Artemis phosphorylated by DNA-dependent protein kinase associates preferentially with discrete regions of chromatin

Artemis is a nuclear phosphoprotein required for genomic integrity whose phosphorylation is increased subsequent to DNA damage. Artemis phosphorylation by the DNA-dependent protein kinase (DNA-PK) and the association of Artemis with DNA-PK catalytic subunit (DNA-PKcs) have been proposed to be crucial for the variable, diversity, joining (V(D)J) reaction, genomic stability and cell survival in response to double-stranded DNA breaks. The exact nature of the effectors of Artemis phosphorylation is presently being debated. Here, we have delimited the interface on Artemis required for its association with DNA-PKcs and present the characterization of six DNA-PK phosphorylation sites on Artemis whose phosphorylation shows dependence on its association with DNA-PKcs and is induced by double-stranded DNA damage. Surprisingly, DNA-PKcs Artemis association appeared to be dispensable in a V(D)J recombination assay with stably integrated DNA substrates. Phosphorylation at two of the sites on Artemis, S516 and S645, was verified in vivo using phosphospecific antibodies. Basal Artemis S516 and S645 phosphorylation in vivo showed a significant dependence on DNA-PKcs association. However, regardless of its association with DNA-PKcs, phosphorylation of Artemis at both S516 and S645 was stimulated in response to the double-stranded DNA-damaging agent bleomycin, albeit to a lesser extent. This suggests that additional factors contribute to promote DNA damage-induced Artemis phosphorylation. Intriguingly, pS516/pS645 Artemis was concentrated in chromatin-associated nuclear foci in na?ve cells. These foci were maintained upon DNA damage but failed to overlap with the damage-induced gammaH2AX. These results provide the expectation of a specific role for DNA-PK-phosphorylated Artemis in both na?ve and damaged cells.     

Linker DNA destabilizes condensed chromatin.

The contribution of the linker region to maintenance of condensed chromatin was examined in two model systems, namely sea urchin sperm nuclei and chicken red blood cell nuclei. Linkerless nuclei, prepared by extensive digestion with micrococcal nuclease, were compared with Native nuclei using several assays, including microscopic appearance, nuclear turbidity, salt stability, and trypsin resistance. Chromatin in the Linkerless nuclei was highly condensed, resembling pyknotic chromatin in apoptotic cells. Linkerless nuclei were more stable in low ionic strength buffers and more resistant to trypsin than Native nuclei. Analysis of histones from the trypsinized nuclei by polyacrylamide gel electrophoresis showed that specific histone H1, H2B, and H3 tail regions stabilized linker DNA in condensed nuclei. Thermal denaturation of soluble chromatin preparations from differentially trypsinized sperm nuclei demonstrated that the N-terminal regions of histones Sp H1, Sp H2B, and H3 bind tightly to linker DNA, causing it to denature at a high temperature. We conclude that linker DNA exerts a disruptive force on condensed chromatin structure which is counteracted by binding of specific histone tail regions to the linker DNA. The inherent instability of the linker region may be significant in all eukaryotic chromatins and may promote gene activation in living cells.     

High concentration of DNA in condensed chromatin.

The lengths of the DNA molecules of eukaryotic genomes are much greater than the dimensions of the metaphase chromosomes in which they are contained during mitosis. From this observation it has been generally assumed that the linear packing ratio of DNA is an adequate measure of the degree of DNA compaction. This review summarizes the evidence suggesting that the local concentration of DNA is more appropriate than the linear packing ratio for the study of chromatin condensation. The DNA concentrations corresponding to most of the models proposed for the 30-40 nm chromatin fiber are not high enough for the construction of metaphase chromosomes. The interdigitated solenoid model has a higher density because of the stacking of nucleosomes in secondary helices and, after further folding into chromatids, it yields a final concentration of DNA that approaches the experimental value found for condensed chromosomes. Since recent results have shown that metaphase chromosomes contain high concentrations of the chromatin packing ions Mg2+ and Ca2+, it is discussed that dynamic rather than rigid models are required to explain the condensation of the extended fibers observed in the absence of these cations. Finally, considering the different lines of evidence demonstrating the stacking of nucleosomes in different chromatin complexes, it is suggested that the face-to-face interactions between nucleosomes may be the driving force for the formation of higher order structures with a high local concentration of DNA.     

Characterisation of DNA from condensed and dispersed human chromatin

Condensed and dispersed chromatin fractions were isolated from human placental nuclei. The DNA of each fraction was purified and characterised by isopycnic centrifugation, thermal fractionation on hydroxylapatite (HAP) and sequence complexity studies. The DNAs had identical buoyant densities in neutral CsCl (1.698 g/cm³) and similar melting profiles on HAP. Analytical ultracentrifugation in Ag⁺-Cs₂SO₄, however, showed that satellite DNAs were present in the condensed fraction DNA (DNA_C) but were not visible in the dispersed fraction DNA (DNA_D). In addition, DNA_C was found to be enriched in highly reiterated sequences (20% reassociated by C₀t 10^?3) which can be correlated with the presence of satellite DNAs, whereas DNA_D contained only 3% of these fast reassociating sequences. In contrast DNA_D contained 30% intermediate sequences (reassociating between C₀t 10^?3 and C₀t 100) which represent only 10% of DNA_C. The reassociated highly repeated sequences of DNA_C showed the presence of two components in both CsCl density gradients and HAP thermal elution studies. This suggests that either there are sequence relationships resulting in partial mismatching between the different highly repeated DNA sequences in this fraction, or that highly repeated sequences are associated with less repetitious DNA. The results are discussed in terms of possible differences in genetic activity between the chromatin fractions.     

Restriction enzyme analysis of DNA methylation in "condensed" chromatin of Ha-ras-transformed NIH 3T3 cells.

Increased amounts of chromatin condensation (i.e., localized areas of high DNA density, or chromatin higher order packing state) have been described in NIH 3T3 cells transformed with the Ha-ras oncogene. The structural basis for this oncogene-mediated alteration in nuclear organization is unknown. Since DNA methylation is likely to be involved in regulating the nucleosomal level of DNA packaging, we studied the role of DNA methylation in higher-order chromatin organization induced by Ha-ras. CpG-methylated DNA content was estimated in "condensed" chromatin of Ha-ras-transformed NIH 3T3 cell lines which differ in ras expression and ras-induced metastatic ability but present approximately the same values of "condensed" chromatin areas. The question posed was that if DNA methylation were involved with the chromatin higher-order organization induced by Ha-ras in these cell lines, the methylated DNA density in the "condensed" chromatin would also be the same. The DNA evaluation was performed by video image analysis in Feulgen-stained cells previously subjected to treatment with Msp I and Hpa II restriction enzymes, which distinguish between methylated and non-methylated DNA. The amount of methylated CpG sequences not digested by Hpa II in "condensed" chromatin regions was found to vary in the studied ras-transformed cell lines. DNA CpG methylation status is thus suggested not to be involved with the higher order chromatin condensation induced by ras transformation in the mentioned NIH 3T3 cell lines.     

PTIP associates with MLL3- and MLL4-containing histone H3 lysine 4 methyltransferase complex

PTIP, a protein with tandem BRCT domains, has been implicated in DNA damage response. However, its normal cellular functions remain unclear. Here we show that while ectopically expressed PTIP is capable of interacting with DNA damage response proteins including 53BP1, endogenous PTIP, and a novel protein PA1 are both components of a Set1-like histone methyltransferase (HMT) complex that also contains ASH2L, RBBP5, WDR5, hDPY-30, NCOA6, SET domain-containing HMTs MLL3 and MLL4, and substoichiometric amount of JmjC domain-containing putative histone demethylase UTX. PTIP complex carries robust HMT activity and specifically methylates lysine 4 (K4) on histone H3. Furthermore, PA1 binds PTIP directly and requires PTIP for interaction with the rest of the complex. Moreover, we show that hDPY-30 binds ASH2L directly. The evolutionarily conserved hDPY-30, ASH2L, RBBP5, and WDR5 likely constitute a subcomplex that is shared by all human Set1-like HMT complexes. In contrast, PTIP, PA1, and UTX specifically associate with the PTIP complex. Thus, in cells without DNA damage agent treatment, the endogenous PTIP associates with a Set1-like HMT complex of unique subunit composition. As histone H3 K4 methylation associates with active genes, our study suggests a potential role of PTIP in the regulation of gene expression.     

Local chromatin microenvironment determines DNMT activity: from DNA methyltransferase to DNA demethylase or DNA dehydroxymethylase

Insights on active DNA demethylation disproved the original assumption that DNA methylation is a stable epigenetic modification. Interestingly, mammalian DNA methyltransferases 3A and 3B (DNMT-3A and -3B) have also been reported to induce active DNA demethylation, in addition to their well-known function in catalyzing methylation. In situations of extremely low levels of S-adenosyl methionine (SAM), DNMT-3A and -3B might demethylate C-5 methyl cytosine (5mC) via deamination to thymine, which is subsequently replaced by an unmodified cytosine through the base excision repair (BER) pathway. Alternatively, 5mC when converted to 5- hydroxymethylcytosine (5hmC) by TET enzymes, might be further modified to an unmodified cytosine by DNMT-3A and -3B under oxidized redox conditions, although exact pathways are yet to be elucidated. Interestingly, even direct conversion of 5mC to cytosine might be catalyzed by DNMTs. Here, we summarize the evidence on the DNA dehydroxymethylase and demethylase activity of DNMT-3A and -3B. Although physiological relevance needs to be demonstrated, the current indications on the 5mC- and 5hmC-modifying activities of de novo DNA C-5 methyltransferases shed a new light on these enzymes. Despite the extreme circumstances required for such unexpected reactions to occur, we here put forward that the chromatin microenvironment can be locally exposed to extreme conditions, and hypothesize that such waves of extremes allow enzymes to act in differential ways.     

Chromosomal localisation of DNA sequences in condensed and dispersed human chromatin.

The DNAs purified from condensed and dispersed human chromatin were used as templates for the in vitro synthesis of ³H-labelled complementary RNAs (cRNAs). These cRNAs were hybridised in situ to preparations of fixed human metaphase chromosomes which had previously been stained with quinacrine and photographed with fluorescent (UV) light. Autoradiographs of the hybridised chromosomes were stained and photographed and the results analysed by comparison of the fluorescence photographs with the autoradiographs. This method allowed positive identification of every chromosomal site of hybridisation and quantitative analysis of grain distribution over a number of metaphase spreads. The cRNA transcribed from condensed chromatin DNA (cRNA_C) hybridised mainly to a limited number of sites close to or including centromeric heterochromatin (C-bands) and also to the brightly fluorescent regions of the Y chromosome. Many of these C-band regions are known to contain satellite DNAs, indicating that the repeated DNA in the condensed chromatin fraction consists largely, if not entirely, of satellite sequences. The cRNA transcribed from dispersed chromatin DNA (cRNA_D) does not contain satellite DNAs and hybridised more generally over the chromosome arms. However, the main sites of hybridisation with cRNA_D included the C-bands in the Y chromosome and autosomes, i.e. those regions which bound cRNA_C. This suggests that nonsatellite repeated DNA sequences may be associated with satellite DNAs in the chromosomes. No general correlation between the distribution of either kind of cRNA and the overall level of quinacrine fluorescence in chromosomes or chromosome arms was detectable, nor could the dispersed fraction be equated with cytological euchromatin, since it hybridised in many sites which appear heterochromatic. However, there was a suggestion that some non-fluorescing Q-bands bound cRNA_D preferentially. The differences which were found between the distribution of the cRNAs from the two chromatin fractions may be associated with differences in genetic activity.     

Proliferating cell nuclear antigen associates with histone deacetylase activity, integrating DNA replication and chromatin modification

Faithful inheritance of the chromatin structure is essential for maintaining the gene expression integrity of a cell. Histone modification by acetylation and deacetylation is a critical control of chromatin structure. In this study, we test the hypothesis that histone deacetylase 1 (HDAC1) is physically associated with a basic component of the DNA replication machinery as a mechanism of coordinating histone deacetylation and DNA synthesis. Proliferating cell nuclear antigen (PCNA) is a sliding clamp that serves as a loading platform for many proteins involved in DNA replication and DNA repair. We show that PCNA interacts with HDAC1 in human cells and in vitro and that a considerable fraction of PCNA and HDAC1 colocalize in the cell nucleus. PCNA associates with histone deacetylase activity that is completely abolished in the presence of the HDAC inhibitor trichostatin A. Trichostatin A treatment arrests cells at the G(2)-M phase of the cell cycle, which is consistent with the hypothesis that the proper formation of the chromatin after DNA replication may be important in signaling the progression through the cell cycle. Our results strengthen the role of PCNA as a factor coordinating DNA replication and epigenetic inheritance.     

Avidin binds to condensed chromatin.

The protein avidin has been conjugated with fluorescein isothiocyanate and then used to treat cells and chromosome preparations. Fluorescence microscopy reveals that this protein is bound by areas of the specimen containing condensed chromatin.     

Nucleosome DNA repeat length and histone complement in a fungus exhibiting condensed chromatin

Fungal chromatins are reported to exhibit unusually short nucleosomal DNA repeat lengths. To test whether this is a phylogenetic feature of fungi or rather is correlated with an apparent absence of condensed chromatin in the organisms studied, we have examined the chromatin organization and the complement of basic nuclear proteins in the fungus Entomophthora, an organism which exhibits marked chromatin condensation. Micrococcal nuclease digestion of Entomophthora chromatin revealed a nucleosomal DNA repeat length of 197 +/- 1.2 base pairs (bp). This repeat length is 20-40 bp longer than that reported for any fungus. Entomophthora nucleosomes exhibited an HI-like protein which was much less basic than the HI histones reported for higher eukaryotes but which was similar in basicity to the HI histone reported for the fungus Neurospora. However, the nucleosomal DNA repeat length of Neurospora chromatin is reported to be unusually short, whereas that of Entomophthora was found to be typical of the repeat lengths observed for chromatins of higher eukaryotes. Thus, repeat length, at least in fungi, would not appear to be directly determined by the basicity of the fungal cognate of histone HI.     

Differential accessibility of DNA in extended and condensed chromatin to pancreatic DNase I

Pancreatic DNase I has been used to study the interaction between DNA and chromosomal proteins in extended and condensed chromatin fractions isolated from mouse and Chinese hamster livers. It was found that DNase digests extended chromatin at a faster rate than condensed chromatin, and the evidence suggests that the chromosomal proteins are more tightly complexed to the DNA in condensed than in extended chromatin. This difference in DNA-protein interaction in extended and condensed chromatin may be related to the functional difference which characterizes these fractions, and might be one of the factors underlying the production of bands on metaphase chromosomes.     

Tel1p preferentially associates with short telomeres to stimulate their elongation

In many organisms, telomeric DNA consists of long tracts of short repeats. Shorter tracts are preferentially lengthened by telomerase, suggesting a conserved mechanism that recognizes and elongates short telomeres. Tel1p, an ATM family checkpoint kinase, plays an important role in telomere elongation, as cells lacking Tel1p have short telomeres and show reduced recruitment of telomerase components to telomeres. We show that Tel1p association increased as telomeres shortened in vivo in the presence or absence of telomerase and that Tel1p preferentially associated with the shortest telomeres. Tel1p association was independent of Tel1p kinase activity and enhanced by Mre11p. Tel1p overexpression simultaneously stimulated telomerase-mediated elongation and Tel1p association with all telomeres. Thus, Tel1p preferentially associates with the shortest telomeres and stimulates their elongation by telomerase.     

Identification of in vivo DNA targets of chromatin proteins using tethered dam methyltransferase

We have developed a novel technique, named DamID, for the identification of DNA loci that interact in vivo with specific nuclear proteins in eukaryotes. By tethering Escherichia coli DNA adenine methyltransferase (Dam) to a chromatin protein, Dam can be targeted in vivo to native binding sites of this protein, resulting in local DNA methylation. Sites of methylation can subsequently be mapped using methylation-specific restriction enzymes or antibodies. We demonstrate the successful application of DamID both in Drosophila cell cultures and in whole flies. When Dam is tethered to the DNA-binding domain of GAL4, targeted methylation is limited to a region of a few kilobases surrounding a GAL4 binding sequence. Using DamID, we identified a number of expected and unexpected target loci for Drosophila heterochromatin protein 1. DamID has potential for genome-wide mapping of in vivo targets of chromatin proteins in various eukaryotes.     

Nonhistone chromatin proteins HMG-14 and HMG-17 bind preferentially to single-stranded DNA.

Proteins extracted from chicken erythrocyte chromatin with 0.35 M NaCl were subjected to sequential chromatography on columns containing immobilized double-stranded and single-stranded DNA's. Two-dimensional electrophoresis of protein fractions revealed that HMG-14 and HMG-17 are among the proteins that are retained by the single-stranded DNA column in 0.2 M NaCl/l mM Tris-Cl (pH 7.5) after having failed to be retained by the double-stranded column under the same conditions. That suggests that those two proteins possess preferential affinity for single-stranded DNA. Further evidence for that was provided by chromatography of purified HMG-14 and of purified HMG-17 on single-stranded and double-stranded DNA columns. We discuss the possible relevance of our results to suggested functions of HMG-14 and HMG-17.