Internucleosomal Linker Regions Research Articles

Refined DNase-seq protocol and data analysis reveals intrinsic bias in transcription factor footprint identification.

DNase-seq is a powerful technique for identifying cis-regulatory elements across the genome. We studied the key experimental parameters to optimize the performance of DNase-seq. We found that sequencing short 50-100bp fragments that accumulate in long inter-nucleosome linker regions is more efficient for identifying transcription factor binding sites than using longer fragments. We also assessed the potential of DNase-seq to predict transcription factor occupancy through the generation of nucleotide-resolution transcription factor footprints. In modeling the sequence-specific DNaseI cutting bias we found a surprisingly strong effect that varied over more than two orders of magnitude. This confounds DNaseI footprint analysis to the extent that the nucleotide resolution cleavage patterns at most transcription factor binding sites are derived from intrinsic DNaseI cleavage bias rather than from specific protein-DNA interactions. In contrast, quantitative comparison of DNaseI hypersensitivity between states can predict transcription factor occupancy associated with particular biological perturbations.

Intrinsic coupling of lagging-strand synthesis to chromatin assembly

Fifty per cent of the genome is discontinuously replicated on the lagging strand as Okazaki fragments. Eukaryotic Okazaki fragments remain poorly characterized and, because nucleosomes are rapidly deposited on nascent DNA, Okazaki fragment processing and nucleosome assembly potentially affect one another. Here we show that ligation-competent Okazaki fragments in Saccharomyces cerevisiae are sized according to the nucleosome repeat. Using deep sequencing, we demonstrate that ligation junctions preferentially occur near nucleosome midpoints rather than in internucleosomal linker regions. Disrupting chromatin assembly or lagging-strand polymerase processivity affects both the size and the distribution of Okazaki fragments, suggesting a role for nascent chromatin, assembled immediately after the passage of the replication fork, in the termination of Okazaki fragment synthesis. Our studies represent the first high-resolution analysis--to our knowledge--of eukaryotic Okazaki fragments in vivo, and reveal the interconnection between lagging-strand synthesis and chromatin assembly.

Diverse Roles of RNA Polymerase II-associated Factor 1 Complex in Different Subpathways of Nucleotide Excision Repair

Transcription-coupled repair (TCR) and global genomic repair (GGR) are two pathways of nucleotide excision repair (NER). In Saccharomyces cerevisiae, Rad26 is important but not absolutely required for TCR. Rpb4, a nonessential RNA polymerase II (Pol II) subunit that forms a subcomplex with Rpb7, and the Spt4-Spt5 complex, a transcription elongation factor, have been shown to suppress Rad26-independent TCR. The Pol II-associated factor 1 complex (Paf1C) has been shown to function in transcription elongation, 3'-processing of mRNAs, and posttranslational modification of histones. Here we show that Paf1C plays a marginal role in facilitating Rad26-dependent TCR but significantly suppresses Rad26-independent TCR. The suppression of Rad26-independent TCR is achieved by cooperating with Spt4-Spt5. We propose a model that, in the absence of Rad26, a lesion is "locked" in the active center of a Pol II elongation complex, which is stabilized by the coordinated interactions of Rpb4-Rpb7, Spt4-Spt5, and Paf1C with each other and with the core Pol II. We also found that Paf1C facilitates GGR, especially in internucleosomal linker regions. The facilitation of GGR is achieved through enabling monoubiquitination of histone H2B lysine 123 by Bre1, which in turn permits di- and trimethylation of histone H3 lysine 79 by Dot1. To our best knowledge, among the NER-modulating factors documented so far, Paf1C appears to have the most diverse functions in different NER pathways or subpathways.

Histone H1 subtype preferences of DFF40 and possible nuclear localization of DFF40/45 in normal and trichostatin A-treated NB4 leukemic cells

A major hallmark of the terminal stages of apoptosis is the internucleosomal DNA fragmentation. The endonuclease responsible for this type of DNA degradation is the DNA fragmentation factor (DFF). DFF is a complex of the endonuclease DFF40 and its chaperone/inhibitor, DFF45. In vitro work has shown that histone H1 and HMGB1/2 recruit/target DFF40 to the internucleosomal linker regions of chromatin and that histone H1 directly interacts with DFF40 conferring DNA binding ability and enhancing its nuclease activity. The histone H1 family is comprised of many subtypes, which recent work has shown may have distinct roles in chromatin function. Thus we studied the binding association of DFF40 with specific H1 subtypes and whether these binding associations are altered after the induction of apoptosis in an in vivo cellular context. The apoptotic agent used in this study is the histone deacetylase inhibitor, trichostatin A (TSA). We separated the insoluble chromatin-enriched fraction from the soluble nuclear fraction of the NB4 leukemic cell line. Using MNase digestion, we provide evidence which strongly suggests that the heterodimer, DFF40-DFF45, is localized to the chromatin fraction under apoptotic as well as non-apoptotic conditions. Moreover, we present results that show that DFF40 interacts with the all H1 subtypes used in this study, but preferentially interacts with specific H1 subtypes after the induction of apoptosis by TSA. These results illustrate for the first time the association of DFF40 with individual H1 subtypes, under a specific apoptotic stimulus in an in vivo cellular context.

Evidence for Long Poly(dA).Poly(dT) Tracts in D. Discoideum DNA at High Frequencies and Their Preferential Avoidance of Nucleosomal DNA Core Regions

The eukaryote, Dictyostelium discoideum, has one of the most (A+T) rich genomes studied to date. Isolated nuclear D. discoideum DNA (AX3 strain) was used to qualitatively determine the frequency and length distribution of long (dA).(dT) homopolymer tracts in this genome, in comparison to the less (A+T) rich calf thymus and Schistosoma mansoni DNAs that had few observable long tracts. These experimental data accurately reflect the significantly elevated frequencies of long tracts found computationally within the D. discoideum intron and flanking sequences, but not exons. PCR amplification of long (dA).(dT) homopolymer tract containing sequences was carried out. Then experimental biotinylated (dT)jg probe hybridization to the PCR amplified DNA showed that the long (dA).(dT) homopolymer tracts were enriched in D. discoideum sequences only hundreds of base pair in length, under conditions where no equivalent hybridization was observed to S. mansoni DNA or calf DNA sequences. Similar probe hybridization to DNA isolated following micrococcal nuclease digestion of D. discoideum chromatin demonstrated that long (dA).(dT) homopolymer tracts were more highly enriched in nucleosomal DNA lengths that included the internucleosomal linker as compared to shorter linker free mononucleosomal lengths. This observation is in agreement with the frequency of tract spacing results calculated from GenBank sequence data. These frequency data indicate that adjacent long tracts plus the intervening spacer DNA are found at peak lengths (average 42bp), exactly characteristic of the internucleosomal spacer region of D. discoideum chromatin and are in sufficient number to be found in nearly half of all nucleosomes. Compared to shuffled tract sequence controls, these lengths of adjacent long tracts plus the intervening spacer DNA were found to be significantly enriched. Lesser enrichments are observed at lengths corresponding to adjacent tracts being separated by nucleosomal core length DNA sequences (145–185bp). These data strongly suggest that adjacent long tracts occur spaced at selected lengths so as to avoid the central core regions of nucleosomes and instead are found localized within internucleosomal DNA linker and core edge regions in D. discoideum chromatin.

Inhibition of myocardial apoptosis reduces infarct size and improves regional contractile dysfunction during reperfusion.

Myocardial apoptosis is primarily triggered during reperfusion (R) through various mechanisms that may involve endonuclease to cleavage genomic DNA in the internucleosomal linker regions. However, the relative contribution of myocardial apoptosis to development of myocardial injury during R remains unknown. In the present study, we examined whether inhibition of apoptosis with aurintricarboxylic acid (ATA), an endonuclease inhibitor, during R reduces infarct size and improves regional contractile function. In two groups of chronically-instrumented dogs, 1 h of left anterior descending (LAD) coronary occlusion was followed by 24 h of R with infusion of saline (control, n=8) or ATA (1 mg/kg/h, n=8) into the left atrium starting 5 min before R and continuing for 2 h. ATA significantly reduced apoptotic cells (TUNEL staining) in the peri-necrotic myocardium (12+/-1%* vs. 36+/-4%), consistent with the absence of DNA laddering. To confirm inhibition of apoptosis with ATA, densitometrically, Bcl-2 (% of normal myocardium) was significantly increased vs. control (102+/-12* vs. 68+/-9) and Bax as well as the activated caspase-3 were significantly reduced vs. control (108+/-17* vs. 194+/-42 and -29+/-4* vs. 174+/-43, respectively). ATA significantly improved segmental shortening (3.3+/-1.2* vs. -1.8+/-0.7%) and segmental work (79.3+/-11.3* vs. 7.1+/-5.8 mmHg/mm) in area at risk myocardium, and reduced infarct size (TTC staining, 27+/-0.2* vs. 37+/-0.5%), confirmed by lower plasma creatine kinase activity. In addition, myocardial blood flow (0.9+/-0.1* vs. 0.4+/-0.1 ml/min/g) and endothelial-dependent maximal vascular relaxation (119+/-6* vs. 49+/-8%) were significantly improved. Myeloperoxidase activity in area at risk myocardium, a marker for neutrophil accumulation, was also significantly reduced (17+/-4* vs. 138+/-28 Delta Abs/min). These data suggest that the inhibition of apoptosis during R is associated with a reduction in infarction, improvement in regional contractile and vascular endothelial functions as well as augmentation in myocardial blood flow. *P<0.05 vs. control group.

The SWI/SNF chromatin-remodeling factor stimulates repair by human excision nuclease in the mononucleosome core particle.

To investigate the role of chromatin remodeling in nucleotide excision repair, we prepared mononucleosomes with a 200-bp duplex containing an acetylaminofluorene-guanine (AAF-G) adduct at a single site. DNase I footprinting revealed a well-phased nucleosome structure with the AAF-G adduct near the center of twofold symmetry of the nucleosome core. This mononucleosome substrate was used to examine the effect of the SWI/SNF remodeling complex on the activity of human excision nuclease reconstituted from six purified excision repair factors. We found that the three repair factors implicated in damage recognition, RPA, XPA, and XPC, stimulate the remodeling activity of SWI/SNF, which in turn stimulates the removal of the AAF-G adduct from the nucleosome core by the excision nuclease. This is the first demonstration of the stimulation of nucleotide excision repair of a lesion in the nucleosome core by a chromatin-remodeling factor and contrasts with the ACF remodeling factor, which stimulates the removal of lesions from internucleosomal linker regions but not from the nucleosome core.

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.

Effect of Hypoxia on Cerebral Cortical Genomic DNA in the Guinea Pig Fetus at Term † 1895

Our previous studies have shown that hypoxia in fetal guinea pigs modifies cortical nuclear membranes resulting in increased high-affinity Ca++-ATPase activity and increased number (Bmax) of nuclear membrane IP3 receptors, resulting in calcium influx into the nucleus. The increased intranuclear Ca++ could activate nuclear Ca++-dependent endonuclease,which cuts genomic DNA at specific cleavage sites, causing specific DNA fragmentation. The present study tests the hypothesis that brain tissue hypoxia results in fragmentation of genomic DNA via endonuclease activity in the cerebral cortex of the guinea pig fetus. Guinea pig fetuses were obtained from anesthetized, normoxic (21% oxygen for 60 min, n=6) and hypoxic (7% oxygen for 60 min, n=6) mothers. Fetal brains were removed immediately after the experimental period. Brain tissue hypoxia was documented biochemically by decreased levels of ATP and phosphocreatine. Cerebral cortical nuclei were isolated and purified using a discontinuous sucrose gradient. DNA was isolated and purity was determined by the ratio of absorbance at 260/280nm. DNA samples were separated by gel electrophoresis on 1% agarose and the DNA bands stained with ethidium bromide. The density of the DNA bands was analyzed by imaging densitometery. In normoxic nuclei, only one DNA band was visualized, whereas DNA from hypoxic nuclei demonstrated six discrete peaks ranging in size from 100 to 2000 basepairs. DNA profile suggests that there is specific fragmentation of DNA in a “ladder” pattern in hypoxic nuclei at the internucleosomal linker regions. No random DNA cleavage (which would appear as a dense “smear” due to randomized fragments) was observed. The data demonstrate that there is fragmentation of nuclear DNA, a hallmark of programmed cell death, in cerebral cortex in the guinea pig fetus during acute hypoxia in utero. We speculate that the specific fragmentation of nuclear DNA in the brain cells of the guinea pig fetus is due to increased endonuclease activity as a result of altered nuclear membrane mechanisms of Ca++ influx, leading to hypoxia induced programmed cell death.

The effect of aurintricarboxylic acid, an endonuclease inhibitor, on ischemia/reperfusion damage in rat retina.

Apoptosis is a form of cell death distinct from necrosis showing distinctive morphologic features and may require energy. It is under various control mechanisms and may involve an endonuclease, which cleavages genomic DNA in the internucleosomal linker regions. Previously, we reported that ischemic/reperfusion injury to rat retina induced endonuclease mediated apoptosis of retinal neurons. In this study, we examined the effect of aurintricarboxylic acid (ATA), an endonuclease inhibitor, on ischemia/reperfusion damage in rat retina in our established rat model. A single intraperitoneal injection of ATA at 2 mg/kg given immediately after 60 minutes of ischemia to the retina showed no observable effect. At 10 mg/kg, there was notable beneficial effect morphologically but not morphometrically. ATA at 100 mg/kg showed significant effect both morphologically and morphometrically. This observation is consistent with the hypothesis that endonuclease mediated apoptosis may be involved in retinal cell loss after ischemia/reperfusion insult.

Directed cell killing (apoptosis) in human lymphoblastoid cells incubated in the presence of farnesol: Effect of phosphatidylcholine

Previously reported observations have shown that trans-trans farnesol inhibits incorporation of choline into phosphatidylcholine and reduces the growth rate of the human acute leukemia CEM-C1 cell line (Melnykovych, G., Haug, J.S. and Goldner, C.M. (1992) Biochem. Biophys. Res. Commun. 186, 543–548). These findings have now been followed up in order to establish a relationship between the inhibition of phosphatidylcholine synthesis and the ensuing cell shrinkage and cell death which takes place at higher concentrations of farnesol or upon long incubation. The present results show that after incubation in the presence of farnesol the cells decrease in viability. Their nuclear DNA becomes fragmented at internucleosomal linker regions, showing characteristic pattern of bands at 180 to 200 base-pair intervals. This farnesol-induced effect was also demonstrated by flow cytometry by staining the cellular DNA with propidium iodide and was partially reversible with phosphatidylcholine.

Study of barley endonucleases and alpha-amylase genes.

We have identified an endonuclease(s) that preferentially cleaves the internucleosomal linker regions in the aleurone chromatin producing mono- and oligonucleosomes. This enzyme(s) has been designated as a "linker"-specific nuclease(s). This nuclease does not require divalent cations for activity, and therefore it is not the "Ca2+-Mg2+-DNase" found in mammalian cells. The linker-specific nuclease activity is not detectable in the dry aleurone tissue and in the tissue treated with 0.5 mM cordycepin. The endonuclease activity of the aleurone tissue incubated with gibberellic acid is higher than the level of this endonuclease in tissue treated with abscisic acid or water alone. Nuclei isolated from embryos have lower levels of endonuclease activities compared to those from aleurone tissue. Digestion of the nuclei from embryos with micrococcal nuclease revealed the subunit structure of chromatin. In Southern blots of the HindIII digests of DNA from embryos, five DNA bands hybridized to a nick-translated alpha-amylase cDNA clone. In similar autoradiograms with aleurone DNA, particular bands are less visible, notably in the DNA isolated from the tissue treated with gibberellic acid. This is the first report of the presence of a linker-specific nuclease activity in plant cells.

Correlation of nitrosourea murine bone marrow toxicity with deoxyribonucleic acid alkylation and chromatin binding sites

All of the clinically available nitrosourea antitumor agents produce serious treatment-limiting bone marrow toxicity. A reduction in this toxicity can be achieved by attaching the chloroethylnitrosourea cytotoxic group to C2 (chlorozotocin) or C1 (1-(2-chloroethyl)-3-(β- d-glucopyranosyl)-1-nitrosourea, GANU) of glucose. Both glucose analogs are less myelotoxic in mice than 1-(2-chloroethyl)-3-cyclohepyl-1-nitrosourea (CCNU) or 1-(4-amino-2-methylpyrimidin-5-yl)methyl-3-(2-chloroethyl)-3-nitrosourea (ACNU), while retaining comparable antitumor activity against the murine L121o leukemia. To define the nuclear mechanisms for this reduced myelotoxicity, alkylation of L1210 and murine bone marrow DNA was quantitated. With the use of the endonucleases micrococcal nuclease and DNase I, the sites of alkylation within the chromatin substructure were determined. Experiments were performed on L1210 leukemia or bone marrow cells that had been incubated in vitro for 2 hr with 0.1 mM [ 14C]chloroethyl drug. The quantitative alkylation of DNA by GANU was 1.3-fold greater in L1210, as compared to bone marrow, cells. This ratio of DNA alkylation is comparable to the 1.3 ratio we previously reported for chlorozotocin [L. C. Panasci, D. Green and P. S. Schein, J. clin. Invest. 64, 1103 (1979)]. In contrast, the ratio of alkylation (L1210: bone marrow DNA) for the myelotoxic ACNU was 0.66, similar to 0.59 for CCNU. Nuclease digestion experiments demonstrated that chlorozotocin and GANU preferentially alkylated internucleosomal linker regions of bone marrow chromatin, while nucleosome core particles were the preferred targets of CCNU and ACNU. The reduced myelotoxicity of chlorozotocin and GANU may be correlated with the advantageous ratio of L1210: bone marrow DNA alkylation and preferential alkylation of internucleosomal regions of bone marrow chromatin.

Changes in chromatin structure associated with Alzheimer's disease.

The enzyme micrococcal nuclease was used to examine the accessibility of chromatin extracted from brains of 13 patients with senile and presenile dementia of the Alzheimer type. Compared with chromatin extracted from brains of 8 patients without neurological signs or brain pathology and brains of 7 patients with nonAlzheimer dementia, Alzheimer chromatin was less accessible to this enzyme. Reduced accessibility was reflected by a reduced yield of mononucleosomes in comparison with dinucleosomes and larger oligomers. Both neuronal and glial chromatin were found to be similarly affected. The reduced yield of mononucleosomes from Alzheimer chromatin is not due to their increased breakdown, but is probably related to protein associated with the internucleosomal linker region that retards nuclease action. Dinucleosomes isolated from control and Alzheimer nuclease digests were examined for their protein complement. Three perchloric acid-soluble proteins situated in the histone H1 region of sodium dodecyl sulfate (SDS) gels were present in elevated levels in Alzheimer dinucleosomes. These results represent the first example of altered chromosomal proteins associated with a diseased state of the brain.

Deoxyribonuclease II as a probe for chromatin structure: I. Location of cleavage sites

DNAase II has been shown to cleave condensed mouse liver chromatin at 100-bp † † Abbreviations used: bp, base-pairs; PDE, phosphodiesterase; PMSF, phenylmethanesulfonyl fluoride; EGTA, ethyleneglycol bis(β-aminoethyl ether)- N,N′-tetraacetic acid. intervals while chromatin in the extended form is cleaved at 200-bp intervals (Altenburger et al., 1976). Evidence is presented here that DNA digestion patterns of a half-nucleosomal periodicity are also obtained upon DNAase II digestion of chicken erythrocyte nuclei and yeast nuclei, both of which differ in their repeat lengths (210 and 165 bp) from mouse liver chromatin. In the digestion of mouse liver nuclei a shift from the 100-bp to the 200-bp cleavage mode takes place when the concentration of monovalent cations present during digestion is decreased below 1 mM. When soluble chromatin prepared by micrococcal nuclease is digested with DNAase II the same type of shift occurs, albeit at higher ionic strength. In order to map the positions of the DNAase II cleavage sites on the DNA relative to the positions of the nucleosome cores, the susceptibility of DNAase II-derived DNA termini to exonuclease III was investigated. In addition, oligonucleosome fractions from HaeIII and micrococcal nuclease digests were end-labelled with polynucleotide kinase and digested with DNAase II under conditions leading to 100 and 200-bp digestion patterns. Analysis of the chain lengths of the resulting radioactively labelled fragments together with the results of the exonuclease assay allow the following conclusions. In the 200-bp digestion mode, DNAase II cleaves exclusively in the internucleosomal linker region. Also in the 100-bp mode cleavage occurs initially in the linker region. Subsequently, DNAase II cleaves at intranucleosomal locations, which are not, however, in the centre of the nucleosome but instead around positions 20 and 125 of the DNA associated with the nucleosome core. At late stages of digestion intranucleosomal cuts predominate and linkers that are still intact are largely resistant to DNAase II due to interactions between adjacent nucleosomes. These findings offer an explanation for the sensitivity of DNAase II to the higher-order structure of chromatin.

Evidence for the location of high mobility group protein T in the internucleosomal linker regions of trout testis chromatin.

Antibodies against the trout testis non"histone chromosomal protein, high mobility group protein T (HMG-T), have been elicited in goats. The antiserum was shown to be specific for HMG-T and did not cross-react with histone 1 or with the other two trout testis HMG proteins, H6 and ubiquitin. Purified anti-HMG-T IgG was used to determine the location of HMG-T within chromatin subunits separated on sucrose gradients. Binding of fluorescent labeled anti-HMG-T to these subunits clearly supports the notion that this protein is associated not with the nucleosome core but rather with the internucleosomal linker regions, and previously suggested (Levy W., B., Wong, N.C.W., and Dixon, G. H. (1977) Proc. Natl. Acad. Sci. U.S.A. 74, 2810-2814).

A nuclear protein-modifying enzyme is responsive to ordered chromatin structure

Poly (ADP-ribose) polymerase, a nuclear protein-modifying enzyme, binds to the internucleosomal linker region of chromatin, although it modifies certain core nucleosomal histones in addition to histone H1. The activity per unit of DNA chromatin changes with the nucleosome repeat number. It reaches a maximum on chromatin of 8-10 nucleosomes in length. As the complexity of chromatin with respect to nucleosome repeat number and compactness increases, a decline and stabilization of specific activity is noted. The difference in specific activity is maintained through resedimentation and dialysis of particles. It does not appear due to differences in polymer chain length or differential degradation of poly (ADP-ribose). The data suggest a relationship between ADP-ribosylation and chromatin organization and vice versa.