Neural induction promotes large-scale chromatin reorganisation of the Mash1 locus.

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.

Heritable gene silencing in lymphocytes delays chromatid resolution without affecting the timing of DNA replication.

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.

Transcription and the territory: the ins and outs of gene positioning.

When cells exit mitosis, the neat rod-like chromosomes decondense into their interphase state. However, the chromatin threads are not randomly dispersed throughout the nucleoplasm. Rather, individual chromosomes appear to be organized into discrete, non-overlapping "territories". Current studies attempt to unravel how gene loci are organized within these territories, whether their subterritorial positions are dependent on transcription, and the extent to which the loci can move.

Antihuman epidermal growth factor receptor 2 antibody herceptin inhibits autocrine motility factor (AMF) expression and potentiates antitumor effects of AMF inhibitors.

Overexpression of the human epidermal growth factor receptor (HER) 2 has been linked to the development and maintenance of malignant phenotypes in breast tumors. In addition, the growth and dissemination of human cancers are regulated in part by the autocrine motility factor (AMF)/phosphoglucose isomerase shown to be up-regulated by heregulin (HRG) in breast cancer cells. This study was undertaken to explore the effect of anti-HER2 monoclonal antibody 4D5 [Herceptin (HCT)] on AMF expression and the potential of its augmentation by specific simple sugar AMF inhibitors. Here we show that HCT treatment of high HER2-expressing breast cancer SK-BR3, BT-474, and ZR-75R cells resulted in down-regulation of AMF mRNA and protein. HCT inhibited the ability of HRG to induce AMF expression in cells with a normal HER2 level, and HCT-mediated down-regulation could be reversed by HRG treatment in breast cancer cells with a high HER2 level. HCT also inhibited transcription from a chimeric pGL3-Luc vector-based reporter system containing the 1.8-kb promoter region of human AMF. Treatment of breast cancer cells with the combination of HCT and specific AMF inhibitors, erythrose 4-phosphate or D-mannose 6-phosphate, resulted in an additive inhibitory effect on both the growth rate and invasiveness of cells as compared with treatment with each agent alone. Results presented here suggest that HCT can effectively block both ligand-induced and constitutive expression of AMF associated with high HER2 overexpression, implying a role of the AMF pathway in the action of HCT. Accordingly, the combination of AMF inhibitor with HCT can potentiate the growth-inhibitory and anti-invasive action of HCT in breast cancer cells.

Subchromosomal positioning of the epidermal differentiation complex (EDC) in keratinocyte and lymphoblast interphase nuclei.

The epidermal differentiation complex (EDC) at 1q21 is host to many structurally and functionally related genes coding for proteins involved in the differentiation process of keratinocytes. The grouping together of these genes which share spatial and temporal expression and interrelated functions is a remarkable genomic feature which has led to suggestions that the region may have a coordinated transcription control mechanism. With the growing awareness that the organization of the genome within the interphase nucleus is relevant to transcriptional activity, we have investigated the spatial organization of the EDC in the nuclei of keratinocytes, where the EDC genes are highly expressed, and lymphoblasts, where they are silent. Using 2D and 3D FISH we find that in keratinocyte nuclei the EDC is frequently positioned external to the chromosome 1 territory compared to lymphoblasts where the EDC more often adopts a peripheral or internal location. It has been previously shown that the MHC region can extend from the chromosome 6 territory in relation to transcriptional activity. This study of the EDC thus provides a further example of a gene-dense complex capable of assuming extraterritorial positioning in relation to cell type/transcription status.

Topographical relationship between the entorhinal cortex and the septotemporal axis of the dentate gyrus in rats: II. Cells projecting from lateral entorhinal subdivisions.

Projections from the rat lateral entorhinal cortex (area 28-l) to the dentate gyrus were traced and then interpreted according to a parcellation scheme that recognized four cytoarchitectonic subdivisions of area 28-l: areas dorsolateral (dl), ventrolateral (vl), ventromedial (vm), and TR. Following lesions of area 28-l, anterograde degeneration was traced with the Fink-Heimer method. In parallel experiments iontophoretic injections of horseradish peroxidase (HRP) were made in the lateral perforant path terminal zone of the dentate molecular layer. Retrograde neuronal labeling patterns within area 28-l were charted following dorsal, midseptotemporal (mid ST), and ventral dentate injections. In two additional cases HRP was deposited in the ventral subiculum. Lesions of area dl (which lies entirely on the posterolateral cortex) produced terminal degeneration that was confined to the dorsal one-half of the dentate gyrus. Lesions involving primarily areas vl and vm (which lie on the posteroinferior cortex) caused a complementary pattern of degeneration; silver grains predominated in the ventral dentate gyrus. Injections of HRP into the outer dentate molecular layer labeled layer II neurons within area 28-l. Deposits of HRP in the dorsal one-third of the dentate gyrus labeled a rostrocaudal strip of neurons within the dorsal one-third of area dl; no other subdivisions of area 28-l contained labeling. After mid-ST deposits of HRP, a rostrocaudally oriented strip of labeled cells appeared in the ventral one-third of area dl. Mid-ST injections also labeled neurons in the caudolateral quadrant of area vl. Injection of HRP into the ventral dentate gyrus labeled neurons in the caudomedial quadrant of area vl as well as a few neurons in caudal area vm. No labeled cells were ever found in area dl following ventral dentate HRP deposits. Neurons within area TR were never retrogradely labeled from injections of HRP into the dentate perforant path zone. However, ventral subicular injections of HRP labeled a few cells in the posterior part of area TR, as well as hundreds of neurons throughout the rostrocaudal extent of area vl. The results indicate a highly organized innervation of the dentate gyrus by several subdivisions of area 28-l. In area dl, rostrocaudal strips of layer II neurons innervate distinct segments of the dorsal ST axis. The posterior half of areas vl and vm innervates the ventral half of the ST axis; a lateromedial gradient there corresponds to increasingly ventral terminations along the dentate ST axis.(ABSTRACT TRUNCATED AT 400 WORDS)

Foreign and endogenous serum protein extravasation during harmaline tremors or kainic acid seizures in the rat: a comparison.

Cerebrovascular permeability to protein (CVP-p) was assessed in rats following the systemic injection of either kainic acid (KA) or harmaline. The extravasation of a foreign (horseradish peroxidase, HRP) or an endogenous (rat immunoglobulin G, IgG) tracer protein was determined using immunohistochemical methods. During KA-induced seizures, an extravasation of both HRP and presumed IgG occurred in similar forebrain loci; a lamina-specific extravasation occurred within the dorsal hippocampus. During harmaline-induced tremors protein extravasation also occurred, but was tracer dependent. HRP reaction product was observed within the inferior olive, the cortex of the cerebellar vermis and the neocortex. However, IgG-like immunoreactivity was only detected within the circumventricular organs of harmaline-treated rats. Because KA, but not harmaline, is neurotoxic, the results are consistent with an influence of endogenous serum protein extravasation on seizure-related hippocampal damage. Possible homeostatic properties of altered CVP-p are also considered.

Dietary pyridoxine and the susceptibility to limbic motor seizures in rats.

Dietary pyridoxine (PN) deficiency in adult female rats produced a 32% decrease in hippocampal gamma-aminobutyric acid content, measured by a radioreceptor assay. No spontaneous seizures were observed in pyridoxine-deficient animals, but the seizure latency after a systemic kainic acid challenge decreased by 35%. The results suggest that latency is a useful measure of limbic seizure susceptibility; this susceptibility can be manipulated by diet; and in adults pyridoxine-dependent mechanisms normally participate in preventing, rather than initiating, limbic seizures.

Increased cerebrovascular permeability to protein during systemic kainic acid seizures.

Cerebrovascular permeability to protein (CVP-p) was assessed during limbic seizures by injecting unrestrained rats intraperitoneally with kainic acid followed by intravenous horseradish peroxidase (HRP); animals survived approximately 1 h after seizure onset. Brains were processed for the blue HRP reaction product followed by light microscopic examination of sequential sagittal sections. In all cases kainate-induced seizures caused increased CVP-p within the thalamus, temporal hippocampal formation, and neocortex. Somewhat less frequently other limbic structures and the striatum were HRP-positive. A lamina-specific extravasation occurred in the dorsal hippocampus; reaction product occupied the mossy fiber zone of field CA3, a likely focus of kainate action. Extravasation of HRP also occurred within, or juxtaposed to, certain myelinated fiber bundles. Brains from animals treated as blanks (kainate but no HRP) were devoid of peroxidase activity, and in nonseizing animals HRP gained access only to circumventricular organs. Although regions of increased CVP-p partially covary with areas of increased electrical activity and glucose metabolism, neuronal activation occurs over a much greater volume of brain tissue than does CVP-p. A close relationship may exist between these circumscribed areas of protein extravasation and seizure foci. Both vasogenic and cytotoxic edema appear to be simultaneously present during kainate seizures.

Topography between the entorhinal cortex and the dentate septotemporal axis in rats: I. Medial and intermediate entorhinal projecting cells.

Retrograde tracing experiments were performed to clarify the topographic projection from medial (area 28m) and intermediate (area 28i) divisions of the entorhinal cortex to the dentate gyrus. Pipets filled with horseradish peroxidase (HRP) were positioned by electrophysiologic guidance at one of several septotemporal (S-T) levels in the dentate molecular layer of anesthetized rats; the tracer was expelled iontrophoretically to minimize its spread. Retrograde labeling of neurons within areas 28m and 28i was analyzed in relation to cytoarchitectonic as well as spatial features of the region (obtained by histologic reconstruction). Regardless of the S-T level, ejections of HRP which were confined to the dentate gyrus labeled only layer II neurons of each area. Following septal pole ejections, labeled neurons were located in the posterolateral, extreme posterior, and posteromedial parts of both areas 28m and 28i. Mid S-T ejections produced not only a ventral, but also an anteromedial, shift in the location of entorhinal projection cells; no cells were labeled posterolaterally. After temporal dentate ejections labeled neurons occupied the most anteromedial part of these entorhinal areas. For both areas, but especially for area 28i, convergence of entorhinal efferents upon a single S-T level in the dentate gyrus occurred from neurons which lay in a dorsoventral (i.e., frontal), and to a lesser extent a rostrocaudal, plane. The efferent axes of both areas 28m and 28i thus appear to be curved and are therefore best described in three dimensions. The entorhinal axes begin in a posterodorsolateral location, wrap around the posterior cortical convexity, and end in an anteroventromedial position. The results provide a useful map for in situ exploration of entorhinodentate connections in the rat, emphasize the parallel innervation of the dentate gyrus by distinct entorhinal fiber systems, and reflect the importance of the S-T axis as a framework for interpreting hippocampal organization.

Interaction between zinc deprivation and acute ethanol intoxication during pregnancy in rats.

A single intraperitoneal injection of ethanol given at gestational day 10 to pregnant rats undergoing transient zinc deprivation increases the probability of complications in the pregnancy, compared to either intoxication in well-fed dams or to zinc deprivation alone. The result suggest that dietary factors may contribute to ethanol-induced terata and that non-abusive or infrequent maternal use of ethanol can be particularly dangerous to the fetus during a dietary deficiency.