Molecular analysis of the human beta-globin locus activation region.

Recently, DNA sequences containing four erythroid-specific DNase I hypersensitive sites within 20 kilobases 5' of the human epsilon-globin gene have been identified as an important cis-acting regulatory element, the locus activation region (LAR). Subfragments of the LAR, containing either all or only the two 5' or two 3' hypersensitive sites were linked to the human beta-globin gene and analyzed for their effect on globin gene expression in stably transformed mouse erythroleukemia (MEL) cells. Constructs containing all four of the hypersensitive sites increase beta-globin mRNA levels 8- to 13-fold, while constructs with only the 5' or 3' sites increase globin expression to a lesser extent. No effect was seen when the constructs were assayed in 3T3 fibroblasts. All of the LAR derivatives form hypersensitive sites at the corresponding sequence position in MEL cells prior to and after induction of MEL cell differentiation. However, in 3T3 fibroblasts only the hypersensitive site corresponding to the previously described erythroid-specific -10.9 site was formed.     

Cloning of the complete Ly-6E.1 gene and identification of DNase I hypersensitive sites corresponding to expression in hematopoietic cells

The Sca-1 antibody recognizes antigens encoded by members of the Ly-6 multigene family. These antigens are expressed on fetal and adult hematopoietic stem cells, progenitor cells, mature activated T cells, and some nonhematopoietic cells and are most likely encoded by the Ly- 6E.1 and Ly-6A.2 genes. Characterization and isolation of regulatory elements of Ly-6E.1 and A.2 genes that govern tissue-specific and high levels of expression in the cells of the hematopoietic system (particularly stem cells) are of considerable interest. To characterize the control elements of this gene, we have cloned a 30-kb fragment encoding a fully functional Ly-6E.1 gene and 13 kb of 5' and 13 kb of 3' flanking sequence. Transfection studies in murine erythroleukemia (MEL) cells show that a 14-kb BamHI fragment from this clone is sufficient to confer Ly-6E.1 gene expression at levels equivalent to those of the endogenous gene. By mapping regions of chromatin sensitive to DNase I digestion, we have located hypersensitive sites in the 5' and 3' regions of the gene in FDCP-1 cells, MEL cells, and various T- cell lines. The appearance of two 5' hypersensitive sites in hematopoietic cells correlates with Ly-6E.1 expression after gamma- interferon induction. We show that the presence of hypersensitive sites in the 5' and 3' regions corresponds to Sca-1 expression, and we also discuss the localization of putative regulatory control elements.     

Changes of DNA methylation and chromatin structure in the human myeloperoxidase gene during myeloid differentiation.

Expression of the myeloperoxidase (MPO) gene is tightly regulated in a tissue- and development-specific manner. Accumulation of MPO messenger RNA (mRNA) occurs only at the late myeloblastic and promyelocytic stages of myeloid differentiation and is negligible at other stages of myeloid development and in other tissues. The goal of our studies was to begin to understand the events that occur to control MPO gene expression during normal granulocytopoiesis. Chromatin structure of the MPO gene was evaluated by DNase I treatment of isolated nuclei and Southern blot analysis. No detectable DNase I hypersensitive sites were found in the region of the MPO gene in non-myeloid cells. One site was present in the 5' upstream region in myeloid cells that are developmentally too immature to transcribe MPO. Three sites of hypersensitivity in the regions of the putative MPO promoter and upstream region occurred in MPO-expressing promyelocytes. These sites were markedly reduced in terminally differentiated, non-expressing myeloid cells. Analysis of DNA methylation of the MPO gene using methylation-sensitive restriction enzymes showed that the gene was highly methylated in non-myeloid cells. Stepwise demethylation occurred in myeloid cells developmentally too immature to transcribe MPO. Maximal demethylation in the 5' gene region occurred in MPO-expressing promyelocytes. This methylation pattern did not change in terminally differentiated, MPO non-expressing myeloid cells. A somatic hybrid cell formed by fusion of HL-60 (MPO-expressing cells) and PUT (MPO non-expressing lymphoid cells) extinguished expression of MPO and showed a chimeric pattern of MPO gene methylation, suggesting that demethylation is necessary but not sufficient for expression of the MPO gene. Our studies show that demethylation and DNase I hypersensitivity of the MPO gene were associated with a tissue-dependent potential for MPO gene expression that preceded the developmental ability to express MPO mRNA.     

Human fetal to adult hemoglobin switching: changes in chromatin structure of the beta-globin gene locus.

We have investigated the chromatin structure of the chromosomal DNA regions containing the human G gamma-, A gamma-, delta-, and beta-globin structural genes in both fetal and adult erythropoietic tissues and in two human erythroleukemia cells lines before and after induction. Our results indicate that DNase I introduces specific cuts into the beta-globin gene cluster in erythroid cells but not in leukocytes. The predominant sites are located at the 5' sides of the G gamma-, A gamma-, delta-, and beta-globin genes, within 200 base pairs of the respective cap sites. Examination of fetal liver cells has revealed the presence of hypersensitive sites at the 5' side of all four genes, whereas analysis of adult bone marrow has revealed the characteristic sites near the delta- and beta-globin genes but no hypersensitive sites at the 5' termini of the G gamma- or A gamma-globin genes. The presence of delta and beta hypersensitive sites in fetal cells suggests that the increment in expression of the delta and beta genes during development most likely involves the modulation of another pathway to gene expression. Using isolated nuclei from HEL and K562 cells, we have found that the G gamma, A gamma, delta, and beta genes are preferentially sensitive [relative to the pro-alpha2(I) collagen gene] to mild digestion with DNase I, whereas these genes are as resistant as collagen genes in cells that do not express globin. These findings are discussed within the context of chromatin structural correlates of hemoglobin switching.     

Murine erythroleukemia cell differentiation: DNase I hypersensitivity and DNA methylation near the globin genes

The sensitivity to digestion by DNase I of chromatin containing the alpha- and beta(major)-globin genes and the pattern of DNA methylation near these genes was examined during hexamethylenebisacetamide (HMBA)-mediated erythroid differentiation of murine erythroleukemia cells (MELC). In uninduced and induced cells, the chromatin regions containing the alpha- and beta-(major)-globin genes are more sensitive to digestion by DNase I than is the region containing an immunoglobulin gene (Igalpha) not expressed during erythroid differentiation. However, at low concentrations of DNase I, a 6- to 10-fold increase in site-specific cleavages was generated in chromatin regions near both the alpha- and beta(major)-globin genes in cells induced to differentiate by HMBA. The DNase I hypersensitive site near the beta(major)-globin gene maps to a small region near the 5' terminus of the gene. No detectable change in the pattern of DNA methylation around either the alpha- or beta-globin genes was observed during HMBA-mediated erythroid differentiation. Of the potentially methylated sites assayed and mapped near the beta(major)-globin gene, one site is fully methylated, one is partially methylated, and one is unmethylated both in uninduced and induced cells. Many (but not all) sites assayed near the alpha-globin genes are unmethylated in both uninduced and induced cells. These results show that specific alterations of chromatin structure occur during MELC differentiation and suggest that these changes may not involve alterations in the pattern of DNA methylation.     

Characterization of the DNase I hypersensitive site 3' of the human beta globin gene domain

The members of the human beta globin gene family are flanked by strong DNase I hypersensitive sites. The collection of sites 5' to the epsilon globin gene is able to confer high levels of expression of linked globin genes, but a function has not been assigned to the site 3' to the beta globin gene (3'HS1). Our analysis of this DNase I super hypersensitive site shows that the region is composed of multiple DNase I sites. By examination of the DNA sequence, we have determined that the region is very A/T-rich and contains topoisomerase II recognition sequences, as well as several consensus binding motifs for GATA-1 and AP-1/NF-E2. Gel mobility shift assays indicate that the region can interact in vitro with GATA-1 and AP-1/NF-E2, and functional studies show that the region serves as a scaffold attachment region in both erythroid and nonerythroid cell lines. Whereas many of the physical features of 3'HS1 are shared by 5'HS2 (a component of the 5' locus control region), transient expression studies show that 3' HS1 does not share the erythroid-specific enhancer activity exhibited by 5'HS2.     

Intronic enhancers coordinate epithelial-specific looping of the active CFTR locus

The regulated expression of large human genes can depend on long-range interactions to establish appropriate three-dimensional structures across the locus. The cystic fibrosis transmembrane conductance regulator (CFTR) gene, which encompasses 189 kb of genomic DNA, shows a complex pattern of expression with both spatial and temporal regulation. The flanking loci, ASZ1 and CTTNBP2, show very different tissue-specific expression. The mechanisms governing control of CFTR expression remain poorly understood, although they are known to involve intronic regulatory elements. Here, we show a complex looped structure of the CFTR locus in cells that express the gene, which is absent from cells in which the gene is inactive. By using chromatin conformation capture (3C) with a bait probe at the CFTR promoter, we demonstrate close interaction of this region with sequences in the middle of the gene about 100 kb from the promoter and with regions 3′ to the locus that are about 200 kb away. We show that these interacting regions correspond to prominent DNase I hypersensitive sites within the locus. Moreover, these sequences act cooperatively in reporter gene constructs and recruit proteins that modify chromatin structure. The model for CFTR gene expression that is revealed by our data provides a paradigm for other large genes with multiple regulatory elements lying within both introns and intergenic regions. We anticipate that these observations will enable original approaches to designing regulated transgenes for tissue-specific gene therapy protocols.     

DNase I hypersensitive sites in Drosophila chromatin occur at the 5' ends of regions of transcription

By using a map of the unique region of DNA encoding the fur small heat-shock proteins of Drosophila melanogaster (hsp 22, hsp 23, hsp 26, and hsp 28), and a simple mapping technique, the positions of the DNase I hypersensitive sites of chromatin in the vicinity of these genes have now been determined. The major chromatin-specific sites occur at the 5' ends of each of the four heat-shock protein genes in embryo nuclei. These genes are not active in the nuclei analyzed but can be quickly induced in these cells by the heat-shock stimulus. The chromatin structure indicated by DNase I hypersensitivity may be a necessary factor in the general mechanism of gene activation.