scholarly journals Formation of stable chromatin structures on the histone H4 gene during differentiation in Tetrahymena thermophila.

1986 ◽  
Vol 6 (8) ◽  
pp. 3014-3017 ◽  
Author(s):  
D S Pederson ◽  
K Shupe ◽  
G A Bannon ◽  
M A Gorovsky
Keyword(s):  
Chromatin Structure ◽  
Transcriptional Control ◽  
Tetrahymena Thermophila ◽  
Micrococcal Nuclease ◽  
Histone H4 ◽  
Hypersensitive Sites ◽  
Nuclear Differentiation ◽  
Histone Gene Transcription ◽  
The Relationship ◽  
I Gene

The relationship between chromatin structure and the transcriptional activity of the histone H4-I gene of Tetrahymena thermophila was explored. Indirect end-labeling studies demonstrated that major DNase I- and micrococcal nuclease-hypersensitive sites flank the active macronuclear genes but not the inactive micronuclear genes. Runon transcription experiments with isolated macronuclei indicated that histone gene transcription rates decreased when cells were starved. However, macronuclear nuclease-hypersensitive sites persisted upon starvation. Thus, one level of transcriptional control of the H4-I gene results in altered chromatin structure and is established during nuclear differentiation. The rate of transcription is also controlled, but not through hypersensitive site-associated structures.

Formation of stable chromatin structures on the histone H4 gene during differentiation in Tetrahymena thermophila

1986 ◽  
Vol 6 (8) ◽  
pp. 3014-3017
Author(s):  
D S Pederson ◽  
K Shupe ◽  
G A Bannon ◽  
M A Gorovsky
Keyword(s):  
Chromatin Structure ◽  
Transcriptional Control ◽  
Tetrahymena Thermophila ◽  
Micrococcal Nuclease ◽  
Histone H4 ◽  
Hypersensitive Sites ◽  
Nuclear Differentiation ◽  
Histone Gene Transcription ◽  
The Relationship ◽  
I Gene

The relationship between chromatin structure and the transcriptional activity of the histone H4-I gene of Tetrahymena thermophila was explored. Indirect end-labeling studies demonstrated that major DNase I- and micrococcal nuclease-hypersensitive sites flank the active macronuclear genes but not the inactive micronuclear genes. Runon transcription experiments with isolated macronuclei indicated that histone gene transcription rates decreased when cells were starved. However, macronuclear nuclease-hypersensitive sites persisted upon starvation. Thus, one level of transcriptional control of the H4-I gene results in altered chromatin structure and is established during nuclear differentiation. The rate of transcription is also controlled, but not through hypersensitive site-associated structures.

scholarly journals Site-specific methylation of adenine in the nuclear genome of a eucaryote, Tetrahymena thermophila.

1986 ◽  
Vol 6 (7) ◽  
pp. 2364-2370 ◽  
Author(s):  
G S Harrison ◽  
R C Findly ◽  
K M Karrer
Keyword(s):  
Heat Shock ◽  
Protein Gene ◽  
De Novo ◽  
Tetrahymena Thermophila ◽  
Nuclear Genome ◽  
Histone H4 ◽  
Ciliated Protozoan ◽  
Site Specific ◽  
Logarithmic Growth ◽  
I Gene

DNA in the polyploid macronucleus of the ciliated protozoan Tetrahymena thermophila contains the modified base N6-methyladenine. We identified two GATC sites which are methylated in most or all of the 45 copies of the macronuclear genome. One site is 2 kilobases 5' to the histone H4-I gene, and the other is 5 kilobases 3' to the 73-kilodalton heat shock protein gene. These sites are de novo methylated between 10 and 16 h after initiation of conjugation, during macronuclear anlage development. The methylation states of these two GATC sites and four other unmethylated GATC sites do not change in the DNA of cells cultured under conditions which change the activity of the genes, including logarithmic growth, starvation, and heat shock.

scholarly journals Long-Range Nucleosome Ordering Is Associated with Gene Silencing in Drosophila melanogaster Pericentric Heterochromatin

2001 ◽  
Vol 21 (8) ◽  
pp. 2867-2879 ◽  
Author(s):  
Fang-Lin Sun ◽  
Matthew H. Cuaycong ◽  
Sarah C. R. Elgin
Keyword(s):  
Drosophila Melanogaster ◽  
Gene Silencing ◽  
Long Range ◽  
Chromatin Structure ◽  
Regulatory Region ◽  
Pericentric Heterochromatin ◽  
Dnase I ◽  
Micrococcal Nuclease ◽  
Hypersensitive Sites ◽  
Nucleosome Array

ABSTRACT We have used line HS-2 of Drosophila melanogaster, carrying a silenced transgene in the pericentric heterochromatin, to investigate in detail the chromatin structure imposed by this environment. Digestion of the chromatin with micrococcal nuclease (MNase) shows a nucleosome array with extensive long-range order, indicating regular spacing, and with well-defined MNase cleavage fragments, indicating a smaller MNase target in the linker region. The repeating unit is ca. 10 bp larger than that observed for bulkDrosophila chromatin. The silenced transgene shows both a loss of DNase I-hypersensitive sites and decreased sensitivity to DNase I digestion within an array of nucleosomes lacking such sites; within such an array, sensitivity to digestion by MNase is unchanged. The ordered nucleosome array extends across the regulatory region of the transgene, a shift that could explain the loss of transgene expression in heterochromatin. Highly regular nucleosome arrays are observed over several endogenous heterochromatic sequences, indicating that this is a general feature of heterochromatin. However, genes normally active within heterochromatin (rolled and light) do not show this pattern, suggesting that the altered chromatin structure observed is associated with regions that are silent, rather than being a property of the domain as a whole. The results indicate that long-range nucleosomal ordering is linked with the heterochromatic packaging that imposes gene silencing.

scholarly journals High-Resolution Structural Analysis of Chromatin at Specific Loci: Saccharomyces cerevisiae Silent Mating-Type Locus HMRa

1999 ◽  
Vol 19 (12) ◽  
pp. 7944-7950 ◽  
Author(s):  
Anish Ravindra ◽  
Kerstin Weiss ◽  
Robert T. Simpson
Keyword(s):  
High Resolution ◽  
Mating Type ◽  
Chromatin Structure ◽  
Micrococcal Nuclease ◽  
Histone H4 ◽  
Type Locus ◽  
Amino Terminal ◽  
Regulator Protein ◽  
Chromosome Iii ◽  
Nuclease Mapping

ABSTRACT Genetic and biochemical evidence implicates chromatin structure in the silencing of the two quiescent mating-type loci near the telomeres of chromosome III in yeast. With high-resolution micrococcal nuclease mapping, we show that the HMRa locus has 12 precisely positioned nucleosomes spanning the distance between the E and I silencer elements. The nucleosomes are arranged in pairs with very short linkers; the pairs are separated from one another by longer linkers of ∼20 bp. Both the basic amino-terminal region of histone H4 and the silent information regulator protein Sir3p are necessary for the organized repressive chromatin structure of the silent locus. Compared to HMRa, only small differences in the availability of the TATA box are present for the promoter in the cassette at the active MATa locus. Features of the chromatin structure of this silent locus compared to the previously studied HMLα locus suggest differences in the mechanisms of silencing and may relate to donor selection during mating-type interconversion.

scholarly journals Site-specific methylation of adenine in the nuclear genome of a eucaryote, Tetrahymena thermophila

1986 ◽  
Vol 6 (7) ◽  
pp. 2364-2370
Author(s):  
G S Harrison ◽  
R C Findly ◽  
K M Karrer
Keyword(s):  
Heat Shock ◽  
Protein Gene ◽  
De Novo ◽  
Tetrahymena Thermophila ◽  
Nuclear Genome ◽  
Histone H4 ◽  
Ciliated Protozoan ◽  
Site Specific ◽  
Logarithmic Growth ◽  
I Gene

DNA in the polyploid macronucleus of the ciliated protozoan Tetrahymena thermophila contains the modified base N6-methyladenine. We identified two GATC sites which are methylated in most or all of the 45 copies of the macronuclear genome. One site is 2 kilobases 5' to the histone H4-I gene, and the other is 5 kilobases 3' to the 73-kilodalton heat shock protein gene. These sites are de novo methylated between 10 and 16 h after initiation of conjugation, during macronuclear anlage development. The methylation states of these two GATC sites and four other unmethylated GATC sites do not change in the DNA of cells cultured under conditions which change the activity of the genes, including logarithmic growth, starvation, and heat shock.

MGA2 and SPT23 Are Modifiers of Transcriptional Silencing in Yeast

Genetics ◽  
2000 ◽  
Vol 156 (3) ◽  
pp. 933-941 ◽  
Author(s):  
Mary Lou Dula ◽  
Scott G Holmes
Keyword(s):  
Transcription Factors ◽  
Chromatin Structure ◽  
Transcriptional Control ◽  
Transcriptional Silencing ◽  
Sir Proteins

Abstract Transcriptional silencing at the HM loci and telomeres in yeast depends on several trans-acting factors, including Rap1p and the Sir proteins. The SUM1-1 mutation was identified by its ability to restore silencing to strains deficient in one or more of these trans-acting factors. The mechanism by which SUM1-1 bypasses the requirement for silencing proteins is not known. We identified four loci that when reduced in dosage in diploid strains increase the ability of SUM1-1 strains to suppress silencing defects. Two of the genes responsible for this effect were found to be MGA2 and SPT23. Mga2p and Spt23p were previously identified as functionally related transcription factors that influence chromatin structure. We find that deletion of MGA2 or SPT23 also increases the efficiency of silencing in haploid SUM1-1 strains. These results suggest that Mga2p and Spt23p are antagonists of silencing. Consistent with this proposal we find that deletion of MGA2 or SPT23 also suppresses the silencing defects caused by deletion of the SIR1 gene or by mutations in the HMR silencer sequences. However, we find that Mga2p and Spt23p can positively affect silencing in other contexts; deletion of either MGA2 or SPT23 decreases mating in strains bearing mutations in the HML-E silencer. Mga2p and Spt23p appear to be a novel class of factors that influence disparate pathways of transcriptional control by chromatin.

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.

Extinction of phosphoenolpyruvate carboxykinase gene expression is associated with loss of a specific chromatin-binding protein from a far upstream domain

1990 ◽  
Vol 10 (7) ◽  
pp. 3782-3787
Author(s):  
Y T Ip ◽  
R E Fournier ◽  
R Chalkley
Keyword(s):  
Chromatin Structure ◽  
Phosphoenolpyruvate Carboxykinase ◽  
Specific Protein ◽  
Gene Activity ◽  
Upstream Region ◽  
Hypersensitive Site ◽  
Chromosome 11 ◽  
Protein Factor ◽  
Hypersensitive Sites ◽  
Liver Gene

We have analyzed the chromatin structure of the phosphoenolpyruvate carboxykinase (PEPCK) gene in hepatoma x fibroblast hybrids with different extinction phenotypes. These hybrids included a karyotypically complete hybrid in which all liver gene activity was extinguished, a microcell hybrid that contained a single mouse chromosome 11 and in which PEPCK gene activity was decreased but inducible by cyclic AMP, and a segregant line that had lost all mouse chromosomes and in which the PEPCK gene was reexpressed. We found that only in the completely extinguished hybrid was PEPCK chromatin structure radically different from that in the parental hepatoma cells. In this hybrid, there was no evidence of any factors binding to the promoter or to the upstream hypersensitive site at -4800 base pairs. In the other cell lines, even when PEPCK gene transcription was low, the PEPCK chromatin showed characteristic structures typical of a transcriptionally competent gene, with hypersensitive sites at positions previously described. Loss of the upstream hypersensitive site was also shown to be correlated with the absence of a liver-specific protein factor that binds specifically to the upstream region.

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