Heterochromatin protein 1 modifies mammalian PEV in a dose- and chromosomal-context-dependent manner.

Locus control regions (LCRs) are gene regulatory elements in mammals that can overcome the highly repressive effects normally associated with heterochromatic transgene locations (for example the centromere) in mice. Deletion of essential LCR sequences renders the cognate gene susceptible to this form of repression, so a proportion of the cells from transgenic mice that would normally express the transgene are silenced-a phenomenon known as position effect variegation (PEV). We show here that PEV can also occur when the transgene is non-centromeric and that the extent of variegation can be developmentally regulated. Furthermore, by overexpressing a mammalian homologue (M31) of Drosophila melanogaster heterochromatin protein 1 (HP1; refs 7,8) in transgenic mouse lines that exhibit PEV, it is possible to modify the proportion of cells that silence the transgene in a dose-dependent manner. Thus, we show M31 overexpression to have two contrasting effects which are dependent on chromosomal context: (i) it enhanced PEV in those lines with centromeric or pericentromeric transgene locations; and (ii) it suppressed PEV when the transgene was non-centromeric. Our results indicate that components or modifiers of heterochromatin may have a chromosomal-context-dependent role in gene silencing and activation decisions in mammals.

Locus control region function and heterochromatin-induced position effect variegation.

Human CD2 locus control region (LCR) sequences are shown here to be essential for establishing an open chromatin configuration. Transgenic mice carrying an hCD2 mini-gene attached only to the 3' CD2 transcriptional enhancer exhibited variegated expression when the transgene integrated in the centromere. In contrast, mice carrying a transgene with additional 3' sequences showed no variegation even when the latter integrated in centromeric positions. This result suggests that LCRs operate by ensuring an open chromatin configuration and that a short region, with no enhancer activity, functions in the establishment, maintenance, or both of an open chromatin domain.

Locus control regions and epigenetic chromatin modifiers.

Locus control regions are defined as gene regulatory sequences that enable chromosomal position-independent gene expression in transgenic mice. Recent studies have shown the ability of such regions to overcome the highly repressive effect of heterochromatin and have identified both trans-acting and cis-acting factors that participate in gene silencing and activation mechanisms.

Locus control regions: overcoming heterochromatin-induced gene inactivation in mammals.

Differentiation of specific cell types during the development of mammals requires the selective silencing or activation of tissue-specific genes. Locus control regions (LCRs) are gene regulatory elements that act in cis to ensure that active transcriptional units are established in all cells of a given cell lineage. Over the past year, it has become clear that this process takes place at the level of chromatin remodelling, and that LCRs ensure that this decision is made by both alleles in every cell. Studies on LCRs and analysis of gene expression in transgenic mice at the single cell level has revealed that the breakdown in LCR function accompanying the deletion of specific sequences results in a phenomenon known as position effect variegation, described in detail in yeast and Drosophila. Thus, when located in close proximity to heterochromatin a transgene linked to a disabled LCR is randomly silenced in a proportion of cells. This finding implies that all subregions within an LCR are necessary to ensure the establishment of an open chromatin configuration of a gene even when the latter is located in a highly heterochromatic region.

Heterochromatin protein 1: a pervasive controlling influence.

Heterochromatin protein 1 (HP1), a component of condensed chromatin, was discovered more than 10 years ago and subsequently found to play important roles in chromosomal biology and gene silencing. Consistent with the hypothesis that post-translational modifications of histones may functionally 'mark' DNA sequences, HP1 was found to bind to 'silent' chromatin via the methylated lysine 9 (K9) residue on the histone H3 tail that protrudes from the nucleosome. The discovery of several HP1-associating proteins has given us insight into how HP1 may function. Although initially found to localise predominantly at heterochromatin, recent data suggest that HP1 also localises and dynamically participates in gene regulation in euchromatin. Moreover, the initial definition of HP1 as a gene repressor may need to be revisited, as HP1 has been shown, in some cases, to localise at transcriptionally active chromosomal sites. Here we review current knowledge on HP1 and explore possible mechanisms whereby HP1 might exert divergent effects on gene regulation.

Random activation of a transgene under the control of a hybrid hCD2 locus control region/Ig enhancer regulatory element.

Locus control regions such as those of human CD2 and beta-globin differ from classical enhancers in that, whereas the former confer high level, copy-dependent, position-independent expression to linked genes in transgenic mice, the latter do not, expression levels being dependent on the site of integration. We report that the position independence of the CD2 locus control region is modified by coupling it to the immunoglobulin heavy chain enhancer. Whilst in the majority of transgenic lines the Ig heavy chain enhancer has little or no effect on T cell expression of the hCD2 transgene, in others transgene expression is non-specifically extinguished in a proportion of lymphoid cells. The transgenic locus chromatin appears inaccessible to DNase I in these cells, which do not express the gene. Furthermore, mice homozygous for the hybrid hCD2-Ig heavy chain enhancer construct contain T cells with both an active and an inactive transgene. The 'decision' to express or repress the gene appears to be a random process which involves each chromosome separately, occurs at early stages in differentiation and is heritable by daughter cells. These data suggest the possibility that stochastic decisions might control a number of biological processes.

A CD8 genomic fragment that directs subset-specific expression of CD8 in transgenic mice.

Helper and cytotoxic T cell subsets require the expression of different coreceptors (CD4 and CD8, respectively) for their development and function. We have cloned the CD8 gene locus from genomic cosmid and P1 libraries and analyzed the region around the CD8alpha and CD8beta genes for gene expression regulatory elements. DNase I (DNase I) hypersensitivity analysis of 80 kb in the CD8 locus identified four clusters of putative regulatory regions, three of which are thymocyte specific. Transgenic mice carrying the cloned CD8alphabeta genomic locus and containing the identified DNase I-hypersensitive site clusters express the transgenic CD8 in a developmentally regulated, tissue-specific, and CD8 T cell subset-specific manner.

Nimodipine in migraine prophylaxis.

Nimodipine is a compound that is thought to block the influx of calcium through channels in vascular smooth muscle. This paper describes a double-blind parallel-group comparison of 40 mg nimodipine three times a day and placebo. Sixty-eight patients received treatment after a run-in period of 2 months, and of these, 57 completed 8 weeks or more of the trial. All but five of these completed the full 6-month trial. The nimodipine and placebo groups showed no significant differences in the frequency of attacks, severity or duration of headache, or gastrointestinal or other symptoms.

Human HMG box transcription factor HBP1: a role in hCD2 LCR function.

The locus control region (LCR) of the human CD2 gene (hCD2) confers T cell-specific, copy-dependent and position-independent gene expression in transgenic mice. This LCR consists of a strong T cell-specific enhancer and an element without enhancer activity (designated HSS3), which is required for prevention of position effect variegation (PEV) in transgenic mice. Here, we identified the HMG box containing protein-1 (HBP1) as a factor binding to HSS3 of the hCD2 LCR. Within the LCR, HBP1 binds to a novel TTCATTCATTCA sequence that is higher in affinity than other recently reported HBP1-binding sites. Mice transgenic for a hCD2 LCR construct carrying a deletion of the HBP1-binding sequences show a propensity for PEV if the transgene integrates in a heterochromatic region of the chromosome such as the centromere or telomere. We propose that HBP1 plays an important role in chromatin opening and remodelling activities by binding to and bending the DNA, thus allowing DNA-protein and/or protein-protein interactions, which increase the probability of establishing an active locus.