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Corticotropin-releasing factor (CRF) plays a central role in the regulation of the hypothalamic-pituitary-adrenal axis, mediating endocrine and behavioral responses to various stressors. Two high-affinity receptors for CRF have been described. Although many of the intracellular signaling pathways activated by CRF have been studied extensively, our knowledge of transcriptional responses downstream of the CRF receptor 1 (CRFR1) is still limited. To elucidate gene networks regulated by CRF and CRFR1, we applied microarray technology to explore transcriptional response to CRF stimulation. Therefore, mouse pituitary-derived AtT-20 cells were exposed continuously to CRF either in the presence or absence of the specific CRFR1 antagonist R121919. Transcriptional responses to different treatments were studied in a time course ranging from 0.5 to 24 h. Microarray data were analyzed using classic microarray data analysis tools such as correspondence factor analysis, cluster analysis, and fold-change filtering. Furthermore, spectral map analysis was applied, a recently introduced unsupervised multivariate analysis method. A broad and transient transcriptional response to CRF was identified that could be blocked by the antagonist. This way, several known CRF-induced target genes and novel CRF responsive genes were identified. These include transcription factors such as cAMP-responsive element modulator (7x increased), secreted peptides such as cholecystokinin (1.5x), and proteins involved in modulating intracellular signaling, such as regulator of G-protein signaling 2 (11x). Up-regulation of many of these genes can be explained as negative feedback, attenuating CRF-activated pathways. In addition, spectral map analysis proved to be a promising new tool for microarray data analysis.

Corticotropin-releasing factor (CRF) plays an important role in mediating central and peripheral responses to stress. Alterations in CRF system activity have been linked to a number of psychiatric disorders, including anxiety and depression. Aim of this study was to elucidate homeostatic mechanisms induced by lifelong elevated CRF levels in the brain. We therefore profiled gene expression in several brain areas of transgenic mice overexpressing CRF (CRF-OE), a model for chronic stress. Several genes showed altered expression levels in CRF-OE mice when compared to their wild type littermates and were confirmed by quantitative PCR. Differences in gene expression profiles revealed the presence of previously unrecognized homeostatic mechanisms in CRF-OE animals. These included changes in glucocorticoid signaling, as exemplified by changes in 11beta-hydroxysteroid dehydrogenase type 1, FK506 binding protein 5 and serum/glucocorticoid kinase. Alterations in expression of genes involved in myelination (myelin, myelin-associated glycoprotein), cell proliferation and extracellular matrix formation (Edg2, Fgfr2, decorin, brevican) suggest changes in the dynamics of neurogenesis in CRF-OE. Pronounced changes in neurotensin (NT) receptors 1 and 2 mRNA were identified. Overall downregulation of NT receptors in CRF-OE animal was substantiated by receptor binding studies. Pronounced neurotensin receptor downregulation was observed for NT type 1 receptors in limbic brain areas, suggesting that NT could be implicated in some of the effects attributed to CRF overexpression. These data show that lifelong exposure to excessive CRF leads to adaptive changes in the brain which could play a role in some of the behavioral and physiological alterations seen in these animals.

The migration of cells and the extension of cellular processes along pathways to their defined destinations are crucial in the development of higher organisms. Caenorhabditis elegans unc-53 plays an important role in cell migration and the outgrowth of cellular processes such as axons. To gain further insight into the biological function of unc53H2, a recently identified mammalian homologue of unc-53, we have generated mice carrying a mutation of unc53H2 and provide evidence that unc53H2 is involved in neuronal development and, more specifically, the development of different sensory systems. The unc53H2 hypomorphic mouse showed a general impaired acuity of several sensory systems (olfactory, auditory, visual and pain sensation) which in case of the visual system was corroborated by the morphological observation of hypoplasia of the optic nerve. We hypothesize that in analogy with its C. elegans homologue, unc53H2 may play a role in the processes of cellular outgrowth and migration.

This article describes three multivariate projection methods and compares them for their ability to identify clusters of biological samples and genes using real-life data on gene expression levels of leukemia patients. It is shown that principal component analysis (PCA) has the disadvantage that the resulting principal factors are not very informative, while correspondence factor analysis (CFA) has difficulties interpreting distances between objects. Spectral map analysis (SMA) is introduced as an alternative approach to the analysis of microarray data. Weighted SMA outperforms PCA, and is at least as powerful as CFA, in finding clusters in the samples, as well as identifying genes related to these clusters. SMA addresses the problem of data analysis in microarray experiments in a more appropriate manner than CFA, and allows more flexible weighting to the genes and samples. Proper weighting is important, since it enables less reliable data to be down-weighted and more reliable information to be emphasized.

DNA methylation at position 5 in the cytosine ring in the sequence CpG can be detrimental to the transcription of a variety of genes in higher eukaryotes (1,2). Although the significance of this transcriptional repression is currently under debate (3,4), there is little disagreement that it plays an important role in genomic imprinting and X-chromosome inactivation (5,6). To study the effects of DNA methylation on transcription in an experimental system, bacterial DNA methyltransferases have been used widely in order to mimic the DNA methylation pattern of eukaryotic genes. However, usually every target site in a given recombinant DNA molecule will be subject to DNA methylation by making use of those enzymes. This might result in an exaggeration of the effects of DNA methylation, as most recombinant DNA molecules contain a high degree of prokaryotic DNA, which is rich in CpGs. This methylated CpGrich DNA can contribute to the effects of DNA methylation by formation of a repressive chromatin structure (7,8). In addition, selective DNA methylation is required to distinguish the effects of DNA methylation on transcription initiation and transcript elongation (8,9). Thus, there is a requirement for a method to generate recombinant DNA molecules that are methylated in a predetermined region. The chapter following this one will describe a method that makes use of ligation of methylated DNA fragments into unmethylated vector DNA. This method relies on the availability of suitable restriction sites, which allow directional cloning of the fragment and, in addition, requires a highly efficient ligation reaction.

To date, seven different human histone deacetylases (HDACs) have been identified, which fall into two distinct classes. We have isolated and characterized a cDNA encoding a novel human HDAC, which we name HDAC8. HDAC8 shows a high degree of sequence similarity to HDAC1 and HDAC2 and thus belongs to the class I of HDACs. HDAC8 is expressed in a variety of tissues. Human cells overexpressing HDAC8 localize the protein in sub-nuclear compartments whereas HDAC1 shows an even nuclear distribution. In addition, the HDAC8 gene is localized on the X chromosome at position q13, which is close to the XIST gene and chromosomal breakpoints associated with preleukemia.

CpG methylation in vertebrates correlates with alterations in chromatin structure and gene silencing. Differences in DNA-methylation status are associated with imprinting phenomena and carcinogenesis. In Xenopus laevis oocytes, DNA methylation dominantly silences transcription through the assembly of a repressive nucleosomal array. Methylated DNA assembled into chromatin binds the transcriptional repressor MeCP2 which cofractionates with Sin3 and histone deacetylase. Silencing conferred by MeCP2 and methylated DNA can be relieved by inhibition of histone deacetylase, facilitating the remodelling of chromatin and transcriptional activation. These results establish a direct causal relationship between DNA methylation-dependent transcriptional silencing and the modification of chromatin.

DNA methylation is intricately involved in a variety of cellular processes, such as differentiation, cell cycle progression, X-chromosome inactivation and genomic imprinting. However, little is known about how specific DNA methylation patterns are established and maintained. Previously one mammalian DNA methyltransferase has been described, but there has been considerable speculation about the presence of a second activity capable of methylation. Here we report the identification and characterization of a novel human putative DNA methyltransferase. Using a bioinformatics screen we have identified several expressed sequence tags which show high sequence similarity to the Schizosaccharomyces pombe gene pmt1+. The cDNA for PuMet (for putative DNA methyltransferase) was cloned and the predicted amino acid sequence deduced. The gene is ubiquitously expressed, albeit at low levels. Like several other DNA methyltransferases, the bacterially overexpressed protein is not active in methylation assays.

The replication of the genome during S phase is a crucial period for the establishment and maintenance of programmes of differential gene activity. Existing chromosomal structures are disrupted during replication and reassembled on both daughter chromatids. The capacity to reassemble a particular chromatin structure with defined functional properties reflects the commitment of a cell type to a particular state of determination. The core and linker histones and their modifications, enzymes that modify the histones, DNA methylation and proteins that recognize methylated DNA within chromatin may all play independent or interrelated roles in defining the functional properties of chromatin. Pre-existing protein-DNA interactions and DNA methylation in a parental chromosome will influence the structure and function of daughter chromosomes generating an epigenetic imprint. In this chapter we consider the events occurring at the eukaryotic replication fork, their consequences for pre-existing chromosomal structures and how an epigenetic imprint might be maintained.

DNA methylation has an essential regulatory function in mammalian development, serving to repress nontranscribed genes stably in differentiated adult somatic cells. Recent data implicate transcriptional repressors specific for methylated DNA and chromatin assembly in this global control of gene activity. The assembly of specialized nucleosomal structures on methylated DNA helps to explain the capacity of methylated DNA segments to silence transcription more effectively than conventional chromatin. Specialized nucleosomes also provide a potential molecular mechanism for the stable propagation of DNA methylation-dependent transcriptional silencing through cell division.

Background: The regulation of DNA methylation is required for differential expression of imprinted genes during vertebrate development. Earlier studies that monitored the activity of the Herpes simplex virus (HSV) thymidine kinase (tk) gene after injection into rodent cells have suggested that assembly of chromatin influences the methylation-dependent repression of gene activity. Here, we examine the mechanism of methylation-dependent HSV tk gene regulation by direct determination of nucleoprotein organization during the establishment of a transcriptionally silenced state after microinjection of templates with defined methylation states into Xenopus oocyte nuclei., Results: The transcriptional silencing conferred by a methylated DNA segment was not immediate, as methylated templates were initially assembled into active transcription complexes. The eventual loss of DNase I hypersenitive sites and inhibition of transcription at the HSV tk promoter only occurred after several hours. Flanking methylated vector DNA silenced the adjacent unmethylated HSV tk promoter, indicative of a dominant transmissible repression originating from a center of methylation. The resulting repressive nucleoprotein structure silenced transcription in the presence of activators that are able to overcome repression of transcription by nucleosomes., Conclusions: Silencing of transcription by DNA methylation is achieved at the level of transcription initiation and involves the removal of transcriptional machinery from active templates. This transcriptional repression can occur by indirect mechanisms involving the time-dependent assembly of repressive nucleoprotein complexes, which are able to inhibit transcription more effectively than nucleosomes alone.

Expansion and methylation of CGG repeat sequences is associated with Fragile X syndrome in humans. We have examined the consequences of CGG repeat expansion and methylation for nucleosome assembly and positioning on the Fragile X Mental Retardation gene 1 (FMR1) gene. Short unmethylated CGG repeats are not particularly favored in terms of affinity for the histone octamer or for positioning of the reconstituted nucleosome. However, upon methylation their affinity for the histone octamer increases and a highly positioned nucleosome assembles with the repeat sequences found adjacent to the nucleosomal dyad. Expansion of these CGG repeats abolishes the preferential nucleosome assembly due to methylation. Thus, the expansion and methylation of these triplet repeats can alter the functional organization of chromatin, which may contribute to alterations in the expression of the FMR1 gene and the disease phenotype.

The detailed mechanisms of inhibition of transcription by DNA methylation are still unknown, but it has become obvious that the formation of chromatin plays an important role in this process. Using an approach enabling us to methylate, in vitro, chosen regions in a plasmid, we now show that specific methylation of nonpromoter sequences results in transcriptional inhibition of a reporter gene construct and that this inhibition is independent of the position of the methylated region within the plasmid. In plasmid minichromosomes containing a short region of methylated DNA, both methylated and unmethylated sequences are protected from limited MspI digestion. Our results show that inactive chromatin is present at unmethylated regions in partially methylated minichromosomes and can thereby inhibit gene expression. Spreading of the inactive chromatin is not inhibited by the presence of active promoters, nor is it a consequence of transcriptional inactivity.