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PubMed2007

High-throughput mapping of the chromatin structure of human promoters.

Our understanding of how chromatin structure influences cellular processes such as transcription and replication has been limited by a lack of nucleosome-positioning data in human cells. We describe a high-resolution microarray approach combined with an analysis algorithm to examine nucleosome positioning in 3,692 promoters within seven human cell lines. Unlike unexpressed genes without transcription-preinitiation complexes at their promoters, expressed genes or genes containing preinitiation complexes exhibit characteristic nucleosome-free regions at their transcription start sites. The combination of these nucleosome data with chromatin immunoprecipitation-chip analyses reveals that the melanocyte master regulator microphthalmia-associated transcription factor (MITF) predominantly binds nucleosome-free regions, supporting the model that nucleosomes limit sequence accessibility. This study presents a global view of human nucleosome positioning and provides a high-throughput tool for analyzing chromatin structure in development and disease.

Algorithms↗
PubMed1985

Transient alterations of the chromatin structure of sea urchin early histone genes during embryogenesis.

We describe features of the chromatin structure of the early histone gene family of Strongylocentrotus purpuratus during development. Before and after the histone genes are transcriptionally active, chromatin structure is quite similar with well-defined spaced nucleosomes and no major 5'-flanking sites hypersensitive to nucleases. During the period when the genes are active, marked changes in chromatin structure occur. Micrococcal nuclease digestion generates monomer nucleosomes and only trace amounts of higher multimers. Regions hypersensitive to an endogenous nuclease and DNAase I appear in the 5'-flanking regions of genes for H2A, H2B and H3. Each region consists of four sites spanning a DNA length of 200-250 base pairs. In each case, one major cutting site is near the TATA box; the bulk of the sensitive region is in the nontranscribed spacer. Other sites, in 3'-flanking regions of the genes, are sensitive to nucleases only when the histone genes are no longer transcribed.

Animals↗
PubMed2001

Chromatin structure and gene regulation in the immune system.

The development of the immune system and the host response to microbial infection rely on the activation and silencing of numerous, differentially expressed genes. Since the mid-1980s, a primary goal has been to identify transcription factors that regulate specific genes and specific immunological processes. More recently, there has been a growing appreciation of the role of chromatin structure in gene regulation. Before most activators of a gene access their binding sites, a transition from a condensed to a decondensed chromatin structure appears to take place. The activation of transcription is then accompanied by the remodeling of specific nucleosomes. Conversely, the acquisition of a more condensed chromatin structure is often associated with gene silencing. Chromatin structure is a particularly significant contributor to gene regulation because it is likely to be a major determinant of cell identity and cell memory. That is, the propagation of decondensed chromatin at specific loci through DNA replication and cell division helps a cell remember which genes are expressed constitutively in that cell type or are poised for expression upon exposure to a stimulus. Here we review recent progress toward understanding the role of chromatin in the immune system. The interleukin-4 gene serves as a primary model for exploring the events involved in the acquisition and heritable maintenance of a decondensed chromatin structure. Studies of the interleukin-12 p40 and interferon-beta genes are then reviewed for insight into the mechanisms by which the remodeling of specific nucleosomes in the vicinity of a promoter can contribute to rapid activation following cell stimulation. Finally, basic principles of gene silencing are discussed.

Alleles↗
PubMed2001

Effect of poly(ADP-ribosyl)ation and Mg2+ ions on chromatin structure revealed by scanning force microscopy.

Poly(ADP-ribosyl)ation of nuclear proteins is responsible for major changes in the high-order chromatin structure. The effects of this post-translation modification on nuclear architecture were examined at different Mg2+ concentrations using scanning force microscopy. A quantitative analysis of the internucleosomal distance, the width, and the volume of chromatin fibers imaged in tapping mode reveals that poly(ADP-ribosyl)ation induces a complete relaxation and decondensation of the chromatin structure. Our data, on the center-to-center distance between adjacent nucleosomes and on the fiber width, indicate that the poly(ADP-ribosyl)ated fibers remain significantly decondensed even in the presence of Mg2+. Our results also show that the Mg2+ assumes an important role in the folding of chromatin structure, but Mg2+ is not able to restore the native feature of chromatin, when the fibers are depleted of H1/H5 histones. The combined effect of post-translation modification and cation ions on the chromatin structure shows that poly(ADP-ribosyl)ation could promote accessibility to DNA even in those nuclear processes that require Mg2+.

Chromatin↗
PubMed1991

Restriction enzymes permit quantitative determination of defined chromatin structures within the chromosome.

Open chromatin structures, operationally defined as nuclease-hypersensitive sites, are frequently found spanning the controlling regions of genes and they may ensure that trans-acting factors have ready access to their genomic substrates. The rapidity and extent of induction of a gene may be dependent on the probability that its promoter is folded into an open structure. We show that restriction enzymes can be used to estimate the probability that a given promoter region is contained within a defined structure in the chromosome. In the case of the Drosophila major heat-shock-protein gene, we show that an individual promoter element is folded in an accessible form in at least 75% of embryonic chromosomes. This efficient maintenance of the hypersensitive region may be a necessary precondition for a rapid heat-shock response.

Animals↗
PubMed1996

Effect of differential gene expression on the chromatin structure of the DHFR gene domain in vivo.

Photoactivated psoralen was used to probe region-specific chromatin structure in Chinese hamster ovary (CHO) cells. Specifically, the chromatin structure of six regions within the dihydrofolate reductase (DHFR) gene was probed with photoactivated psoralen in cells cultured in such ways as to differentially express the DHFR gene. Cells were irradiated with X-rays prior to the psoralen photocross-linking reaction in order to eliminate the influence of any DNA torsional tension on the psoralen binding and the sequence-specificity of psoralen binding was adjusted for. It was found that a region encompassing the promoter of the serum-regulated DHFR gene was about 50% more accessible to psoralen photocross-linking in serum-stimulated cells and about 90% more accessible in serum-starved cells than the other five regions of the DHFR gene analyzed and the genome overall. Treating serum-stimulated cells with the RNA polymerase II transcriptional inhibitor 5,6-dichloro-1-beta-D-ribofuranosyl-benzimidazole (DRB) or the topoisomerase I inhibitor camptothecin reversed the elevated accessibility of the DHFR promoter region. These results suggest that the accessible chromatin structure of the DHFR promoter is not dependent on serum-stimulated poising of the gene for transcription, but may reflect the ability of the RNA polymerase to clear the promoter.

Animals↗
PubMed1988

Expression of human cytomegalovirus immediate early antigens is responsible for a novel chromatin structure of infected human embryonic lung cells.

Whereas human embryonic lung (HEL) cells displayed chromatin fibers composed of a repeat of conventional nucleosomes of 15 nm in diameter, human cytomegalovirus (HCMV) infection induced transient appearance of a novel chromatin structure composed of a repeat of large ellipsoids of 45-65 nm X 15-30 nm with linkers of 50-60 nm long and 6-7 nm thick. Essentially the same change in chromatin structure could be induced when uninfected HEL cell nuclei were incubated in vitro with a 0.4 M NaCl nuclear extract from HCMV-infected HEL cells expressing immediate early antigens (IEA's) or with a similar nuclear extract from NIH/3T3 cells constitutively expressing HCMV IEA's. The latter cell line was established by transformation of the mouse cells with a plasmid carrying the HCMV major immediate early and immediate early 2 genes. These results together with those of control experiments suggest that the expression of IEA's is directly or indirectly responsible for the appearance of the novel chromatin structure in HCMV-infected HEL cells.

Animals↗
PubMed1990

Periodicity of DNA folding in higher order chromatin structures.

Each level of DNA folding in cells corresponds to a distinct chromatin structure. The basic chromatin units, nucleosomes, are arranged into solenoids which form chromatin loops. To characterize better the loop organization of chromatin we have assumed that the accessibility of DNA inside these structures is lower than on the outside and examined the size distribution of high mol. wt DNA fragments obtained from cells and isolated nuclei after digestion with endogenous nuclease or topoisomerase II. The largest discrete fragments obtained contain 300 kbp of DNA. Their further degradation proceeds through another discrete size step of 50 kbp. This suggests that chromatin loops contain approximately 50 kbp of DNA and that they are grouped into hexameric rosettes at the next higher level of chromatin structure. Based upon these observations a model by which the 30 nm chromatin fibre can be folded up into compact metaphase chromosomes is also described.

Animals↗
PubMed1993

Recovery of transcriptionally active chromatin restriction fragments by binding to organomercurial-agarose magnetic beads. A rapid and sensitive method for monitoring changes in higher order chromatin structure during gene activation and repression.

The unfolding of nucleosomes along transcriptionally active DNA sequences uncovers previously shielded cysteinyl-thiol groups of histone H3 molecules located at the center of the nucleosome core. This change in conformation and SH reactivity of nucleosomes along transcribed DNA sequences makes it possible to separate active from inactive nucleosomes by mercury affinity chromatography. The binding of thiol-reactive nucleosomes to an organomercurial-agarose column has been shown previously to reflect, with accuracy, both the timing and extent of transcription of the associated DNA sequences (Chen, T. A., and Allfrey, V. G. (1987) Proc. Natl. Acad. Sci. U. S. A. 84, 5252-5256). Here, we extend this experimental approach to the analysis of higher order chromatin structures. Large chromatin fragments released by treating isolated nuclei with restriction endonucleases are fractionated on mercurated agarose magnetic beads that capture nucleosomes with accessible histone H3 thiols, but do not react with the hidden H3 thiols of the compactly beaded nucleosomes of inactive genes. The SH-reactive domains of c-myc and other genes are rapidly separated from the non-SH-reactive restriction fragments by the magnetic bead technique. The new method also overcomes a major limitation of mercurated agarose column chromatography, which is not suitable for studies of higher order chromatin structure because large chromatin fragments occlude the mercury column; occlusion is not a problem in magnetic separations using suspended mercurated agarose beads. Here, we describe the synthesis of mercurated agarose magnetic beads with high capacity for SH groups and test their application to the recovery of chromatin restriction fragments of c-myc and the growth arrest gene gas1.

Animals↗
PubMed1997

Interplay of yeast global transcriptional regulators Ssn6p-Tup1p and Swi-Snf and their effect on chromatin structure.

Transcriptional regulation in yeast involves a number of general trans-acting factors affecting chromatin structure. The Swi-Snf complex is required for expression of a large number of genes and has the ability to remodel chromatin in vitro. The Ssn6p-Tup1p repressor complex may be involved in chromatin organization through the interaction with pathway-specific DNA-binding proteins. To study the interplay of these factors and their effect on chromatin we have analyzed SUC2 chromatin structure in wild-type cells and in strains bearing combinations of ssn6/tup1 and swi1 mutations. We have mapped nucleosome positioning of the repressed gene in wild-type cells using primer extension methodology, allowing base pair resolution, and have analyzed details of chromatin remodeling in the derepressed state. In ssn6 or tup1 mutants under repressing conditions the observed changes in SUC2 chromatin structure may be suppressed by the swi1 mutation, suggesting that Ssn6p-Tup1p is not required for the establishment of nucleosome positioning at the SUC2 promoter. Our data indicate the involvement of chromatin remodeling factors distinct from the Swi-Snf complex in SUC2 transcriptional regulation and suggest that Swi-Snf may antagonize Ssn6p-Tup1p by controlling remodeling activity. We also show that a relatively high level of SUC2 transcription can coexist with positioned nucleosomes.

Chromatin↗
PubMed1988

Determinants for the DNase I-hypersensitive chromatin structure 5' to a human HSP70 gene.

A DNase I hypersensitive site was detected in chromatin formed over a human hsp70 gene segment after amplification in COS7 cells. Deletion mutant analysis was used to evaluate the sequence requirements for this chromatin structure. Determinants sufficient to form the hypersensitive site are contained in a 280 base-pair sequence corresponding approximately to the region that is hypersensitive. Deletion of sequences from either end of this region resulted in reduced hypersensitivity, suggesting that multiple genetic elements contribute to the formation of this chromatin structure. As has been reported for other heat shock genes, the hypersensitive chromatin structure is present prior to heat treatment and does not change in intensity or position after heat shock, in spite of the fact that hsp70 gene expression is completely dependent on heat induction. Sequence requirements for hypersensitivity were generally similar to those for heat-induced gene expression when mutant plasmids were tested at low copy number (e.g. in HeLa cells or in COS cells without amplification); however, deletion of sequences between -223 and -162 with respect to the start of transcription abolished the hypersensitive site but had no effect on gene expression. A barrier to exonuclease III digestion was detected within this region (near an imperfect inverted repeat sequence centered at position -202), suggesting that proteins are tightly bound to the DNA at this location.

Animals↗
PubMed1991

The chromatin structure of Saccharomyces cerevisiae autonomously replicating sequences changes during the cell division cycle.

The chromatin structures of two well-characterized autonomously replicating sequence (ARS) elements were examined at their chromosomal sites during the cell division cycle in Saccharomyces cerevisiae. The H4 ARS is located near one of the duplicate nonallelic histone H4 genes, while ARS1 is present near the TRP1 gene. Cells blocked in G1 either by alpha-factor arrest or by nitrogen starvation had two DNase I-hypersensitive sites of about equal intensity in the ARS element. This pattern of DNase I-hypersensitive sites was altered in synchronous cultures allowed to proceed into S phase. In addition to a general increase in DNase I sensitivity around the core consensus sequence, the DNase I-hypersensitive site closest to the core consensus became more nuclease sensitive than the distal site. This change in chromatin structure was restricted to the ARS region and depended on replication since cdc7 cells blocked near the time of replication initiation did not undergo the transition. Subsequent release of arrested cdc7 cells restored entry into S phase and was accompanied by the characteristic change in ARS chromatin structure.

Cell Cycle↗
PubMed1987

Tissue-specific changes in chromatin structure of the rat aldolase B locus.

Chromatin structures of the aldolase B gene locus in repressed and derepressed states were examined by DNase I digestion. Within the gene locus, several structural features were observed with respect to the sensitivity to DNase I; hypersensitive sites, relatively resistant regions, and preferential cleavage sites within the resistant regions. The hypersensitive sites and the resistant regions are tissue- or cell-specifically distributed, but are not simply related to the active or inactive state chromatin. Among these structural features, however, a DNase I-hypersensitive site located about 0.3 kilobase pairs (kb) upstream from the transcription-initiation site is characteristic only in transcriptionally active tissues or cells (liver, kidney and Morris hepatoma 5123D). In addition, analysis with nuclei of fetal liver cells indicated that this hypersensitive site is constructed prior to the transcriptional activation of the aldolase B gene during development. These results may indicate that the structural alteration in chromatin at the 0.3 kb upstream site is related to the regulation of the aldolase B gene expression.

Animals↗
PubMed2001

Genomic structure of the promoters of the human estrogen receptor-alpha gene demonstrate changes in chromatin structure induced by AP2gamma.

Expression of human estrogen receptor-alpha (ERalpha) involves the activity from several promoters that give rise to alternate untranslated 5' exons. However, the genomic locations of the alternate 5' exons have not been reported previously. We have developed a contig map of the human ERalpha gene that includes all of the known alternate 5' exons. By using S1 nuclease and 5'- rapid amplification of cDNA ends, the cap sites for the alternate ERalpha transcripts E and H were identified. DNase I-hypersensitive sites specific to ERalpha-positive cells were associated with each of the cap sites. A DNase I-hypersensitive site, HS1, was localized to binding sites for AP2 in the untranslated region of exon 1 and was invariably present in the chromatin structure of ERalpha-positive cells. Overexpression of AP2gamma in human mammary epithelial cells generated the HS1-hypersensitive site. The ERalpha promoter was induced by AP2gamma in mammary epithelial cells, and trans-activation was dependent upon the region of the promoter containing the HS1 site. These results demonstrate that AP2gamma trans-activates the ERalpha gene in hormone-responsive tumors by inducing changes in the chromatin structure of the ERalpha promoter. These data are further evidence for a critical role for AP2 in the oncogenesis of hormone-responsive breast cancers.

Alternative Splicing↗
PubMed1990

The differentially expressed 16-kD heat shock genes of Caenorhabditis elegans exhibit differential changes in chromatin structure during heat shock.

The 16-kD heat shock genes of Caenorhabditis elegans are encoded by four highly similar genes, arranged as divergently transcribed pairs. In spite of the high level of identity that exists between the HSP16 genes, after 2 hr of heat shock the mRNA from one locus accumulates at 7-14 times the level of that from the other locus. To determine if differential HSP16 gene transcriptional activity contributes to these differences, we examined the chromatin structure of the HSP16 genes in nonshocked embryos and in embryos undergoing both the initial phases of heat shock and after 2 hr of heat shock. To carry out these studies, we developed a nuclei isolation procedure that has allowed us to prepare large amounts of nuclei from C. elegans embryos, larvae, and adults that are essentially free of endogenous nuclease and protease activities and appear to be an excellent substrate for investigating chromatin structure in C. elegans. This procedure has enabled us to report the first observations of C. elegans basic chromatin structure, as well as characterize HSP16 chromatin structure in detail. The data suggest that differential HSP16 RNA accumulation following 2 hr of heat shock appears to be correlated with a change in the chromatin structure of one of the HSP16 loci to a preinduction, transcriptionally inactive configuration.

Animals↗
PubMed1986

Study of chromatin structure in ataxia-telangiectasia cells.

Micrococcal nuclease was used as a probe to study chromatin structure in control and ataxia-telangiectasia cells. The rate and extent of release of acid-soluble nucleotide was similar in both cell types. Production of mono- and oligonucleosomes by micrococcal nuclease as determined by gel electrophoresis also failed to reveal differences in chromatin structure between control and ataxia-telangiectasia cells. Radiation exposure did not significantly alter the kinetics of digestion. These results indicate that there are no gross alterations in chromatin structure in ataxia-telangiectasia cells.

Ataxia Telangiectasia↗
PubMed1986

Chromatin structure of the telomeric region and 3'-nontranscribed spacer of Tetrahymena ribosomal RNA genes.

The chromatin structure of the 3'-nontranscribed spacer of the linear rRNA gene molecules of Tetrahymena thermophila was examined. This region includes the transcription termination site, two sets of recently identified conserved spacer repeats (Type IV and V repeats (Challoner, P. B., Amin, A. A., Pearlman, R. E., and Blackburn, E. H. (1985) Nucleic Acids Res. 13, 2661-2680], and the terminus of the molecule. Using sensitivity to nucleases as a probe, a unique chromatin structure was found in this rDNA region. Proceeding from the end of the rDNA molecule, the telomeric repeated sequence, (CCCCAA)n, was packaged in a non-nucleosomal complex adjacent to three phased nucleosomes. This nucleosomal structure was disrupted at the Type V repeat region, which, compared with the neighboring nucleosomal region, was more accessible to nucleases and, from both micrococcal nuclease and DNase I digestion results, was packaged in chromatin differently from the sequences flanking it on both sides. The region between the Type V repeats and adjacent to the transcription termination site was in yet another distinguishable chromatin structure as judged by its sensitivity to nucleases. It includes sites protected in chromatin and sites which were cleaved in chromatin but not detectably digested in DNA controls, suggesting that specific proteins are also associated with this region.

Animals↗
PubMed1988

Chromatin structure of altered yeast centromeres.

We have investigated the chromatin structure of wild-type and mutationally altered centromere sequences in the yeast Saccharomyces cerevisiae by using an indirect end-labeling mapping strategy. Wild-type centromere DNA from chromosome III (CEN3) exhibits a nuclease-resistant chromatin structure 220-250 base pairs long, centered around the conserved centromere DNA element (CDE) III. A point mutation in CDE III that changes a central cytidine to a thymidine and completely disrupts centromere function has lost the chromatin conformation typically associated with the wild-type centromere. A second conserved DNA element, CDE I, is spatially separated from CDE III by 78-86 A + T-rich base pairs, which is termed CDE II. The sequence and spatial requirements for CDE II are less stringent; alterations in CDE II length and sequence can be tolerated to a limited extent. Nuclease-resistant cores are altered in dimension in two CDE II CEN3 mutations. Two CDE I deletion mutations that retain partial centromere function also show nuclease-resistant regions of reduced size and intensity. The results from a number of such altered centromeres indicate a correlation between the presence of a protected core and centromere function.

Base Sequence↗