Repressive Chromatin Structure Research Articles

Yeast ASF1 protein is required for cell cycle regulation of histone gene transcription.

Transcription of the four yeast histone gene pairs (HTA1-HTB1, HTA2-HTB2, HHT1-HHF1, and HHT2-HHF2) is repressed during G1, G2, and M. For all except HTA2-HTB2, this repression requires several trans-acting factors, including the products of the HIR genes, HIR1, HIR2, and HIR3. ASF1 is a highly conserved protein that has been implicated in transcriptional silencing and chromatin assembly. In this analysis, we show that HIR1 interacts with ASF1 in a two-hybrid analysis. Further, asf1 mutants, like hir mutants, are defective in repression of histone gene transcription during the cell cycle and in cells arrested in early S phase in response to hydroxyurea. asf1 and hir1 mutations also show very similar synergistic interactions with mutations in cac2, a subunit of the yeast chromatin assembly factor CAF-I. The results suggest that ASF1 and HIR1 function in the same pathway to create a repressive chromatin structure in the histone genes during the cell cycle.

Methylation and colorectal cancer.

Statistics rate colorectal adenocarcinoma as the most common cause of cancer death on exclusion of smoking-related neoplasia. However, the reported accumulation of genetic lesions over the adenoma to adenocarcinoma sequence cannot wholly account for the neoplastic phenotype. Recently, heritable, epigenetic changes in DNA methylation, in association with a repressive chromatin structure, have been identified as critical determinants of tumour progression. Indeed, the transcriptional silencing of both established and novel tumour suppressor genes has been attributed to the aberrant cytosine methylation of promoter-region CpG islands. This review aims to set these epigenetic changes within the context of the colorectal adenoma to adenocarcinoma sequence. The role of cytosine methylation in physiological and pathological gene silencing is discussed and the events behind aberrant cytosine methylation in ageing and cancer are appraised. Emphasis is placed on the interrelationships between epigenetic and genetic lesions and the manner in which they cooperate to define a CpG island methylator phenotype at an early stage in tumourigenesis. Finally, the applications of epigenetics to molecular pathology and patient diagnosis and treatment are reviewed.

RNA polymerase II elongation through chromatin.

The machinery that transcribes protein-coding genes in eukaryotic cells must contend with repressive chromatin structures in order to find its target DNA sequences. Diverse arrays of proteins modify the structure of chromatin at gene promoters to help transcriptional regulatory proteins access their DNA recognition sites. The way in which disruption of chromatin structure at a promoter is transmitted through a whole gene has not been defined. Recent breakthroughs suggest that the passage of an RNA polymerase through a gene is coupled to mechanisms that propagate the breakdown of chromatin.

A histone deacetylase inhibitor potentiates estrogen receptor activation of a stably integrated vitellogenin promoter in HepG2 cells.

To compare the role of histone deactylation in estrogen activation of a transiently transfected vitellogenin (VIT) promoter and an integrated VIT promoter in the same cells, we produced three HepG2, human hepatoma, cell lines (HepG2ERV cells) stably expressing human estrogen receptor alpha (hERalpha) and containing an integrated VIT promoter-chloramphenicol acetyltransferase (VIT-CAT) reporter gene. The three ER-positive HepG2ERV cell lines and wild-type, ER-negative, HepG2 cells cotransfected with cytomegalovirus-hERalpha exhibited similar MOX-dependent inductions of 20- to 50-fold with a transiently transfected VIT-luciferase reporter and 15- to 50-fold with a transfected 4-estrogen response element-TATA-luciferase reporter gene. The histone deacetylase inhibitor, trichostatin A, did not enhance MOX induction of the transiently transfected VIT promoter in the HepG2ERV cells. In contrast, trichostatin A dramatically potentiated MOX induction of the stably integrated VIT-CAT reporter gene, resulting in MOX-ER-dependent increases in CAT activity of up to 600-fold. These data demonstrate that although liganded ER exhibits the capacity to fully activate a transiently transfected VIT promoter, under some circumstances the ability to reorganize a repressive chromatin structure may be limiting for steroid receptor action.

Transposons and genome evolution in plants.

Although it is known today that transposons comprise a significant fraction of the genomes of many organisms, they eluded discovery through the first half century of genetic analysis and even once discovered, their ubiquity and abundance were not recognized for some time. This genetic invisibility of transposons focuses attention on the mechanisms that control not only transposition, but illegitimate recombination. The thesis is developed that the mechanisms that control transposition are a reflection of the more general capacity of eukaryotic organisms to detect, mark, and retain duplicated DNA through repressive chromatin structures.

Transcriptional repression by Pax5 (BSAP) through interaction with corepressors of the Groucho family.

Pax5 (BSAP) functions as both a transcriptional activator and repressor during midbrain patterning, B-cell development and lymphomagenesis. Here we demonstrate that Pax5 exerts its repression function by recruiting members of the Groucho corepressor family. In a yeast two-hybrid screen, the groucho-related gene product Grg4 was identified as a Pax5 partner protein. Both proteins interact cooperatively via two separate domains: the N-terminal Q and central SP regions of Grg4, and the octapeptide motif and C-terminal transactivation domain of Pax5. The phosphorylation state of Grg4 is altered in vivo upon Pax5 binding. Moreover, Grg4 efficiently represses the transcriptional activity of Pax5 in an octapeptide-dependent manner. Similar protein interactions resulting in transcriptional repression were also observed between distantly related members of both the Pax2/5/8 and Groucho protein families. In agreement with this evolutionary conservation, the octapeptide motif of Pax proteins functions as a Groucho-dependent repression domain in Drosophila embryos. These data indicate that Pax proteins can be converted from transcriptional activators to repressors through interaction with corepressors of the Groucho protein family.

Initiation of a chromatin-based transcriptionally repressive state in the preimplantation mouse embryo: lack of a primary role for expression of somatic histone H1.

A chromatin-based transcriptionally repressive state develops during the two-cell stage in preimplantation mouse embryos. Correlated with the initial formation of this state is the expression of somatic histone H1, which could confer repression by promoting the formation of a transcriptionally repressive chromatin structure. To ascertain if the expression of histone H1 could play such a primary role in initiating the formation of this transcriptionally repressive state, the endogenous pool of somatic histone H1 in the two-cell embryo was greatly expanded by injection of 25 or 100 pg of histone H1 at the one-cell stage. The expression of the transcription-requiring complex, which is an accepted marker for genome activation, was then assessed during the two-cell stage. No significant inhibition was noted following the injection of 25 pg of histone H1. A transient inhibition was observed following injection of 100 pg, but this was likely due to a delay in cleavage to the two-cell stage. We conclude that it is unlikely that the expression of somatic histone H1 is a major factor in the initial establishment of the chromatin-based transcriptionally repressive state that accompanies genome activation.

Sp1 and chromatin environment are important contributors to the formation of repressive chromatin structures on the transfected human adenine nucleotide translocase-2 promoter

Sp1 and chromatin environment are important contributors to the formation of repressive chromatin structures on the transfected human adenine nucleotide translocase-2 promoter

Sp1 and chromatin environment are important contributors to the formation of repressive chromatin structures on the transfected human adenine nucleotide translocase-2 promoter

The influence of chromatin on the human adenine nucleotide translocase isoform 2 (ANT2) promoter was investigated in transfected cells treated with the deacetylase inhibitors butyrate and trichostatin A (TSA). Both inhibitors activated the expression of reporter plasmids transfected into HeLa cells, indicating that the promoter was suppressed by hypoacetylated chromatin and activated by hyperacetylation. Inhibitor-dependent activation was traced to the two Sp1-activation elements within the proximal promoter region, indicating that the Sp1 elements are repressed by chromatin structure. Repressive chromatin structures were also formed on the promoter integrated into a stable chromatin environment, as shown by the effects of TSA and butyrate on 14 single-cell-derived NIH3T3 clones bearing the stable integrated ANT2 promoter. Both the basal expression of the luciferase reporter gene and the response to TSA and butyrate varied widely between clones. The range of basal expression (4000-fold) was due partially to variation in the formation of repressive chromatin, since clones with low basal expression were induced by TSA, but those with high basal expression were less effected. These data indicate that chromatin environment surrounding the integrated DNA exerts a strong influence on chromatin-dependent repression of the ANT2 promoter, and that the ability of Sp1 to activate ANT2 expression is compromised in the repressed state.

Chromatin-remodeling complexes involved in gene activation by the glucocorticoid receptor

Publisher Summary This chapter provides an overview of the glucocorticoid receptor (GR) and general mechanisms of gene regulation and also describes the selected aspects of the molecular mechanisms by which the GR, once bound to DNA, activates the expression of genes through its N-terminal transactivation domain τ1. GR belongs to a large family of ligand-inducible nuclear receptors and consists of a ligand-binding domain, a DNA-binding domain, and transactivation domains. The main transcriptional activation domain, τ1, is located in the N terminus of the receptor. Current models for many nuclear receptors suggest that gene activation involves both derepression of a repressive chromatin structure within promoters and subsequent activation of transcription, involving recruitment of the transcriptional machinery. Activators can modulate chromatin structure by interacting with histone acetyltransferases (HATs) and/or adenosine triphosphate (ATP)-dependent chromatin remodeling complexes. Preinitiation complex formation can be influenced by direct interactions of activators with basal transcription factors or indirect interactions by coactivators.

High-resolution structural analysis of chromatin at specific loci: Saccharomyces cerevisiae silent mating-type locus HMRa.

Genetic and biochemical evidence implicates chromatin structure in the silencing of the two quiescent mating-type loci near the telomeres of chromosome III in yeast. With high-resolution micrococcal nuclease mapping, we show that the HMRa locus has 12 precisely positioned nucleosomes spanning the distance between the E and I silencer elements. The nucleosomes are arranged in pairs with very short linkers; the pairs are separated from one another by longer linkers of approximately 20 bp. Both the basic amino-terminal region of histone H4 and the silent information regulator protein Sir3p are necessary for the organized repressive chromatin structure of the silent locus. Compared to HMRa, only small differences in the availability of the TATA box are present for the promoter in the cassette at the active MATa locus. Features of the chromatin structure of this silent locus compared to the previously studied HMLalpha locus suggest differences in the mechanisms of silencing and may relate to donor selection during mating-type interconversion.

A Hypothesis for the Transcriptional Control of Amphibian Metamorphosis by the Thyroid Hormone Receptor

SYNOPSIS. Amphibian metamorphosis is controlled by thyroid hormone and the thyroid hormone receptor. The thyroid hormone receptor functions as a master control factor that can both activate and repress genes in controlling the transformation of the larval tadpole to the adult frog. We have made use of the microinjection of both Xenopus laevis oocytes and embryos to investigate the molecular mechanisms involved in this regulation of transcription. We define three steps in the regulation of transcription by thyroid hormone receptor: the establishment of a repressive chromatin structure, disruption of chromatin and transcriptional activation. Our results also suggest that there are four major stages in the developmental control of gene expression during metamorphosis 1) Tissue and stage specific expression during embryonic development prior to tadpole hatching independent of thyroid hormone receptor. 2) The general repression of transcription of these genes by thyroid hormone receptor after tadpole hatching (stage 36), but prior to the production of thyroid hormone, which is first detectible in the circulating plasma around stage 55. 3) The activation of transcription dependent on thyroid hormone and receptor at metamorphosis (stages 55-66). 4) The acquisition of tissue specific and possibly thyroid hormone-independent patterns of gene activity.

An insulator element and condensed chromatin region separate the chicken beta-globin locus from an independently regulated erythroid-specific folate receptor gene.

We have identified a folate receptor gene upstream of the chicken beta-globin locus and separated from it by a 16 kbp region of silent chromatin. We find that this receptor is expressed only at a stage of erythroid differentiation (CFU-E) preceding the activation of beta-globin genes, consistent with the role of folate receptors in proliferation. This discovery raises the question of how these two loci are regulated during erythropoiesis. Our data suggest that the folate receptor gene and the beta-globin locus are regulated independently. We show that a 3.3 kbp DNA region upstream of the folate receptor gene is sufficient to induce strong expression of a transgene in CFU-E stage cells. We also find that the region between the beta-globin locus and the folate receptor gene is fully methylated and condensed at this stage of differentiation. Its 3' boundary coincides with the 5' beta-globin insulator. We speculate that the 5' beta-globin boundary element might be important for the proper regulation of two adjacent domains activated at two different stages during differentiation.

Beta-Phaseolin gene activation is a two-step process: PvALF- facilitated chromatin modification followed by abscisic acid-mediated gene activation.

We have shown previously that a rotationally and translationally positioned nucleosome is responsible for the absence of transcriptional expression from the phaseolin (phas) gene promoter in leaf tissue and that the repressive chromatin structure is disrupted on transcriptional activation during embryogenesis. To investigate how the chromatin structure is modified, we ectopically expressed PvALF, a putative seed-specific phas activator, in leaf tissue of a tobacco line transgenic for a chimeric phas/uidA construct. DNase I footprinting in vivo revealed that the ectopic expression of PvALF resulted in remodeling of the chromatin architecture over the TATA region of the phas promoter but did not lead to transcriptional activation in the absence of abscisic acid (ABA). Treatment of the transgenic tobacco leaves with ABA in the absence of PvALF neither alleviated the repressive chromatin architecture nor activated transcription. However, in the presence of PvALF, high levels of beta-glucuronidase expression were obtained on exposure of leaves to ABA. These results reveal that expression from the phas promoter involves at least two discrete steps: chromatin potentiation by PvALF followed by ABA-mediated transcriptional activation.

Transcriptional repression of the yeast CHA1 gene requires the chromatin-remodeling complex RSC.

In eukaryotes, DNA is packaged into chromatin, a compact structure that must be disrupted when genes are transcribed by RNA polymerase II. For transcription to take place, chromatin is remodeled via nucleosome disruption or displacement, a fundamental transcriptional regulatory mechanism in eukaryotic organisms. Here we show that the yeast chromatin-remodeling complex, RSC (remodels the structure of chromatin), isolated on the basis of homology to the SWI/SNF complex, is required for proper transcriptional regulation and nucleosome positioning in the highly inducible CHA1 promoter. In the absence of Sth1p/Nps1p (a homolog of Swi2p/Snf2p) or of Swh3p (a homolog of Swi3p), expression of CHA1 in non-induced cells is increased to a level comparable with that of fully induced cells. Furthermore, in non-induced cells depleted for Sth1p/Nps1p or Swh3p, a nucleosome positioned over the TATA box of the CHA1 promoter is disrupted, an architectural change normally only observed during transcriptional induction. In addition, deletion of the gene-specific activator Cha4p did not affect derepression of CHA1 in cells depleted for Swh3p. Thus, CHA1 constitutes a target for the RSC complex, and we propose that RSC is essential for maintaining a repressive chromatin structure at the CHA1 promoter.

Tissue-specific and developmental stage-specific DNA binding by a mammalian SWI/SNF complex associated with human fetal-to-adult globin gene switching

SWI/SNF complexes in yeast and higher eukaryotes are thought to facilitate gene activation and transcription factor binding by disrupting repressive chromatin structures. Little is known, however, about how these complexes target specific genes for activation. We now have purified a specialized SWI/SNF-related complex (PYR complex) from murine erythroleukemia (MEL) cell nuclear extract that binds pyrimidine-rich elements at the human and murine beta-globin loci. PYR complex DNA-binding activity is restricted to definitive hematopoietic cells and is both DNA sequence- and length-dependent. Mass spectrometric identification of purified peptides and antibody supershift assays indicate that PYR complex contains at least four known mammalian SWI/SNF subunits: BAF57, INI1, BAF60a, and BAF170. PYR complex broadly footprints a 250-bp pyrimidine-rich element between the human fetal and adult beta-globin genes. A short intergenic deletion that removes this element from a human globin locus cosmid construct results in delayed human fetal-to-adult globin gene switching in transgenic mice. Taken together, the data suggest that PYR complex may act through this intergenic element to facilitate human fetal-to-adult globin gene switching, presumably by opening the locus in the region of the adult genes to permit the binding of beta-globin transcriptional activators.

Transcription factors in DNA replication.

Accumulating evidence suggests the involvement of transcription factors in the regulation of DNA replication in eukaryotic cells. Almost all eukaryotic DNA viruses contain binding sites for transcription factors which function as auxiliary elements for DNA replication initiation at replication origins, and, indeed, the binding of transcription factors to these elements has been shown to stimulate DNA replication. Transcription factors also regulate some of the chromosome DNA replication origins of budding yeast, indicating that transcription factor involvement in DNA replication is not restricted to viruses. Consistent with this notion, recently determined replication origins of higher eukaryotes have been found occasionally to associate with transcription factor binding sites, although there is no direct evidence for the involvement of the factors that bind to these sequences in DNA replication. Analyses using viral and yeast systems have suggested that transcription factors stimulate the formation of the replication initiation complex by engaging in specific interactions with proteins of the initiation complex and/or by modulating the repressive chromatin structure around origins of replication. These mechanisms are analogous to those advanced to explain stimulation of transcription by transcription factors. The accumulated data suggests that transcription factors play a general role in the formation of functional complexes on chromosomes.

Mechanisms of Chromatin Alteration in Transcriptional Regulation.

Gene expression in eukatyotic cells involves the interaction of proteins with DNA packaged into nucleosomes: the primary structural units of chromatin. Recent sutudies show that transcription is regulated by multiple-steps of chromatin alteration, and that chromatin remodeling complexes are mainly responsible for the chromatin alteration from inactive state to destabilized state. Destabilization of nucleosomes was also found to occur in vivo at specific DNA sequences which can adopt non-B DNA structures. Hence, the nucleosome destabilizing sequences could be involved in the establishment of promoters that preset chromatin structure accessible for trans-acting factors prior to activation. Finally, organization of repressive chromatin structure by repressors in conjunction with global transcriptional regulators is described.

Silent chromatin in yeast: an orchestrated medley featuring Sir3p

Extensive regions of chromosomes can be transcriptionally repressed through silencing mechanisms mediated by complex chromatin structures. One of the most refined molecular portraits of silenced chromatin comes from studies of the silent mating-type loci and telomeres of S. cerevisiae. In this budding yeast, the Sir3p silent information regulator emerges as a critically important silencing component that interacts with nucleosomes and other silencing proteins. Not only is it essential for silencing, but Sir3p is also capable of spreading silenced chromatin when its dosage is increased. Sir3p is a target of mitogen-activated protein (MAP) kinase cascade regulation and has significant similarity to the Orc1p subunit of the DNA replication origin recognition complex. Thus, in concert with other silencing proteins, Sir3p appears poised to respond to cellular signals and reprogram silencing through replication-associated assembly of repressive chromatin structures.

Nerve Growth Factor Up-regulates the Transcriptional Activity of CBP through Activation of the p42/p44MAPK Cascade

Cyclic AMP response element-binding protein-binding protein (CBP) functions as a transcriptional coactivator through interactions with a number of cellular and viral transcription factors. It has been suggested to play a central integrative role in gene regulation. However, little is known about signal cascades that can regulate CBP activity. Here we show that either nerve growth factor (NGF) or cAMP treatment led to enhanced activity of CBP in PC12 cells. The C-terminal glutamine-rich activation domain of CBP was shown to be responsible for induction by NGF and cAMP. NGF-induced enhancement of CBP activity was also observed in protein kinase A (PKA)-deficient PC12 cells, whereas cAMP failed to increase the transcriptional activity of CBP in these cells. Moreover, the specific PKA inhibitor H-89 blocked cAMP-induced but not NGF-induced up-regulation of CBP activity. The up-regulation of CBP transcriptional activity in response to NGF was, however, prevented by the specific inhibitor of mitogen-activated protein kinase (p42/44(MAPK)) activation, PD98059, which had no effect on the up-regulation induced by cyclic AMP, indicating that activation of the mitogen-activated protein kinase signal pathway is specifically involved in the NGF-induced activation of CBP. In addition, expression of a dominant-negative interfering mutant of p42/44(MAPK) can prevent the NGF-mediated induction of the CBP activity, whereas expression of a p42/44(MAPK) constitutively active mutant can enhance the transcriptional activity of CBP. These data indicate that activation of the p42/p44(MAPK) cascade mediates the up-regulation of the transcriptional activity of CBP by NGF, whereas the similar up-regulation induced by cyclic AMP is mediated by PKA activation.