A Contigs Strategy of Southern Blot Hybridization: Screening for DNase I Hypersensitive Sites in the 200 Kb Region 5′ to the B-Globin Locus LCR.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 1219-1219
Author(s):  
Ping Xiang ◽  
Hemei Han ◽  
Xiangdong Fang ◽  
George Stamatoyannopoulos ◽  
Qiliang Li
Keyword(s):  
Southern Blot ◽  
Dna Sequences ◽  
Globin Gene ◽  
Blot Hybridization ◽  
Restriction Enzymes ◽  
Southern Blot Hybridization ◽  
Dnase I ◽  
Screening Process ◽  
Dnase I Hypersensitive Sites ◽  
Hypersensitive Sites

Abstract Formation of DNase I hypersensitive sites is an indication of local disruption of chromatin conformation. It has been documented that HS sites are frequently associated with functional DNA sequences, such as, promoters, enhancers, and insulators. While Southern blot hybridization is the standard method to detect HS sites, this procedure is time-consuming and labor intensive. To improve the efficiency of HS detection through Southern blot hybridization, we designed a contigs strategy of Southern blot hybridization and test it in the 200 kb region 5′ to the LCR in the b-globin locus. Based on the human genome sequence we made physical maps of seven 6-bp-cut restriction enzymes in the 200 kb region. From the map we selected continuous contigs of 10 to 15 kb fragment; and designed hybridization probes for the 5′ and 3′ ends of each fragment (some probes can be used in two neighboring fragments). The screening was performed on erythroid (K562) and non-erythroid (Jurkat) cell lines. We found about 40 HS sites within the region. The major sites were either erythroid specific (for instance, HSs at −66 kb, −142 kb, and −236 kb, the cap site of the e-globin gene is +1), or non-erythroid specific (for instances, HSs at −111 kb, −164 kb, and −205 kb). These HS sites will be investigated for enhancer, promoter, and insulator function using transient and stable transfection studies. Due to the limited number of enzyme required and the fact that each blot could be used several times, this strategy can greatly expedite the screening process for presence of DNase I hypersensitive sites. Estimated efficiency of this screening approach is about 0.5 to1 Mb per person per year.

Correlation between patterns of DNase I-hypersensitive sites and upstream promoter activity of the human epsilon-globin gene at different stages of erythroid development

1990 ◽  
Vol 10 (3) ◽  
pp. 1199-1208
Author(s):  
P Bushel ◽  
K Rego ◽  
L Mendelsohn ◽  
M Allan
Keyword(s):  
Globin Gene ◽  
K562 Cells ◽  
Dnase I ◽  
Base Pairs ◽  
Dnase I Hypersensitive Sites ◽  
Hypersensitive Sites ◽  
Hel Cells ◽  
Globin Synthesis ◽  
Erythroleukemic Cell ◽  
Erythroid Development

DNA 5' to the human epsilon-globin gene exhibits unique patterns of DNase I-hypersensitive sites (DHS) in three human erythroleukemic cell lines which represent the embryonic (K562), fetal (HEL), and adult (KMOE) stages of erythroid development. We have mapped 10 epsilon-globin DHS in K562 cells, in which the epsilon-globin gene is maximally active. Major sites are located -11.7, -10.5, -6.5, -2.2 kilobase pairs (kbp) and -200 base pairs (bp) upstream of the gene and directly over the major cap site. Minor sites are located -5.5, -4.5, and -1.48 kbp and -900 bp upstream of the cap site. In HEL cells, in which the epsilon-globin gene is expressed at extremely low levels, the -11.7-, -10.5-, -5.5-, -4.5-, and -2.2-kbp DHS are no longer detectable; the -200-bp site is approximately 300-fold less sensitive to DNase I; and the -1.48-kbp, -900-bp, and major cap site DHS are 3- to 4-fold less sensitive. Only the DHS located -6.5 kbp relative to the major cap site is detectable at all three stages of erythroid development, including KMOE cells in which epsilon-globin synthesis is undetectable. We suggest that this site may be implicated in maintaining the entire beta-globin cluster in an active chromatin conformation. The five DHS downstream of the -6.5-kbp element possess associated promoters. Thus two distinct types of DHS exist--promoter positive and promoter negative. In HEL cells, all the upstream promoters are inactivated, although the -1.48-kbp and -900- and -200-bp DHS are still present. This suggests that the maintenance of DHS and regulation of their associated promoters occur by independent mechanisms. The inactivation of the upstream promoters in HEL cells while the major cap site remains active represents a unique pattern of expression and suggests that HEL cells possess regulatory factors which specifically down regulate the epsilon-globin upstream promoters.

scholarly journals Synergistic and Additive Properties of the Beta-Globin Locus Control Region (LCR) Revealed by 5′HS3 Deletion Mutations: Implication for LCR Chromatin Architecture

2005 ◽  
Vol 25 (16) ◽  
pp. 7033-7041 ◽  
Author(s):  
Xiangdong Fang ◽  
Jin Sun ◽  
Ping Xiang ◽  
Man Yu ◽  
Patrick A. Navas ◽  
...  
Keyword(s):  
Histone Acetylation ◽  
Control Region ◽  
Chromatin Structure ◽  
Globin Gene ◽  
Dnase I ◽  
Beta Globin ◽  
Pol Ii ◽  
Dnase I Hypersensitive Sites ◽  
Chromatin Architecture ◽  
Hypersensitive Sites

ABSTRACT Deletion of the 234-bp core element of the DNase I hypersensitive site 3 (5′HS3) of the locus control region (LCR) in the context of a human beta-globin locus yeast artificial chromosome (β-YAC) results in profound effects on globin gene expression in transgenic mice. In contrast, deletion of a 2.3-kb 5′HS3 region, which includes the 234-bp core sequence, has a much milder phenotype. Here we report the effects of these deletions on chromatin structure in the beta-globin locus of adult erythroblasts. The 234-bp 5′HS3 deletion abolished histone acetylation throughout the β-globin locus; recruitment of RNA polymerase II (pol II) to the LCR and beta-globin gene promoter was reduced to a basal level; and formation of all the 5′ DNase I hypersensitive sites of the LCR was disrupted. The 2.3-kb 5′HS3 deletion mildly reduced the level of histone acetylation but did not change the profile across the whole locus; the 5′ DNase I hypersensitive sites of the LCR were formed, but to a lesser extent; and recruitment of pol II was reduced, but only marginally. These data support the hypothesis that the LCR forms a specific chromatin structure and acts as a single entity. Based on these results we elaborate on a model of LCR chromatin architecture which accommodates the distinct phenotypes of the 5′HS3 and HS3 core deletions.

scholarly journals DHSpred: support-vector-machine-based human DNase I hypersensitive sites prediction using the optimal features selected by random forest

2017 ◽  
Author(s):  
Balachandran Manavalan ◽  
Tae Hwan Shin ◽  
Gwang Lee
Keyword(s):  
Support Vector Machine ◽  
Random Forest ◽  
Dna Sequences ◽  
Feature Selection Method ◽  
Regulatory Elements ◽  
Dnase I ◽  
Support Vector ◽  
Large Set ◽  
Dnase I Hypersensitive Sites ◽  
Hypersensitive Sites

AbstractDNase I hypersensitive sites (DHSs) are genomic regions that provide important information regarding the presence of transcriptional regulatory elements and the state of chromatin. Therefore, identifying DHSs in uncharacterized DNA sequences is crucial for understanding their biological functions and mechanisms. Although many experimental methods have been proposed to identify DHSs, they have proven to be expensive for genome-wide application. Therefore, it is necessary to develop computational methods for DHS prediction. In this study, we proposed a support vector machine (SVM)-based method for predicting DHSs, called DHSpred (DNase I Hypersensitive Site predictor in human DNA sequences), which was trained with 174 optimal features. The optimal combination of features was identified from a large set that included nucleotide composition and di- and trinucleotide physicochemical properties, using a random forest algorithm. DHSpred achieved a Matthews correlation coefficient and accuracy of 0.660 and 0.871, respectively, which were 3% higher than those of control SVM predictors trained with non-optimized features, indicating the efficiency of the feature selection method. Furthermore, the performance of DHSpred was superior to that of state-of-the-art predictors. An online prediction server has been developed to assist the scientific community, and is freely available at:http://www.thegleelab.org/DHSpred.html.

Characterization of the Opisthorchis viverrini genome

1991 ◽  
Vol 65 (1) ◽  
pp. 51-54 ◽  
Author(s):  
R. Sermswan ◽  
S. Mongkolsuk ◽  
S. Sirisinha
Keyword(s):  
Southern Blot ◽  
Genomic Dna ◽  
Blot Hybridization ◽  
Restriction Enzymes ◽  
Southern Blot Hybridization ◽  
Fasciola Gigantica ◽  
Opisthorchis Viverrini ◽  
Repeated Dna ◽  
Dna Elements

ABSTRACTThe methylations of trematode genomic DNA were analyzed using restriction enzymes and Southern blot hybridization. Restriction enzymes MspI, HpaII, HhaI were used to probe CpG methylation while MboI, Sau3A, DpnI were used for A methylation. The results revealed that Opisthorchis viverrini, Fasciola gigantica and Gigantocotyle siamensis had neither CpG nor A methylations. The presence of highly repeated DNA elements was also demonstrated in O. viverrini genomic DNA.

scholarly journals Correlation between patterns of DNase I-hypersensitive sites and upstream promoter activity of the human epsilon-globin gene at different stages of erythroid development.

1990 ◽  
Vol 10 (3) ◽  
pp. 1199-1208 ◽  
Author(s):  
P Bushel ◽  
K Rego ◽  
L Mendelsohn ◽  
M Allan
Keyword(s):  
Globin Gene ◽  
K562 Cells ◽  
Dnase I ◽  
Base Pairs ◽  
Dnase I Hypersensitive Sites ◽  
Hypersensitive Sites ◽  
Hel Cells ◽  
Globin Synthesis ◽  
Erythroleukemic Cell ◽  
Erythroid Development

DNA 5' to the human epsilon-globin gene exhibits unique patterns of DNase I-hypersensitive sites (DHS) in three human erythroleukemic cell lines which represent the embryonic (K562), fetal (HEL), and adult (KMOE) stages of erythroid development. We have mapped 10 epsilon-globin DHS in K562 cells, in which the epsilon-globin gene is maximally active. Major sites are located -11.7, -10.5, -6.5, -2.2 kilobase pairs (kbp) and -200 base pairs (bp) upstream of the gene and directly over the major cap site. Minor sites are located -5.5, -4.5, and -1.48 kbp and -900 bp upstream of the cap site. In HEL cells, in which the epsilon-globin gene is expressed at extremely low levels, the -11.7-, -10.5-, -5.5-, -4.5-, and -2.2-kbp DHS are no longer detectable; the -200-bp site is approximately 300-fold less sensitive to DNase I; and the -1.48-kbp, -900-bp, and major cap site DHS are 3- to 4-fold less sensitive. Only the DHS located -6.5 kbp relative to the major cap site is detectable at all three stages of erythroid development, including KMOE cells in which epsilon-globin synthesis is undetectable. We suggest that this site may be implicated in maintaining the entire beta-globin cluster in an active chromatin conformation. The five DHS downstream of the -6.5-kbp element possess associated promoters. Thus two distinct types of DHS exist--promoter positive and promoter negative. In HEL cells, all the upstream promoters are inactivated, although the -1.48-kbp and -900- and -200-bp DHS are still present. This suggests that the maintenance of DHS and regulation of their associated promoters occur by independent mechanisms. The inactivation of the upstream promoters in HEL cells while the major cap site remains active represents a unique pattern of expression and suggests that HEL cells possess regulatory factors which specifically down regulate the epsilon-globin upstream promoters.

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