Erin O'Shea (Harvard): Large-scale characterization of nucleosome positioning in Saccharomyces cerevisiae has revealed a stereotyped promoter organization in which a nucleosome-free region (NFR) is present within several hundred base pairs upstream of the translation start site. Many transcription factors bind within NFRs and nucleate chromatin remodelling events which then expose other cis-regulatory elements. However, it is not clear how transcription-factor binding and chromatin influence quantitative attributes of gene expression. Here we show that nucleosomes function largely to decouple the threshold of induction from dynamic range. With a series of variants of one promoter, we establish that the affinity of exposed binding sites is a primary determinant of the level of physiological stimulus necessary for substantial gene activation, and sites located within nucleosomal regions serve to scale expression once chromatin is remodelled. Furthermore, we find that the S. cerevisiae phosphate response (PHO) pathway exploits these promoter designs to tailor gene expression to different environmental phosphate levels. Our results suggest that the interplay of chromatin and binding-site affinity provides a mechanism for fine-tuning responses to the same cellular state. Moreover, these findings may be a starting point for more detailed models of eukaryotic transcriptional control.

Malcolm Walkinshaw (University of Edinburgh): MeCP2 is an essential transcriptional repressor that mediates gene silencing through binding to methylated DNA. Binding specificity has been thought to depend on hydrophobic interactions between cytosine methyl groups and a hydrophobic patch within the methyl-CpG-binding domain (MBD). X-ray analysis of a methylated DNA-MBD cocrystal reveals, however, that the methyl groups make contact with a predominantly hydrophilic surface that includes tightly bound water molecules. This suggests that MeCP2 recognizes hydration of the major groove of methylated DNA rather than cytosine methylation per se. The MeCP2-DNA complex also identifies a unique structural role for T158, the residue most commonly mutated in Rett syndrome.

Eric Nestler (The University of Texas Southwestern Medical Center): Here, we identify the activity-dependent class II histone deacetylase, HDAC5, as a central integrator of these stimuli with changes in chromatin structure and gene expression. Chronic, but not acute, exposure to cocaine or stress decreases HDAC5 function in the nucleus accumbens (NAc), a major brain reward region, which allows for increased histone acetylation and transcription of HDAC5 target genes. This regulation is behaviorally important, as loss of HDAC5 causes hypersensitive responses to chronic, not acute, cocaine or stress. These findings suggest that proper balance of histone acetylation is a crucial factor in the saliency of a given stimulus and that disruption of this balance is involved in the transition from an acute adaptive response to a chronic psychiatric illness.

Horace Loh (University of Minnesota Medical School): MOR expression could be induced by a demethylating agent (5'-aza-2'-deoxycytidine) or histone deacetylase inhibitors in the P19 cells, suggesting involvement of DNA methylation and histone deacetylation for MOR gene silencing. Analysis of CpG DNA methylation revealed that the proximal promoter region was unmethylated in differentiated cells compared to its hypermethylation in undifferentiated cells. In contrast, the methylation of other regions was not changed in either cell type. In vitro methylation of the MOR promoters suppressed promoter activity in the reporter assay. Methyl-CpG-binding proteins (MBPs), such as MBP 2 (MeCP2), bind preferentially to methylated DNA and directly repress transcription, inhibit the binding of other trans factors, structurally modify the DNA, or recruit corepressor complexes. Upon differentiation, the in vivo interaction of MeCP2 was reduced in the MOR promoter region, coincident with histone modifications that are relevant to active transcription. When MeCP2 was disrupted using MeCP2 small interfering RNA, the endogenous MOR gene was increased. These data suggest that DNA methylation is closely linked to the MeCP2-mediated chromatin structure of the MOR gene.

Schahram Akbarian (University of Massachusetts Medical School): We show that histone H3-lysine 4 methylation progressively increased at GAD1 and other GABAergic gene promoters (GAD2, NPY, SST) in human prefrontal cortex (PFC) from prenatal to peripubertal ages and throughout adulthood. Alterations in schizophrenia included decreased GAD1 expression and H3K4-trimethylation, predominantly in females and in conjunction with a risk haplotype at the 5' end of GAD1. Heterozygosity for a truncated, lacZ knock-in allele of mixed-lineage leukemia 1 (Mll1), a histone methyltransferase expressed in GABAergic and other cortical neurons, resulted in decreased H3K4 methylation at GABAergic gene promoters. In contrast, Gad1 H3K4 (tri)methylation and Mll1 occupancy was increased in cerebral cortex of mice after treatment with the atypical antipsychotic, clozapine. These effects were not mimicked by haloperidol or genetic ablation of dopamine D2 and D3 receptors, suggesting that blockade of D2-like signaling is not sufficient for clozapine-induced histone methylation.

Laura Landweber (Princeton University): During development of the somatic macronucleus, Oxytricha trifallax destroys 95% of its germ line, severely fragmenting its chromosomes, and then unscrambles hundreds of thousands of remaining fragments by permutation or inversion. Here we demonstrate that DNA or RNA templates can orchestrate these genome rearrangements in Oxytricha, supporting an epigenetic model for sequence-dependent comparison between germline and somatic genomes. A complete RNA cache of the maternal somatic genome may be available at a specific stage during development to provide a template for correct and precise DNA rearrangement. We show the existence of maternal RNA templates that could guide DNA assembly, and that disruption of specific RNA molecules disables rearrangement of the corresponding gene. Injection of artificial templates reprogrammes the DNA rearrangement pathway, suggesting that RNA molecules guide genome rearrangement.

Enrico Avvedimento (Università degli Studi, Naples, Italy): We analyzed how H3 histone methylation and demethylation control expression of estrogen-responsive genes and show that a DNA-bound estrogen receptor directs transcription by participating in bending chromatin to contact the RNA polymerase II recruited to the promoter. This process is driven by receptor-targeted demethylation of H3 lysine 9 at both enhancer and promoter sites and is achieved by activation of resident LSD1 demethylase. Localized demethylation produces hydrogen peroxide, which modifies the surrounding DNA and recruits 8-oxoguanine–DNA glycosylase 1 and topoisomerase IIbeta, triggering chromatin and DNA conformational changes that are essential for estrogen-induced transcription. Our data show a strategy that uses controlled DNA damage and repair to guide productive transcription.

Wolfgang Sadée (The Ohio State University): To explore the effect of polymorphisms and epigenetic factors on mRNA expression, we have measured allelic expression imbalance (AEI) in female human brain tissue, employing two frequent marker single nucleotide polymorphisms (SNPs) in exon 8 (T890G) and exon 14 (C1409T) of MAOA. AEI ratios ranged from 0.3 to 4 in prefrontal cortex, demonstrating the presence of strong cis-acting factors in mRNA expression. Analysis of CpG methylation in the MAOA promoter region revealed substantial methylation in females but not in males. MAOA methylation ratios for the three- and four-repeat pVNTR alleles of MAOA did not correlate with X-chromosome inactivation ratios, determined at the X-linked androgen receptor locus, suggesting an alternative process of dosage compensation in females. The extent of allelic MAOA methylation was highly variable and correlated with AEI (R2=0.5 and 0.7 at two CpG loci), indicating that CpG methylation regulates gene expression. Genetic factors appeared also to contribute to the AEI ratios. Genotyping of 13 MAOA polymorphisms in female subjects showed strong association with a haplotype block spanning from the pVNTR to the marker SNP. Therefore, allelic mRNA expression is affected by genetic and epigenetic events, both with the potential to modulate biogenic amine tone in the CNS.

Carmen Sapienza (Temple University School of Medicine): We show that imprinted regions contain significantly more LD units (LDU) and have significantly more haplotype blocks of smaller sizes than flanking nonimprinted regions. There is also an excess of hot-spots of recombination at imprinted regions, and this is likely to do with the presence of imprinted genes, per se. These findings indicate that imprinted chromosomal regions are historical “hot-spots” of recombination. Interestingly, fine-mapping of recombination events within the most male meiosis–specific recombination hot-spot of Chromosome 11p15.5 indicates that many events may occur within or directly adjacent to regions that are differentially methylated in somatic cells. Taken together, these findings support the involvement of a combination of specific DNA sequences and epigenetic factors as major determinants of hot-spots of recombination at imprinted chromosomal regions.