Determining how genes are epigenetically regulated to ensure their correct spatial and temporal expression during development is key to our understanding of cell lineage commitment. Here we examined epigenetic changes at an important proneural regulator gene Mash1 (Ascl1), as embryonic stem (ES) cells commit to the neural lineage. In ES cells where the Mash1 gene is transcriptionally repressed, the locus replicated late in S phase and was preferentially positioned at the nuclear periphery with other late-replicating genes (Neurod, Sprr2a). This peripheral location was coupled with low levels of histone H3K9 acetylation at the Mash1 promoter and enhanced H3K27 methylation but surprisingly location was not affected by removal of the Ezh2/Eed HMTase complex or several other chromatin-silencing candidates (G9a, SuV39h-1, Dnmt-1, Dnmt-3a and Dnmt-3b). Upon neural induction however, Mash1 transcription was upregulated (>100-fold), switched its time of replication from late to early in S phase and relocated towards the interior of the nucleus. This spatial repositioning was selective for neural commitment because Mash1 was peripheral in ES-derived mesoderm and other non-neural cell types. A bidirectional analysis of replication timing across a 2 Mb region flanking the Mash1 locus showed that chromatin changes were focused at Mash1. These results suggest that Mash1 is regulated by changes in chromatin structure and location and implicate the nuclear periphery as an important environment for maintaining the undifferentiated state of ES cells.

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Early differentiation of B lymphocytes requires the function of multiple transcription factors that regulate the specification and commitment of the lineage. Loss-and gain-of-function experiments have provided important insight into the transcriptional control of B lymphopoiesis, whereby E2A was suggested to act upstream of EBF1 and Pax5 downstream of EBF1. However, this simple hierarchy cannot account for all observations, and our understanding of a presumed regulatory network, in which transcription factors and signaling pathways operate, is limited. Here, we show that the expression of the Ebf1 gene involves two promoters that are differentially regulated and generate distinct protein isoforms. We find that interleukin-7 signaling, E2A, and EBF1 activate the distal Ebf1 promoter, whereas Pax5, together with Ets1 and Pu.1, regulates the stronger proximal promoter. In the absence of Pax5, the function of the proximal Ebf1 promoter and accumulation of EBF1 protein are impaired and the replication timing and subcellular localization of the Ebf1 locus are altered. Taken together, these data suggest that the regulation of Ebf1 via distinct promoters allows for the generation of several feedback loops and the coordination of multiple determinants of B lymphopoiesis in a regulatory network.

Temporal control of DNA replication has been implicated in epigenetic regulation of gene expression on the basis of observations that certain tissue-specific genes replicate earlier in expressing than non-expressing cells. Here, we show evidence that several leukocyte-specific genes replicate early in lymphocytes regardless of their transcription and also in fibroblasts, where these genes are never normally expressed. Instead, the heritable silencing of some genes (Rag-1, TdT, CD8alpha and lambda5) and their spatial recruitment to heterochromatin domains within the nucleus of lymphocytes resulted in a markedly delayed resolution of sister chromatids into doublet signals discernable by 3D fluorescence in situ hybridization (FISH). Integration of transgenes within heterochromatin (in cis) did, however, confer late replication and this was reversed after variegated transgene expression. These findings emphasise that chromosomal location is important for defining the replication timing of genes and show that retarded sister-chromatid resolution is a novel feature of inactive chromatin.

Post-translational modifications of histone amino termini are thought to convey epigenetic information that extends the coding potential of DNA. In particular, histone lysine methylation has been implicated in conveying transcriptional memory and maintaining lineage fidelity. Here an analysis of histone lysine methylation in quiescent (G(0)) and cycling lymphocytes showed that methylation of histone H3 at lysines 4 (H3K4), 9 (H3K9), 27 (H3K27) and histone H4 at lysine 20 is markedly reduced in resting B lymphocytes as compared with cycling cells. Quiescent B cells also lacked heterochromatin-associated HP1beta and Ikaros at pericentric chromatin and expressed low levels of Ezh2 and ESET histone methyl transferases (HMTases). Nuclei from resting B or T cells were approximately three times more efficiently reprogrammed in nuclear transfer assays than cells in which HMTase expression, histone methylation and HP1beta binding had been restored following mitotic stimulation. These results showing local and global changes in histone lysine methylation levels in vivo demonstrate that constitutive heterochromatin organization is modified in resting lymphocytes and suggest that histone hypomethylation is a useful indicator of epigenetic plasticity.

Clinical reports of cases with deletions in chromosome 6p are relatively rare. We present a detailed study by fluorescent in situ hybridisation (FISH) of six new cases with distinct but overlapping 6p deletions involving the 6p24 -pter chromosomal segment. Chromosomal breakpoints in individual cases were investigated using a large panel of probes previously mapped and characterised in our laboratory to cover the distal region of 6p. These cases have allowed refinement of genotype -phenotype correlations and strongly suggest a gene involved in regulating the development of hearing is localised within 6p25. There is also evidence for one or more loci involved in heart, skeletal and craniofacial development in the 6p24-p25 region. Furthermore, the Dandy -Walker malformation is associated with deletion of 6p24 -pter.

BackgroundProviders of psychological therapies are encouraged to offer patients choice about their treatment, but there is very little information about what preferences people have or the impact that meeting these has on treatment outcomes.MethodCross-sectional survey of people receiving psychological treatment from 184 NHS services in England and Wales. 14,587 respondents were asked about treatment preferences and the extent to which these were met by their service. They were also asked to rate the extent to which therapy helped them cope with their difficulties.ResultsMost patients (12,549–86.0 %, 95 % CI: 85.5–86.6) expressed a preference for at least one aspect of their treatment. Of these, 4,600 (36.7 %, 95 % CI: 35.8–37.5) had at least one preference that was not met. While most patients reported that their preference for appointment times, venue and type of treatment were met, only 1,769 (40.5 %) of the 4,253 that had a preference for gender had it met. People who expressed a preference that was not met reported poorer outcomes than those with a preference that was met (Odds Ratios: appointment times = 0.29, venue = 0.32, treatment type = 0.16, therapist gender = 0.32, language in which treatment was delivered = 0.40).ConclusionsMost patients who took part in this survey had preferences about their treatment. People who reported preferences that were not met were less likely to state that treatment had helped them with their problems. Routinely assessing and meeting patient preferences may improve the outcomes of psychological treatment.

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