969 resultados para Chromatin.
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In Saccharomyces cerevisiae, TBF1, an essential gene, influences telomere function but also has other roles in the global regulation of transcription. We have identified a new member of the tbf1 gene family in the mammalian pathogen Pneumocystis carinii. We demonstrate by transspecies complementation that its ectopic expression can provide the essential functions of Schizosaccharomyces pombe tbf1 but that there is no rescue between fission and budding yeast orthologues. Our findings indicate that an essential function of this family of proteins has diverged in the budding and fission yeasts and suggest that effects on telomere length or structure are not the primary cause of inviability in S. pombe tbf1 null strains.
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The need for better gene transfer systems towards improved risk=benefit balance for patients remains a major challenge in the clinical translation of gene therapy (GT). We have investigated the improvement of integrating vectors safety in combining (i) new short synthetic genetic insulator elements (GIE) and (ii) directing genetic integration to heterochromatin. We have designed SIN-insulated retrovectors with two candidate GIEs and could identify a specific combination of insulator 2 repeats which translates into best functional activity, high titers and boundary effect in both gammaretro (p20) and lentivectors (DCaro4) (see Duros et al, abstract ibid). Since GIEs are believed to shield the transgenic cassette from inhibitory effects and silencing, DCaro4 has been further tested with chimeric HIV-1 derived integrases which comprise C-ter chromodomains targeting heterochromatin through either histone H3 (ML6chimera) or methylatedCpGislands (ML10). With DCaro4 only and both chimeras, a homogeneous expression is evidenced in over 20% of the cells which is sustained over time. With control lentivectors, less than 2% of cells express GFP as compared to background using a control double-mutant in both catalytic and ledgf binding-sites; in addition, a two-times increase of expression can be induced with histone deacetylase inhibitors. Our approach could significantly reduce integration into open chromatin sensitive sites in stem cells at the time of transduction, a feature which might significantly decrease subsequent genotoxicity, according to X-SCIDs patients data.Work performed with the support of EC-DG research within the FP6-Network of Excellence, CLINIGENE: LSHB-CT-2006-018933
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RNA polymerase III (Pol III) occurs in two versions, one containing the POLR3G subunit and the other the closely related POLR3GL subunit. It is not clear whether these two Pol III forms have the same function, in particular whether they recognize the same target genes. We show that the POLR3G and POLR3GL genes arose from a DNA-based gene duplication, probably in a common ancestor of vertebrates. POLR3G- as well as POLR3GL-containing Pol III are present in cultured cell lines and in normal mouse liver, although the relative amounts of the two forms vary, with the POLR3G-containing Pol III relatively more abundant in dividing cells. Genome-wide chromatin immunoprecipitations followed by high-throughput sequencing (ChIP-seq) reveal that both forms of Pol III occupy the same target genes, in very constant proportions within one cell line, suggesting that the two forms of Pol III have a similar function with regard to specificity for target genes. In contrast, the POLR3G promoter-not the POLR3GL promoter-binds the transcription factor MYC, as do all other promoters of genes encoding Pol III subunits. Thus, the POLR3G/POLR3GL duplication did not lead to neo-functionalization of the gene product (at least with regard to target gene specificity) but rather to neo-functionalization of the transcription units, which acquired different mechanisms of regulation, thus likely affording greater regulation potential to the cell.
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Repression and activation of gene transcription involves multiprotein complexes that modify chromatin structure. The integration of these complexes at regulatory sites can be assisted by co-factors that link them to DNA-bound transcriptional regulators. In humans, one such co-factor is the herpes simplex virus host-cell factor 1 (HCF-1), which is implicated in both activation and repression of transcription. We show here that disruption of the gene encoding the Drosophila melanogaster homolog of HCF-1, dHCF, leads to a pleiotropic phenotype involving lethality, sterility, small size, apoptosis, and morphological defects. In Drosophila, repressed and activated transcriptional states of cell fate-determining genes are maintained throughout development by Polycomb Group (PcG) and Trithorax Group (TrxG) genes, respectively. dHCF mutant flies display morphological phenotypes typical of TrxG mutants and dHCF interacts genetically with both PcG and TrxG genes. Thus, dHCF inactivation enhances the mutant phenotypes of the Pc PcG as well as brm and mor TrxG genes, suggesting that dHCF possesses Enhancer of TrxG and PcG (ETP) properties. Additionally, dHCF interacts with the previously established ETP gene skd. These pleiotropic phenotypes are consistent with broad roles for dHCF in both activation and repression of transcription during fly development.
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The ultrastructure and distribution of gonial and somatic cells in the ovary of Dermatobia hominis was studied during the 3rd larval instar. In larvae weighing between 400 and 500 mg, the ovary is partially divided into basal and apical regions by oblong somatic cells that penetrate from the periphery; these cells show ovoid nucleus and cytoplasm full of microtubules. In both regions, gonial cells with regular outlines, large nucleus and low electron-density cytoplasm are scattered among the interstitial somatic cells. These later cells have small nucleus and electrodense cytoplasm. Clear somatic cells with small nucleus and cytoplasm of very low electron-density are restrict to the apical region of the gonad. Degenerating interstitial somatic cells are seen in the basal portion close to the ovary peduncle. During all this larval period the morphological features of the ovary remain almost the same. At the end of the period there is a gradual deposition of glycogen in the cytoplasm of the somatic cells, increase in the number and density of their mitochondria plus nuclear modification as membrane wrinkling and chromatin condensation in masses.
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BACKGROUND: Regulation of genes transferred to eukaryotic organisms is often limited by the lack of consistent expression levels in all transduced cells, which may result in part from epigenetic gene silencing effects. This reduces the efficacy of ligand-controlled gene switches designed for somatic gene transfers such as gene therapy. METHODS: A doxycycline-controlled transgene was stably introduced in human cells, and clones were screened for epigenetic silencing of the transgene. Various regulatory proteins were targeted to the silent transgene, to identify those that would mediate regulation by doxycycline. RESULTS: A doxycycline-controlled minimal promoter was found to be prone to gene silencing, which prevents activation by a fusion of the bacterial TetR DNA-binding domain with the VP16 activator. DNA modification studies indicated that the silenced transgene adopts a poorly accessible chromatin structure. Several cellular transcriptional activators were found to restore an accessible DNA structure when targeted to the silent transgene, and they cooperated with Tet-VP16 to mediate regulation by doxycycline. CONCLUSIONS: Reversal of the silencing of a tetracycline-regulated minimal promoter requires a chromatin-remodeling activity for subsequent promoter activation by the Tet-VP16 fusion protein. Thus, distinct regulatory elements may be combined to obtain long-term regulation and persistent expression of exogenous genes in eukaryotic cells.
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The Myc proto-oncoproteins are transcription factors that recognize numerous target genes through hexameric DNA sequences called E-boxes. The mechanism by which they then activate the expression of these targets is still under debate. Here, we use an RNAi screen in Drosophila S2 cells to identify Drosophila host cell factor (dHCF) as a novel co-factor for Myc that is functionally required for the activation of a Myc-dependent reporter construct. dHCF is also essential for the full activation of endogenous Myc target genes in S2 cells, and for the ability of Myc to promote growth in vivo. Myc and dHCF physically interact, and they colocalize on common target genes. Furthermore, down-regulation of dHCF-associated histone acetyltransferase and histone methyltransferase complexes in vivo interferes with the Myc biological activities. We therefore propose that dHCF recruits such chromatin-modifying complexes and thereby contributes to the expression of Myc targets and hence to the execution of Myc biological activities.
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SUMMARY Genomic imprinting is an epigenetic mechanism of transcriptional regulation that ensures restriction of expression of a subset of mammalian genes to a single parental allele. The best studied example of imprinted gene regulation is the Igf2/H19 locus, which is also the most commonly altered by loss of imprinting (LOT) in cancer. LOT is associated with numerous hereditary diseases and several childhood, and adult cancers. Differential expression of reciprocal H19 and 1gf2 alleles in somatic cells depends on the methylation status of the imprinting control region (ICR) which regulates binding of CTCF, an ubiquitously expressed 11-zinc finger protein that binds specifically to non-methylated maternal ICR and thereby attenuates expression of Igf2, while it does not bind to methylated paternal ICR, which enables Igf2 expression. Initial ICR methylation occurs during gametogenesis by an as yet unknown mechanism. The accepted hypothesis is that the event of differential maternal and paternal DNA methylation depends on germ-line specific proteins. Our Laboratory identified a novel 11-zinc-finger protein CTCF-T (also known as CTCFL and BORIS) that is uniquely expressed in the male germ-line and is highly homologous within its zinc-finger region with CTCF. The amino-acid sequences flanking the zinc-finger regions of CTCF and CTCF-T have widely diverged, suggesting that though they could bind to the same DNA targets (ICRs) they are likely to have different functions. Interestingly, expression of CTCF-T and CTCF is mutually exclusive; CTCF-T-positive (CTCF-negative) cells occur in the stage of spermatogenesis that coincides with epigenetic reprogramming, including de novo DNA methylation. In our study we demonstrate the role that CTCF-T plays in genomic imprinting. Here we show that CTCF-T binds in vivo to the ICRs of Igf2/H19 and Dlk/Gt12 imprinted genes. In addition, we identified two novel proteins interacting with CTCF-T: a protein arginine methyltransferase PRMT7 and an arginine-rich histone H2A variant that we named trH2A. These interactions were confirmed and show that the two proteins interact with the amino-teiminal region of CTCF-T. Additionally, we show interaction of the amino- terminal region of CTCF-T with histones H1, H2A and H3. These results suggest that CTCF-T is a sequence-specific DNA (ICR) binding protein that associates with histones and recruits PRMT7. Interestingly, PRMT7 has a histone-methyltransferase activity. It has been shown that histone methylation can mark chromatin regions thereby directing DNA-methylation; thus, our hypothesis is that the CTCF-T protein-scaffold directs PRMT7 to methylate histone(s) assembled on ICRs, which marks chromatin for the recruitment of the de novo DNA methyltransferases to methylate DNA. To test this hypothesis, we developed an in vivo DNA-methylation assay using Xenopus laevis' oocytes, where H19 ICR and different expression cDNAs, including CTCF-T, PRMT7 and the de novo DNA methyltransferases (Dnmt3a, Dnmt3b and Dnmt3L) are microinjected into the nucleus. The methylation status of CpGs within the H19 ICR was analysed 48 or 72 hours after injection. Here we demonstrate that CpGs in the ICR are methylated in the presence of both CTCF-T and PRMT7, while control oocytes injected only with ICR did not show any methylation. Additionally, we showed for the first time that Dnmt3L is crucial for the establishment of the imprinting marks on H19 ICR. Moreover, we confirmed that Dnmt3a and Dnmt3b activities are complementary. Our data indicate that all three Dnmt3s are important for efficient de novo DNA methylation. In conclusion, we propose a mechanism for the establishment of de novo imprinting marks during spermatogenesis: the CTCF-T/PRMT7 protein complex directs histone methylation leading to sequence-specific de novo DNA methylation of H19 ICR. RESUME L'empreinte génomique parentale est un mécanisme épigénétique de régulation transcriptionelle qui se traduit par une expression différentielle des deux allèles de certains gènes, en fonction de leur origine parentale. L'exemple le mieux caractérisé de gènes soumis à l'empreinte génomique parentale est le locus Igf2/H19, qui est aussi le plus fréquemment altéré par relaxation d'empreinte (en anglais: loss of imprinting, LOI) dans les cancers. Cette relaxation d'empreinte est aussi associée à de nombreuses maladies héréditaires, ainsi qu'à de nombreux cancers chez l'enfant et l'adulte. Dans les cellules somatiques, les différences d'expression des allèles réciproques H19 et Ig12 est sous le contrôle d'une région ICR (Imprinting Control Region). La méthylation de cette région ICR régule l'ancrage de la protéine à douze doigts de zinc CTCF, qui se lie spécifiquement à l'ICR maternel non-méthylé, atténuant ainsi l'expression de Igf2, alors qu'elle ne s'ancre pas à l'ICR paternel méthyle. Le mécanisme qui accompagne la méthylation initiale de la région ICR durant la gamétogenèse n'a toujours pas été élucidé. L'hypothèse actuelle propose que la différence de méthylation entre l'ADN maternel et paternel résulte de l'expression de protéines propres aux zones germinales. Notre laboratoire a récemment identifié une nouvelle protéine à douze doigts de zinc, CTCF-T (aussi dénommée CTCFL et BORRIS), qui est exprimée uniquement dans les cellules germinales mâles, dont la partie à douze doigts de zinc est fortement homologue à la protéine CTCF. La séquence d'acides aminés de part et d'autre de cette région est quant à elle très divergente, ce qui implique que CTCF-T se lie sans doute au même ADN cible que CTCF, mais possède des fonctions différentes. De plus, l'expression de CTCF-T et de CTCF s'oppose mutuellement; l'expression de la protéine CTCF-T (cellules CTCF-T positives, CTCF negatives) qui a lieu pendant la spermatogenèse coïncide avec la reprogrammation épigénétique, notamment la méthylation de novo de l'ADN. La présente étude démontre le rôle essentiel joué par la protéine CTCF-T dans l'acquisition de l'empreinte génomique parentale. Nous montrons ici que CTCF-T s'associe in vivo avec les régions ICR des loci Igf2/H19 et Dlk/Gt12. Nous avons également identifié deux nouvelles protéines qui interagissent avec CTCF-T : une protéine arginine méthyl transférase PRMT7, et un variant de l'histone H2A, riche en arginine, que nous avons dénommé trH2A. Ces interactions ont été analysées plus en détail, et confinnent que ces deux protéines s'associent avec la région N-terminale de CTCF-T. Aussi, nous présentons une interaction de la région N-terminale de CTCF-T avec les histones H1, H2, et H3. Ces résultats suggèrent que CTCF-T est une protéine qui se lie spécifiquement aux régions ICR, qui s'associe avec différents histones et qui recrute PRMT7. PRMT7 possède une activité méthyl-tansférase envers les histones. Il a été montré que la méthylation des histones marque certains endroits de la chromatine, dirigeant ainsi la méthylation de l'ADN. Notre hypothèse est donc la suivante : la protéine CTCF-T sert de base qui dirige la méthylation des histones par PRMT7 dans les régions ICR, ce qui contribue à marquer la chromatine pour le recrutement de nouvelles méthyl transférases pour méthyler l'ADN. Afin de valider cette hypothèse, nous avons développé un système de méthylation de l'ADN in vivo, dans des oeufs de Xenopus laevis, dans le noyau desquels nous avons mico-injecté la région ICR du locus H19, ainsi que différents vecteurs d'expression pour CTCF-T, PRMT7, et les de novo méthyl transférases (Dnmt3a, Dnmt3b et Dnmt3L). Les CpGs méthyles de la région ICR du locus H19 ont été analysé 48 et 72 heures après l'injection. Cette technique nous a permis de démontrer que les CpGs de la région ICR sont méthyles en présence de CTCF-T et de PRMT7, tandis que les contrôles injectés seulement avec la région ICR ne présentent aucun signe de méthylation. De plus, nous démontrons pour la première fois que la protéine méthyl transférase Dnmt3L est déterminant pour l'établissement de l'empreinte génomique parentale au niveau de la région ICR du locus H19. Aussi, nous confirmons que les activités méthyl transférases de Dnmt3a et Dnmt3b sont complémentaires. Nos données indiquent que les trois protéines Dnmt3 sont impliquées dans la méthylation de l'ADN. En conclusion, nous proposons un mécanisme responsable de la mise en place de nouvelles empreintes génomiques pendant la spermatogenèse : le complexe protéique CTCF-T/PRMT7 dirige la méthylation des histones aboutissant à la méthylation de novo de l'ADN au locus H19.
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By interacting with MHC class II molecules, CD4 facilitates lineage development as well as activation of Th cells. Expression of physiological levels of CD4 requires a proximal CD4 enhancer to stimulate basic CD4 promoter activity. T cell factor (TCF)-1/beta-catenin pathway has previously been shown to regulate thymocyte survival via up-regulating antiapoptotic molecule Bcl-xL. By both loss and gain of function studies, in this study we show additional function of TCF-1/beta-catenin pathway in the regulation of CD4 expression in vivo. Mice deficient in TCF-1 displayed significantly reduced protein and mRNA levels of CD4 in CD4+ CD8+ double-positive (DP) thymocytes. A transgene encoding Bcl-2 restored survival but not CD4 levels of TCF-1(-/-) DP cells. Thus, TCF-1-regulated survival and CD4 expression are two separate events. In contrast, CD4 levels were restored on DP TCF-1(-/-) cells by transgenic expression of a wild-type TCF-1, but not a truncated TCF-1 that lacks a domain required for interacting with beta-catenin. Furthermore, forced expression of a stabilized beta-catenin, a coactivator of TCF-1, resulted in up-regulation of CD4. TCF-1 or stabilized beta-catenin greatly stimulated activity of a CD4 reporter gene driven by a basic CD4 promoter and the CD4 enhancer. However, mutation of a potential TCF binding site located within the enhancer abrogated TCF-1 and beta-catenin-mediated activation of CD4 reporter. Finally, recruitment of TCF-1 to CD4 enhancer was detected in wild-type but not TCF-1 null mice by chromatin-immunoprecipitation analysis. Thus, our results demonstrated that TCF/beta-catenin pathway enhances CD4 expression in vivo by recruiting TCF-1 to stimulate CD4 enhancer activity.
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SUMMARY : Eukaryotic DNA interacts with the nuclear proteins using non-covalent ionic interactions. Proteins can recognize specific nucleotide sequences based on the sterical interactions with the DNA and these specific protein-DNA interactions are the basis for many nuclear processes, e.g. gene transcription, chromosomal replication, and recombination. New technology termed ChIP-Seq has been recently developed for the analysis of protein-DNA interactions on a whole genome scale and it is based on immunoprecipitation of chromatin and high-throughput DNA sequencing procedure. ChIP-Seq is a novel technique with a great potential to replace older techniques for mapping of protein-DNA interactions. In this thesis, we bring some new insights into the ChIP-Seq data analysis. First, we point out to some common and so far unknown artifacts of the method. Sequence tag distribution in the genome does not follow uniform distribution and we have found extreme hot-spots of tag accumulation over specific loci in the human and mouse genomes. These artifactual sequence tags accumulations will create false peaks in every ChIP-Seq dataset and we propose different filtering methods to reduce the number of false positives. Next, we propose random sampling as a powerful analytical tool in the ChIP-Seq data analysis that could be used to infer biological knowledge from the massive ChIP-Seq datasets. We created unbiased random sampling algorithm and we used this methodology to reveal some of the important biological properties of Nuclear Factor I DNA binding proteins. Finally, by analyzing the ChIP-Seq data in detail, we revealed that Nuclear Factor I transcription factors mainly act as activators of transcription, and that they are associated with specific chromatin modifications that are markers of open chromatin. We speculate that NFI factors only interact with the DNA wrapped around the nucleosome. We also found multiple loci that indicate possible chromatin barrier activity of NFI proteins, which could suggest the use of NFI binding sequences as chromatin insulators in biotechnology applications. RESUME : L'ADN des eucaryotes interagit avec les protéines nucléaires par des interactions noncovalentes ioniques. Les protéines peuvent reconnaître les séquences nucléotidiques spécifiques basées sur l'interaction stérique avec l'ADN, et des interactions spécifiques contrôlent de nombreux processus nucléaire, p.ex. transcription du gène, la réplication chromosomique, et la recombinaison. Une nouvelle technologie appelée ChIP-Seq a été récemment développée pour l'analyse des interactions protéine-ADN à l'échelle du génome entier et cette approche est basée sur l'immuno-précipitation de la chromatine et sur la procédure de séquençage de l'ADN à haut débit. La nouvelle approche ChIP-Seq a donc un fort potentiel pour remplacer les anciennes techniques de cartographie des interactions protéine-ADN. Dans cette thèse, nous apportons de nouvelles perspectives dans l'analyse des données ChIP-Seq. Tout d'abord, nous avons identifié des artefacts très communs associés à cette méthode qui étaient jusqu'à présent insoupçonnés. La distribution des séquences dans le génome ne suit pas une distribution uniforme et nous avons constaté des positions extrêmes d'accumulation de séquence à des régions spécifiques, des génomes humains et de la souris. Ces accumulations des séquences artéfactuelles créera de faux pics dans toutes les données ChIP-Seq, et nous proposons différentes méthodes de filtrage pour réduire le nombre de faux positifs. Ensuite, nous proposons un nouvel échantillonnage aléatoire comme un outil puissant d'analyse des données ChIP-Seq, ce qui pourraient augmenter l'acquisition de connaissances biologiques à partir des données ChIP-Seq. Nous avons créé un algorithme d'échantillonnage aléatoire et nous avons utilisé cette méthode pour révéler certaines des propriétés biologiques importantes de protéines liant à l'ADN nommés Facteur Nucléaire I (NFI). Enfin, en analysant en détail les données de ChIP-Seq pour la famille de facteurs de transcription nommés Facteur Nucléaire I, nous avons révélé que ces protéines agissent principalement comme des activateurs de transcription, et qu'elles sont associées à des modifications de la chromatine spécifiques qui sont des marqueurs de la chromatine ouverte. Nous pensons que lés facteurs NFI interagir uniquement avec l'ADN enroulé autour du nucléosome. Nous avons également constaté plusieurs régions génomiques qui indiquent une éventuelle activité de barrière chromatinienne des protéines NFI, ce qui pourrait suggérer l'utilisation de séquences de liaison NFI comme séquences isolatrices dans des applications de la biotechnologie.
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Background: The transcription factor IRF4 is involved in several T-cell-dependent chronic inflammatory diseases. To elucidate the mechanisms for pathological cytokine production in colitis, we addressed the role of the IRF transcription factors in human inflammatory bowel disease (IBD) and experimental colitis.Methods: IRF levels and cytokine production in IBD patients were studied as well as the effects of IRF4 deficiency in experimental colitis.Results: In contrast to IRF1, IRF5, and IRF8, IRF4 expression in IBD was augmented in the presence of active inflammation. Furthermore, IRF4 levels significantly correlated with IL-6 and IL-17 mRNA expression and to a lesser extent with IL-22 mRNA expression in IBD. To further explore the role of IRF4 under in vivo conditions, we studied IRF4-deficient and wildtype mice in experimental colitis. In contrast to DSS colitis, IRF4 deficiency was protective in T-cell-dependent transfer colitis associated with reduced ROR alpha/gamma t levels and impaired IL-6, IL-17a, and IL-22 production, suggesting that IRF4 acts as a master regulator of mucosal Th17 cell differentiation. Subsequent mechanistic studies using database analysis, chromatin immunoprecipitation, and electrophoretic mobility shift assays identified a novel IRF4 binding site in the IL-17 gene promoter. Overexpression of IRF4 using retroviral infection induced IL-17 production and IL-17 together with IL-6 induced ROR gamma t expression.Conclusions: IRF4 can directly bind to the IL-17 promotor and induces mucosal ROR gamma t levels and IL-17 gene expression thereby controlling Th17-dependent colitis. Targeting of this molecular mechanism may lead to novel therapeutic approaches in human IBD.
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BACKGROUND/AIMS: The Peroxisome Proliferator-Activated Receptor (PPAR) alpha belongs to the superfamily of Nuclear Receptors and plays an important role in numerous cellular processes, including lipid metabolism. It is known that PPARalpha also has an anti-inflammatory effect, which is mainly achieved by down-regulating pro-inflammatory genes. The objective of this study was to further characterize the role of PPARalpha in inflammatory gene regulation in liver. RESULTS: According to Affymetrix micro-array analysis, the expression of various inflammatory genes in liver was decreased by treatment of mice with the synthetic PPARalpha agonist Wy14643 in a PPARalpha-dependent manner. In contrast, expression of Interleukin-1 receptor antagonist (IL-1ra), which was acutely stimulated by LPS treatment, was induced by PPARalpha. Up-regulation of IL-1ra by LPS was lower in PPARalpha -/- mice compared to Wt mice. Transactivation and chromatin immunoprecipitation studies identified IL-1ra as a direct positive target gene of PPARalpha with a functional PPRE present in the promoter. Up-regulation of IL-1ra by PPARalpha was conserved in human HepG2 hepatoma cells and the human monocyte/macrophage THP-1 cell line. CONCLUSIONS: In addition to down-regulating expression of pro-inflammatory genes, PPARalpha suppresses the inflammatory response by direct up-regulation of genes with anti-inflammatory properties.
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We report the generation and analysis of functional data from multiple, diverse experiments performed on a targeted 1% of the human genome as part of the pilot phase of the ENCODE Project. These data have been further integrated and augmented by a number of evolutionary and computational analyses. Together, our results advance the collective knowledge about human genome function in several major areas. First, our studies provide convincing evidence that the genome is pervasively transcribed, such that the majority of its bases can be found in primary transcripts, including non-protein-coding transcripts, and those that extensively overlap one another. Second, systematic examination of transcriptional regulation has yielded new understanding about transcription start sites, including their relationship to specific regulatory sequences and features of chromatin accessibility and histone modification. Third, a more sophisticated view of chromatin structure has emerged, including its inter-relationship with DNA replication and transcriptional regulation. Finally, integration of these new sources of information, in particular with respect to mammalian evolution based on inter- and intra-species sequence comparisons, has yielded new mechanistic and evolutionary insights concerning the functional landscape of the human genome. Together, these studies are defining a path for pursuit of a more comprehensive characterization of human genome function.
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AbstractBACKGROUND: KRAB-ZFPs (Krüppel-associated box domain-zinc finger proteins) are vertebrate-restricted transcriptional repressors encoded in the hundreds by the mouse and human genomes. They act via an essential cofactor, KAP1, which recruits effectors responsible for the formation of facultative heterochromatin. We have recently shown that KRAB/KAP1 can mediate long-range transcriptional repression through heterochromatin spreading, but also demonstrated that this process is at times countered by endogenous influences.METHOD: To investigate this issue further we used an ectopic KRAB-based repressor. This system allowed us to tether KRAB/KAP1 to hundreds of euchromatic sites within genes, and to record its impact on gene expression. We then correlated this KRAB/KAP1-mediated transcriptional effect to pre-existing genomic and chromatin structures to identify specific characteristics making a gene susceptible to repression.RESULTS: We found that genes that were susceptible to KRAB/KAP1-mediated silencing carried higher levels of repressive histone marks both at the promoter and over the transcribed region than genes that were insensitive. In parallel, we found a high enrichment in euchromatic marks within both the close and more distant environment of these genes.CONCLUSION: Together, these data indicate that high levels of gene activity in the genomic environment and the pre-deposition of repressive histone marks within a gene increase its susceptibility to KRAB/KAP1-mediated repression.
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Ultrastructural aspects of spermatogenesis, spermiogenesis and of the mature spermatozoon of a microcotylid monogenean Metamicrocotyla macracantha parasite from Mugil liza, are described. The irregularly-shaped spermatogonia divides by successive mitoses, forming the primary spermatocytes, identified by the presence of synaptonemal complexes in their nuclei. The spermatids formed by meiotic cell divisions of the secondary spermatocytes, differentiate into a mature spermatozoon. Cross sections of the head and the middle region of mature spermatozoa show the nucleus with strong condensed chromatin, the mitochondria with short cristae, peripheral microtubules and two axonemes with a 9+1 pattern, confirming the characteristics of this genus.