Analysis of chromatin of skeletal muscle of developing rats using micrococcal nuclease and DNase I

1984 ◽  
Vol 9 (4) ◽  
pp. 245-251 ◽  
Author(s):  
R. S. Pandey ◽  
M. S. Kanungo
Keyword(s):  
Skeletal Muscle ◽  
Dnase I ◽  
Micrococcal Nuclease

scholarly journals Maintenance of Open Chromatin and Selective Genomic Occupancy at the Cell Cycle-Regulated Histone H4 Promoter during Differentiation of HL-60 Promyelocytic Leukemia Cells

2003 ◽  
Vol 23 (4) ◽  
pp. 1460-1469 ◽  
Author(s):  
Hayk Hovhannisyan ◽  
Brian Cho ◽  
Partha Mitra ◽  
Martin Montecino ◽  
Gary S. Stein ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Kinase Inhibitor ◽  
Restriction Enzymes ◽  
Dnase I ◽  
Promyelocytic Leukemia ◽  
Micrococcal Nuclease ◽  
Leukemia Cells ◽  
N Gene ◽  
Open Chromatin ◽  
Histone H4

ABSTRACT During the shutdown of proliferation and onset of differentiation of HL-60 promyelocytic leukemia cells, expression of the cell cycle-dependent histone genes is downregulated at the level of transcription. To address the mechanism by which this regulation occurs, we examined the chromatin structure of the histone H4/n (FO108, H4FN) gene locus. Micrococcal nuclease, DNase I, and restriction enzymes show similar cleavage sites and levels of sensitivity at the H4/n locus in both proliferating and differentiated HL-60 cells. In contrast, differentiation-related activation of the cyclin-dependent kinase inhibitor p21cip1/WAF1 gene is accompanied by increased nuclease hypersensitivity. Chromatin immunoprecipitation assays of the H4/n gene reveal that acetylated histones H3 and H4 are maintained at the same levels in proliferating and postproliferative cells. Thus, the chromatin of the H4/n locus remains in an open state even after transcription ceases. Using ligation-mediated PCR to visualize genomic DNase I footprints at single-nucleotide resolution, we find that protein occupancy at the site II cell cycle element is selectively diminished in differentiated cells while the site I element remains occupied. Decreased occupancy of site II is reflected by loss of the site II binding protein HiNF-P. We conclude that H4 gene transcription during differentiation is downregulated by modulating protein interaction at the site II cell cycle element and that retention of an open chromatin conformation may be associated with site I occupancy.

Major histocompatibility complex class I genes in murine fibrosarcoma IC9 are down regulated at the level of the chromatin structure

1989 ◽  
Vol 9 (7) ◽  
pp. 3136-3142
Author(s):  
U Maschek ◽  
W Pülm ◽  
S Segal ◽  
G J Hämmerling
Keyword(s):  
Major Histocompatibility Complex ◽  
Major Histocompatibility Complex Class ◽  
Chromatin Structure ◽  
Dnase I ◽  
Class I ◽  
Micrococcal Nuclease ◽  
Complex Class ◽  
Major Histocompatibility ◽  
Histocompatibility Complex ◽  
Nuclear Extracts

The fibrosarcoma IC9 is deficient in the expression of the major histocompatibility complex class I genes Kb, Kk, and Dk and expresses only the Db molecule. Because class I deficiency may enable tumor cells to escape the immune response by cytotoxic T lymphocytes, we investigated why the class I genes are not expressed. Expression of the silent class I genes could not be induced, but all known DNA-binding factors specific for class I genes could be detected in nuclear extracts of IC9 cells. After cloning of the silent Kb gene from the IC9 cells and subsequent transfection of this cloned Kb gene into LTK- and IC9 cells, normal Kb antigens were expressed on the cell surface of both cell lines. Digestion of the chromatin of IC9 cells with micrococcal nuclease and DNase I showed a decreased nuclease sensitivity of the silent class I genes in comparison with active genes and the absence of DNase I hypersensitive sites in the promoter region of the silent Dk gene. These findings demonstrate that class I expression is turned off by a cis-acting regulatory mechanism at the level of the chromatin structure.

scholarly journals DNase I- and micrococcal nuclease-hypersensitive sites in the human apolipoprotein B gene are tissue specific.

1988 ◽  
Vol 8 (1) ◽  
pp. 71-80 ◽  
Author(s):  
B Levy-Wilson ◽  
C Fortier ◽  
B D Blackhart ◽  
B J McCarthy
Keyword(s):  
Hela Cells ◽  
Apolipoprotein B ◽  
Hepg2 Cells ◽  
Transcriptional Start Site ◽  
Dnase I ◽  
Micrococcal Nuclease ◽  
Apo B ◽  
Human Apolipoprotein ◽  
Hypersensitive Sites ◽  
High Degree

We have mapped the DNase I- and micrococcal nuclease-hypersensitive sites present in the 5' end of the human apolipoprotein B (apo-B) gene in nuclei from cells expressing or not expressing the gene. Four DNase I-hypersensitive sites were found in nuclei from liver-derived HepG2 cells and intestine-derived CaCo-2 cells, which express the apo-B gene, but not in HeLa cells, which do not. These sites are located near positions -120, -440, -700, and +760 base pairs relative to the transcriptional start site. Undifferentiated CaCo-2 cells exhibited another site, near position -540. Six micrococcal nuclease-hypersensitive sites were found in nuclei from HepG2 and CaCo-2 cells, but not in HeLa cells or free DNA. These sites are located near positions -120, -390, -530, -700, -850, and +210. HepG2 cells exhibited another site, near position +460. Comparison of the DNA sequence of the 5' flanking regions of the human and mouse apo-B genes revealed a high degree of evolutionary conservation of short stretches of sequences in the immediate vicinity of each of the DNase I- and most of the micrococcal nuclease-hypersensitive sites.

scholarly journals The Forkhead Transcription Factor FoxI1 Remains Bound to Condensed Mitotic Chromosomes and Stably Remodels Chromatin Structure

2006 ◽  
Vol 26 (1) ◽  
pp. 155-168 ◽  
Author(s):  
Jizhou Yan ◽  
Lisha Xu ◽  
Gregory Crawford ◽  
Zenfeng Wang ◽  
Shawn M. Burgess
Keyword(s):  
Chromatin Structure ◽  
Fluorescent Protein ◽  
Living Cells ◽  
Dnase I ◽  
Higher Order ◽  
Micrococcal Nuclease ◽  
Stable Cell Line ◽  
Order Structure ◽  
Dna Fragments ◽  
Mitotic Chromosomes

ABSTRACT All forkhead (Fox) proteins contain a highly conserved DNA binding domain whose structure is remarkably similar to the winged-helix structures of histones H1 and H5. Little is known about Fox protein binding in the context of higher-order chromatin structure in living cells. We created a stable cell line expressing FoxI1-green fluorescent protein (GFP) or FoxI1-V5 fusion proteins under control of the reverse tetracycline-controlled transactivator doxycycline inducible system and found that unlike most transcription factors, FoxI1 remains bound to the condensed chromosomes during mitosis. To isolate DNA fragments directly bound by the FoxI1 protein within living cells, we performed chromatin immunoprecipitation assays (ChIPs) with antibodies to either enhanced GFP or the V5 epitope and subcloned the FoxI1-enriched DNA fragments. Sequence analyses indicated that 88% (106/121) of ChIP sequences contain the consensus binding sites for all Fox proteins. Testing ChIP sequences with a quantitative DNase I hypersensitivity assay showed that FoxI1 created stable DNase I sensitivity changes in condensed chromosomes. The majority of ChIP targets and random targets increased in resistance to DNase I in FoxI1-expressing cells, but a small number of targets became more accessible to DNase I. Consistently, the accessibility of micrococcal nuclease to chromatin was generally inhibited. Micrococcal nuclease partial digestion generated a ladder in which all oligonucleosomes were slightly longer than those observed with the controls. On the basis of these findings, we propose that FoxI1 is capable of remodeling chromatin higher-order structure and can stably create site-specific changes in chromatin to either stably create or remove DNase I hypersensitive sites.

scholarly journals Major histocompatibility complex class I genes in murine fibrosarcoma IC9 are down regulated at the level of the chromatin structure.

1989 ◽  
Vol 9 (7) ◽  
pp. 3136-3142 ◽  
Author(s):  
U Maschek ◽  
W Pülm ◽  
S Segal ◽  
G J Hämmerling
Keyword(s):  
Major Histocompatibility Complex ◽  
Major Histocompatibility Complex Class ◽  
Chromatin Structure ◽  
Dnase I ◽  
Class I ◽  
Micrococcal Nuclease ◽  
Complex Class ◽  
Major Histocompatibility ◽  
Histocompatibility Complex ◽  
Nuclear Extracts

The fibrosarcoma IC9 is deficient in the expression of the major histocompatibility complex class I genes Kb, Kk, and Dk and expresses only the Db molecule. Because class I deficiency may enable tumor cells to escape the immune response by cytotoxic T lymphocytes, we investigated why the class I genes are not expressed. Expression of the silent class I genes could not be induced, but all known DNA-binding factors specific for class I genes could be detected in nuclear extracts of IC9 cells. After cloning of the silent Kb gene from the IC9 cells and subsequent transfection of this cloned Kb gene into LTK- and IC9 cells, normal Kb antigens were expressed on the cell surface of both cell lines. Digestion of the chromatin of IC9 cells with micrococcal nuclease and DNase I showed a decreased nuclease sensitivity of the silent class I genes in comparison with active genes and the absence of DNase I hypersensitive sites in the promoter region of the silent Dk gene. These findings demonstrate that class I expression is turned off by a cis-acting regulatory mechanism at the level of the chromatin structure.

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