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PubMed2006

Preferential in vivo DNA repair of melphalan-induced damage in human genes is greatly affected by the local chromatin structure.

To investigate the molecular mechanisms of action of the nitrogen mustard melphalan in patients treated for multiple myeloma, the in vivo induction and repair of melphalan-induced DNA damage was measured in genes with different transcriptional activity (b-actin>p53>N-ras>d-globin) from leukocytes of 20 multiple myeloma patients following chemotherapeutic administration of high-dose melphalan (200mg/m(2)) and autologous blood stem cell transplantation. Heterogeneous repair was found among the studied genes. The extent of repair was always in the order: b-actin>p53>N-ras>d-globin, correlating with the gene transcriptional state. Similar findings were obtained using peripheral blood mononuclear cells (PBMC) from healthy volunteers following in vitro treatment with melphalan, indicating that these results are not malignant disease-specific. Following in vitro treatment of PBMC from healthy volunteers with alpha-amanitin, an inhibitor of RNA polymerase II that can also induce condensation of chromatin structure, a significant inhibition of the removal of melphalan-induced damage in the three active genes but not in the silent d-globin gene was found, suggesting that transcription and/or chromatin structure may play important roles in the preferential DNA repair. When the in vivo DNA damage formation and repair in multiple myeloma patients following chemotherapeutic administration of melphalan was measured in the two strands of the active genes, no strand bias was found, indicating that the global genome repair subpathway of nucleotide excision repair may play a crucial role in the repair of these adducts. These results were also confirmed in PBMC from healthy volunteers following in vitro treatment with melphalan. Using micrococcal nuclease digestion of nuclei isolated from PBMC of multiple myeloma patients before the chemotherapeutic treatment, as well as from PBMC of healthy volunteers, we probed the chromatin structure in each gene and found that the "looseness" of the chromatin structure correlated with the levels of the gene-specific repair, being again in the order: b-actin>p53>N-ras>d-globin. To conclude, the in vivo gene-specific repair of melphalan-induced damage in humans is greatly affected by the local chromatin structure.

Actins↗
PubMed2002

The impact of testicular and accessory sex gland function on sperm chromatin integrity as assessed by the sperm chromatin structure assay (SCSA).

BACKGROUND: The sperm chromatin structure assay (SCSA) provides an objective assessment of sperm chromatin integrity, which is essential for normal sperm function. SCSA is valuable as a fertility marker in epidemiological studies and in the clinical situation. Little is known about the impact of testicular and post-testicular function on SCSA parameters. METHODS: Ejaculates from 278 military conscripts of median age 18.1 (range 18-21) years were included. Levels of reproductive hormones, the length of the CAG repeat of the androgen receptor gene, sperm concentration, abstinence period and biochemical parameters of epididymal and accessory sex gland secretions were correlated to the SCSA parameters, DNA fragmentation index (DFI) and highly DNA stainable (HDS) cells. RESULTS: Negative correlations were found between sperm concentration and DFI (r = -0.119, P = 0.049) and HDS (r = -0.513, P < 0.0001). DFI was negatively correlated with levels of estradiol (r = -0.19, P = 0.002) and free testosterone (r = -0.13, P = 0.03). DFI also correlated positively with abstinence time (r = 0.17, P = 0.005), and with seminal concentrations of fructose (r = 0.18, P = 0.004) and zinc (r = 0.12, P = 0.04). CONCLUSIONS: Sex steroid production, spermatogenic function, abstinence time and seminal vesicle function appear to impact on sperm chromatin integrity and thereby on sperm fertilizing capacity. These findings may improve present understanding of the pathophysiology of male infertility.

Adolescent↗
PubMed1981

Chromatin structure of the histone genes of D. melanogaster.

We have examined the chromatin structure of the histone gene repeat of D. melanogaster using an indirect end-labeling technique. Our results show that each DNA segment of the repeat is packaged into a precisely defined and characteristic structure, as follows: the nontranscribed spacers display a "normal" chromatin arrangement, with each nucleosome precisely positioned on the underlying DNA sequence; the 5' ends of all five histone genes are in an exposed configuration, highly sensitive to both micrococcal nuclease and DNAase I; and the genes have an "altered" chromatin structure, as indicated by the weak and irregularly spaced nuclease cuts. This well-defined chromatin arrangement is established early in development and is stably maintained throughout the remainder of the D. melanogaster life cycle.

Animals↗
PubMed1992

Chromatin structure of the yeast SUC2 promoter in regulatory mutants.

We have previously suggested that two positioned nucleosomes are removed from the promoter of the Saccharomyces cerevisiae SUC2 gene upon depression by glucose starvation. To gain further insight into the changes accompanying derepression at the chromatin level we have studied the chromatin structure of the SUC2 promoter in several mutants affecting SUC2 expression. The non-derepressible mutants snf1, snf2 and snf5 present a chromatin structure characteristic of the repressed state, irrespective of the presence or absence of glucose. The non-repressible mutants, mig1 and ssn6, as well as the double mutant snfs sn6 exhibit an opened chromatin structure even in the presence of glucose. These results suggest that the DNA-binding protein encoded by MIG1 is necessary to produce the characteristic pattern of repressed chromatin and that the SNF1 protein kinase is sufficient to produce the derepressed chromatin pattern. A model is presented for the transitions that result in opening up of the chromatin structure.

Base Sequence↗
PubMed2001

Chromatin structure mapping in Saccharomyces cerevisiae in vivo with DNase I.

Most methods for assessment of chromatin structure involve chemical or nuclease damage to DNA followed by analysis of distribution and susceptibility of cutting sites. The agents used generally do not permeate cells, making nuclear isolation mandatory. In vivo mapping strategies might allow detection of labile constituents and/or structures that are lost when chromatin is swollen in isolated nuclei at low ionic strengths. DNase I has been the most widely used enzyme to detect chromatin sites where DNA is active in transcription, replication or recombination. We have introduced the bovine DNase I gene into yeast under control of a galactose-responsive promoter. Expression of the nuclease leads to DNA degradation and cell death. Shorter exposure to the active enzyme allows mapping of chromatin structure in whole cells without isolation of nuclei. The validity and efficacy of the strategy are demonstrated by footprinting a labile repressor bound to its operator. Investigation of the inter-nucleosome linker regions in several types of repressed domains has revealed different degrees of protection in cells, relative to isolated nuclei.

Animals↗
PubMed1995

Steroid hormone receptor status defines the MMTV promoter chromatin structure in vivo.

The ability to respond to small signalling molecules such as steroid hormones is important for many physiological processes. Steroid hormones act through a group of high affinity receptors that regulate transcription by binding to hormone response elements (HREs) located within the promoters of target genes, which themselves are organized with nuclear proteins to form chromatin. To dissect the mechanisms(s) of steroid hormone action we have used the steroid inducible mouse mammary tumor virus (MMTV) promoter as a model system. The MMTV promoter is assembled into a phased array of nucleosomes that are specifically positioned in rodent cells. Induction of transcription by glucocorticoids is accompanied by the appearance of a hypersensitive region in the proximal promoter which allows the hormone dependent assembly of a preinitiation complex including transcription factors such as nuclear factor 1 (NF1) and the octamer transcription factor (OTF). Surprisingly, when introduced by transient transfection, the progesterone receptor (PR) is unable to activate this promoter in vivo, a finding that may result from its inability to alter MMTV promoter chromatin. In an attempt to investigate the failure of the PR to activate the promoter, we have stably introduced the MMTV promoter into human T47D breast cancer cells that express high levels of the PR. In contrast to what has been observed previously in rodent cells, the MMTV templates resident in human breast cancer cells adopt a novel and constitutively open chromatin structure. The constitutively open chromatin structure is accompanied by the hormone independent loading of transcription factors including the PR and NF1. In T47D cells that stably express the glucocorticoid receptor, the MMTV promoter responds to glucocorticoids, but not progestins, and displays glucocorticoid induced restriction enzyme hypersensitivity and transcription factor loading. These findings suggest that the organization of the MMTV chromatin structure is dependent upon the cell type and receptor status of the recipient cell into which the MMTV promoter is stably introduced.

Animals↗
PubMed1997

Direct evidence for SIR2 modulation of chromatin structure in yeast rDNA.

The yeast SIR2 gene maintains inactive chromatin domains required for transcriptional repression at the silent mating-type loci and telomeres. We previously demonstrated that SIR2 also acts to repress mitotic and meiotic recombination between the tandem ribosomal RNA gene array (rDNA). Here we address whether rDNA chromatin structure is altered by loss of SIR2 function by in vitro and in vivo assays of sensitivity to micrococcal nuclease and dam methyltransferase, respectively, and present the first chromatin study that maps sites of SIR2 action within the rDNA locus. Control studies at the MAT alpha locus also revealed a previously undetected MNase-sensitive site at the a1-alpha 2 divergent promoter which is protected in sir2 mutant cells by the derepressed a1-alpha 2 regulator. In rDNA, SIR2 is required for a more closed chromatin structure in two regions: SRR1, the major SIR-Responsive Region in the non-transcribed spacer, and SRR2, in the 18S rRNA coding region. None of the changes in rDNA detected in sir2 mutants are due to the presence of the a1-alpha 2 repressor. Reduced recombination in the rDNA correlates with a small, reproducible transcriptional silencing position effect. Deletion and overexpression studies demonstrate that SIR2, but not SIR1, SIR3 or SIR4, is required for this rDNA position effect. Significantly, rDNA transcriptional silencing and rDNA chromatin accessibility respond to SIR2 dosage, indicating that SIR2 is a limiting component required for chromatin modeling in rDNA.

Chromatin↗
PubMed1994

Methylation status and chromatin structure of an early response gene (ornithine decarboxylase) in resting and stimulated NIH-3T3 fibroblasts.

The early response gene ornithine decarboxylase (odc) is indispensable for normal and malignant cell growth. Although DNA methylation is generally associated with chromatin condensation and gene inactivation, the odc gene is heavily methylated at CCGG-sequences in animal cell lines. In this work we analyzed the chromatin structure and the DNA methylation status at the CpG-rich promoter sequences at the odc locus in mouse 3T3 fibroblasts. We show that the proximal promoter region of the odc locus is not hypermethylated, while the distal promoter sequences appear to have a few methylated CCGG-sites and display methylation polymorphism. Furthermore, it was found that the 5' promoter region of odc is constitutively more sensitive to micrococcal nuclease than the coding and 3' regions of the odc gene. Stimulation of the cells with serum resulted in an appearance of a DNase I sensitive site at the promoter region. The chromatin structure of the mid-coding and 3' regions of the odc gene also underwent structural changes that were accompanied by the rapid accumulation of odc mRNA. Such changes were not detected in the chromatin structure of glyceraldehyde-3-phosphate dehydrogenase (gadph) gene, whose expression remains invariant upon serum stimulation. These data suggest that the chromatin structure may play an important role in the rapid transcriptional activation of odc and other immediate early genes during serum stimulation.

3T3 Cells↗
PubMed1993

(CT)n (GA)n repeats and heat shock elements have distinct roles in chromatin structure and transcriptional activation of the Drosophila hsp26 gene.

Previous analysis of the hsp26 gene of Drosophila melanogaster has shown that in addition to the TATA box and the proximal and distal heat shock elements (HSEs) (centered at -59 and -340, relative to the start site of transcription), a segment of (CT)n repeats at -135 to -85 is required for full heat shock inducibility (R.L. Glaser, G.H. Thomas, E.S. Siegfried, S.C.R. Elgin, and J.T. Lis, J. Mol. Biol. 211:751-761, 1990). This (CT)n element appears to contribute to formation of the wild-type chromatin structure of hsp26, an organized nucleosome array that leaves the HSEs in nucleosome-free, DNase I-hypersensitive (DH) sites (Q. Lu, L.L. Wallrath, B.D. Allan, R.L. Glaser, J.T. Lis, and S.C.R. Elgin, J. Mol. Biol. 225:985-998, 1992). Inspection of the sequences upstream of hsp26 has revealed an additional (CT)n element at -347 to -341, adjacent to the distal HSE. We have analyzed the contribution of this distal (CT)n element (-347 to -341), the proximal (CT)n element (-135 to -85), and the two HSEs both to the formation of the chromatin structure and to heat shock inducibility. hsp26 constructs containing site-directed mutations, deletions, substitutions, or rearrangements of these sequence elements have been fused in frame to the Escherichia coli lacZ gene and reintroduced into the D. melanogaster genome by P-element-mediated germ line transformation. Chromatin structure of the transgenes was analyzed (prior to gene activation) by DNase I or restriction enzyme treatment of isolated nuclei, and heat-inducible expression was monitored by measuring beta-galactosidase activity. The results indicate that mutations, deletions, or substitutions of either the distal or the proximal (CT)n element affect the chromatin structure and heat-inducible expression of the transgenes. These (CT)n repeats are associated with a nonhistone protein(s) in vivo and are bound by a purified Drosophila protein, the GAGA factor, in vitro. In contrast, the HSEs are required for heat-inducible expression but play only a minor role in establishing the chromatin structure of the transgenes. Previous analysis indicates that prior to heat shock, these HSEs appear to be free of protein. Our results suggest that GAGA factor, an abundant protein factor required for normal expression of many Drosophila genes, and heat shock factor, a specific transcription factor activated upon heat shock, play distinct roles in gene regulation: the GAGA factor establishes and/or maintains the DH sites prior to heat shock induction, while the activated heat shock factor recognizes and binds HSEs located within the DH sites to trigger transcription.

Animals↗
PubMed1976

Chromatin structure and function in proliferating cells.

The conclusions that we would like to draw from this review are the following: (a) Chromatin structure and function are exceedingly sensitive to changes in the proliferative state of a cell. Differences can be detected between cells in mitosis, G1 and S, and even between G0 and G1 cells. (b)These differences are very unlikely to be artifactual, since similar changes can also be demonstrated in intact nuclei. (c) Some of these differences can be abolished by extraction of chromatins with low concentrations of salt. (d) Differences between chromatins of normal and neoplastic cells can also be detected, but they are largely related to differences in the extent of cell proliferation. (e) A number of laboratories have been very busy in trying to elucidate chromatin structure with different technologies. Sometimes a change in a macromolecule cause by a physiological stimulus can tell us as much about its structure as a thousand instruments. The changes occuring in chromatin of proliferating cells could perhaps be profitably used to know more about chromatin structure.

Animals↗
PubMed1985

Drug action and chromatin structure. I. Adriamycin binding to core particle of nucleosomes and subsequent enhanced DNA fragmentation induced by micrococcal nuclease.

The relevance of the subunit structure of chromatin to the mode of action of Adriamycin in Novikoff hepatoma had been investigated. To elucidate whether drug binding takes place at random along the chromatin fiber or not Novikoff hepatoma nuclei treated with Adriamycin in vivo were digested with Micrococcal nuclease and were subsequently separated into three fractions, S1, S2 and P2. In these chromatin fraction Adriamycin and the DNA content was determined. DNA were isolated from the above fractions and were analyzed by polyacrylamide gel electrophoresis, furthermore the S2 fraction was sedimented on sucrose density gradient. The results indicate that binding of Adriamycin takes place preferentially on the core particle inducing structural alterations. Adriamycin binding not only enhanced DNA nuclease digestion but altered the size of the fragments.

Animals↗
PubMed1986

Chromatin structure of the chicken lysozyme gene domain as determined by chromatin fractionation and micrococcal nuclease digestion.

The chromatin structure encompassing the lysozyme gene domain in hen oviduct nuclei was studied by measuring the partitioning of coding and flanking sequences during chromatin fractionation and by analyzing the nucleosome repeat in response to micrococcal nuclease digestion. Following micrococcal nuclease digestion, nuclei were sedimented to obtain a chromatin fraction released during digestion (S1) and then lysed in tris(hydroxymethyl)aminomethane-(ethylenedinitrilo)tetraacetic acid-[ethylenebis(oxyethylenenitrilo)]tetraacetic acid and centrifuged again to yield a second solubilized chromatin fraction (S2) and a pelleted fraction (P2). By dot-blot hybridization with 14 specific probes, it is found that the fractionation procedure defines three classes of sequences within the lysozyme gene domain. The coding sequences, which partition with fraction P2, are flanked by class I flanking sequences, which partition with fractions S1 and P2 and which extend over 11 kilobases (kb) on the 5'side and probably over about 4 kb on the 3' side. The partitioning of class II flanking sequences, which are located distal of class I flanking sequences, is different from that of class I flanking sequences. Coding sequences lack a canonical nucleosome repeat, class I flanking sequences possess a disturbed nucleosome repeat, and class II flanking sequences generate an extended nucleosomal ladder. Coding and class I flanking sequences are more readily digested by micrococcal nuclease than class II flanking sequences and the inactive beta A-globin gene. In hen liver, where the lysozyme gene is inactive, coding and class I flanking sequences fractionate into fractions S2 and P2.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMedDate not supplied

Relation between carcinogenesis, chromatin structure and poly(ADP-ribosylation) (review).

Poly(ADP-ribose) is a naturally occurring nuclear macromolecule resembling nucleic acids. It is synthesized from NAD+ on histones and a few other nuclear proteins. Its function, although not completely understood, might be to alter chromatin structure and to regulate the activity of proteins involved in the metabolism of DNA strand breaks such as ligase II, and topoisomerase I. In addition, poly(ADP-ribose) modifies proteins involved in gene expression such as acetylated histones. HMG proteins, and T antigen. The enzyme poly(ADP-ribose) polymerase responsible for this modification has the unique property of requiring nicks or free ends on the DNA for its activity and of being automodified. The automodified enzyme, presumably found at the vicinity of DNA strand breaks at damaged chromatin sites, could remove histones from DNA and attract enzymes that have an affinity for poly(ADP-ribose) such as ligase II or poly(ADP-ribose) glycohydrolase, the polymer-degrading enzyme. Alterations in chromatin structure alter gene expression and seem to be involved in repair, replication, and recombination and in changing DNA superhelical density, intermediate steps in molecular carcinogenesis. Experiments with cells in culture and laboratory animals show that inhibition of poly(ADP-ribosylation) alters transformation and tumorigenicity brought about by a great number of carcinogenic agents. Cancer can be caused by the accumulation of unrepaired DNA strand breaks in the cell accelerating gene rearrangements, deletions, insertions and amplifications. Repair of DNA strand breaks shows an absolute dependence upon the rapid synthesis and degradation of poly(ADP-ribose). The polymer has a very short half life indeed. Data are reviewed on changes in chromatin structure and function caused by histone and nonhistone poly(ADP-ribosylation). The link of this modification to transformation, tumorigenesis, development, replication and gene expression is examined. A model is proposed to explain the effect of poly(ADP-ribosylation) on chromatin structure at the molecular level. Mono- and oligo(ADP-ribosylated) histones present in nuclei under physiological conditions are proposed to functions, like acetylated histones, in maintaining chromatin loops into transcriptionally active structures. On the other hand, poly(ADP-ribosylated) histones and poly(ADP-ribosylated) enzymes such as DNA and RNA polymerases, suggested to be modified from in vitro studies, might only appear in cells that have been heavily damaged by carcinogen. Their function might be to remove histones from DNA in order to facilitate repair and to shut down transcription and replication.

Animals↗
PubMed1988

The chromatin structure at the promoter of a glyceraldehyde phosphate dehydrogenase gene from Saccharomyces cerevisiae reflects its functional state.

The chromatin structure of TDH3, one of three genes encoding glyceraldehyde phosphate dehydrogenases in Saccharomyces cerevisiae, was analyzed by nuclease digestion. A large hypersensitive region was found at the TDH3 promoter extending from the RNA initiation site at position -40 to position -560. This hypersensitive domain is nucleosome free and includes all putative cis-acting regulatory DNA elements. It is equally present in cells grown on fermentable as well as nonfermentable carbon sources. In a mutant which lacks the trans-activating protein GCR1 and which as a consequence expresses TDH3 at less than 5% of the wild-type level, the chromatin structure is different. Hypersensitivity between -40 and -370 is lost, due to the deposition of nucleosomes on a stretch that is nucleosome free in wild-type cells. Hypersensitivity is retained, however, further upstream (from -370 to -560). A similarly altered chromatin structure, as in a ger1 mutant, is found in wild-type cells when they approach stationary phase. This is the first evidence for a growth-dependent regulation of the TDH3 promoter.

Chromatin↗
PubMed2001

Regulation of bleomycin-induced DNA breakage and chromatin structure in lung endothelial cells by integrins and poly(ADP-ribose) polymerase.

Activation of endothelial cell integrins inhibits DNA breakage by diverse agents, including the DNA-damaging agent bleomycin. DNA breaks activate nuclear poly(ADP-ribose) polymerase (PARP), which regulates chromatin structure and DNA repair. We determined the role of PARP in suppression of bleomycin genotoxicity by integrins using wild-type and PARP knockout mouse lung endothelial cells (MLEC), and the PARP inhibitor, 3-aminobenzamide (3AB). Activation of beta1 integrins by antibody clustering enhanced the sensitivity of wild-type nuclei to digestion with micrococcal nuclease and deoxyribonuclease I, indicating that chromatin structure was altered. 3AB blocked this effect. Knockout and 3AB-treated wild-type MLEC were hypersensitive to deoxyribonuclease I compared with wild-type cells, demonstrating that PARP regulates chromatin structure. Integrin clustering reduced the hypersensitivity of knockout cells, suggesting additional, PARP-independent mechanisms that inhibit nuclease interaction with chromatin. Bleomycin caused DNA breakage in wild-type and knockout MLEC. Breaks were eliminated after 60 min incubation of wild-type cells in drug-free medium, whereas 3AB or PARP knockout inhibited DNA repair. Integrin clustering protected wild-type cells from DNA breakage, and 3AB and PARP knockout inhibited this protection. Bleomycin caused large increases in PARP activity in wild-type but not knockout MLEC, and integrin clustering inhibited the activation of PARP. The results indicate that the antigenotoxic effects of integrin activation require PARP and that integrins alter chromatin structure by PARP-dependent and -independent mechanisms.

Animals↗
PubMed1992

Effect of polyamine depletion on chromatin structure in U-87 MG human brain tumour cells.

The chromatin structure of polyamine-depleted U-87 MG human brain tumour cells was studied by following the kinetics of digestion of cell nuclei by micrococcal nuclease and bovine pancreatic DNAase I. Cells growing in monolayers were treated with either alpha-difluoromethylornithine (DFMO), to deplete putrescine and spermidine, or N1,N14-bis(ethyl)homospermine (BE-4-4-4), to deplete putrescine, spermidine and spermine. BE-4-4-4 increased the initial rates of digestion and the magnitudes of limit digest by both enzymes; DFMO increased the limit digests without affecting initial digestion rates. Addition of 1 mM-putrescine 1 day after addition of DFMO reversed the effect of DFMO on limit digests. (Because polyamine uptake is low in cells treated with BE-4-4-4, and because putrescine does not reverse the growth-inhibitory effects of BE-4-4-4, reversal of the effects of BE-4-4-4 with putrescine was not attempted.) The increases in initial rates and limit digests did not result from changes in the lengths of nucleosomal or linker DNA, from blocks in cell-cycle progression, or from growth inhibition caused by DFMO or BE-4-4-4. Thus, because the limit digest is highest in cells with the lowest polyamine levels, it seems clear that the enhanced enzymic digestion of nuclei is caused by polyamine depletion and its possible effect on chromatin structure.

Brain Neoplasms↗
PubMed2004

Human cervical mucus can act in vitro as a selective barrier against spermatozoa carrying fragmented DNA and chromatin structural abnormalities.

PURPOSE: We have carried out experiments to determine if human cervical mucus can act as an in vitro selective barrier against spermatozoa morphologically normal that carry genetic structural abnormalities. METHODS: Sperm chromatin abnormalities have been evaluated by Chromomycin A3 and "endogenous" nick translation. RESULTS: The data obtained have shown that spermatozoa possessing higher levels of DNA protamination are more proficient in crossing the cervical mucus barrier. Moreover, the levels of positivity to endogenous nick translation treatment was practically zero in such spermatozoa. CONCLUSIONS: We suggest that sperm penetration of cervical mucus could be used to select sperm preparations free of fragmented DNA or chromatin structural abnormalities for assisted reproduction.

Cervix Mucus↗
PubMed2000

Developmental patterns of chromatin structure and DNA methylation responsible for epigenetic expression of a maize regulatory gene.

Epigenetic regulatory mechanisms heritably alter patterns of gene expression without changes in DNA sequence. Epigenetic states are often correlated with developmentally imposed alterations in genomic DNA methylation and local chromatin structure. Pl-Blotched is a stable epigenetic allele of the maize anthocyanin regulatory gene, purple plant1(pl). Pl-Blotched plants display a variegated pattern of pigmentation that contrasts sharply with the uniformly dark purple pigmentation of plants carrying the dominant Pl-Rhoades allele. Previously, we showed that the lower level of pigmentation in Pl-Blotched is correlated with lower pl mRNA levels and increased DNA methylation at some sites. To explore how DNA methylation, chromatin structure, and developmental stage might contribute to the expression of Pl-Blotched, we used methylation-sensitive restriction enzymes and DNaseI sensitivity assays to compare the methylation status and chromatin structure of Pl-Blotched and Pl-Rhoades at different stages in development. Both alleles exhibit developmentally sensitive changes in methylation. In Pl-Blotched, methylation of two diagnostic HpaII/MspI sites increases progressively, coincident with the juvenile-to-adult transition in growth. In seedlings, the chromatin encompassing the coding region of the gene is less sensitive to DNaseI digestion in Pl-Blotched than in Pl-Rhoades. Developmental maturation from seedling to adult is accompanied by expansion of this closed chromatin domain to include the promoter and downstream flanking sequences. We provide evidence to show that chromatin structure, rather than DNA methylation, is the primary epigenetic determinant for the phenotypic differences between Pl-Blotched and Pl-Rhoades.

Alleles↗