Chromatin structure, epigenetic mechanisms and long-range interactions in the human insulin locus.

Regulation of gene expression in eukaryotes is largely dependent on variations in chromatin structure. More recently, it has become clear that this may involve not only local chromatin organization but also distant regulatory elements that participate in large-scale chromatin architecture within the nucleus. We describe recent methods that make possible the detection of such structures and apply them to analysis of the human insulin (INS) locus in pancreatic islets. We show that the INS gene is part of an extended 'open' chromatin domain that includes adjacent genes as well. We also find that in islets, the INS promoter is in physical contact with distant sites on the same human chromosome and notably, with the SYT8 gene, located nearly 300 kb away. The strength of the contact between INS and SYT8 is increased by glucose, and this results in stimulation of SYT8 expression. Inhibition of INS transcription decreases SYT8 expression. Furthermore, downregulation of SYT8 results in decreased secretion of insulin. Our results thus establish the existence of a regulatory network between the INS gene and other distant genes through long-range physical interactions, and suggest that such networks may have general importance for insulin biology and diabetes.

Chromatin boundaries, insulators, and long-range interactions in the nucleus.

Within the genome, expressed genes marked by "open" chromatin are often adjacent to silent, heterochromatic regions. There are also regions containing neighboring active genes with different programs of expression. In both cases, DNA sequence elements may function as insulators, either providing barriers that prevent the incursion of heterochromatic signals into open domains or acting to block inappropriate contact between the enhancer of one gene and the promoter of another. The mechanisms associated with insulation are diverse: Enhancer-blocking insulation is largely associated with the ability to stabilize the formation of loop domains within the nucleus. Barrier insulation is often associated with the ability to block propagation of silencing histone modifications. Here, we provide examples of both kinds of insulator action, derived initially from studies of the compound insulator element at the 5' end of the chicken ß-globin locus. Such elements appear to have more general regulatory roles in the genome that have been exploited to provide insulator function where necessary to demarcate separate domains within the nucleus.

Chromatin goes global.

The latest findings on the structure of chromatin, its organization in the nucleus, and its involvement in regulating gene expression were presented at a recent meeting at the Juan March Foundation in Madrid, Spain.

Correlation between histone lysine methylation and developmental changes at the chicken beta-globin locus.

Methylation of histones at specific residues plays an important role in transcriptional regulation. Chromatin immunoprecipitation of dimethylated lysine 9 on histone H3 across 53 kilobases of the chicken beta-globin locus during erythropoiesis shows an almost complete anticorrelation between regions of elevated lysine 9 methylation and acetylation. Lysine 9 is methylated most over constitutive condensed chromatin and developmentally inactive globin genes. In contrast, lysine 4 methylation of histone H3 correlates with H3 acetylation. These results lead us to propose a mechanism by which the insulator in the beta-globin locus can protect the globin genes from being silenced by adjacent condensed chromatin.

Transitions in histone acetylation reveal boundaries of three separately regulated neighboring loci.

We have studied developmentally regulated patterns of histone acetylation at high resolution across approximately 54 kb of DNA containing three independently regulated but neighboring genetic loci. These include a folate receptor gene, a 16 kb condensed chromatin region, the chicken beta-globin domain and an adjacent olfactory receptor gene. Within these regions the relative levels of acetylation appear to fall into three classes. The condensed chromatin region maintains the lowest acetylation at every developmental stage. Genes that are inactive show similarly low levels, but activation results in a dramatic increase in acetylation. The highest levels of acetylation are seen at regulatory sites upstream of the genes. These patterns imply the action of more than one class of acetylation. Notably, there is a very strong constitutive focus of hyperacetylation at the 5' insulator element separating the globin locus from the folate receptor region, which suggests that this insulator element may harbor a high concentration of histone acetylases.

A large upstream region is not necessary for gene expression or hypersensitive site formation at the mouse beta -globin locus.

Developmental expression at the beta-globin locus is regulated in part by the locus control region, a region upstream of the genes containing at least five major DNase I hypersensitive sites (HSs) in mammalian erythrocytes. Sequences farther 5' of these HSs are conserved in mouse and human, and both loci are embedded within a cluster of functional odorant receptor genes. In humans, distant upstream sequences have been implicated in regulation of the beta-globin genes. In this study, the role of the 5'-most HSs and their adjacent sequence was investigated by deletion of an 11-kb region from the mouse locus, including 5'HS 4.2, 5'HS 5, 5'HS 6, and the 5'beta1 odorant receptor gene. Mice that were homozygous for this deletion were fully viable, and no significant effect on adult beta-globin gene expression was seen. 5'HSs 1-4, which are located downstream of the deletion, were still present in the mutant mice. In addition, two new upstream HSs, HS -60.7 and HS -62.5, were found in erythroid tissue of both wild-type and mutant mice. Therefore, although the possibility of a minor role still exists, neither the HSs nor the other regions deleted in this study are essential for beta-globin gene expression, and it is unlikely that chromatin structure is affected either upstream or downstream of the deletion. This is the largest deletion at the mouse locus control region to show no apparent phenotype, and focuses attention on the possible contribution of sequences even farther upstream.

Comparative structural and functional analysis of the olfactory receptor genes flanking the human and mouse beta-globin gene clusters.

By sequencing regions flanking the beta-globin gene complex in mouse (Hbbc) and human (HBBC), we have shown that the beta-globin gene cluster is surrounded by a larger cluster of olfactory receptor genes (ORGs). To facilitate sequence comparisons and to investigate the regulation of ORG expression, we have mapped 5' sequences of mRNA from olfactory epithelium encoding beta-globin-proximal ORGs. We have found that several of these genes contain multiple noncoding exons that can be alternatively spliced. Surprisingly, the only common motifs found in the promoters of these genes are a "TATA" box and a purine-rich motif. Sequence comparisons between human and mouse reveal that most of the conserved regions are confined to the coding regions and transcription units of the genes themselves, but a few blocks of conserved sequence also are found outside of ORG transcription units. The possible influence of beta-globin regulatory sequences on ORG expression in olfactory epithelium was tested in mice containing a deletion of the endogenous beta-globin locus control region, but no change in expression of the neighboring ORGs was detected. We evaluate the implications of these results for possible mechanisms of regulation of ORG transcription.

Transcription through nucleosomes.

Transcriptionally active genes in eukaryotes still retain most of the Chromatin packaging that is characteristic of eukaryotic DNA. Nucleosomes and even some higher order structure are present, although the histones may be chemically modified, for example by acetylation or phosphorylation, as part of the activation process. The presence of nucleosomes on the coding region of active genes raises the question: How does an RNA polymerase transcribe such a template? We have attempted to answer this question with relatively simple model systems involving a template carrying a single positioned nucleosome. We have shown that when a phage polymerase, SP6, transcribes such a template, the histone octamer of the nucleosome is not released into solution. Instead it is retained on the same DNA molecule, but displaced from its original binding site. Further studies have allowed us to propose a detailed model, which appears to hold not only for SP6 but also for transcription by the much larger RNA polymerase III from yeast. Our most recent results, obtained by electron cryomicroscopy, confirm and refine this model.

Methylation of a CTCF-dependent boundary controls imprinted expression of the Igf2 gene.

The expression of the insulin-like growth factor 2 (Igf2) and H19 genes is imprinted. Although these neighbouring genes share an enhancer, H19 is expressed only from the maternal allele, and Igf2 only from the paternally inherited allele. A region of paternal-specific methylation upstream of H19 appears to be the site of an epigenetic mark that is required for the imprinting of these genes. A deletion within this region results in loss of imprinting of both H19 and Igf2 (ref. 5). Here we show that this methylated region contains an element that blocks enhancer activity. The activity of this element is dependent upon the vertebrate enhancer-blocking protein CTCF. Methylation of CpGs within the CTCF-binding sites eliminates binding of CTCF in vitro, and deletion of these sites results in loss of enhancer-blocking activity in vivo, thereby allowing gene expression. This CTCF-dependent enhancer-blocking element acts as an insulator. We suggest that it controls imprinting of Igf2. The activity of this insulator is restricted to the maternal allele by specific DNA methylation of the paternal allele. Our results reveal that DNA methylation can control gene expression by modulating enhancer access to the gene promoter through regulation of an enhancer boundary.

Structural and functional conservation at the boundaries of the chicken beta-globin domain.

We show that the 3' boundary of the chicken beta-globin locus bears striking structural similarities to the 5' boundary. In erythroid cells a clear transition in DNase I sensitivity of chromatin at the 3' end of the locus is observed, the location of this transition is marked by a constitutive DNase I hypersensitive site (HS), and DNA spanning this site has the enhancer-blocking capacity of an insulator. This HS contains a binding site for the transcription factor CTCF. As in the case of the 5' insulator, the CTCF site is both necessary and sufficient for the enhancer-blocking activity of the 3' boundary. The position of this insulator is consistent with our proposal that it may function to maintain the distinct regulatory programs of the globin genes and their closely appended 3' neighbor, an odorant receptor gene. We conclude that both boundaries of the chicken beta-globin domain are capable of playing functionally similar roles and that the same protein is a necessary component of the molecular mechanism through which these boundaries are defined.

Positional enhancer-blocking activity of the chicken beta-globin insulator in transiently transfected cells.

It is thought that insulators demarcate transcriptionally and structurally independent chromatin domains. Insulators are detected by their ability to block enhancer-promoter interactions in a directional manner, and protect a transgene from position effects. Most studies are performed in stably transformed cells or organisms. Here we analyze the enhancer-blocking activity of the chicken beta-globin insulator in transient transfection experiments in both erythroid and nonerythroid cell lines. We show that four tandem copies of a 90-bp fragment of this insulator were able to block an enhancer in these experiments. In circular plasmids, placement on either side of the enhancer reduced activity, but when the plasmid was linearized, the enhancer-blocking activity was observed only when the insulator was placed between the promoter and the enhancer. These observations are consistent with the position-dependent enhancer-blocking activity of the insulator observed in stable transformation experiments.

The nature of the nucleosomal barrier to transcription: direct observation of paused intermediates by electron cryomicroscopy.

Transcribing SP6 RNA polymerase was arrested at unique positions in the nucleosome core, and the complexes were analyzed using biochemical methods and electron cryomicroscopy. As the polymerase enters the nucleosome, it disrupts DNA-histone interactions behind and up to approximately 20 bp ahead of the elongation complex. After the polymerase proceeds 30-40 bp into the nucleosome, two intermediates are observed. In one, only the DNA ahead of the polymerase reassociates with the octamer. In the other, DNA both ahead of and behind the enzyme reassociates. These intermediates present a barrier to elongation. When the polymerase approaches the nucleosome dyad, it displaces the octamer, which is transferred to promoter-proximal DNA.

The protein CTCF is required for the enhancer blocking activity of vertebrate insulators.

An insulator is a DNA sequence that can act as a barrier to the influences of neighboring cis-acting elements, preventing gene activation, for example, when located between an enhancer and a promoter. We have identified a 42 bp fragment of the chicken beta-globin insulator that is both necessary and sufficient for enhancer blocking activity in human cells. We show that this sequence is the binding site for CTCF, a previously identified eleven-zinc finger DNA-binding protein that is highly conserved in vertebrates. CTCF sites are present in all of the vertebrate enhancer-blocking elements we have examined. We suggest that directional enhancer blocking by CTCF is a conserved component of gene regulation in vertebrates.

An insulator element and condensed chromatin region separate the chicken beta-globin locus from an independently regulated erythroid-specific folate receptor gene.

We have identified a folate receptor gene upstream of the chicken beta-globin locus and separated from it by a 16 kbp region of silent chromatin. We find that this receptor is expressed only at a stage of erythroid differentiation (CFU-E) preceding the activation of beta-globin genes, consistent with the role of folate receptors in proliferation. This discovery raises the question of how these two loci are regulated during erythropoiesis. Our data suggest that the folate receptor gene and the beta-globin locus are regulated independently. We show that a 3.3 kbp DNA region upstream of the folate receptor gene is sufficient to induce strong expression of a transgene in CFU-E stage cells. We also find that the region between the beta-globin locus and the folate receptor gene is fully methylated and condensed at this stage of differentiation. Its 3' boundary coincides with the 5' beta-globin insulator. We speculate that the 5' beta-globin boundary element might be important for the proper regulation of two adjacent domains activated at two different stages during differentiation.

Stopped at the border: boundaries and insulators.

Boundaries in chromatin are often marked by the presence of insulator elements. New results in Drosophila have identified an insulator with a proven boundary function essential for development. Other studies suggest a connection between the activity of some insulators and Drosophila trithorax-Group and Polycomb-Group genes. Several examples of vertebrate insulators have now been found; their locations suggest important boundary functions. Enhancer-blocking studies in oocytes and position-effect studies in transformed cells shed new light on insulator mechanisms.

Conservation of sequence and structure flanking the mouse and human beta-globin loci: the beta-globin genes are embedded within an array of odorant receptor genes.

In mouse and human, the beta-globin genes reside in a linear array that is associated with a positive regulatory element located 5' to the genes known as the locus control region (LCR). The sequences of the mouse and human beta-globin LCRs are homologous, indicating conservation of an essential function in beta-globin gene regulation. We have sequenced regions flanking the beta-globin locus in both mouse and human and found that homology associated with the LCR is more extensive than previously known, making up a conserved block of approximately 40 kb. In addition, we have identified DNaseI-hypersensitive sites within the newly sequenced regions in both mouse and human, and these structural features also are conserved. Finally, we have found that both mouse and human beta-globin loci are embedded within an array of odorant receptor genes that are expressed in olfactory epithelium, and we also identify an olfactory receptor gene located 3' of the beta-globin locus in chicken. The data demonstrate an evolutionarily conserved genomic organization for the beta-globin locus and suggest a possible role for the beta-globin LCR in control of expression of these odorant receptor genes and/or the presence of mechanisms to separate regulatory signals in different tissues.

Alternate promoters and developmental modulation of expression of the chicken GATA-2 gene in hematopoietic progenitor cells.

We have isolated and characterized the chicken GATA-2 (cGATA-2) gene. We show that, as in the case of some other members of the GATA gene family, the gene is expressed from alternative first exons. One of the resulting mRNAs represents only a minor form of the GATA-2 mRNA in the cells and tissues we analyzed; the other is ubiquitously expressed. We have defined the minimal promoter that controls expression of this most abundant mRNA and that is necessary for full activity in hematopoietic progenitor cells. The activity of this promoter in transient assays is consistent with developmental differences of expression levels in these cells. We identify within the promoter a previously unrecognized extended CCAAT motif essential for its activity. The organization of the cGATA-2 gene, with alternative first exons and a CCAAT box in the proximal promoter, is similar to that recently described for mouse GATA-2, and the proximal promoter also resembles the only promoter so far described in Xenopus. Nonetheless, the roles of the promoters in development and tissue-specific expression are quite different in these organisms, most strikingly in the mouse, which assigns developmental roles to its proximal and distal promoters that are quite different from those in the chicken. We suggest that although the overall organization may remain the same, the role assigned to each promoter varies among organisms. We identify distant upstream regulatory elements in the cGATA-2 gene that modulate expression from the proximal promoter and that may be responsible for this variation.

Loss of transcriptional activity of a transgene is accompanied by DNA methylation and histone deacetylation and is prevented by insulators.

The constitutive DNase I hypersensitive site at the 5' end of the chicken beta-globin locus marks the boundary of the active chromatin domain in erythroid cells. The DNA sequence containing this site has the properties of an insulator, as shown by its ability in stable transformation experiments to block enhancer-promoter interaction when it lies between the two, but not when it lies outside, and to protect against position effects in Drosophila. We now show that the chicken insulator can protect a stably integrated gene, which is otherwise subject to great variability of expression, from chromatin-mediated repression in cell culture. When the integrated reporter gene is surrounded by insulator elements, stably transformed cell lines display consistent enhancer-dependent expression levels, in accord with the strength of the enhancer. In the absence of insulators, long-term nonselective propagation of cells carrying the integrated reporter gene results in gradual extinction of the reporter's expression, with expression patterns from tandemly repeated inserted genes suggesting that the extinction of adjacent genes is coupled. We show that the uninsulated reporter genes, in addition to becoming transcriptionally inactive, lose several epigenetic hallmarks of active chromatin, including nuclease accessibility, DNA hypomethylation, and histone hyperacetylation during time in culture. Treatment with inhibitors of histone deacetylase or DNA methylation reverses the extinction of the uninsulated genes. Extinction is completely prevented by flanking the reporter construct with insulators. Furthermore, in contrast to the uninsulated reporter genes, chromatin over the insulated genes retains nuclease accessibility and histone hyperacetylation. However, there is no clear correlation between the presence of the insulators and the level of DNA methylation. This leads us to propose a model for the insulator's ability to protect against extinction in the transformed cell lines and to function as a chromatin boundary for the chicken beta-globin locus in normal erythroid cells.