DNAase I-hypersensitive Sites Research Articles

Analysis of human breast cancer nuclear proteins binding to the promoter elements of the c-myc gene.

The expression of the c-myc gene is essential for the proliferation of both hormone-dependent and -independent human breast cancer cells. The regulation of c-myc gene expression in MCF-7 (hormone-dependent, estrogen-receptor (ER)-positive) and MDA MB 231 (hormone-independent, ER-negative) human breast cancer cells differs, with the c-myc gene of MCF-7 but not MDA MB 231 cells being regulated at the transcriptional level by estrogen. We have shown previously that the DNAase I hypersensitive (DH) sites in the c-myc chromatin of hormone-dependent and -independent human breast cancer cells were similar, with the exception of DH site II2. DH site II2, which maps near the P0 promoter, was less sensitive in hormone-dependent than in hormone-independent cells. As DH sites generally indicate the presence of sequence-specific DNA-binding proteins, we undertook a study to identify the nuclear proteins isolated from MCF-7 and MDA MB 231 cells that bound to the P0 and P2 promoter regions of the c-myc gene in vitro. The studies presented here provide evidence that Sp1 and/or Sp1-like proteins bind to the P0 and P2 promoter regions of the c-myc gene of MCF-7 and MDA MB 231 cells. Furthermore, evidence is presented for the presence of several previously unidentified sequence-specific DNA-binding proteins binding to these promoters. The DNA-binding activities of these latter proteins differed in the nuclear extracts of the MCF-7 and MDA MB 231 human breast cancer cells.

Identification of two elements involved in regulating expression of the murine leukaemia inhibitory factor gene.

Mouse leukaemia inhibitory factor (LIF) is a polyfunctional cytokine which exhibits multiple functions in vitro and in vivo. Two forms of LIF cDNA, differing at their 5' ends, have been described encoding either diffusible (D-LIF) or matrix-associated (M-LIF) forms of the protein [Rathjen, Toth, Willis, Heath and Smith (1990) (Cell 62, 1105-1114]. The present report describes the DNA sequence and functional characterization of the murine LIF gene and its surrounding transcriptional regulatory elements. Transient transfection of constructs containing the LIF gene and various amounts of 5'-non-coding sequence failed to give detectable levels of expression, suggesting the presence of inhibitory sequences within the LIF gene. Stable cell lines were produced by transfection of experimental constructs containing various lengths of 5'-non-coding sequence of the LIF gene, or the heterologous phosphoglycerate kinase promoter, linked to an LIF/neomycin-resistance-hybrid-coding sequence. The frequency of recovery of stable clones indicated that sequences located in the first intron between the transcriptional start sites for D-LIF and M-LIF act to suppress expression of the gene in most genomic locations. This region is rich in GC residues and has been shown to be hypomethylated in vitro [Kaspar, Dvorak and Bartunek (1993) FEBS Lett. 319, 159-162]. Analysis of the LIF/neomycin-resistance transgene expression in these stable cell clones demonstrated that transcripts containing the M-LIF or D-LIF exons required the presence of sequences located between -1200 and -3200 in the LIF gene. In the absence of these sequences, transcription is initiated elsewhere within the first intron. These sequences can be replaced by the heterologous phosphoglycerate kinase promoter. Deletion of the GC-rich region between the D-LIF and M-LIF transcriptional start sites results in the appearance of transcripts that do not splice out the first intron of the LIF gene. These may result from gene or promoter trapping of the LIF gene. Sequence analysis of the region between -1200 and -3200 revealed a number of minimal steroid-response elements, regions of similarity to DNAase I-hypersensitive sites in the uteroglobin gene and a region of alternating purine/pyrimidine sequence. This study therefore defines two important regulatory regions in the LIF gene: a GC-rich region in the first intron and a distal 'enhancer' located between -3200 and -1200.

4991587 Adaptive filtering of physiological signals in physiologically gated magnetic resonance imaging

Two distinct nonoverlapping enhancer elements can be defined within the polyoma enhancer region. In mouse fibroblasts, element A provides a 3-fold higher enhancement of the α2-collagen promoter than element B. In mouse embryonal carcinoma cells, element B shows the same efficiency as in fibroblasts, whereas that of element A decreases by a factor of 3.5. Moreover, a single point mutation (PyEC F9.1) increases the efficiency of element B in both cell types, making it superior to element A in embryonal carcinoma cells. The core of element A is located within a 35 bp region tandem duplicated in several wild-type strains, mostly homologous to a crucial repeated sequence of adenovirus E1a enhancer (Hearing and Shenk, 1983). Element B contains the consensus sequence of Weiher et al. (1982). These two homologies are precisely adjacent to the two DNAase I-hypersensitive sites present in viral chromatin.

Somatic diversification of the chicken immunoglobulin light chain gene is limited to the rearranged variable gene segment

Previous studies have shown that the chicken λ immunoglobulin light chain gene undergoes a single rearrangement that results in functional VJ joining of the unique variable (V λ1) and joining (J λ) coding regions. The immunologic repertoire of λ genes is created through extensive sequence diversification within the rearranged locus during B cell development in the bursa of Fabricius. This sequence diversification was detected only at the rearranged V λ1 segment and not within the 5′ leader sequence, the J λ segment, or the unrearranged V λ1 segment. The selective diversification of the rearranged V λ1 segment was associated with unique DNAase I-hypersensitive sites on the rearranged allele. While probes for V λ1 sequences detect multiple homologous V λ segments, probes for both the 5′ leader and J λ segments fail to detect homologous sequences. Taken together, these results suggest that a highly selective process, possibly gene conversion, operates during B cell ontogeny to generate diversity within the λ gene.

Two distinct enhancers with different cell specificities coexist in the regulatory region of polyoma

Two distinct nonoverlapping enhancer elements can be defined within the polyoma enhancer region. In mouse fibroblasts, element A provides a 3-fold higher enhancement of the α2-collagen promoter than element B. In mouse embryonal carcinoma cells, element B shows the same efficiency as in fibroblasts, whereas that of element A decreases by a factor of 3.5. Moreover, a single point mutation (PyEC F9.1) increases the efficiency of element B in both cell types, making it superior to element A in embryonal carcinoma cells. The core of element A is located within a 35 bp region tandem duplicated in several wild-type strains, mostly homologous to a crucial repeated sequence of adenovirus E1a enhancer (Hearing and Shenk, 1983). Element B contains the consensus sequence of Weiher et al. (1982). These two homologies are precisely adjacent to the two DNAase I-hypersensitive sites present in viral chromatin.

Nuclease-hypersensitive sites in the chromatin domain of the chicken lysozyme gene.

We have examined the chromatin structure of a 22 kilobase-pair chromosomal region containing the lysozyme gene in laying hen. Nuclease-hypersensitive sites were probed with DNAase I by using an indirect end-labeling technique. Eight DNAase I-hypersensitive sites could be mapped in the flanking regions of the gene in oviduct cells, in which the gene is expressed. The same sensitive sites were detected by utilization of an endogenous nuclease activity present in oviduct nuclei. Only one hypersensitive site was detected in the chromatin from erythrocytes, in which the gene is not expressed. The 3'-terminus of the lysozyme gene is highly exposed in nuclei from both tissues. Of special interest is the hypersensitive site at the 5'-terminus of the actively transcribed gene since it maps at the region of multiple initiation sites of transcription and the putative control regions of steroid hormones. DNAase I-hypersensitive chromatin structures also at a greater distance from the gene may take part in the control of gene expression.

Propagation of globin DNAase i-hypersensitive sites in absence of factors required for induction: A possible mechanism for determination

We tested whether DNAase I-hypersensitive sites, once induced, can be propagated to daughter cells in the absence of the original inducer. Chicken embryo fibroblasts infected with a temperature-sensitive Rous sarcoma virus at 41 °C and then shifted to 36°C become transformed and begin to transcribe globin RNA. DNAase I-hypersensitive sites appear in the α- and β-globin chromatin domains. Neither transcription nor hypersensitive sites are detected in cells infected and maintained at 41°C. Activation of the globin hypersensitive sites occurs within 30 min of a temperature shift to 36°C and does not require new protein synthesis. To test for the self-propagation of these hypersensitive structures, we inactivated the v-src gene product by a shift back up to 41°C, and allowed the cells to divide 20 times at 41°C. Although transcription of the globin genes was minimal after this treatment, the DNAase I-hypersensitive sites remained. The same sites can be induced by NaCI shock of cells. After the cells are returned to normal medium and allowed to grow for 20 doublings, the hypersensitive sites remain. This suggests that once formed, DNAase I-hypersensitive sites have the capacity to template their own structure independent of the initial “inductive” event. The single-stranded character of these DNAase I-hypersensitive sites could explain these results.

An expandable gene that encodes a Drosophila glue protein is not expressed in variants lacking remote upstream sequences

The Drosophila melanogaster gene Sgs4 encodes one of the glue polypeptides, sgs-4, synthesized in the larval salivary gland. We have examined the structure and expression of Sgs4 in five strains that produce abundant amounts of sgs-4 and its mRNA and in four that do not. The nonproducers include three Japanese strains that accumulate trace amounts of mRNA and one strain, BER-1, that contains no detectable Sgs4 RNA. Sgs4 carries a tandem array of repeated 21 bp elements within its coding sequence. The number of elements per array varies, causing considerable differences in the lengths of Sgs4 and its mRNA among the strains. These differences in length are not correlated with differences in mRNA abundance; rather, the low or zero abundance in nonproducers correlates with the loss of DNA upstream from the gene. The Japanese nonproducers carry a 52 bp deletion 305 bp upstream from the 5′ end of Sgs4, and BER-1 carries a 95 bp deletion 392 bp upstream. Curiously, each deletion encompasses one or more of the salivary-gland-specific DNAase I-hypersensitive sites which are known to flank the Sgs4 gene.

A complex of interacting DNAase I-hypersensitive sites near the Drosophila glue protein gene, Sgs4.

The chromatin structure adjacent to the Drosophila glue protein gene Sgs4 changes drastically when the gene is active. In nuclei from embryos or tissue culture cells in which Sgs4 is inactive, there are three DNAase I-hypersensitive sites 3' to the gene, but none near its 5' end. In the nuclei of late third instar salivary glands, Sgs4 is actively transcribed, and a complex of five DNAase I-hypersensitive sites appears 5' to the gene. Two of the sites are near the point of transcription initiation, at -70 and +30. The others are much farther from the gene at -330, -405 and -480 and are affected by small deletions: one deletion reduces expression about 50-fold and removes sequences corresponding to the -330 hypersensitive site; another makes no Sgs4 RNA and removes sequences corresponding to two hypersensitive sites, -405 and -480. Thus the hypersensitive sites, or DNA sequences within 50 bp of them, seem to be required for normal gene expression Distinct changes are seen in the chromatin from salivary glands of these mutant strains. The first strain is simply missing the -330 hypersensitive site, while the second is missing all of the tissue-specific 5' sites, even though sequences corresponding to three of them remain. This suggests that hierarchical interactions among the regions 5' to Sgs4 are required for its full expression. A sequence of 14 bp at the most prominent hypersensitive site (-405) is closely related to sequences 5' to several other eucaryotic genes.

An altered DNA conformation detected by S1 nuclease occurs at specific regions in active chick globin chromatin.

The single-stranded activity of S1-nuclease cleaves globin chromatin in red cell nuclei in specific regions. The cleavages are observed only in tissues in which the globin genes are active, and they "switch" to reflect the switching pattern of globin-gene expression in embryonic and adult red cells. The positions of the S1 cleavages in the beta- and alpha-globin chromatin correspond to the general region of known DNAase I-hypersensitive sites, but can be distinguished in detail. When DNA segments containing these regions are subcloned into pBR322 and the supercoiled molecules are treated with S1, similar sites are cleaved in the purified supercoiled (but not linear) recombinant plasmid DNA. However, the dominant S1 cutting sites are shifted in the plasmid vis-a-vis the chromatin. We believe that some aspect of DNA sequence is translated into an altered DNA structure in chromatin and that it is this altered structure that is recognized by s1 nuclease and possibly by certain chromosomal proteins. Several physical properties reflected in the S1 digestion of supercoiled plasmids suggest a mechanism for generating differences in daughter cells during development.

Temperature-sensitive changes in the structure of globin chromatin in lines of red cell precursors transformed by ts-AEV

Chicken bone marrow cells infected in vitro with a temperature-sensitive avian erythroblastosis virus fail to produce hemoglobin at 36°C. When the product or products of the transforming gene ( erb) are inactivated by a temperature shift to 42°C in culture, several different cloned lines of cells infected with the temperature-sensitive avian erythroblastosis virus begin to make hemoglobin. This shift in phenotype correlates with an increase in hemoglobin mRNA specific to both adult and embryonic α and β globin. The switch is accompanied by the acquisition of DNAase I-hypersensitive sites in one cell line (clone 2); however, a hypothetically more mature line (clone 3) has already acquired globin DNAase-hypersensitive sites but does not express hemoglobin until the temperature shift. Several (but not all) specific restriction sites associated with both the α and β domains become unmethylated after the switch from 36°C to 42°C. The magnitude of these methylation switches is small compared with changes that occur in these genes during normal avian erythropoiesis. The results suggest that changes in chromosomal structure precede transcription and are not a consequence of transcription. Since (presumptive) precursor cloned lines can be established with some, but not all, of the structural properties of active globin chromatin, it is likely that many of these properties can be independently established and are not obligatorily related.

DNAase I-hypersensitive sites of chromatin

During the last few years nucleases have been found to be effective tools for the dissection of the structure of chromatic. DNAase I (or pancreatic DNAase, E.C.3.1.4.5) is an endonuclease with little DNA-sequence specificity. When eucaryotic nuclei are partially digested with DNAase I, the resultant DNA fragments form a continuum of sizes, perceived as a uniform smear on agarose gels. Nonetheless, work with DNAase I has been of continuing interest since the finding of Weintraub and Groudine (Science 193, 848-856, 1976) that active chromatin is preferentially digested by this enzyme. The data now in hand indicate that a region of the chromatin, including the region of transcription and extending many kilobases beyond it, is generally very sensitive to DNAase I for a gene in the active state. In contrast with these broad patterns of differential DNAase I sensitivity, we have recently recognized and studied specific sites in chromatin that are even more sensitive to DNAase I, now referred to as DNAase l-hypersensitive sites. Such sites were first observed when the gel blot-hybridization technique of Southern was used to analyze the consequence of a very light digestion of Drosophila nuclei by DNAase I. Using a specific recombinant plasmid probe, we observed a series of discrete bands, indicating specific cleavage of the DNA in chromatin. Control experiments showed that no such bands were observed following digestion of naked DNA with DNAase I, demonstrating that the hypersensitive sites exist as a consequence of the chromatin structure. The pattern of bands obtained from chromatin was unique for each of five cloned Drosophila loci examined, suggesting that the sites recognized by DNAase I were specifically and nonuniformly distributed along the chromatin fiber (Wu et al., Cell 76, 797-806,1979). The smear of fragments seen as the digestion pattern for the genome as a whole apparently represents the sum of thousands of discrete patterns, rather than indicating a lack of specificity in the digestion of chromatin by DNAase I. A number of such nuclease-sensitive sites have now been mapped precisely with different approaches, most frequently an indirect end-labeling technique first applied by Nedospasov and Georgiev using micrococcal nuclease (BBRC 92, 532-539, 1980) and by Wu using DNAase I (Nature 286, 854-860,198O). Nuclei are digested lightly with the nuclease to introduce an average of one cut within a region of interest, bounded but not intersected by a particular restriction site. A hypothetical case is shown in the figure (A), in which we wish to examine the region between the two Barn sites. DNA is purified from the digested chromatin Minireviews

Activation of globin genes during chicken development

Hb first appears in the developing chicken blastoderm at 35 hr of incubation. We have isolated the presumptive Hb-forming area of the embryo at 20–23 hr and shown that most of the cells in this region are red cell precursors. The structure and activity of the globin genes in these precursors have been assayed in three ways: by runoff nuclear transcription to measure the number of initiated RNA polymerases present on the globin genes; by measuring the degree of methylation of these genes with methyl-sensitive restriction enzymes; and by monitoring the DNAase I sensitivity and the generation of DNAase I-hypersensitive sites in the globin chromatin. All these assays show the globin genes to be inactive in precursor cells. Additional experiments focusing on the mechanism of the transition from an inactive to an active chromosome structure indicate that DNA replication but not cytokinesis is intimately coupled to the transition process.