An Improved Restriction Enzyme Accessibility Assay for Analyzing Changes in Chromatin Structure in Samples of Limited Cell Number

Abstract Renin cells are crucial for the regulation of blood pressure and fluid electrolyte homeostasis. We have recently shown that renin cells possess unique chromatin features at regulatory regions throughout the genome that may determine the identity and memory of the renin phenotype. The 3-D structure of chromatin may be equally important in the determination of cell identity and fate. CCCTC-binding factor (Ctcf) is a highly conserved chromatin organizer that may regulate the renin phenotype by controlling chromatin structure. We found that Ctcf binds at several conserved DNA sites surrounding and within the renin locus, suggesting that Ctcf may regulate the transcriptional activity of renin cells. In fact, deletion of Ctcf in cells of the renin lineage led to decreased endowment of renin-expressing cells accompanied by decreased circulating renin, hypotension, and severe morphological abnormalities of the kidney, including defects in arteriolar branching, and ultimately renal failure. We conclude that control of chromatin architecture by Ctcf is necessary for the appropriate expression of renin, control of renin cell number and structural integrity of the kidney.

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DNA accessibility is not the primary determinant of chromatin-mediated gene regulation

10.1101/639971 ◽

2019 ◽

Cited By ~ 1

Author(s):

Răzvan V. Chereji ◽

Peter R. Eriksson ◽

Josefina Ocampo ◽

David J. Clark

Keyword(s):

Gene Expression ◽

Gene Regulation ◽

Transcription Factors ◽

Restriction Enzyme ◽

Chromatin Structure ◽

Mouse Liver ◽

Digestion Rate ◽

Dna Accessibility ◽

Primary Determinant ◽

Liver Nuclei

ABSTRACTDNA accessibility is thought to be of major importance in regulating gene expression. We test this hypothesis using a restriction enzyme as a probe of chromatin structure and as a proxy for transcription factors. We measured the digestion rate and the fraction of accessible DNA at all genomicAluI sites in budding yeast and mouse liver nuclei. Hepatocyte DNA is more accessible than yeast DNA, consistent with longer linkers between nucleosomes, and indicating that nucleosome spacing is a major determinant of accessibility. DNA accessibility varies from cell to cell, such that essentially no sites are accessible or inaccessible in every cell.AluI sites in inactive mouse promoters are accessible in some cells, implying that transcription factors could bind without activating the gene. Euchromatin and heterochromatin have very similar accessibilities, suggesting that transcription factors can penetrate heterochromatin. Thus, DNA accessibility is not likely to be the primary determinant of gene regulation.

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A Human Globin Enhancer Causes both Discrete and Widespread Alterations in Chromatin Structure

Molecular and Cellular Biology ◽

10.1128/mcb.23.22.8099-8109.2003 ◽

2003 ◽

Vol 23 (22) ◽

pp. 8099-8109 ◽

Cited By ~ 32

Author(s):

AeRi Kim ◽

Ann Dean

Keyword(s):

Histone Modification ◽

Restriction Enzyme ◽

Chromatin Structure ◽

Globin Gene ◽

Gene Activation ◽

Proximal Promoter ◽

Coding Region ◽

Nucleosome Remodeling ◽

Active Locus

ABSTRACT Gene activation requires alteration of chromatin structure to facilitate active transcription complex formation at a gene promoter. Nucleosome remodeling complexes and histone modifying complexes each play unique and interdependent roles in bringing about these changes. The role of distant enhancers in these structural alterations is not well understood. We studied nucleosome remodeling and covalent histone modification mediated by the β-globin locus control region HS2 enhancer at nucleosome-level resolution throughout a 5.5-kb globin gene model locus in vivo in K562 cells. We compared the transcriptionally active locus to one in which HS2 was inactivated by mutations in the core NF-E2 sites. In contrast to inactive templates, nucleosomes were mobilized in discrete areas of the active locus, including the HS2 core and the proximal promoter. Large differences in restriction enzyme accessibility between the active and inactive templates were limited to the regions of nucleosome mobilization, which subsumed the DNase I hypersensitive sites. In contrast to this discrete pattern, histone H3 and H4 acetylation and H3 K4 methylation were elevated across the entire active locus, accompanied by depletion of linker histone H1. The coding region of the gene differed from the regulatory regions, demonstrating both nucleosome mobilization and histone hyperacetylation, but lacked differences in restriction enzyme accessibility between transcriptionally active and inactive genes. Thus, although the histone modification pattern we observe is consistent with the spreading of histone modifying activity from the distant enhancer, the pattern of nucleosome mobilization is more compatible with direct contact between an enhancer and promoter.

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Glucocorticoid Receptor Activation of the IκBα Promoter within Chromatin

Molecular Biology of the Cell ◽

10.1091/mbc.12.11.3365 ◽

2001 ◽

Vol 12 (11) ◽

pp. 3365-3374 ◽

Cited By ~ 44

Author(s):

Bonnie J. Deroo ◽

Trevor K. Archer

Keyword(s):

Transcription Factor ◽

Glucocorticoid Receptor ◽

Restriction Enzyme ◽

Chromatin Structure ◽

Receptor Activation ◽

Model Systems ◽

Micrococcal Nuclease ◽

Open Chromatin ◽

Glucocorticoid Treatment

The glucocorticoid receptor (GR) is a ligand-activated transcription factor that induces expression of many genes. The GR has been useful for understanding how chromatin structure regulates steroid-induced transcription in model systems. However, the effect of glucocorticoids on chromatin structure has been examined on few endogenous mammalian promoters. We investigated the effect of glucocorticoids on the in vivo chromatin structure of the glucocorticoid-responsive IκBα gene promoter, the inhibitor of the ubiquitous transcription factor, nuclear factor kappa B (NFκB). Glucocorticoids inhibit NFκB activity in some tissues by elevating the levels of IκBα. We found that glucocorticoids activated the IκBα promoter in human T47D/A1-2 cells containing the GR. We then investigated the chromatin structure of the IκBα promoter in the absence and presence of glucocorticoids with the use of micrococcal nuclease, restriction enzyme, and deoxyribonuclease (DNaseI) analyses. In untreated cells, the promoter assembles into regularly positioned nucleosomes, and glucocorticoid treatment did not alter nucleosomal position. Restriction enzyme accessiblity studies indicated that the IκBα promoter is assembled as phased nucleosomes that adopt an “open” chromatin architecture in the absence of hormone. However, glucocorticoids may be required for transcription factor binding, because DNaseI footprinting studies suggested that regulatory factors bind to the promoter upon glucocorticoid treatment.

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The ultrastructure of acinar cells in the external orbital gland of young and old male rats

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100082248 ◽

1978 ◽

Vol 36 (2) ◽

pp. 276-277

Author(s):

R. Carriere

Keyword(s):

Young Adulthood ◽

Acinar Cells ◽

Cell Number ◽

Male Rats ◽

Polyploid Cell ◽

Secretory Cells ◽

Age Changes ◽

Albino Rat ◽

Golgi Membranes ◽

Diploid Cells

The external orbital gland of the albino rat exhibits both sexual dimorphism and histological age changes. In males, many cells attain a remarkable degree of polyploidy and an increase of polyploid cell number constitutes the major age change until young adulthood. The acini of young adults have a small lumen and are composed of tall serous cells. Subsequently, many acini acquire a larger lumen with an irregular outline while numerous vacuoles accumulate throughout the secretory cells. At the same time, vesicular acini with a large lumen surrounded by pale-staining low cuboidal diploid cells begin to appear and their number increases throughout old age. The fine structure of external orbital glands from both sexes has been explored and in considering acinar cells from males, emphasis was given to the form of the Golgi membranes and to nuclear infoldings of cytoplasmic constituents.

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Effects of the pseudomonad phytotoxin coronatine on tomato leaf structure and ultrastructure

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100140683 ◽

1995 ◽

Vol 53 ◽

pp. 862-863

Author(s):

D.A. Palmer ◽

C.L. Bender

Keyword(s):

Pseudomonas Syringae ◽

Membrane Integrity ◽

Leaf Tissue ◽

Cell Number ◽

Cell Ultrastructure ◽

Response To Treatment ◽

Prominent Symptom ◽

Chlorophylls A And B ◽

Cell Dimensions ◽

Structure And Ultrastructure

Coronatine is a non-host-specific phytotoxin produced by several members of the Pseudomonas syringae group of pathovars. The toxin acts as a virulence factor in P. syringae pv. tomato, allowing the organism to multiply to a higher population density and develop larger lesions than mutant strains unable to produce the toxin. The most prominent symptom observed in leaf tissue treated with coronatine is an intense spreading chlorosis; this has been attributed to a loss of chlorophylls a and b in tobacco. Coronatine's effects on membrane integrity and cell ultrastructure have not been previously investigated. The present study describes changes in tomato leaves in response to treatment with purified coronatine, infection by a coronatine-producing strain of P. syringae pv. tomato, and infection by a cor" mutant.In contrast to H2O-treated tissue, coronatine-treated tissue showed a diffuse chlorosis extending approximately 5 mm from the inoculation site. Leaf thickness, cell number, and cell dimensions were similar for both healthy and coronatine-treated, chlorotic tissue; however, the epidermal cell walls were consistently thicker in coronatine-treated leaves (Figs, la and lb).

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Effects of Mg2+ on Chromatic Structure in Isolated Chicken Liver Nuclei

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100158194 ◽

1990 ◽

Vol 48 (3) ◽

pp. 130-131

Author(s):

Soichiro Arai ◽

Yuh H. Nakanishi

Keyword(s):

Chromatin Structure ◽

Room Temperature ◽

Osmium Tetroxide ◽

Divalent Cations ◽

Chromatin Condensation ◽

Chicken Liver ◽

Electron Microscopic ◽

Razor Blade ◽

Microscopic Studies ◽

Liver Nuclei

Although many electron microscopic studies on extracted chromatin have provided considerable information on chromatin condensation induced by divalent cations, there is only a little literature available on the effects of divalent cations on chromatin structure in intact nuclei. In the present study, the effects of Mg2+ on chromatin structure in isolated chicken liver nuclei were examined over a wide concentration range of Mg2+ by scanning electron microscopy.Nuclei were prepared from chicken liver by the method of Chauveau et al. with some modifications. The nuclei were suspended in 25 mM triethanolamine chloride buffer (pH7.4) with 1 mM EDTA or in the buffer with concentrations of MgCl2 varying from 1 to 50 mM. After incubation for 1 min at 0°C, glutaraldehyde was added to 1.8% and the nuclei were fixed for 1 h at 4°C. The fixed nuclei were mixed with 15% gelatin solution warmed at about 40°C, and kept at room temperature until the mixture set. The gelatin containing the nuclei was fixed with 2% glutaraldehyde for 2-4 h, and cut into small blocks. The gelatin blocks were conductive-stained with 2% tannic acid and 2% osmium tetroxide, dehydrated in a graded series of ethanol, and freeze-cracked with a razor blade in liquid nitrogen.

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Lamin B receptor: role on chromatin structure, cellular senescence and possibly aging

Biochemical Journal ◽

10.1042/bcj20200165 ◽

2020 ◽

Vol 477 (14) ◽

pp. 2715-2720

Author(s):

Susana Castro-Obregón

Keyword(s):

Cellular Senescence ◽

Nuclear Membrane ◽

Chromatin Structure ◽

Cholesterol Synthesis ◽

Reductase Activity ◽

Chimeric Protein ◽

Nuclear Lamina ◽

Lamin B ◽

Inner Nuclear Membrane ◽

Lamin B Receptor

The nuclear envelope is composed by an outer nuclear membrane and an inner nuclear membrane, which is underlain by the nuclear lamina that provides the nucleus with mechanical strength for maintaining structure and regulates chromatin organization for modulating gene expression and silencing. A layer of heterochromatin is beneath the nuclear lamina, attached by inner nuclear membrane integral proteins such as Lamin B receptor (LBR). LBR is a chimeric protein, having also a sterol reductase activity with which it contributes to cholesterol synthesis. Lukasova et al. showed that when DNA is damaged by ɣ-radiation in cancer cells, LBR is lost causing chromatin structure changes and promoting cellular senescence. Cellular senescence is characterized by terminal cell cycle arrest and the expression and secretion of various growth factors, cytokines, metalloproteinases, etc., collectively known as senescence-associated secretory phenotype (SASP) that cause chronic inflammation and tumor progression when they persist in the tissue. Therefore, it is fundamental to understand the molecular basis for senescence establishment, maintenance and the regulation of SASP. The work of Lukasova et al. contributed to our understanding of cellular senescence establishment and provided the basis that lead to the further discovery that chromatin changes caused by LBR reduction induce an up-regulated expression of SASP factors. LBR dysfunction has relevance in several diseases and possibly in physiological aging. The potential bifunctional role of LBR on cellular senescence establishment, namely its role in chromatin structure together with its enzymatic activity contributing to cholesterol synthesis, provide a new target to develop potential anti-aging therapies.

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Apoptosis and development

Essays in Biochemistry ◽

10.1042/bse0390011 ◽

2003 ◽

Vol 39 ◽

pp. 11-24 ◽

Cited By ~ 6

Author(s):

Justin V McCarthy

Keyword(s):

Drosophila Melanogaster ◽

Mammalian Cells ◽

Cell Number ◽

Model Organisms ◽

Biological Processes ◽

Nematode Caenorhabditis Elegans ◽

Apoptotic Pathway ◽

Genetic Pathways ◽

Evolutionarily Conserved ◽

Multicellular Organisms

Apoptosis is an evolutionarily conserved process used by multicellular organisms to developmentally regulate cell number or to eliminate cells that are potentially detrimental to the organism. The large diversity of regulators of apoptosis in mammalian cells and their numerous interactions complicate the analysis of their individual functions, particularly in development. The remarkable conservation of apoptotic mechanisms across species has allowed the genetic pathways of apoptosis determined in lower species, such as the nematode Caenorhabditis elegans and the fruitfly Drosophila melanogaster, to act as models for understanding the biology of apoptosis in mammalian cells. Though many components of the apoptotic pathway are conserved between species, the use of additional model organisms has revealed several important differences and supports the use of model organisms in deciphering complex biological processes such as apoptosis.

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