Richard Festenstein

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 minigene 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.

Unravelling heterochromatin: competition between positive and negative factors regulates accessibility

Heterochromatin mediates many diverse functions in the cell nucleus, including centromere function, gene silencing and nuclear organization. The condensed structure of pericentromeric heterochromatin is associated with the presence of a regular arrangement of nucleosomes, which might be due in part to the underlying sequence of the satellite repeats. Recent studies identified methylation of the histone H3 tail as an epigenetic mark that affects acetylation and phosphorylation of histone tail residues and also acts as a recognition signal for binding of heterochromatin protein 1 (HP1). The decision to silence or activate heterochromatic genes appears to be the result of a balance between negative factors that promote the formation of condensed higher-order chromatin structure, and positively acting transcription factors that bind to regulatory sequences and activate gene expression.

DNA triplet repeats mediate heterochromatin-protein-1-sensitive variegated gene silencing.

Gene repression is crucial to the maintenance of differentiated cell types in multicellular organisms, whereas aberrant silencing can lead to disease. The organization of DNA into chromatin and heterochromatin is implicated in gene silencing. In chromatin, DNA wraps around histones, creating nucleosomes. Further condensation of chromatin, associated with large blocks of repetitive DNA sequences, is known as heterochromatin. Position effect variegation (PEV) occurs when a gene is located abnormally close to heterochromatin, silencing the affected gene in a proportion of cells. Here we show that the relatively short triplet-repeat expansions found in myotonic dystrophy and Friedreichs ataxia confer variegation of expression on a linked transgene in mice. Silencing was correlated with a decrease in promoter accessibility and was enhanced by the classical PEV modifier heterochromatin protein 1 (HP1). Notably, triplet-repeat-associated variegation was not restricted to classical heterochromatic regions but occurred irrespective of chromosomal location. Because the phenomenon described here shares important features with PEV, the mechanisms underlying heterochromatin-mediated silencing might have a role in gene regulation at many sites throughout the mammalian genome and modulate the extent of gene silencing and hence severity in several triplet-repeat diseases.

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 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.

H3 Lysine 4 Is Acetylated at Active Gene Promoters and Is Regulated by H3 Lysine 4 Methylation

Methylation of histone H3 lysine 4 (H3K4me) is an evolutionarily conserved modification whose role in the regulation of gene expression has been extensively studied. In contrast, the function of H3K4 acetylation (H3K4ac) has received little attention because of a lack of tools to separate its function from that of H3K4me. Here we show that, in addition to being methylated, H3K4 is also acetylated in budding yeast. Genetic studies reveal that the histone acetyltransferases (HATs) Gcn5 and Rtt109 contribute to H3K4 acetylation in vivo. Whilst removal of H3K4ac from euchromatin mainly requires the histone deacetylase (HDAC) Hst1, Sir2 is needed for H3K4 deacetylation in heterochomatin. Using genome-wide chromatin immunoprecipitation (ChIP), we show that H3K4ac is enriched at promoters of actively transcribed genes and located just upstream of H3K4 tri-methylation (H3K4me3), a pattern that has been conserved in human cells. We find that the Set1-containing complex (COMPASS), which promotes H3K4me2 and -me3, also serves to limit the abundance of H3K4ac at gene promoters. In addition, we identify a group of genes that have high levels of H3K4ac in their promoters and are inadequately expressed in H3-K4R, but not in set1Δ mutant strains, suggesting that H3K4ac plays a positive role in transcription. Our results reveal a novel regulatory feature of promoter-proximal chromatin, involving mutually exclusive histone modifications of the same histone residue (H3K4ac and H3K4me).

Evidence for Distinct CD4 Silencer Functions at Different Stages of Thymocyte Differentiation.

An intronic silencer within the CD4 gene is the critical cis regulatory element for T cell subset-specific expression of CD4. We have combined transfection studies with gene targeting in mice to identify several key sequences within the silencer core that are required for gene silencing during thymocyte development. In mice, mutations in individual sites resulted in variegated, but heritable, derepression of CD4 in mature CD8(+) T lymphocytes, whereas compound mutations resulted in full derepression. These results indicate that there is partial redundancy in recruiting a chromatin remodeling machinery that results in epigenetic silencing. Mutations in single sites also resulted in partial derepression of CD4 in immature double-negative thymocytes, but there was no apparent variegation. These findings suggest two distinct modes of CD4 silencer function at different developmental stages: active repression in CD4(-)CD8(-) thymocytes, in which silencing must be reversible, and epigenetic gene silencing upon differentiation to the CD8(+) cytotoxic T cell lineage.

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, CD8α and λ5) 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.

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.

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.