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Angiogenic factors are necessary for tumor proliferation and thus are attractive therapeutic targets. In this study, we have used engineered zinc finger protein (ZFP) transcription factors (TFs) to repress expression of vascular endothelial growth factor (VEGF)-A in human cancer cell lines. We create potent transcriptional repressors by fusing a designed ZFP targeted to the VEGF-A promoter with either the ligand-binding domain of thyroid hormone receptor alpha or its viral relative, vErbA. Moreover, this ZFP-vErbA repressor binds its intended target site in vivo and mediates the specific deacetylation of histones H3 and H4 at the targeted promoter, a result that emulates the natural repression mechanism of these domains. The potential therapeutic relevance of ZFP-mediated VEGF-A repression was addressed using the highly tumorigenic glioblastoma cell line U87MG. Despite the aberrant overexpression of VEGF-A in this cell line, engineered ZFP TFs were able to repress the expression of VEGF-A by >20-fold. The VEGF-A levels observed after ZFP TF-mediated repression were comparable to those of a nonangiogenic cancer line (U251MG), suggesting that the degree of repression obtained with the ZFP TF would be sufficient to suppress tumor angiogenesis. Thus, engineered ZFP TFs are shown to be potent regulators of gene expression with therapeutic promise in the treatment of disease.

Epigenetic control of transcription is essential for mammalian development and its deregulation causes human disease. For example, loss of proper imprinting control at the IGF2-H19 domain is a hallmark of cancer and Beckwith-Wiedemann syndrome, with no targeted therapeutic approaches available. To address this deficiency, we engineered zinc-finger transcription proteins (ZFPs) that specifically activate or repress the IGF2 and H19 genes in a domain-dependent manner. Importantly, we used these ZFPs successfully to reactivate the transcriptionally silent IGF2 and H19 alleles, thus overriding the natural mechanism of imprinting and validating an entirely novel avenue for 'transcription therapy' of human disease.

Stably integrated transgenes flanked by the chicken beta-globin HS4 insulator are protected against chromosomal position effects and gradual extinction of expression during long-term propagation in culture. To investigate the mechanism of action of this insulator, we used bisulfite genomic sequencing to examine the methylation of individual CpG sites within insulated transgenes, and compared this with patterns of histone acetylation. Surprisingly, although the histones of the entire insulated transgene are highly acetylated, only a specific region in the promoter, containing binding sites for erythroid-specific transcription factors, is highly protected from DNA methylation. This critical region is methylated in noninsulated and inactive lines. MBD3 and Mi-2, subunits of the Mi-2/NuRD repressor complex, are bound in vivo to these silenced noninsulated transgenes. In contrast, insulated cell lines do not show any enrichment of Mi-2/NuRD proteins very late in culture. In addition to the high levels of histone acetylation observed across the entire insulated transgene, significant peaks of H3 acetylation are present over the HS4 insulator elements. Targeted histone acetylation by the chicken beta-globin insulator occurs independently of gene transcription and does not require the presence of a functional enhancer. We suggest that this acetylation is in turn responsible for the maintenance of a region of unmethylated DNA over the promoter. Whereas DNA methylation often leads to histone deacetylation, here acetylation appears to prevent methylation.

Unregulated expression of vascular endothelial growth factor-A (VEGF-A) plays an important role in tumor growth. We have identified a cell type-specific enhancer, HS-1100, that contributes to VEGF-A transcriptional activation in tumorigenic glioblastoma cell lines. This enhancer exhibits increased accessibility to DNase I in glioblastoma cell lines that express high levels of VEGF-A but not in several other cell lines that express much lower levels of VEGF-A. HS-1100 contains a number of sequence elements that are highly conserved among human, mouse, and rat, including the hypoxia-response element (HRE). We show that the HRE contributes significantly to the cell type-specific enhancer activity of HS-1100 in U87MG glioblastoma cells. We use chromatin immunoprecipitation assays to show that endothelial PAS domain protein 1 (EPAS1) can efficiently bind to the endogenous HRE in U87MG cells but not in HEK293 cells in which the chromosomal HS-1100 is not accessible to DNase I. A dominant negative EPAS1 significantly reduces HS-1100 enhancer activity and VEGF-A levels in U87MG cells. Our results provide insight into the molecular mechanisms of VEGF-A up-regulation during cancer development.

Overexpression of the human multidrug resistance gene 1 (MDR1) is a negative prognostic factor in leukemia. Despite intense efforts to characterize the gene at the molecular level, little is known about the genetic events that switch on gene expression in P-glycoprotein-negative cells. Recent studies have shown that the transcriptional competence of MDR1 is often closely associated with DNA methylation. Chromatin remodeling and modification targeted by the recognition of methylated DNA provide a dominant mechanism for transcriptional repression. Consistent with this epigenetic model, interference with DNA methyltransferase and histone deacetylase activity alone or in combination can reactivate silent genes. In the present study, we used chromatin immunoprecipitation to monitor the molecular events involved in the activation and repression of MDR1. Inhibitors of DNA methyltransferase (5-azacytidine [5aC]) and histone deacetylase (trichostatin A [TSA]) were used to examine gene transcription, promoter methylation status, and the chromatin determinants associated with the MDR1 promoter. We have established that methyl-CpG binding protein 2 (MeCP2) is involved in methylation-dependent silencing of human MDR1 in cells that lack the known transcriptional repressors MBD2 and MBD3. In the repressed state the MDR1 promoter is methylated and assembled into chromatin enriched with MeCP2 and deacetylated histone. TSA induced significant acetylation of histones H3 and H4 but did not activate transcription. 5aC induced DNA demethylation, leading to the release of MeCP2, promoter acetylation, and partial relief of repression. MDR1 expression was significantly increased following combined 5aC and TSA treatments. Inhibition of histone deacetylase is not an overriding mechanism in the reactivation of methylated MDR1. Our results provide us with a clearer understanding of the molecular mechanism necessary for repression of MDR1.

To determine functional differences between the two splice variants of PPARgamma (gamma1 and gamma2), we sought to selectively repress gamma2 expression by targeting engineered zinc finger repressor proteins (ZFPs) to the gamma2-specific promoter, P2. In 3T3-L1 cells, expression of ZFP55 resulted in >50% reduction in gamma2 expression but had no effect on gamma1, whereas adipogenesis was similarly reduced by 50%. However, ZFP54 virtually abolished both gamma2 and gamma1 expression, and completely blocked adipogenesis. Overexpression of exogenous gamma2 in the ZFP54-expressing cells completely restored adipogenesis, whereas overexpression of gamma1 had no effect. This finding clearly identifies a unique role for the PPARgamma2 isoform.

As molecular biologists, we are continuing to unravel the interactions by which DNA binding proteins mediate the expression of genes. The chromatin immunoprecipitation (ChIP) technique provides us with an exquisite tool to investigate the interplay between chromatin structure and its role in regulating transcription, replication, and recombination in vivo. We describe a robust assay used to identify the molecular determinants associated with chromatin. In this article we illustrate the ChIP technique and use the transcriptionally silent-hypermethylated multidrug resistance (MDR1) gene as the platform for methyl-CpG binding protein 2 (MeCP2) localization on chromatin. Driven by the hypothesis that repression is strongly dependent on the methylation profile of the endogenous promoter, we demonstrate that MDR1 is targeted by MeCP2. Methylated MDR1 chromatin is highly enriched with MeCP2 and is in striking contrast to localization observed in cells in which MDR1 is transcriptionally active. In a distinct model system we discuss experimental methods used to immunopurify the MeCP2 repressor complex on chromatin and quantify protein-DNA association by competitive PCR approach., ((c) 2001 Elsevier Science.)

Innumerable transcription factors integrate cellular and intercellular signals to generate a profile of expressed genes that is characteristic of the biochemical and cellular properties of the cell. This profile of expressed genes changes dynamically along with the developmental stage and differentiation state of the cell. The biochemical machinery upon which transcription factors integrate their signals is referred to as the general transcription machinery. However, this machinery is not of universal composition, and variants of the general transcription factors play specific roles in embryonic development, reflecting the constraints and requirements of developmental gene regulation.

Methylation of CpG dinucleotides is a predominant modification of genomic DNA in many species, especially in vertebrates. This modification, generally associated with transcriptional repression, is rapidly and globally lost during mammalian pre-implantation development. This loss of methylation is gradually reversed during subsequent stages of development. Here we show that the amphibian Xenopus laevis maintains high levels of DNA methylation during early embryonic development. The methylation status of specific loci is independent of the temporal expression profile. The observations have profound implications for the regulation of early embryonic gene regulation and genome function.

DNA methylation in Drosophila melanogaster is restricted temporally during development and occurs at a significantly lower frequency than in mammals. Thus, the regulatory functions, if any, of this form of DNA modification in Drosophila are unclear. However, the presence of homologs of vertebrate methyl-CpG-binding proteins implies functional consequences for DNA methylation in flies. This work describes the properties of dMBD-like, a Drosophila homolog of vertebrate MBD2 and MBD3. dMBD-like and dMBD-likeDelta (a splice variant) failed to bind model methylated DNA probes, inconsistent with their function as mediators of methyl CpG-directed transcriptional repression. However, the MBD-like proteins exhibit transcriptional and biochemical properties consistent with roles as components of a histone deacetylase-dependent corepressor complex similar to the vertebrate Mi-2 complex. The two proteins are differentially expressed during development, suggesting functional specialization. dMBD-like and/or dMBD-likeDelta is present at the chromocenter on larval polytene chromosomes as well as at discrete bands interspersed along the euchromatic chromosome arms, many of which are coincident with known ecdysone-induced loci. This banding pattern suggests gene-specific regulatory functions for dMBD-like and the Drosophila Mi-2 complex.

The chromatin architecture of a promoter is an important determinant of its transcriptional response. For most target genes, the thyroid hormone receptor (TR) activates gene expression in response to thyroid hormone (T(3)). In contrast, the thyroid-stimulating hormone alpha-subunit (TSH alpha) gene promoter is down-regulated by TR in the presence of T(3). Here we utilize the capacity for the Xenopus oocyte to chromatinize exogenous nuclear- injected DNA to analyze the chromatin architecture of the TSH alpha promoter and how this changes upon TR-mediated regulation. Interestingly, in the oocyte, the TSH alpha promoter was positively regulated by T(3). In the inactive state, the promoter contained six loosely positioned nucleosomes. The addition of TR/retinoid X receptor together had no effect on the chromatin structure, but the inclusion of T(3) induced strong positioning of a dinucleosome in the TSH alpha proximal promoter that was bordered by regions that were hypersensitive to cleavage by methidiumpropyl EDTA. We identified a novel thyroid response element that coincided with the proximal hypersensitive region. Furthermore, we examined the consequences of mutations in TR that impaired coactivator recruitment. In a comparison with the Xenopus TR beta A promoter, we found that the effects of these mutations on transactivation and chromatin remodeling were significantly more severe on the TSH alpha promoter.

The assembly of the genome into chromatin imposes a poorly understood set of rules and constraints on action by regulatory factors. We investigated the role played by chromatin infrastructure in enabling an acute response of the Xenopus TRbetaA gene to thyroid hormone receptor (TR), an extensively studied member of the nuclear hormone receptor superfamily. We found that in addition to the known TR response element (TRE) in the promoter, full range regulation required an upstream enhancer that contained multiple nonconsensus TREs and augmented ligand action at high receptor levels. An array of translationally positioned nucleosomes formed over the TRbetaA locus in vivo; unliganded TR engaged this array in linker DNA between two nucleosomes and via TREs on the surface of histone octamers. Remarkably, assembly of enhancer DNA into mature chromatin potentiated binding by TR to its target response elements and enabled a greater range of regulation by TR than was observed on immature chromatin templates. Because assembly of enhancer DNA into chromatin increased TR binding to the nonconsensus TREs, we hypothesize that chromatin disruption targeted by liganded TR to the enhancer may lead to receptor release from the template and to an attenuation of response to hormone.

CpG methylation is maintained in daughter chromatids by the action of DNA methyltransferase at the replication fork. An opportunity exists for transcription factors at replication forks to bind their cognate sequences and thereby prevent remethylation by DNA methyltransferase. To test this hypothesis, we injected a linearized, methylated, and partially single-stranded reporter plasmid into the nuclei of Xenopus oocytes and followed changes in the transcriptional activity after DNA replication. We find that dependent on Gal4-VP16, the action of DNA methyltransferase, and replication-coupled chromatin assembly DNA replication provides a window of time in which regulatory factors can activate or repress gene activity. Demethylation in the promoter region near the GAL4 binding sites of the newly synthesized DNA did not occur even though the Gal4 binding sites were occupied and transcription was activated. We conclude that "passive" demethylation at the replication fork is not simply dependent on the presence of DNA binding transcriptional activators.

A typical human cell expresses only a few thousand of the more than 30 000 genes contained within our chromosomes. The chromosomal infrastructure is essential for gene control, determining both active and repressed states. It is important not only to turn the right genes on but also to turn the right genes off. Histones and chromatin components have key roles in this decision making process. Mistakes have severe consequences. If as few as three inappropriate genes are turned off, a normal cell can be converted into a cancer cell. This epigenetic silencing of genes underlies a new approach to cancer therapy. Advances in the biochemistry and genetics of chromatin remodeling reveal that gene inactivation depends on the recruitment of enzymes that control the display of DNA within the chromosome. Mistargeting of these enzymes leads to tumorigenesis, but inhibition of their activity presents a novel approach to therapy.

The number of chromatin modifying and remodeling complexes implicated in genome control is growing faster than our understanding of the functional roles they play. We discuss recent in vitro experiments with biochemically defined chromatin templates that illuminate new aspects of action by histone acetyltransferases and ATP-dependent chromatin remodeling engines in facilitating transcription. We review a number of studies that present an 'ordered recruitment' view of transcriptional activation, according to which various complexes enter and exit their target promoter in a set sequence, and at specific times, such that action by one complex sets the stage for the arrival of the next one. A consensus emerging from all these experiments is that the joint action by several types of chromatin remodeling machines can lead to a more profound alteration of the infrastructure of chromatin over a target promoter than could be obtained by these enzymes acting independently. In addition, it appears that in specific cases one type of chromatin structure alteration (e.g., histone hyperacetylation) is contingent upon prior alterations of a different sort (i.e., ATP-dependent remodeling of histone-DNA contacts). The striking differences between the precise sequence of action by various cofactors observed in these studies may be - at least in part - due to differences between the specific promoters studied, and distinct requirements exhibited by specific loci for chromatin remodeling based on their pre-existing nucleoprotein architecture.

Chromatin structure, the organized packaging of DNA with histones in the nucleus, is now seen as a dynamic fabric that changes with development. Here, we use studies on the phaseolin (phas) gene that encodes a seed protein to show how chromatin structure interacts with the transcription machinery to accomplish rigorous spatial regulation of expression. In leaf and other vegetative tissues, a nucleosome is rotationally and translationally positioned over an ensemble of three phased TATA boxes, denying access to TBP. Current interest focuses on the mechanisms by which this architecture is remodeled during embryogenesis. The transcription factor PvALF is intrinsically involved, as are other non-histone proteins and abscisic acid. These concepts, and the possible modular nature of phas expression, are summarized together with speculations concerning the re-establishment of the nucleosome over the phas promoter during terminal stages of embryogenesis.

DNA methylation of tumor suppressor genes is a common feature of human cancer. The cyclin-dependent kinase inhibitor gene p16/Ink4A is hypermethylated in a wide range of malignant tissues and the p14/ARF gene located 20 kb upstream on chromosome 9p21 is also methylated in carcinomas. p14/ARF (ARF, alternative reading frame) does not inhibit the activities of cyclins or cyclin-dependent kinase complexes; however, the importance of the two gene products in the etiology of cancer resides in their involvement in two major cell cycle regulatory pathways: p53 and the retinoblastoma protein, Rb, respectively. Distinct first exons driven from separate promoters are spliced onto the common exons 2 and 3 and the resulting proteins are translated in different reading frames. Both genes are expressed in normal cells but can be alternatively or coordinately silenced when their CpG islands are hypermethylated. Herein, we examined the presence of methyl-CpG binding proteins associated with aberrantly methylated promoters, the distribution of acetylated histones H3 and H4 by chromatin immunoprecipitation assays, and the effect of chemical treatment with 5-aza-2'-deoxycytidine (5aza-dC) and trichostatin A on gene induction in colon cell lines by quantitative reverse transcriptase-PCR. We observed that the methyl-CpG binding protein MBD2 is targeted to methylated regulatory regions and excludes the acetylated histones H3 and H4, resulting in a localized inactive chromatin configuration. When methylated, the genes can be induced by 5aza-dC but the combined action of 5aza-dC and trichostatin A results in robust gene expression. Thus, methyl-CpG binding proteins and histone deacetylases appear to cooperate in vivo, with a dominant effect of DNA methylation toward histone acetylation, and repress expression of tumor suppressor genes hypermethylated in cancers.

We have mapped conserved regions of enhanced DNase I accessibility within the endogenous chromosomal locus of vascular endothelial growth factor A (VEGF-A). Synthetic zinc finger protein (ZFP) transcription factors were designed to target DNA sequences contained within the DNase I-hypersensitive regions. These ZFPs, when fused to either VP16 or p65 transcriptional activation domains, were able to activate expression of the VEGF-A gene as assayed by mRNA accumulation and VEGF-A protein secretion through a range exceeding that induced by hypoxic stress. Importantly, multiple splice variants of VEGF-A mRNA with defined physiological functions were induced by a single engineered ZFP transcription factor. We present evidence for an enhanced activation of VEGF-A gene transcription by ZFP transcription factors fused to VP16 and p65 targeted to two distinct chromosomal sites >500 base pairs upstream or downstream of the transcription start site. Our strategy provides a novel approach for dissecting the requirements for gene regulation at a distance without altering the DNA sequence of the endogenous target locus.

Epigenetic regulation involves the maintenance of a particular state of gene expression--most commonly, repression--in the face of repeated mitosis, and frequently meiosis. Remarkably, changes in such heritable expression states occur without an alteration of the primary DNA sequence. We present a brief history of research in epigenetics, beginning with pioneering work in the 1950s by B. McClintock and R. A. Brink on maize kernel color inheritance. We describe the complex biochemistry of DNA methylation--the molecular basis of most epigenetic regulation in mammalian genomes--and review data connecting it to targeted modification and remodeling of chromatin structure. Several prominent examples of epigenetically regulated loci--X chromosome inactivation, imprinting, repetitive DNA silencing, and aberrant methylation patterns in neoplasia--are reviewed along with a description of our current understanding of the underlying molecular mechanisms. A common theme that emerges is the complex integration of epigenetic regulatory pathways with the chromatin infrastructure over target DNA loci.

A wide variety of histone-like proteins can be assembled into nucleosomal structures. Core and linker histone variants, proteins of the histone-fold and winged-helix families can all contribute to the local differentiation of functional chromosomal domains. It is very difficult to disrupt core histone interactions within a nucleosome in vivo. Histones H3 and H4 do not exchange out of chromatin outside S-phase. Histones H2A and H2B do exchange out of chromatin, but do so predominantly during transcription. This confers stability on the nucleosome during the cell cycle. Linker histones have a much less stable association with nucleosomal DNA, allowing for reversible activation of transcription. A distinct feature of histone interactions with nucleosomal DNA is the exposure of DNA on the surface of the nucleosome. One side of DNA is occluded on the histone surface, but the other is exposed and potentially accessible to other regulatory proteins. A major contributory factor to the functional specialization of chromatin is the capacity to target nucleosome modification and disruption.

DNA methylation has long been associated with stable transcriptional silencing and a repressive chromatin structure (reviewed in refs. 1,2). Differential methylation is associated with imprinting, carcinogenesis, silencing of repetitive DNA, and allows for differentiating cells to efficiently shut off unnecessary genes. In vertebrates, where 60-90% of genomic CpG dinucleotides are methylated, methylation-dependent repression is vital for proper embryonic development (3). Microinjection experiments using methylated DNA templates implicate chromatin structure as an underlying mechanism of methylation-dependent silencing (4,5). Methyl-specific transcriptional repression requires chromatin assembly, and can be partially relieved by the histone deacetylase inhibitor Trichostatin A. In addition, several proteins have been identified that specifically bind to methylated DNA (6-8). Two of these methyl-DNA binding proteins, MeCP1 and MeCP2, have been shown to mediate transcriptional repression (6,7). MeCP1 is a relatively uncharacterized complex that requires at least 12 symmetrical methyl-CpGs for DNA binding (6). MeCP2 is a single polypeptide containing a methyl-binding domain capable of binding a single methyl-CpG, and a transcriptional repression domain (9). Recently MeCP2 was shown to interact with the Sin3 corepressor and histone deacetylase (10,11). Changes in the acetylation state of the core histone tails correlates with changes in transcription (reviewed in refs. 12,13), and several transcriptional repression complexes containing histone deacetylases have recently been described (10,14,15).

Transcriptional activity is closely associated with DNA methylation and chromatin remodelling. Evidence is emerging that a family of methylation specific (methyl-CpG binding domain, MBD) proteins have the capacity to bind to methylated sequences and repress transcription. Recent advances in this area reveal that many of the MBD proteins are associated with histone deacetylase (HDAC) dependant repression. The capacity of MBD association to repress transcription would largely be defined by promoter structure and this is best explained by the position and density of DNA methylation. The mechanism of specific targeting of MBD family members to methylated sequences remains largely unknown. In order to understand the mechanistic details of silencing the current challenge is to identify and map these molecular determinants assembled on native chromatin in model systems of human development and disease. Downstream targets such as the methylated Fragile X Mental Retardation gene 1 (FMR1) gene and tumour suppressor genes are likely candidates. In this article, we describe a powerful strategy that involves the immunoprecipitation of in vivo formaldehyde fixed chromatin to identify MBD binding complexes directly isolated from the natural chromosomal environment. We demonstrate the methylated human Multidrug Resistance gene 1 (MDR1) is enriched with transcriptional repressors that belong to the MBD family and this would account for transcriptional silencing.

CpG methylation, the most common epigenetic modification of vertebrate genomes, is primarily associated with transcriptional repression. MeCP2, MBD1, MBD2, MBD3 and MBD4 constitute a family of vertebrate proteins that share the methyl-CpG-binding domain (MBD). The MBD, consisting of about 70 residues, possesses a unique alpha/beta-sandwich structure with characteristic loops, and is able to bind single methylated CpG pairs as a monomer. All MBDs except MBD4, an endonuclease that forms a complex with the DNA mismatch-repair protein MLH1, form complexes with histone deacetylase. It has been established that MeCP2, MBD1 and MBD2 are involved in histone deacetylase-dependent repression and it is likely that this is also the case for MBD3. The current model proposes that MBD proteins are involved in recruiting histone deacetylases to methyl CpG-enriched regions in the genome to repress transcription. The lack of selectivity for MBD association with particular DNA sequences indicates that other mechanisms account for their recruitment to particular regions in the genome.

The TATA-binding protein (TBP) is believed to function as a key component of the general transcription machinery. We tested the role of TBP during the onset of embryonic transcription by antisense oligonucleotide-mediated turnover of maternal TBP messenger RNA. Embryos without detectable TBP initiated gastrulation but died before completing gastrulation. The expression of many genes transcribed by RNA polymerase II and III was reduced; however, some genes were transcribed with an efficiency identical to that of TBP-containing embryos. Using a similar antisense strategy, we found that the TBP-like factor TLF/TRF2 is essential for development past the mid-blastula stage. Because TBP and a TLF factor play complementary roles in embryonic development, our results indicate that although similar mechanistic roles exist in common, TBP and TLF function differentially to control transcription of specific genes.

The nucleosomal ATPase ISWI is the catalytic subunit of several protein complexes that either organize or perturb chromatin structure in vitro. This work reports the cloning and biochemical characterization of a Xenopus ISWI homolog. Surprisingly, whereas we find four complex forms of ISWI in egg extracts, we find no functional homolog of NURF. One of these complexes, xACF, consists of ISWI, Acf1, and a previously uncharacterized protein of 175 kDa. Like both ACF and CHRAC, this complex organizes randomly deposited histones into a regularly spaced array. The remaining three forms include two novel ISWI complexes distinct from known ISWI complexes plus a histone-dependent ATPase complex. This comprehensive biochemical characterization of ISWI underscores the evolutionary conservation of the ACF/CHRAC family.

The neurodevelopmental disorder known as Rett syndrome has recently been linked to the methyl-CpG-binding transcriptional repressor, MeCP2. In this report we examine the consequences of these mutations on the function of MeCP2. The ability to bind specifically to methylated DNA and the transcription repression capabilities are tested, as well as the stability of proteins in vivo. We find that all missense mutations (R106W, R133C, F155S, T158M) within the methyl-binding domain impair selectivity for methylated DNA, and that all nonsense mutations (L138X, R168X, E235X, R255X, R270X, V288X, R294X) that truncate all or some of the transcriptional repression domain (TRD) affect the ability to repress transcription and have decreased levels of stability in vivo. Two missense mutations, one in the TRD (R306C) and one in the C-terminus (E397K), had no noticeable effects on MeCP2 function. Together, these results provide evidence of how Rett syndrome mutations can affect distinct functions of MeCP2 and give insight into these mutations that may contribute to the disease.

We have targeted the activation of an endogenous chromosomal locus including the human erythropoietin gene using synthetic transcription factors. These transcription factors are targeted to particular DNA sequences in the 5'-flanking region of the erythropoietin gene through engineering of a zinc finger DNA binding domain. The DNA binding domain is linked to a VP16 transcriptional activation domain. We find that these synthetic transcription factors invariably activate transiently transfected templates in which sequences within the 5' flank of the erythropoietin gene are fused to a luciferase reporter. The efficiency of activation under these circumstances at a defined site is dependent on DNA binding affinity. In contrast, only a subset of these same zinc finger proteins is able to activate the endogenous chromosomal locus. The activity of these proteins is influenced by their capacity to gain access to their recognition elements within the chromatin infrastructure. Zinc finger transcription factors will provide a powerful tool to probe the determinants of chromatin accessibility and remodeling within endogenous chromosomal loci.

DNA methylation has recently moved to centre stage in the aetiology of human neurodevelopmental syndromes such as the fragile X, ICF and Rett syndromes. These diseases result from the misregulation of genes that occurs with the loss of appropriate epigenetic controls during neuronal development. Recent advances have connected DNA methylation to chromatin-remodelling enzymes, and understanding this link will be central to the design of new therapeutic tools.

Cloning by the transplantation of somatic nuclei into unfertilized eggs requires a dramatic remodeling of chromosomal architecture. Many proteins are specifically lost from nuclei, and others are taken up from the egg cytoplasm. Recreating this exchange in vitro, we identified the chromatin-remodeling nucleosomal adenosine triphosphatase (ATPase) ISWI as a key molecule in this process. ISWI actively erases the TATA binding protein from association with the nuclear matrix. Defining the biochemistry of global nuclear remodeling may facilitate the efficiency of cloning and other dedifferentiation events that establish new stem cell lineages.

The liver-specific vitellogenin B1 promoter is efficiently activated by estrogen within a nucleosomal environment after microinjection into Xenopus laevis oocytes, consistent with the hypothesis that significant nucleosome remodeling over this promoter is not a prerequisite for the activation by the estrogen receptor (ERalpha). This observation lead us to investigate determinants other than ERalpha of chromatin structure and transcriptional activation of the vitellogenin B1 promoter in this system and in vitro. We find that the liver-enriched transcription factor HNF3 has an important organizational role for chromatin structure as demonstrated by DNase I-hypersensitive site mapping. Both HNF3 and the estrogen receptor activate transcription synergistically and are able to interact with chromatin reconstituted in vitro with three positioned nucleosomes. We propose that HNF3 is the cellular determinant which establishes a promoter environment favorable to a rapid transcriptional activation by the estrogen receptor.

Acetylation of nucleosome core histones, which is positively correlated with transcriptional activity, is developmentally regulated in Xenopus. Here we have used the specific histone deacetylase (HDAC)-inhibitor trichostatin A (TSA) to induce precocious histone hyperacetylation in the early frog embryo in order to investigate the potential role of the endogenous changes in chromatin acetylation for the temporally programmed induction of skeletal myogenesis. We show that TSA-treatment (i) selectively blocked the transcriptional induction of the myoD gene, and (ii) severely reduced subsequent muscle differentiation. Both phenotypes required TSA application before gastrulation. This indicates that HDAC activity is required early for the formation of the frog embryonic musculature, apparently for the induction of the MyoD-dependent muscle cell lineage.

Transcriptional repression by nuclear hormone receptors is thought to result from a unison of targeting chromatin modification and disabling the basal transcriptional machinery. We used Xenopus oocytes to compare silencing effected by the thyroid hormone receptor (TR) and its mutated version, the oncoprotein v-ErbA, on partly and fully chromatinized TR-responsive templates in vivo. Repression by v-ErbA was not as efficient as that mediated by TR, was significantly more sensitive to histone deacetylase (HDAC) inhibitor treatment and, unlike TR, v-ErbA required mature chromatin to effect repression. We find that both v-ErbA and TR can recruit the corepressor N-CoR, but, in contrast to existing models, show a concomitant enrichment for HDAC3 that occurs without an association with Sin3, HDAC1/RPD3, Mi-2 or HDAC5. We propose a requirement for chromatin infrastructure in N-CoR/HDAC3-effected repression and suggest that the inability of v-ErbA to silence on partly chromatinized templates may stem from its impaired capacity to interfere with basal transcriptional machinery function. In support of this notion, we find v-ErbA to be less competent than TR for binding to TFIIB in vitro and in vivo.

Methylation of CpG islands is associated with transcriptional silencing and the formation of nuclease-resistant chromatin structures enriched in hypoacetylated histones. Methyl-CpG-binding proteins, such as MeCP2, provide a link between methylated DNA and hypoacetylated histones by recruiting histone deacetylase, but the mechanisms establishing the methylation patterns themselves are unknown. Whether DNA methylation is always causal for the assembly of repressive chromatin or whether features of transcriptionally silent chromatin might target methyltransferase remains unresolved. Mammalian DNA methyltransferases show little sequence specificity in vitro, yet methylation can be targeted in vivo within chromosomes to repetitive elements, centromeres and imprinted loci. This targeting is frequently disrupted in tumour cells, resulting in the improper silencing of tumour-suppressor genes associated with CpG islands. Here we show that the predominant mammalian DNA methyltransferase, DNMT1, co-purifies with the retinoblastoma (Rb) tumour suppressor gene product, E2F1, and HDAC1 and that DNMT1 cooperates with Rb to repress transcription from promoters containing E2F-binding sites. These results establish a link between DNA methylation, histone deacetylase and sequence-specific DNA binding activity, as well as a growth-regulatory pathway that is disrupted in nearly all cancer cells.

ATP-dependent chromatin remodeling enzymes antagonize the inhibitory effects of chromatin. We compare six different remodeling complexes: ySWI/SNF, yRSC, hSWI/SNF, xMi-2, dCHRAC, and dNURF. We find that each complex uses similar amounts of ATP to remodel nucleosomal arrays at nearly identical rates. We also perform assays with arrays reconstituted with hyperacetylated or trypsinized histones and isolated histone (H3/H4)(2) tetramers. The results define three groups of the ATP-dependent family of remodeling enzymes. In addition we investigate the ability of an acidic activator to recruit remodeling complexes to nucleosomal arrays. We propose that ATP-dependent chromatin remodeling enzymes share a common reaction mechanism and that a key distinction between complexes is in their mode of regulation or recruitment.

We have investigated the properties of mutant forms of the methyl-CpG binding transcriptional repressor MeCP2 associated with Rett syndrome, a childhood neurodevelopmental disorder. We find that four Rett syndrome mutations at known sites within the methyl-CpG binding domain (MBD) impair binding to methylated DNA, but have little effect on nonspecific interactions with unmethylated DNA. Three of these mutations (R106W, R133C, and F155S) have their binding affinities for methylated DNA reduced more than 100-fold; this is consistent with the hypothesis that impaired selectivity for methylated DNA of mutant MeCP2 contributes to Rett syndrome. However, a fourth mutant, T158M, has its binding affinity for methylated DNA reduced only 2-fold, indicative either of additional distinct regulatory functions associated with the MBD or of an exquisite sensitivity of developing neurons to the selective association of MeCP2 with methylated DNA.

Recent studies on the transcriptional regulation of two linked, imprinted genes, Igf2 and H19, have provided evidence for a novel mechanism of epigenetic control. DNA methylation controls the activity of an insulator element located between the two linked genes by regulating the binding of the zinc-finger protein CTCF.

The Mi-2 complex has been implicated in chromatin remodeling and transcriptional repression associated with histone deacetylation. Here, we use a purified Mi-2 complex containing six components, Mi-2, Mta 1-like, p66, RbAp48, RPD3, and MBD3, to investigate the capacity of this complex to destabilize histone-DNA interactions and deacetylate core histones. The Mi-2 complex has ATPase activity that is stimulated by nucleosomes but not by free histones or DNA. This nucleosomal ATPase is relatively inefficient, yet is essential to facilitate both translational movement of histone octamers relative to DNA and the efficient deacetylation of the core histones within a mononucleosome. Surprisingly, ATPase activity had no effect on deacetylation of nucleosomal arrays.

The pathways for selective transcriptional repression of methylated DNA templates by the methyl-CpG-binding protein MeCP2 have been investigated using a purified in vitro transcription system that does not assemble chromatin. MeCP2 selectively inhibits transcription complex assembly on methylated DNA but does not destabilize a pre-assembled transcription complex. MeCP2 functions to repress transcription at a distance of >500 bp from the transcription start site. The transcription repression domain (TRD) of MeCP2 will repress transcription in vitro when fused to a heterologous Gal4 DNA-binding domain. The TRD associates with TFIIB. Exogenous TFIIB does not relieve transcriptional repression established by either intact MeCP2 or a Gal4-TRD fusion protein under these in vitro conditions, nor does the addition of histone deacetylase inhibitors. We find that the transcriptional repression established by both MeCP2 and the Gal4-TRD fusion protein in vitro also correlates with selective assembly of large nucleoprotein complexes. The formation of such complexes reflects a local concentration of DNA-bound transcriptional repressor that may stabilize a state of repression even in the presence of exogenous transcriptional machinery.

The nucleosome and chromatin fiber provide the common structural framework for transcriptional control in eukaryotes. The folding of DNA within these structures can both promote and impede transcription dependent on structural context. Importantly, neither the nucleosome nor the chromatin fiber is a static structure. Histone dissociation, histone modification, nucleosome mobility, and assorted allosteric transitions contribute to transcriptional control. Chromatin remodeling is associated with gene activation and repression. Energy-dependent processes mediate the assembly of both activating and repressive proteins into the nucleosomal infrastructure. Recent progress allows the structural consequences of these processes to be visualized at the chromosomal level. DNA and RNA polymerase, SWI/SNF complexes, histone deacetylases, and acetyltransferases are targeted by gene-specific regulators to mediate these structural transitions. The mistargeting of these enzymes contributes to human developmental abnormalities and tumorigenesis. These observations illuminate the roles of chromatin and chromosomal structural biology in human disease.

Histone acetyltransferases (HATs) play important roles in the regulation of gene expression. In this report, we describe the design, synthesis, and application of peptide CoA conjugates as selective HAT inhibitors for the transcriptional coactivators p300 and PCAF. Two inhibitors (Lys-CoA for p300 and H3-CoA-20 for PCAF) were found to be potent (IC(50) approximately = 0.5 microM) and selective (approximately 200-fold) in blocking p300 and PCAF HAT activities. These inhibitors were used to probe enzymatic and transcriptional features of HAT function in several assay systems. These compounds should be broadly useful as biological tools for evaluating the roles of HATs in transcriptional studies and may serve as lead agents for the development of novel antineoplastic therapeutics.