The chromatin fingerprint of gene enhancer elements

Different cell types within a single organism are generally distinguished by strikingly different patterns of gene expression, which are dynamic throughout development and adult life. Distal enhancer elements are key drivers of spatiotemporal specificity in gene regulation. Often located tens of kilobases from their target promoters and functioning in an orientation-independent manner, the identification of bona fide enhancers has proved a formidable challenge. With the development of ChIP-seq, global cataloging of putative enhancers has become feasible. Here, we review the current understanding of the chromatin landscape at enhancers and how these chromatin features enable robust identification of tissue-specific enhancers.     

Human immunoglobulin kappa gene enhancer: chromatin structure analysis at high resolution.

The murine immunoglobulin kappa gene enhancer has previously been found to coincide with a region of altered chromatin structure reflected in a DNase I hypersensitivity site detectable on Southern blots of B-cell DNA. We examined the chromatin structure of the homologous region of human DNA using the high-resolution electroblotting method originally developed for genomic sequence analysis by G. Church and W. Gilbert (Proc. Natl. Acad. Sci. USA 81:1991-1995, 1984). Analysis of DNA isolated from cells treated in vivo with dimethyl sulfate revealed two B-cell-specific sites of enhanced guanine methylation. Both sites are located within perfect inverted repeats theoretically capable of forming cruciform structures; one of these repeats overlaps an enhancer core sequence. No enhancement or protection of guanine methylation was observed within sequences similar to sites of altered methylation previously described in the immunoglobulin heavy-chain enhancer. Treatment of isolated nuclei with DNase I or a variety of restriction endonucleases defined a B-cell-specific approximately 0.25-kilobase region of enhanced nuclease susceptibility similar to that observed in the murine kappa enhancer. The 130-base-pair DNA segment that shows high sequence conservation between human, mouse, and rabbit DNAs lies at the 5' end of the nuclease-susceptible region.     

Genetic analysis of the enhancer requirements for polyomavirus DNA replication in mice.

In this report, we describe the first systematic analysis of the genetic requirements for polyomavirus (Py) enhancer-activated viral DNA replication during the acute phase of infection in mice. Four mutants were made which substituted XhoI sites for conserved enhancer consensus sequences (adenovirus type 5 E1A, c-fos, simian virus 40, and a glucocorticoidlike consensus sequence). Viral DNA replication in infected mouse organs was measured by DNA blot analysis. Only the loss of the glucocorticoidlike consensus sequence element significantly reduced Py DNA replication in the kidneys, the primary target organ for viral replication. The loss of the c-fos, adenovirus type 5 E1A, or simian virus 40 consensus sequences, however, expanded organ-specific viral DNA replication, relative to wild-type Py, by allowing high-level replication in the pancreas or heart or both. Analysis of Py variants selected for replication in undifferentiated embryonal carcinoma cell lines (PyF441, PyF111) showed that there was little change in levels of viral DNA replication in kidneys and other organs as compared with those in the wild-type virus. If the entire B enhancer is deleted, only low overall levels of viral replication are observed. Wild-type levels of replication in the kidneys can be reconstituted by addition of a single domain from within the A enhancer (nucleotides 5094 to 5132) to the B enhancer deletion virus, suggesting that a single domain from the A enhancer can functionally substitute for the entire B enhancer. This also indicates that the determinants for kidney-specific replication are not found in the B enhancer.     

Interaction of nuclear factor EF-1A with the polyomavirus enhancer region.

Enhancer factor 1A (EF-1A) is a mammalian nuclear protein that previously was shown to bind cooperatively to the repeated core enhancer element I sequence in the adenovirus E1A enhancer region. We now have characterized three binding sites for EF-1A in the polyomavirus A2 (Py) enhancer region. Site 1 resides in the Py A enhancer domain, and sites 2 and 3 reside in the Py B enhancer domain. EF-1A binding to Py site 1 is independent of cooperation with other EF-1A sites or the adjacent binding sites for PEA-1 and PEA-2, two murine nuclear factors that bind in the Py A enhancer domain. EF-1A binding to Py sites 2 and 3, in contrast, is cooperative, similar to the situation previously observed with binding sites in the adenovirus E1A enhancer region. In a transient replication assay, EF-1A site 1 functions synergistically with the PEA-1 and PEA-2 sites in the A enhancer domain to enhance Py replication. The functional cooperativity observed with the EF-1A, PEA-1, and PEA-2 sites in vivo does not reflect cooperative DNA binding interactions, as detected in vitro. Py EF-1A site 1 alone is capable of weakly stimulating Py replication. EF-1A site 1 overlaps with the binding sites for the murine nuclear protein PEA-3 and the ets family of oncoproteins.     

Point mutation in the polyomavirus enhancer alters local DNA conformation

A point mutation in the enhancer of polyomavirus host range mutant, PyEC F441, permits productive infection of the murine embryonal carcinoma cell line, F9. This mutation at nucleotide position 5258 introduces a local conformational change in naked viral DNA. The effect of all four possible nucleotide sequences at position 5258 on local DNA conformation was analyzed by gel electrophoresis of fragments produced by ligation of synthetic oligonucleotides having these sequences. The results indicated that both the wild-type and the F441 sequences introduced local structural polymorphism that can lead to DNA bending. The wild-type sequence had a greater effect on DNA curvature than the F441 sequence. The two other sequences at nucleotide 5258 did not appear to introduce detectable amounts of DNA curvature.     

Enhancer sequences responsible for DNase I hypersensitivity in polyomavirus chromatin.

DNase I preferentially cleaves polyomavirus minichromosomes at two sites in the enhancer, each of which comprises the sequence AAGCAPuPuAAG flanked by short inverted repeats. A tandem duplication of this sequence generates an additional hypersensitive locus. Mutations which alter either the AAGCAPuPuAAG or flanking repeats diminish hypersensitivity. This region must determine the chromatin conformation recognized by DNase I.     

DNA methylation pattern and restriction endonuclease accessibility in chromatin of a germ-line specific gene, the rainbow trout protamine gene.

The chromatin structure of a germ-line specific gene, the TPG-3 gene, one of the rainbow trout protamine genes was analyzed in various tissues. The protamine genes are expressed in early stage testis but not in late stage testis, liver or erythrocyte. Five potential CpG methylation sites in the coding and flanking regions of the TPG-3 protamine gene were monitored in early and late stage testis, nucleoprotamine, liver and erythrocyte. In all cases the patterns of methylation were identical with only one CpG site at position -740 being methylated. Thus, the methylation pattern of this protamine gene remained the same independently of the expression of the gene. Two Msp I sites at positions -293 and/or -275 and +155 were accessible to the enzyme in the TPG-3 chromatin of early stage testis. Since the Msp I site at position -293 and/or -275 was also present in the TPG-3 chromatin of liver, only the site at position +155 within the transcribed region correlated with the expression of the protamine gene.