Permissive Structure Research Articles

The Rtt106 Histone Chaperone Is Functionally Linked to Transcription Elongation and Is Involved in the Regulation of Spurious Transcription from Cryptic Promoters in Yeast*♦

Rtt106 is a histone chaperone that has been suggested to play a role in heterochromatin-mediated silencing in Saccharomyces cerevisiae. It interacts physically and functionally with the chromatin assembly factor-1 (CAF-1), which is associated with replication-coupled nucleosomal deposition. In this work, we have taken several approaches to study Rtt106 in greater detail and have identified a previously unknown function of Rtt106. We found genetic interactions between rtt106Delta and mutations in genes encoding transcription elongation factors, including Spt6, TFIIS, and members of the PAF and yeast DSIF complexes. In addition, chromatin immunoprecipitation analysis indicates that Rtt106 is associated with transcribed regions of active genes. Furthermore, our results show that Rtt106 is required for the repression of transcription from a cryptic promoter within a coding region. This observation strongly suggests that Rtt106 is involved in the regulation of chromatin structure of transcribed regions. Finally, we provide evidence that Rtt106 plays a role in regulating the levels of histone H3 transcription-coupled deposition over transcribed regions. Taken together, our results indicate a direct link for Rtt106 with transcription elongation and the chromatin dynamics associated with RNA polymerase II passage.

Lymphoid-affiliated genes are associated with active histone modifications in human hematopoietic stem cells

AbstractTo address the role of chromatin structure in the establishment of hematopoietic stem cell (HSC) multilineage potential and commitment to the lymphoid lineage, we have analyzed histone modifications at a panel of lymphoid- and myeloid-affiliated genes in multipotent and lineage-committed hematopoietic cells isolated from human cord blood. Our results show that many B- and T-lymphoid genes, although silent in HSCs, are associated with acetylated histones H3 and H4. We also detected histone H3 lysine 4 methylation but not repressive lysine 9 or 27 methylation marks at these loci, indicative of an open chromatin structure. Interestingly, the relative level of H3 lysine 4 dimethylation to trimethylation at B-specific loci was high in multipotent CD34+CD38lo progenitors and decreased as they become actively transcribed in B-lineage cells. In vitro differentiation of CD34+ cells toward the erythroid, granulocyte, and T-cell lineages resulted in a loss of histone acetylation at nonlineage-associated genes. This study provides evidence that histone modifications involved in chromatin decondensation are already in place at lymphoid-specific genes in primary human HSCs, supporting the idea that these genes are “primed” for expression before lineage commitment. This permissive chromatin structure is progressively lost as the stem cell differentiates.

Prostaglandin H synthase-2 gene regulation in the amnion at labour: histone acetylation and nuclear factor kappa B binding to the promoter in vivo

Increased prostaglandin H synthase-2 (PGHS-2) expression in the amnion is critical for the production of prostaglandins that induce labour. The aim of the present investigation was to determine whether PGHS-2 gene activity is controlled by NFkappaB transcription factors in term amnion in vivo as suggested by in vitro findings. Amnion membranes were collected after elective Caesarean section (n = 14) or spontaneous labour (n = 12) at term, and histone acetylation and transcription factor binding to the PGHS-2 and IkappaBalpha promoters were determined in fresh tissues by chromatin immunoprecipitation. High level of histone-3 and -4 acetylation was detected in the proximal 1000 bp region of the PGHS-2 promoter indicating permissive chromatin structure in an area that contains two consensus NFkappaB binding sites and other transcription factor binding motifs. The TATA-box was occupied by TATA-binding protein (TBP) demonstrating that the PGHS-2 gene was transcriptionally active before and after labour. NFkappaB (p65 and p50) binding to the consensus sites, however, was detected only before, but not after, labour. Moreover, NFkappaB factor binding before labour was unrelated to TBP binding to the PGHS-2 TATA-box in the same tissues. Further, p65 binding to the NFkappaB-responsive IkappaBalpha promoter increased at labour and correlated strongly with TBP binding to the TATA-box of this gene. We conclude that the proximal 1000 bp region is involved in PGHS-2 promoter regulation in term amnion. The NFkappaB system is activated at labour and stimulates the IkappaBalpha gene, but the NFkappaB factors do not drive PGHS-2 transcription using consensus promoter sites in normal term amnion in vivo.

The nucleosome surface regulates chromatin compaction and couples it with transcriptional repression

Although it is believed that the interconversion between permissive and refractory chromatin structures is important in regulating gene transcription, this process is poorly understood. Central to addressing this issue is to elucidate how a nucleosomal array folds into higher-order chromatin structures. Such findings can then provide new insights into how the folding process is regulated to yield different functional states. Using well-defined in vitro chromatin-assembly and transcription systems, we show that a small acidic region on the surface of the nucleosome is crucial both for the folding of a nucleosomal template into the 30-nm chromatin fiber and for the efficient repression of transcription, thereby providing a mechanistic link between these two essential processes. This structure-function relationship has been exploited by complex eukaryotic cells through the replacement of H2A with the specific variant H2A.Bbd, which naturally lacks an acidic patch.

A Functional Link between Rhythmic Changes in Chromatin Structure and theArabidopsisBiological Clock

Circadian clocks rhythmically coordinate biological processes in resonance with the environmental cycle. The clock function relies on negative feedback loops that generate 24-h rhythms in multiple outputs. In Arabidopsis thaliana, the clock component TIMING OF CAB EXPRESSION1 (TOC1) integrates the environmental information to coordinate circadian responses. Here, we use chromatin immunoprecipitation as well as physiological and luminescence assays to demonstrate that proper photoperiodic phase of TOC1 expression is important for clock synchronization of plant development with the environment. Our studies show that TOC1 circadian induction is accompanied by clock-controlled cycles of histone acetylation that favor transcriptionally permissive chromatin structures at the TOC1 locus. At dawn, TOC1 repression relies on the in vivo circadian binding of the clock component CIRCADIAN CLOCK ASSOCIATED1 (CCA1), while histone deacetylase activities facilitate the switch to repressive chromatin structures and contribute to the declining phase of TOC1 waveform around dusk. The use of cca1 late elongated hypocotyl double mutant and CCA1-overexpressing plants suggests a highly repressing function of CCA1, antagonizing H3 acetylation to regulate TOC1 mRNA abundance. The chromatin remodeling activities relevant at the TOC1 locus are distinctively modulated by photoperiod, suggesting a mechanism by which the clock sets the phase of physiological and developmental outputs.

Globular structure of human ovulatory cervical mucus

Human cervical mucus is a heterogeneous mixture of mucin glycoproteins whose relative concentration changes during the ovulatory phases, thereby producing different mucus aggregation structures that can periodically permit the transit of spermatozoa for fertilization. In preovulatory phase, mucus is arranged in compact fiber-like structures where sperm transit is hindered. Previously, through observations made of fixed and dehydrated samples, a permissive structure in the ovulatory phase was attributed to the larger diameters of pores in the mucus network. Instead, by means of atomic force microscopy, we can show, for the first time, that unfixed ovulatory mucus is composed by floating globules of mucin aggregates. This finding sheds new light on the mechanism that governs spermatozoa transit toward the uterine cavity. In addition, we demonstrate that the switch from globular ovulatory to fibrous preovulatory mucus largely depends on a pH-driven mechanism. Analysis of mucin 5B primary sequence, the main mucin in ovulatory mucus, highlights pH-sensitive domains that are associated to flexible regions prone to drive aggregation. We suggest an involvement of these domains in the fiber-to-globule switch in cervical mucus.

Decentralized supervisory control of discrete event systems with communication delays based on conjunctive and permissive decision structures

In many practical discrete event systems (DESs), some unexpected and uncontrollable events can subsequently occur before a proper control action is actually applied to a plant due to communication delays. For such DESs, this paper investigates necessary and sufficient conditions for the existence of a nonblocking decentralized supervisor that can correctly achieve a given language specification when the decentralized supervisor is assumed to have a conjunctive and permissive decision structure. In particular, this paper presents a notion of delay-coobservability for a given language specification and shows that it is a key condition for the existence of such a decentralized supervisor.

Histone Modification Pattern of the T-Cellular Herpesvirus Saimiri Genome in Latency

Herpesvirus saimiri (HVS) subgroup C strains are able to growth transform human T lymphocytes in vitro. The stably persisting and nonintegrating HVS episome represents an optimal prerequisite for the investigation of the epigenetic state of latent herpesvirus genomes in vitro. Quantitative chromatin immunoprecipitation experiments using seven different histone acetylation- or methylation-specific antibodies revealed repressive marks at four lytic gene promoters and a variable pattern at the weakly transcribed LANA/orf73 promoter. The constitutive stpC/tip promoter regulating the viral oncoproteins and, more interestingly, the noncoding repetitive H-DNA elements flanking the coding region, showed a permissive chromatin structure. This study provides an appropriate model for the analysis of epigenetic herpesvirus genome modifications and their dynamics in T cells.

Mapping of the juxtacentromeric heterochromatin-euchromatin frontier of human chromosome 21

Euchromatin and heterochromatin are functional compartments of the genome. However, little is known about the structure and the precise location of the heterochromatin-euchromatin boundaries in higher eukaryotes. Constitutive heterochromatin in centromeric regions is associated with (1) specific histone methylation patterns, (2) high levels of DNA methylation, (3) low recombination frequency, and (4) the repression of transcription. All of this contrasts with the permissive structure of euchromatin found along chromosome arms. On the sequence level, the transition between these two domains consists most often of patchworks of segmental duplications. We present here a comprehensive analysis of gene expression, DNA methylation in CpG islands, distribution of histone isoforms, and recombination activity for the juxtacentromeric (or pericentromeric) region of the long arm of human chromosome 21. We demonstrate that most HapMap data are reliable within this region. We show that high linkage disequilibrium between pairs of SNPs extends 719-737 kb from the centromeric alpha-satellite. In the same region we find a peak of histone isoforms H3K9Me3 and H3K27Me (715-822 kb distal to the alpha-satellite). In normal somatic cells, CpG islands proximal to this peak are highly methylated, whereas distal CpG islands are not or very little methylated. This methylation profile undergoes dramatic changes in cancer cells and during spermatogenesis. As a consequence, transcription from heterochromatic genes is activated in the testis, and aberrant gene activation can occur during neoplastic transformation. Our data indicate that the frontier between the juxtacentromeric heterochromatic domain and euchromatic domain of the long arm of chromosome 21 is marked by a heterochromatic peak located approximately 750 kb distal to the alpha-satellite.

The Mobilisation of Native Canadians During the Second World War

Historians have paid scant attention to the compulsory conscription of men under the National Resources Mobilisation Act (NRMA) in Canada during the Second World War. This paper uses the mobilisation of Native Canadians as a case-study to determine the depth and extent of human resource mobilisation policies between 1940 and 1945. Government mobilisation departments and agencies relied on a remarkably decentralised and permissive administrative structure to carry out the NRMA mobilisation mandate. These organizational traits were exacerbated by active Native Canadian opposition to conscription and other factors, such as the geographic isolation and poor health of many Native men. As a result, a patchwork of disparate, inconsistent and ineffectual mobilisation policies affecting Canadian Indians was adopted during the course of the war.

The Extent of Histone Acetylation Correlates with the Differential Rearrangement Frequency of Individual VH Genes in Pro-B Cells

During B lymphocyte development, Ig heavy and L chain genes are assembled by V(D)J recombination. Individual V, D, and J genes rearrange at very different frequencies in vivo, and the natural variation in recombination signal sequence does not account for all of these differences. Because a permissive chromatin structure is necessary for the accessibility of VH genes for VH to DJH recombination, we hypothesized that gene rearrangement frequency might be influenced by the extent of histone modifications. Indeed, we found in freshly isolated pro-B cells from muMT mice a positive correlation between the level of enrichment of VHS107 genes in the acetylated histone fractions as assayed by chromatin immunoprecipitation, and their relative rearrangement frequency in vivo. In the VH7183 family, the very frequently rearranging VH81X gene showed the highest association with acetylated histones, especially in the newborn. Together, our data show that the extent of histone modifications in pro-B cells should be considered as a mechanism by which accessibility and the rearrangement level of individual VH genes is regulated.

Gγ-Globin Promoter Trans-Activation: Characterization of Transcription Factor Interactions in the Upstream cAMP Response Element (CRE).

Erythroid differentiation recapitulates the temporal switch from γ to β-globin gene expression that occurs during fetal development. We and others have demonstrated a p38 mitogen activated protein kinase (MAPK)-dependent mechanism for γ-globin activation by fetal hemoglobin inducers in the setting of permissive chromatin structure. Furthermore, by electrophoretic mobility shift assay we confirmed that CREB and ATF-2 bind the CRE at nucleotide −1222 in the Gγ-promoter (G-CRE). A naturally occurring C→G mutation in the CRE occurs is associated with the Benin haplotype, producing lower fetal hemoglobin levels in sickle cell patients. To understand the functional relevance of the G-CRE we used a luciferase reporter system established with promoter mutants at nucleotides −1500, −1350 and −1180 to produce the respective reporters -1500Gγluc, −1350Gγluc and −1180Gγluc. Transient transfections were performed in K562 cells by electroporation. In the −1180Gγluc reporter there was a loss of the 15 to 20-fold activation induced by 2mM butyrate and 0.5μM trichostatin in the −1500Gγluc reporter . The loss of trans-activation when the G-CRE was deleted in the −1180 mutant confirms functional relevance for this element. Trans-activation studies were also completed with a wtCREB (pCMV-CREB) and dominant negative, pKCREB expression vector. The latter forms an inactive heterodimer with CREB by blocking the DNA binding domain. To determine the direct trans-activation potential of wtCREB, co-transfection studies were completed with 10 to 40μg of either pCMV-CREB or pKCREB alone. We observed a dose-dependent 1.5 to 11-fold increase in luciferase activity with wtCREB compare to a robust 18 to 100-fold Gγ-promoter induction by pkCREB. This ability of both vectors to activate was reversed when the G-CRE was deleted. Interestingly, when an equal concentration of both vectors was co-transfected, a 40-fold increase in promoter activity was observed. Additional studies with pkCREB and butyrate or trichostatin inductions produced a similar synergistic effect. Collectively, the findings suggest that other transcription factors with greater trans-activation potential bind the G-CRE when CREB is inactivated by its dominant negative protein. We will test the two most likely candidate transcription factors, cJun and ATF-2 which bind the G-CRE and are downstream effectors of p38 MAPK signaling. Transfection experiments with cJun confirmed a dose-dependent 80-fold activation of −1500Gγluc. Interestingly, when cJun was combined with wtCREB, γ-promoter trans-activation dropped 60-fold supporting two possible interactions between cJun and CREB: 1) competitive binding to the G-CRE or 2) the formation of an inactive or low activity heterodimer between the two proteins. Additional studies with site-directed mutants of the G-CRE will be performed to ferret out the DNA-protein interactions in this region, thereby providing gene-based approaches for fetal hemoglobin induction.

Deletion of a conserved Il4 silencer impairs T helper type 1–mediated immunity

Helper T cell differentiation involves silencing as well as activation of gene expression. We have identified a conserved silencer of the gene encoding interleukin 4 (Il4) marked by DNase I hypersensitivity (HS IV) and permissive chromatin structure in all helper T cells. Deletion of HS IV increased Il4 and Il13 transcription by naive T cells and led to T helper type 2 skewing in vitro. HS IV controlled Il4 silencing during T helper type 1 differentiation, as HS IV-deficient T helper type 1 cells that expressed interferon-gamma also produced abundant interleukin 4 in vitro and in vivo. Despite mounting a vigorous interferon-gamma response, HS IV-deficient mice were more susceptible to Leishmania major infection than were wild-type littermate control mice, showing a critical function for Il4 silencing in T helper type 1-mediated immunity.

The locus of evaluation: becoming a professional counsellor

Students who register for counselling courses in higher education are mature adults who come from diverse disciplines. They are rich in experience and during the process of counselling education become proficient in adapting to changing circumstances, and working with personal and professional challenge. The requirements for studentship in the field of counselling demand stringent self-examination in group settings, and surpass those required for courses which require only subject knowledge and academic skills. Because of their willingness to pursue self-discovery, counselling students provide a rich source of data for researchers interested in lifelong learning from an holistic perspective of achieving UNESCO's vision of the ‘full development of the personality’ (cited in Darkenwald and Merriam 1982: 13). Student counsellors must demonstrate a willingness to work with uncertainty, an ability to develop personal learning goals and have the capacity to appraise personal development (Mearns 1997: 99). This study examines how student counsellors discover strategies for learning, and how they manage the personal/professional divide. Existing intimate partnerships are viewed as supportive, but suspicious of the learning process. The narratives of students indicate that ‘professionalism’ is about acceptance of self, as much as about qualifications and practice. Participants describe the process of becoming professional, alongside the process of struggling to fit in with the academic requirements of a university. Lifelong learning is described as lacking a framework for acceptance by others, and therefore seen merely as a diversion in the lives of adults. From a counselling perspective, learning requires opportunities for employment so that professional and personal development can continue. Such courses at present appear to offer only a short-term ‘permissive structure’, for learning to take place.

Histone acetylation: a switch between repressive and permissive chromatin. Second in review series on chromatin dynamics.

The organization of eukaryotic chromatin has a major impact on all nuclear processes involving DNA substrates. Gene expression is affected by the positioning of individual nucleosomes relative to regulatory sequence elements, by the folding of the nucleosomal fiber into higher-order structures and by the compartmentalization of functional domains within the nucleus. Because site-specific acetylation of nucleosomal histones influences all three aspects of chromatin organization, it is central to the switch between permissive and repressive chromatin structure. The targeting of enzymes that modulate the histone acetylation status of chromatin, in synergy with the effects mediated by other chromatin remodeling factors, is central to gene regulation.

Selective SiGe epitaxy by rtcvd for new device architectures

CMOS technologies and devices are approaching their theoretical limits such as optical lithography, gate oxide thickness, etc. Consequently, new efforts are made in order to improve or modify device architecture. New epitaxial films grown at low temperature have potential of improving both electrical performances and integration of advanced devices. Firstly, we investigate the selective SiGe epitaxy with high Ge content based on the DCS/GeH 4/HCl/H 2 chemistry using an industrial 200 mm single wafer chemical vapour deposition at reduced pressure (<40 Torr) and low temperature (600–750 °C). We present new results about global loading effects in terms of growth and Ge content (15–40%) of perfectly strained SiGe films used for their selective etch properties in new device architectures. Secondly, we report on morphological results of the novel CMOS device architecture called silicon-on-nothing (SON). They allow extremely thin (a few nanometers) buried dielectric and silicon films to be fabricated with high resolution and uniformity guaranteed by SiGe and Si selective epitaxial processes. It is a very permissive structure as first approach of ultra thin SOI films.

Second messenger specificity of the inositol trisphosphate receptor: reappraisal based on novel inositol phosphates

To further understand how the second messenger D-myo-inositol 1,4,5-trisphosphate [Ins(1,4,5)P3] interacts with its intracellular receptor, we injected 47 highly purified inositol phosphate (InsP) positional isomers in Xenopus oocytes and compared their potency in releasing intracellular Ca2+. The potency of the Ca(2+)-releasing InsPs spanned four orders of magnitude. Seven compounds, including the novel inositol 1,2,4,5-tetrakisphosphate [D/L-Ins (1,2,4,5)P4] and D/L-Ins(1,4,6)P3, had a very high potency. All of these highly active InsPs shared the following structure: two D-trans-equatorial phosphates (eq-P) and one equatorial hydroxyl (eq-OH) attached to ring carbons D-4, D-5, and D-6 (or to the structurally equivalent D-1, D-6, and D-5 carbons). This permissive structure was not sufficient for Ca2+ release, because it was also found in two inactive compounds, Ins(1,6)P2 and Ins(1,3,6)P3. To be active, InsPs also required the structural equivalent of a D-3 eq-OH and/or a D-1 eq-P. Together, our data reveal how the structure of the InsP molecule affects its ability to release Ca2+.