Tissue-specific DNase I-hypersensitive Sites Research Articles

Genome-wide DNase I-hypersensitive site assay reveals distinct genomic distributions and functional features of open chromatin in autopolyploid sugarcane.

The characterization of cis-regulatory DNA elements (CREs) is essential for deciphering the regulation of gene expression in eukaryotes. Although there have been endeavors to identify CREs in plants, the properties of CREs in polyploid genomes are still largely unknown. Here, we conducted the genome-wide identification of DNase I-hypersensitive sites (DHSs) in leaf and stem tissues of the auto-octoploid species Saccharum officinarum. We revealed that DHSs showed highly similar distributions in the genomes of these two S. officinarum tissues. Notably, we observed that approximately 74% of DHSs were located in distal intergenic regions, suggesting considerable differences in the abundance of distal CREs between S. officinarum and other plants. Leaf- and stem-dependent transcriptional regulatory networks were also developed by mining the binding motifs of transcription factors (TFs) from tissue-specific DHSs. Four TEOSINTE BRANCHED 1, CYCLOIDEA, and PCF1 (TCP) TFs (TCP2, TCP4, TCP7, and TCP14) and two ethylene-responsive factors (ERFs) (ERF109 and ERF03) showed strong causal connections with short binding distances from each other, pointing to their possible roles in the regulatory networks of leaf and stem development. Through functional validation in transiently transgenic protoplasts, we isolate a set of tissue-specific promoters. Overall, the DHS maps presented here offer a global view of the potential transcriptional regulatory elements in polyploid sugarcane and can be expected to serve as a valuable resource for both transcriptional network elucidation and genome editing in sugarcane breeding.

Structural switching/polymorphism by sequential base substitution at quasi-palindromic SNP site (G → A) in LCR of human β-globin gene cluster

The human β-globin gene Locus Control Region (LCR), a dominant regulator of globin gene expression contains five tissue-specific DNase I-hypersensitive sites (HSs). A single nucleotide polymorphism (SNP) (A → G) present in HS4 region of locus control region (LCR), have shown a notable association between the G allele and the occurrence of β-thalassemia. This SNP site exhibiting a hairpin − duplex equilibrium manifested in A → B like DNA transition has previously been reported from this laboratory. Since, DNA is a dynamic and adaptable molecule, so any change of a single base within a primary DNA sequence can produce major biological consequences commonly manifested in genetic disorders such as sickle cell anemia and β-thalassemia. Herein, the differential behavior of sequential single base substitutions G → A on the quasi-palindromic sequence (d-TGGGGGCCCCA; HPG11) has been explored. A combination of native gel electrophoresis, circular dichroism (CD), and UV-thermal denaturation (Tm) techniques have been used to investigate the structural polymorphism associated with various variants of HPG11 i.e. HPG11A2 to HPG11A5. The CD spectra confirmed that all the HPG11 variants exhibit a hairpin − duplex equilibrium. Oligomer concentration dependence on CD spectra has been correlated with A → B DNA conformational transition. However, as revealed in gel electrophoresis, HPG11A2 → A5 exhibit the formation of a tetramolecular structure (four-way junction) at higher oligomer concentration. UV-melting studies also supported the melting of hairpin, duplex and four-way junction structure. This polymorphism pattern may possibly be significant for DNA-protein recognition, in the process of regulation of LCR in the β-globin gene.

Methylated Cytosines Mutate to Transcription Factor Binding Sites that Drive Tetrapod Evolution.

In mammals, the cytosine in CG dinucleotides is typically methylated producing 5-methylcytosine (5mC), a chemically less stable form of cytosine that can spontaneously deaminate to thymidine resulting in a T•G mismatched base pair. Unlike other eukaryotes that efficiently repair this mismatched base pair back to C•G, in mammals, 5mCG deamination is mutagenic, sometimes producing TG dinucleotides, explaining the depletion of CG dinucleotides in mammalian genomes. It was suggested that new TG dinucleotides generate genetic diversity that may be critical for evolutionary change. We tested this conjecture by examining the DNA sequence properties of regulatory sequences identified by DNase I hypersensitive sites (DHSs) in human and mouse genomes. We hypothesized that the new TG dinucleotides generate transcription factor binding sites (TFBS) that become tissue-specific DHSs (TS-DHSs). We find that 8-mers containing the CG dinucleotide are enriched in DHSs in both species. However, 8-mers containing a TG and no CG dinucleotide are preferentially enriched in TS-DHSs when compared with 8-mers with neither a TG nor a CG dinucleotide. The most enriched 8-mer with a TG and no CG dinucleotide in tissue-specific regulatory regions in both genomes is the AP-1 motif (TGAC/GTCAN), and we find evidence that TG dinucleotides in the AP-1 motif arose from CG dinucleotides. Additional TS-DHS-enriched TFBS containing the TG/CA dinucleotide are the E-Box motif (GCAGCTGC), the NF-1 motif (GGCA—TGCC), and the GR (glucocorticoid receptor) motif (G-ACA—TGT-C). Our results support the suggestion that cytosine methylation is mutagenic in tetrapods producing TG dinucleotides that create TFBS that drive evolution.

Mechanisms of Transcriptional Regulation of the Human IL-3/GM-CSF Locus by Inducible Tissue-Specific Promoters and Enhancers

The IL-3 and GM-CSF genes are closely linked in the genome and reside within a cluster of cytokine genes. IL-3 and GM-CSF are expressed in a highly inducible and tissue-specific pattern, and this review attempts to provide a comprehensive description of the key regulatory elements in this locus that control their expression. Although these genes are typically coexpressed in T cells, they are differentially regulated in many other cell types, whereby cells such as monocytes and endothelial cells express GM-CSF, but not IL-3. This suggests that they are likely to be regulated by distinct mechanisms. This view is reinforced by the identification of three inducible enhancers in the locus that have different specificities. These enhancers are embedded within arrays of tissue-specific DNaseI hypersensitive sites that most likely play additional roles in establishing distinct patterns of gene expression. This locus also represents a valuable model system for studying the role of chromatin remodeling in cytokine gene activation. NFAT is one inducible factor that appears to play a major role in the formation of DNaseI hypersensitive sites within enhancers, and which functions in a highly cooperative manner with other classes of transcription factors to direct specific patterns of cytokine gene expression.

A complex network of regulatory elements in Ikaros and their activity during hemo-lymphopoiesis.

Regulated expression of Ikaros is critical for normal hemopoiesis and lymphocyte development. To elucidate the mechanisms underlying transcription of Ikaros, tissue-specific DNase I-hypersensitive sites (DHS) were mapped throughout the Ikaros locus, and several promoters were identified. The activity of these regulatory regions was elucidated using an enhanced green fluorescent protein (EGFP) reporter in transgenic mice. Two genomic fragments, each containing a distinct promoter and its associated DHS cluster, were found to be active in the myeloid (DHS-C2 and DHS-C3) and B-cell (DHS-C3) lineages. Although neither of these regulatory regions was active within the majority of differentiating thymocytes and mature T cells, the DHS-C3 region was active at the earliest stages (DN1-DN3) of T-cell differentiation. However, when the DHS-C3 region was combined with the downstream intronic DHS-C6 cluster, its activity was maintained and raised to higher levels at subsequent stages of T-cell differentiation. This combination of regulatory elements provided reporter expression that closely resembles that of endogenous Ikaros during hemo-lymphopoiesis, and it decreased (but did not alleviate) position effect variegation within the expressing cell types.

Regulatory systems of genome domains with vague boundaries

The specific features of genome domains lacking distinct boundaries are considered. These domains cannot be mapped by testing extended genome regions for nuclease sensitivity and thereby differ from structural domains determined at the level of DNA folding in chromatin. Yet they possess the properties of typical functional domains, containing a gene or several coordinated genes along with a complex of cis-regulatory elements, which control these genes. Domains with vague boundaries may be mapped with certain structural tests, e.g., by assessing histone acetylation or the distribution of tissue-specific DNase I-hypersensitive sites through extended genome regions. The mechanisms are described in detail that regulate the function of genes in domains with vague boundaries, including overlapping domains with genes differing in tissue specificity of expression.

DNase I-hypersensitive sites I and II of the human growth hormone locus control region are a major developmental activator of somatotrope gene expression.

High-level expression of the human growth hormone (hGH) gene is limited to somatotrope and lactosomatotrope cells of the anterior pituitary. We previously identified a locus control region (LCR) for the hGH gene composed of four tissue-specific DNase I-hypersensitive sites (HS) located between -14.6 kb and -32 kb 5' to the hGH transcription start site that is responsible for establishing a physiologically regulated chromatin domain for hGH transgene expression in mouse pituitary. In the present study we demonstrated that the LCR mediates somatotrope and lactosomatotrope restriction on an otherwise weakly and diffusely expressed hGH transgene. The subregion of the LCR containing the two pituitary-specific HS, HSI and HSII (-14.6 to -16.2 kb relative to the hGH promoter and denoted HSI,II), was found to be sufficient for mediating somatotrope and lactosomatotrope restriction, for appropriately timed induction of hGH transgene expression between embryonic days 15.5 and 16.5, and for selective extinction of hGH expression in mature lactotropes. When studied by cell transfection, the HSI,II fragment selectively enhanced transcription in a presomatotrope-derived cell line, although at levels (2- to 3-fold) well below that seen in vivo. The LCR activity of the HSI,II element was therefore localized by scoring transgene expression in fetal founder pituitaries at embryonic day 18.5. The data from these studies indicated that a 404-bp segment of the HSI,II region encodes a critical subset of LCR functions, including the establishment of a productive chromatin environment, cell-specific restriction and enhancement of expression, and appropriately timed induction of the hGH transgene during embryonic development.

Appropriate developmental expression of human CD8 beta in transgenic mice.

The human CD8 glycoprotein is expressed either as an alpha beta heterodimer or as an alpha alpha homodimer on thymocytes, mature T cells, and subpopulations of intestinal intraepithelial lymphocytes (IELs). The homodimeric form of CD8 is exclusively expressed on TCR gamma delta IELs and on subsets of NK cells and TCR alpha beta IELs. To understand the molecular mechanisms by which these genes are regulated, we created transgenic mice with a 95-kb human genomic DNA fragment containing the entire CD8 beta gene as well as a cluster of tissue-specific DNase I-hypersensitive sites 7 to 10 kb upstream of the gene. These sites were present in CD8 alpha beta+- but not CD8 alpha beta- T cell lines nor in a B cell line. We found that transgenic mice had correct developmental expression of human CD8 beta on thymocytes and mature CD8+ cells and no expression on mature CD4+ T cells or B cells. Interestingly, the percentage of mouse CD8 alpha+ cells that were human CD8 beta+ varied, depending on the founder line, from 4 to 88%, whereas the percentage among siblings was similar, indicative of a variegated phenotype resulting from site of integration effects. Expression was also observed on intestinal IELs, but only on those expressing the TCR alpha beta receptor and not the TCR gamma delta cells, which exclusively express CD8 alpha alpha. Of the TCR alpha beta+ cells, the transgene was expressed in both the CD8 alpha alpha and alpha beta subpopulations. These results indicate that this 95-kb fragment affords developmentally correct expression of the human CD8 beta gene on thymus-derived T cells in transgenic animals. Therefore, CD8 lineage-specific regulatory sequences must be located within the fragment.

Multiple nuclear factors interact with sequences within the J beta 2-C beta 2 intron of the murine T cell receptor beta-chain gene.

Identification of tissue-specific DNaseI hypersensitive sites in the TCR J beta 2-C beta 2 intron has suggested the presence of sequences involved in the regulation of gene expression. Therefore, we have searched for protein-DNA interactions within a 930-bp fragment derived from the J beta 2-C beta 2 intron by in vitro DNaseI protection experiments and electrophoretic mobility-shift assays. This analysis has revealed, in addition to a previously characterized NF-kappa B binding site, the presence of seven potential protein-DNA interaction sites within this fragment. Interestingly, they are clustered in the regions where in vivo T cell-specific nuclease hypersensitive sites have been previously identified. Binding sites for four potential transcription factors have been mapped precisely by methylation-interference experiments. Sequence comparisons show that one of them is homologous to the Y box present in the promoter regions of MHC class II genes. Identification of several protein-DNA interactions clustered within the J beta 2-C beta 2 intron and the presence of binding sites for two well-characterized transcription factors suggest a transcriptional regulatory function for this region.

Chromatin configuration of the human CD2 gene locus during T-cell development.

To investigate the molecular basis for the tissue-specific expression of the human CD2 gene, its chromatin configuration was assessed by determining DNase I hypersensitivity and the degree of methylation during T-cell lineage commitment and development. Tissue-specific DNase I-hypersensitive sites were found within the 5' promoter region and a region 3' of the gene essential for gene expression. DNase I hypersensitivity of the 5' region correlated strictly with transcriptional activity, whereas hypersensitivity of the 3' region correlated with T-cell progenitor activity or lineage commitment but not necessarily with transcription. Hha I and Hpa II sites around the 5' and 3' regions were undermethylated in CD2-expressing T cells but were more extensively methylated in other cell types. These results define likely regulatory elements both upstream and downstream of the CD2 gene that control its tissue-specific expression. Further, they show that the 3' regulatory region adopts an open chromatin configuration prior to lineage commitment and during early stages of T-cell development before the CD2 gene is transcribed.

Tissue-specific DNase I-hypersensitive sites and hypomethylation in the chicken cardiac myosin light chain gene (L2-A).

As an approach to determine DNA elements involved in tissue-specific gene expression, the functional promoter of the cardiac myosin light chain gene (MLC 2-A) was identified. Chromatin structure of the MLC 2-A gene was examined for tissue specific DNase I-hypersensitive and hypomethylated sites. Heart specific DNase I-hypersensitive sites were detected in the promoter region of the gene as well as in flanking DNA sequences upstream and downstream of the gene. In expressing heart nuclei a region located about 1.5 kilobase pairs upstream of the transcription start site was found to contain both a hypersensitive and an undermethylated site. These structural chromatin changes of the MLC 2-A gene may be related to the expression of the gene.

Albumin and α-Fetoprotein Gene Transcription in Rat Hepatoma Cell Lines is Correlated With Specific DNA Hypomethylation and Altered Chromatin Structure in the 5′ Region

We examined DNA methylation and DNase I hypersensitivity of the alpha-fetoprotein (AFP) and albumin gene region in hepatoma cell lines which showed drastic differences in the level of expression of these genes. We assayed for methylation of the CCGG sequences by using the restriction enzyme isoschizomers HpaII and MspI. We found two methylation sites located in the 5' region of the AFP gene and one in exon 1 of the albumin gene for which hypomethylation is correlated with gene expression. Another such site, located about 4,000 base pairs upstream from the AFP gene, seems to be correlated with the tissue specificity of the cells. DNase I-hypersensitive sites were mapped by using the indirect end-labeling technique with cloned genomic DNA probes. Three tissue-specific DNase I-hypersensitive sites were mapped in the 5' flanking region of the AFP gene when this gene was transcribed. Similarly, three tissue-specific DNase I-hypersensitive sites were detected upstream from the albumin gene in producing cell lines. In both cases, the most distal sites were maintained after cessation of gene activity and appear to be correlated with the potential expression of the gene. Interestingly, specific methylation sites are localized in the same DNA region as DNase I hypersensitive sites. This suggests that specific alterations of chromatin structure and changes in methylation pattern occur in specific critical regulatory regions upstream from the albumin and AFP genes in rat hepatoma cell lines.

Albumin and alpha-fetoprotein gene transcription in rat hepatoma cell lines is correlated with specific DNA hypomethylation and altered chromatin structure in the 5' region.

We examined DNA methylation and DNase I hypersensitivity of the alpha-fetoprotein (AFP) and albumin gene region in hepatoma cell lines which showed drastic differences in the level of expression of these genes. We assayed for methylation of the CCGG sequences by using the restriction enzyme isoschizomers HpaII and MspI. We found two methylation sites located in the 5' region of the AFP gene and one in exon 1 of the albumin gene for which hypomethylation is correlated with gene expression. Another such site, located about 4,000 base pairs upstream from the AFP gene, seems to be correlated with the tissue specificity of the cells. DNase I-hypersensitive sites were mapped by using the indirect end-labeling technique with cloned genomic DNA probes. Three tissue-specific DNase I-hypersensitive sites were mapped in the 5' flanking region of the AFP gene when this gene was transcribed. Similarly, three tissue-specific DNase I-hypersensitive sites were detected upstream from the albumin gene in producing cell lines. In both cases, the most distal sites were maintained after cessation of gene activity and appear to be correlated with the potential expression of the gene. Interestingly, specific methylation sites are localized in the same DNA region as DNase I hypersensitive sites. This suggests that specific alterations of chromatin structure and changes in methylation pattern occur in specific critical regulatory regions upstream from the albumin and AFP genes in rat hepatoma cell lines.

Tissue-specific DNaseI hypersensitive sites in the 5'-flanking sequences of the tryptophan oxygenase and the tyrosine aminotransferase genes.

The genes for tryptophan oxygenase (TO) and tyrosine aminotransferase (TAT) are expressed in a tissue- and development-specific manner and are regulated by glucocorticoids (TO and TAT) and glucagon or its intracellular mediator cAMP (TAT) in rat liver. We have analyzed the chromatin structure of these genes in the vicinity of the 5' ends with regard to DNaseI hypersensitivity and have found DNaseI hypersensitive sites upstream of each of the promoters. Mapping of this region reveals three closely spaced cleavage sites near the TO promoter and a doublet of sites near the TAT promoter. In both genes additional cleavage sites are found further upstream. All hypersensitive sites of both genes are absent in kidney nuclei and therefore appear to be specific for the tissue expressing the genes. A correlation of expression and modified chromatin structure was also observed in a hepatoma cell line expressing TAT but not TO: hypersensitive sites are present in TAT but not in TO chromatin. Upon glucocorticoid induction an additional hypersensitive site is detected approximately 2 kb upstream of the TAT promoter in liver and hepatoma cells.

DNase I-hypersensitive sites in the chromatin of immunoglobulin kappa light chain genes.

We have used DNase I as a probe to examine the chromatin structure of mouse immunoglobulin kappa light chain genes in rearranged and unrearranged chromosomes--i.e., in nuclei from myeloma cells and from brain and liver cells. Tissue-specific DNase I-hypersensitive sites are observed 0.7 and 1.7 kilobases upstream from the 5' end of the C kappa gene in the J kappa -C kappa intron region in myeloma nuclei but not in naked DNA or in brain or liver nuclei. In myeloma cells expressing one functional kappa light chain polypeptide, but with more than one rearranged allele, one DNase I-hypersensitive site is found 0.3 kilobases upstream from the start of the coding region of the V kappa sequence in both functionally rearranged and nonfunctionally rearranged alleles but not in cross-hybridizing V kappa genes in the germ line context. Thus, during the development of B lymphocytes, the commitment of immunoglobulin kappa light chain gene expression seems to be associated with the presence of DNase I hypersensitivities that reflect changes of chromosomal structure surrounding the single copy C kappa gene. In contrast, the germ line V kappa multigene family seems to be located in a chromosomal region that does not exhibit change of DNase I hypersensitivity in response to commitment of immunoglobulin expression; a V kappa gene acquires DNase I hypersensitivity only after it is translocated adjacent to the J kappa -C kappa intron region. The DNase I-hypersensitive site 5' to the V kappa sequence is similar in location to hypersensitive sites found for other eukaryotic genes and is probably associated with the promoter region. However, the DNase I-hypersensitive sites in the J kappa -C kappa intron region are not associated with any known promoters. In addition, the DNA sequences surrounding the C kappa-proximal DNase I-hypersensitive site have several stretches of homology with sequences within the 72-base-pair tandem repeat of simian virus 40, which has been shown to modulate the transcriptional activity of neighboring genes. This DNase I-hypersensitive site in the intron region may be significant for the differential expression of the translocated V kappa genes.