Genome-scale mapping of DNase I sensitivity in vivo using tiling DNA microarrays

2006 ◽  
Vol 3 (7) ◽  
pp. 511-518 ◽  
Author(s):  
Peter J Sabo ◽  
Michael S Kuehn ◽  
Robert Thurman ◽  
Brett E Johnson ◽  
Ericka M Johnson ◽  
...  
Keyword(s):  
Dna Microarrays ◽  
Dnase I ◽  
Dnase I Sensitivity ◽  
Genome Scale

scholarly journals Gamma rays and bleomycin nick DNA and reverse the DNase I sensitivity of beta-globin gene chromatin in vivo.

1987 ◽  
Vol 7 (5) ◽  
pp. 1917-1924 ◽  
Author(s):  
B Villeponteau ◽  
H G Martinson
Keyword(s):  
Globin Gene ◽  
Dnase I ◽  
Globin Genes ◽  
Torsional Stress ◽  
Beta Globin ◽  
Cell Penetration ◽  
Dnase I Sensitivity ◽  
Chicken Erythrocytes ◽  
Active Genes

The active beta-globin genes in chicken erythrocytes, like all active genes, reside in large chromatin domains which are preferentially sensitive to digestion by DNase I. We have recently proposed that the special structure of chromatin in active domains is maintained by torsional stress in the DNA (Villeponteau et al., Cell 39:469-478, 1984). This hypothesis predicts that nicking of the DNA within any such chromosomal domain in vivo will relax the DNA and lead to loss of the special DNase I-sensitive state. Here we have tested this prediction by using gamma irradiation and bleomycin treatment to cleave DNA within intact chicken embryo erythrocytes. Both treatments cause reversal of DNase I sensitivity. Moreover, reversal occurs at approximately one nick per 150 kilobase pairs for both agents despite their entirely unrelated modes of cell penetration and DNA attack. These results suggest that the domain of DNase I sensitivity surrounding the beta-globin genes comprises 150 kilobase pairs of chromatin under torsional stress and that a single DNA nick in this region is sufficient to reverse the DNase I sensitivity throughout the entire domain.

scholarly journals Role of the Promoter in Maintaining Transcriptionally Active Chromatin Structure and DNA Methylation Patterns In Vivo

2003 ◽  
Vol 23 (12) ◽  
pp. 4150-4161 ◽  
Author(s):  
Sung-Hae Lee Kang ◽  
Christine Mione Kiefer ◽  
Thomas P. Yang
Keyword(s):  
Dna Methylation ◽  
Chromatin Structure ◽  
Dnase I ◽  
Dnase I Hypersensitivity ◽  
Translation Start ◽  
Dnase I Sensitivity ◽  
Active Transcription ◽  
Methylation Patterns

ABSTRACT Establishment and maintenance of differential chromatin structure between transcriptionally competent and repressed genes are critical aspects of transcriptional regulation. The elements and mechanisms that mediate formation and maintenance of these chromatin states in vivo are not well understood. To examine the role of the promoter in maintaining chromatin structure and DNA methylation patterns of the transcriptionally active X-linked HPRT locus, 323 bp of the endogenous human HPRT promoter (from position −222 to +102 relative to the translation start site) was replaced by plasmid sequences by homologous recombination in cultured HT-1080 male fibrosarcoma cells. The targeted cells, which showed no detectable HPRT transcription, were then assayed for effects on DNase I hypersensitivity, general DNase I sensitivity, and DNA methylation patterns across the HPRT locus. In cells carrying the deletion, significantly diminished DNase I hypersensitivity in the 5′ flanking region was observed compared to that in parental HT-1080 cells. However, general DNase I sensitivity and DNA methylation patterns were found to be very similar in the mutated cells and in the parental cells. These findings suggest that the promoter and active transcription play a relatively limited role in maintaining transcriptionally potentiated epigenetic states.

Gamma rays and bleomycin nick DNA and reverse the DNase I sensitivity of beta-globin gene chromatin in vivo

1987 ◽  
Vol 7 (5) ◽  
pp. 1917-1924
Author(s):  
B Villeponteau ◽  
H G Martinson
Keyword(s):  
Globin Gene ◽  
Dnase I ◽  
Globin Genes ◽  
Torsional Stress ◽  
Beta Globin ◽  
Cell Penetration ◽  
Dnase I Sensitivity ◽  
Chicken Erythrocytes ◽  
Active Genes

The active beta-globin genes in chicken erythrocytes, like all active genes, reside in large chromatin domains which are preferentially sensitive to digestion by DNase I. We have recently proposed that the special structure of chromatin in active domains is maintained by torsional stress in the DNA (Villeponteau et al., Cell 39:469-478, 1984). This hypothesis predicts that nicking of the DNA within any such chromosomal domain in vivo will relax the DNA and lead to loss of the special DNase I-sensitive state. Here we have tested this prediction by using gamma irradiation and bleomycin treatment to cleave DNA within intact chicken embryo erythrocytes. Both treatments cause reversal of DNase I sensitivity. Moreover, reversal occurs at approximately one nick per 150 kilobase pairs for both agents despite their entirely unrelated modes of cell penetration and DNA attack. These results suggest that the domain of DNase I sensitivity surrounding the beta-globin genes comprises 150 kilobase pairs of chromatin under torsional stress and that a single DNA nick in this region is sufficient to reverse the DNase I sensitivity throughout the entire domain.

scholarly journals Structural Features of Nucleosomes Reorganized by Yeast FACT and Its HMG Box Component, Nhp6

2004 ◽  
Vol 24 (9) ◽  
pp. 3907-3917 ◽  
Author(s):  
Alison R. Rhoades ◽  
Susan Ruone ◽  
Tim Formosa
Keyword(s):  
Dna Sequences ◽  
Structural Features ◽  
Dnase I ◽  
Minor Role ◽  
Entry And Exit ◽  
Hmg Box ◽  
Dnase I Sensitivity ◽  
Nuclease Sensitivity

ABSTRACT The Saccharomyces cerevisiae Spt16/Cdc68, Pob3, and Nhp6 proteins (SPN or yFACT) bind to and alter nucleosomes in vitro, providing a potential explanation for their importance in both transcription and replication in vivo. We show that nucleosomes bound by either Nhp6 alone or the yFACT complex remain largely intact and immobile but are significantly reorganized, as indicated by changes in the pattern of sensitivity to DNase I and enhanced digestion by some restriction endonucleases. In contrast, yFACT enhanced access to exonuclease III only at very high levels of enzyme, suggesting that the DNA near the entry and exit sites of nucleosomes is largely unperturbed and that the position of the histone octamers relative to the DNA is not altered during reorganization. DNase I sensitivity was enhanced at sites clustered near the center of the nucleosomal DNA, away from the entry and exit points, and the pattern of nuclease sensitivity was only mildly affected by the configuration of linker extensions, further indicating that linkers play only a minor role in the reorganization of nucleosomes by yFACT. The DNA in contact with H2A-H2B dimers is therefore the region whose nuclease sensitivity was the least affected by yFACT reorganization. The most dramatic changes in nucleosome structure occurred when Spt16-Pob3 and the HMG box protein Nhp6 were both present, but Nhp6 alone altered DNase I sensitivity at some specific sites, supporting an independent role for this class of proteins in the general management of chromatin properties. yFACT activity does not require ATP hydrolysis and does not alter the position of nucleosomes, indicating that it acts through a mechanism distinct from chromatin remodeling. The results presented here suggest instead that yFACT promotes polymerase progression by reorganizing nucleosome cores into a less inhibitory conformation in which the properties of DNA sequences near the center of the nucleosomes are altered.

DNase I sensitivity of ribosomal RNA Genes in chromatin and nucleolar dominance in wheat

1988 ◽  
Vol 204 (3) ◽  
pp. 535-548 ◽  
Author(s):  
W.F. Thompson ◽  
R.B. Flavell
Keyword(s):  
Ribosomal Rna ◽  
Dnase I ◽  
Ribosomal Rna Genes ◽  
Nucleolar Dominance ◽  
Dnase I Sensitivity ◽  
Rna Genes

scholarly journals Genetic stability of genome-scale deoptimized RNA virus vaccine candidates under selective pressure

2017 ◽  
Vol 114 (3) ◽  
pp. E386-E395 ◽  
Author(s):  
Cyril Le Nouën ◽  
Thomas McCarty ◽  
Michael Brown ◽  
Melissa Laird Smith ◽  
Roberto Lleras ◽  
...  
Keyword(s):  
Virus Vaccine ◽  
Genetic Stability ◽  
Selective Pressure ◽  
Rna Virus ◽  
Temperature Sensitive ◽  
Phenotypic Analysis ◽  
Vaccine Candidates ◽  
Synonymous Mutations ◽  
Genome Scale

Recoding viral genomes by numerous synonymous but suboptimal substitutions provides live attenuated vaccine candidates. These vaccine candidates should have a low risk of deattenuation because of the many changes involved. However, their genetic stability under selective pressure is largely unknown. We evaluated phenotypic reversion of deoptimized human respiratory syncytial virus (RSV) vaccine candidates in the context of strong selective pressure. Codon pair deoptimized (CPD) versions of RSV were attenuated and temperature-sensitive. During serial passage at progressively increasing temperature, a CPD RSV containing 2,692 synonymous mutations in 9 of 11 ORFs did not lose temperature sensitivity, remained genetically stable, and was restricted at temperatures of 34 °C/35 °C and above. However, a CPD RSV containing 1,378 synonymous mutations solely in the polymerase L ORF quickly lost substantial attenuation. Comprehensive sequence analysis of virus populations identified many different potentially deattenuating mutations in the L ORF as well as, surprisingly, many appearing in other ORFs. Phenotypic analysis revealed that either of two competing mutations in the virus transcription antitermination factor M2-1, outside of the CPD area, substantially reversed defective transcription of the CPD L gene and substantially restored virus fitness in vitro and in case of one of these two mutations, also in vivo. Paradoxically, the introduction into Min L of one mutation each in the M2-1, N, P, and L proteins resulted in a virus with increased attenuation in vivo but increased immunogenicity. Thus, in addition to providing insights on the adaptability of genome-scale deoptimized RNA viruses, stability studies can yield improved synthetic RNA virus vaccine candidates.

Genomic footprinting detects factors bound to major late and IVa2 promoters in adenovirus-infected HeLa cells

1988 ◽  
Vol 8 (4) ◽  
pp. 1534-1539
Author(s):  
G Albrecht ◽  
B Devaux ◽  
C Kedinger
Keyword(s):  
Protein Interactions ◽  
Adenovirus Type ◽  
Nuclease Activity ◽  
Dnase I ◽  
Promoter Activation ◽  
Promoter Elements ◽  
Dnase I Footprinting ◽  
Infected Cells ◽  
Adenovirus Type 5

We used DNase I footprinting assays on nuclei isolated from adenovirus-infected cells to examine the nucleoprotein configuration of a 250-base-pair segment which encompasses the adenovirus type 5 major late (ML) and IVa2 promoters. At 12 and 20 h postinfection (p.i.), fine DNase I digestion mapping of wild-type adenovirus-infected cells revealed specific sequences protected from digestion which corresponded to promoter elements required for expression of the ML gene in vivo. At 12 h p.i., a G+C-rich region which lies upstream of the IVa2 cap site and is important for maximal IVa2 activity was also found masked to nuclease activity. At 20 h p.i., however, this element became more sensitive to nuclease attack, while the ML promoter elements stayed protected. No major changes in DNA-protein interactions were detected in the region spanning the ML and IVa2 cap sites upon promoter activation, suggesting that the binding properties of the cognate factors for this region are not modified during the process.

UHF-1, a factor required for maximal transcription of early and late sea urchin histone H4 genes: analysis of promoter-binding sites

1991 ◽  
Vol 11 (2) ◽  
pp. 1048-1061
Author(s):  
I J Lee ◽  
L Tung ◽  
D A Bumcrot ◽  
E S Weinberg
Keyword(s):  
Binding Site ◽  
Sea Urchin ◽  
Transcriptional Start Site ◽  
Sodium Dodecyl ◽  
Nuclear Extract ◽  
Dnase I ◽  
Histone H4 ◽  
Band Shift

A protein, denoted UHF-1, was found to bind upstream of the transcriptional start site of both the early and late H4 (EH4 and LH4) histone genes of the sea urchin Strongylocentrotus purpuratus. A nuclear extract from hatching blastulae contained proteins that bind to EH4 and LH4 promoter fragments in a band shift assay and produced sharp DNase I footprints upstream of the EH4 gene (from -133 to -106) and the LH4 gene (from -94 to -66). DNase I footprinting performed in the presence of EH4 and LH4 promoter competitor DNAs indicated that UHF-1 binds more strongly to the EH4 site. A sequence match of 11 of 13 nucleotides was found within the two footprinted regions: [sequence: see text]. Methylation interference and footprinting experiments showed that UHF-1 bound to the two sites somewhat differently. DNA-protein UV cross-linking studies indicated that UHF-1 has an electrophoretic mobility on sodium dodecyl sulfate-acrylamide gels of approximately 85 kDa and suggested that additional proteins, specific to each promoter, bind to each site. In vitro and in vivo assays were used to demonstrate that the UHF-1-binding site is essential for maximal transcription of the H4 genes. Deletion of the EH4 footprinted region resulted in a 3-fold decrease in transcription in a nuclear extract and a 2.6-fold decrease in expression in morulae from templates that had been injected into eggs. In the latter case, deletion of the binding site did not grossly disrupt the temporal program of expression from the injected EH4 genes. LH4 templates containing a 10-bp deletion in the consensus region or base substitutions in the footprinted region were transcribed at 14 to 58% of the level of the wild-type LH4 template. UHF-1 is therefore essential for maximal expression of the early and late H4 genes.

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