonic Differentiation at Gastrulation.

Epigenetic post-transcriptional modifications of histone tails are thought to help in coordinating gene expression during development. An epigenetic signature is set in pluripotent cells and interpreted later at the onset of differentiation. In pluripotent cells, epigenetic marks normally associated with active genes (H3K4me3) and with silent genes (H3K27me3) atypically co-occupy chromatin regions surrounding the promoters of important developmental genes. However, it is unclear how these epigenetic marks are recognized when cell differentiation starts and what precise role they play. Here, we report the essential role of the nuclear receptor peroxisome proliferator-activated receptor β (PPARβ, NR1C2) in Xenopus laevis early development. By combining loss-of-function approaches, large throughput transcript expression analysis by the mean of RNA-seq and intensive chromatin immunoprecipitation experiments, we unveil an important cooperation between epigenetic marks and PPARβ. During Xenopus laevis gastrulation PPARβ recognizes H3K27me3 marks that have been deposited earlier at the pluripotent stage to activate early differentiation genes. Thus, PPARβis the first identified transcription factor that interprets an epigenetic signature of pluripotency, in vivo, during embryonic development. This work paves the way for a better mechanistic understanding of how the activation of hundreds of genes is coordinated during early development.

Association of nuclear matrix proteins with granular and threaded nuclear bodies in cell lines undergoing apoptosis.

The granules which appear in the nucleolar area in apoptotic HL-60 cells after camptothecin administration (Zweyer et al., Exp. Cell Res. 221,27-40, 1995) were detected also in several other cell lines induced to undergo apoptosis by different stimuli, such as MOLT-4 treated with staurosporine, K-562 incubated with actinomycin D, P-815 exposed to temperature causing heat shock, Jurkat cells treated with EGTA, U-937 growing in the presence of cycloheximide and tumor necrosis factor-alpha, and HeLa cells treated with etoposide. Using immunoelectron microscopy techniques, we demonstrate that, besides the already described nuclear matrix proteins p125 and p160, these granules contain other nucleoskeletal polypeptides such as proliferating cell nuclear antigen, a component of ribonucleoprotein particles, a 105-kDa constituent of nuclear spliceosomes, and the 240-kDa nuclear mitotic apparatus-associated protein referred to as NuMA. Moreover, we also found in the granules SAF-A/hn-RNP-U and SATB1 proteins, two polypeptides that have been reported to bind scaffold-associated regions DNA sequences in vitro, thus mediating the formation of looped DNA structures in vivo. Fibrillarin and coilin are not present in these granules or the PML protein. Thus, the granules seen during the apoptotic process apparently are different from coiled bodies or other types of nuclear bodies. Furthermore, these granules do not contain chromatin components such as histones and DNA. Last, Western blotting analysis revealed that nuclear matrix proteins present in the granules are not proteolytically degraded except for the NuMA polypeptide. We propose that these granules might represent aggregates of nuclear matrix proteins forming during the apoptotic process. Moreover, since the granules are present in several cell lines undergoing apoptosis, they could be considered a previously unrecognized morphological hallmark of the apoptotic process.

Mutation spectrum of MLL2 in a cohort of Kabuki syndrome patients.

ABSTRACT: BACKGROUND: Kabuki syndrome (Niikawa-Kuroki syndrome) is a rare, multiple congenital anomalies/mental retardation syndrome characterized by a peculiar face, short stature, skeletal, visceral and dermatoglyphic abnormalities, cardiac anomalies, and immunological defects. Recently mutations in the histone methyl transferase MLL2 gene have been identified as its underlying cause. METHODS: Genomic DNAs were extracted from 62 index patients clinically diagnosed as affected by Kabuki syndrome. Sanger sequencing was performed to analyze the whole coding region of the MLL2 gene including intron-exon junctions. The putative causal and possible functional effect of each nucleotide variant identified was estimated by in silico prediction tools. RESULTS: We identified 45 patients with MLL2 nucleotide variants. 38 out of the 42 variants were never described before. Consistently with previous reports, the majority are nonsense or frameshift mutations predicted to generate a truncated polypeptide. We also identified 3 indel, 7 missense and 3 splice site. CONCLUSIONS: This study emphasizes the relevance of mutational screening of the MLL2 gene among patients diagnosed with Kabuki syndrome. The identification of a large spectrum of MLL2 mutations possibly offers the opportunity to improve the actual knowledge on the clinical basis of this multiple congenital anomalies/mental retardation syndrome, design functional studies to understand the molecular mechanisms underlying this disease, establish genotype-phenotype correlations and improve clinical management.

Fatty acids, eicosanoids, and hypolipidemic agents identified as ligands of peroxisome proliferator-activated receptors by coactivator-dependent receptor ligand assay.

Peroxisome proliferator-activated receptors (PPARs) are nuclear hormone receptors controlling the expression of genes involved in lipid homeostasis. PPARs activate gene transcription in response to a variety of compounds including hypolipidemic drugs as well as natural fatty acids. From the plethora of PPAR activators, Scatchard analysis of receptor-ligand interactions has thus far identified only four ligands. These are the chemotactic agent leukotriene B4 and the hypolipidemic drug Wy 14,643 for the alpha-subtype and a prostaglandin J2 metabolite and synthetic antidiabetic thiazolidinediones for the gamma-subtype. Based on the hypothesis that ligand binding to PPAR would induce interactions of the receptor with transcriptional coactivators, we have developed a novel ligand sensor assay, termed coactivator-dependent receptor ligand assay (CARLA). With CARLA we have screened several natural and synthetic candidate ligands and have identified naturally occurring fatty acids and metabolites as well as hypolipidemic drugs as bona fide ligands of the three PPAR subtypes from Xenopus laevis. Our results suggest that PPARs, by their ability to interact with a number of structurally diverse compounds, have acquired unique ligand-binding properties among the superfamily of nuclear receptors that are compatible with their biological activity.

p21WAF1/Cip1 is a negative transcriptional regulator of Wnt4 expression downstream of Notch1 activation.

In keratinocytes, the cyclin/CDK inhibitor p21(WAF1/Cip1) is a direct transcriptional target of Notch1 activation; loss of either the p21 or Notch1 genes expands stem cell populations and facilitates tumor development. The Notch1 tumor-suppressor function was associated with down-regulation of Wnt signaling. Here, we show that suppression of Wnt signaling by Notch1 activation is mediated, at least in part, by down-modulation of Wnts gene expression. p21 is a negative regulator of Wnts transcription downstream of Notch1 activation, independently of effects on the cell cycle. More specifically, expression of the Wnt4 gene is under negative control of endogenous p21 both in vitro and in vivo. p21 associates with the E2F-1 transcription factor at the Wnt4 promoter and causes curtailed recruitment of c-Myc and p300, and histone hypoacetylation at this promoter. Thus, p21 acts as a selective negative regulator of transcription and links the Notch and Wnt signaling pathways in keratinocyte growth control.

Transcription factor CTF1 acts as a chromatin domain boundary that shields human telomeric genes from silencing.

Telomeres are associated with chromatin-mediated silencing of genes in their vicinity. However, how epigenetic markers mediate mammalian telomeric silencing and whether specific proteins may counteract this effect are not known. We evaluated the ability of CTF1, a DNA- and histone-binding transcription factor, to prevent transgene silencing at human telomeres. CTF1 was found to protect a gene from silencing when its DNA-binding sites were interposed between the gene and the telomeric extremity, while it did not affect a gene adjacent to the telomere. Protein fusions containing the CTF1 histone-binding domain displayed similar activities, while mutants impaired in their ability to interact with the histone did not. Chromatin immunoprecipitation indicated the propagation of a hypoacetylated histone structure to various extents depending on the telomere. The CTF1 fusion protein was found to recruit the H2A.Z histone variant at the telomeric locus and to restore high histone acetylation levels to the insulated telomeric transgene. Histone lysine trimethylations were also increased on the insulated transgene, indicating that these modifications may mediate expression rather than silencing at human telomeres. Overall, these results indicate that transcription factors can act to delimit chromatin domain boundaries at mammalian telomeres, thereby blocking the propagation of a silent chromatin structure.

PPARs: nuclear receptors for fatty acids, eicosanoids, and xenobiotics.

The peroxisome proliferator-activated receptors have enjoyed the spotlight for many reasons. These transcription factors are ligand-inducible nuclear receptors that modulate gene expression in response to a broad spectrum of compounds. The recognition that PPARs are indeed nuclear receptors for polyunsaturated fatty acids, some eicosanoids and also lipid-lowering and antidiabetic drugs, has opened many exciting avenues of research and drug discovery. Recent studies on the PPAR function have extended the role of these transcription factors beyond energy homeostasis to master gene in adipogenesis and also determinants in inflammation control. While rapid advances have been made, it is clear that we are far from a global understanding of the mechanisms and functions of PPARs.

Extracellular histones in tissue injury and inflammation.

Neutrophil NETosis is an important element of host defense as it catapults chromatin out of the cell to trap bacteria, which then are killed, e.g., by the chromatin's histone component. Also, during sterile inflammation TNF-alpha and other mediators trigger NETosis, which elicits cytotoxic effects on host cells. The same mechanism should apply to other forms of regulated necrosis including pyroptosis, necroptosis, ferroptosis, and cyclophilin D-mediated regulated necrosis. Beyond these toxic effects, extracellular histones also trigger thrombus formation and innate immunity by activating Toll-like receptors and the NLRP3 inflammasome. Thereby, extracellular histones contribute to the microvascular complications of sepsis, major trauma, small vessel vasculitis as well as acute liver, kidney, brain, and lung injury. Finally, histones prevent the degradation of extracellular DNA, which promotes autoimmunization, anti-nuclear antibody formation, and autoimmunity in susceptible individuals. Here, we review the current evidence on the pathogenic role of extracellular histones in disease and discuss how to target extracellular histones to improve disease outcomes.

Expression analyses of three members of the AtPHO1 family reveal differential interactions between signaling pathways involved in phosphate deficiency and the responses to auxin, cytokinin, and abscisic acid.

The PHO1 protein is involved in loading inorganic phosphate (Pi) to the root xylem. Ten genes homologous to AtPHO1 are present in the Arabidopsis thaliana (L.) Heyn genome. From this gene family, transcript levels of only AtPHO1, AtPHO1;H1 and AtPHO1;H10 were increased by Pi-deficiency. While the up-regulation of AtPHO1;H1 and AtPHO1;H10 by Pi deficiency followed the same rapid kinetics and was dependent on the PHR1 transcription factor, phosphite only strongly suppressed the expression of AtPHO1;H1 and had a minor effect on AtPHO1;H10. Addition of sucrose was found to increase transcript levels of both AtPHO1 and AtPHO1;H1 in Pi-sufficient or Pi-deficient plants, but to suppress AtPHO1:H10 under the same conditions. Treatments of plants with auxin or cytokinin had contrasting effect depending on the gene and on the Pi status of the plants. Thus, while both hormones down-regulated expression of AtPHO1 independently of the plant Pi status, auxin and cytokinin up-regulated AtPHO1;H1 and AtPHO1;H10 expression in Pi-sufficient plants and down-regulated expression in Pi-deficient plants. Treatments with abscisic acid inhibited AtPHO1 and AtPHO1;H1 expression in both Pi-sufficient and Pi-deficient plants, but increased AtPHO1;H10 expression under the same conditions. The inhibition of expression by abscisic acid of AtPHO1 and AtPHO1;H1, and of the Pi-starvation responsive genes AtPHT1;1 and AtIPS1, was dependant on the ABI1 type 2C protein phosphatase. These results reveal that various levels of cross talk between the signal transduction pathways to Pi, sucrose and phytohormones are involved in the regulation of expression of the three AtPHO1 homologues.

A nucleosome-dependent static loop potentiates estrogen-regulated transcription from the Xenopus vitellogenin B1 promoter in vitro.

We describe the transcriptional potentiation in estrogen responsive transcription extracts of the Xenopus vitellogenin B1 gene promoter through the formation of a positioned nucleosome. Nuclease digestion and hydroxyl radical cleavage indicate that strong, DNA sequence-directed positioning of a nucleosome occurs between -300 and -140 relative to the start site of transcription. Deletion of this DNA sequence abolishes the potentiation of transcription due to nucleosome assembly. The wrapping of DNA around the histone core of the nucleosome positioned between -300 and -140 creates a static loop in which distal estrogen receptor binding sites are brought close to proximal promoter elements. This might facilitate interactions between the trans-acting factors themselves and/or RNA polymerase. Such a nucleosome provides an example of how chromatin structure might have a positive effect on the transcription process.