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Homology-directed repair is a powerful mechanism for maintaining and altering genomic structure. We asked how chromatin structure contributes to the use of homologous sequences as donors for repair using the chicken B cell line DT40 as a model. In DT40, immunoglobulin genes undergo regulated sequence diversification by gene conversion templated by pseudogene donors. We found that the immunoglobulin Vλ pseudogene array is characterized by histone modifications associated with active chromatin. We directly demonstrated the importance of chromatin structure for gene conversion, using a regulatable experimental system in which the heterochromatin protein HP1 (Drosophila melanogaster Su[var]205), expressed as a fusion to Escherichia coli lactose repressor, is tethered to polymerized lactose operators integrated within the pseudo-Vλ donor array. Tethered HP1 diminished histone acetylation within the pseudo-Vλ array, and altered the outcome of Vλ diversification, so that nontemplated mutations rather than templated mutations predominated. Thus, chromatin structure regulates homology-directed repair. These results suggest that histone modifications may contribute to maintaining genomic stability by preventing recombination between repetitive sequences.
Author Summary
Homologous recombination promotes genetic exchange between regions containing identical or highly related sequences. This is useful in repairing damaged DNA, or in reassorting genes in meiosis, but uncontrolled homologous recombination can create genomic instability. Chromosomes are made up of a complex of DNA and protein, called chromatin. DNA within chromatin is packed tightly in order to fit the entire genome inside a cell; but chromatin structure may become relaxed to allow access to enzymes that regulate gene expression, transcribe genes into mesenger RNA, or carry out gene replication. We asked if chromatin packing regulates homologous recombination. To do this, we tethered a factor associated with compact chromatin, called HP1, adjacent to an immunoglobulin gene locus at which homologous recombination occurs constitutively, in order to produce a diverse repertoire of antibodies. We found that the compact, repressive chromatin structure produced by HP1 prevents homologous recombination. This finding suggests that regulated changes in chromatin structure may contribute to maintaining genomic stability by preventing recombination between repetitive sequences.
Citation: Cummings WJ, Yabuki M, Ordinario EC, Bednarski DW, Quay S, Maizels N (2007) Chromatin Structure Regulates Gene Conversion. PLoS Biol 5(10): e246. doi:10.1371/journal.pbio.0050246
Academic Editor: James E. Haber, Brandeis University, United States of America
Received: March 26, 2007; Accepted: July 17, 2007; Published: September 18, 2007
Copyright: © 2007 Cummings et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: This work was supported by US National Institutes of Health grants R01 GM41712 and P01 CA77852.
Competing interests: The authors have declared that no competing interests exist.
Abbreviations: ψV, pseudo-variable; AcH3, acetylated histone H3; AcH4, acetylated histone H4; ChIP, chromatin immunoprecipitation; diMeK4(H3), histone H3 dimethylated at lysine 4; DSB, double-strand break; GFP, green fluorescent protein; Ig, immunoglobulin; Igλ, immunoglobulin light chain; IPTG, isopropyl-β-D-thiogalactoside; LOH, loss of heterozygosity; PolyLacO, polymerized lactose operator; sIgM, surface immunoglobulin M; V, variable
Homologous recombination provides a pathway for restoring or altering DNA sequence and structure [1–7]. Homologous recombination can recreate the original DNA sequence at a DNA break, and predominates in S/G2 phases of cell cycle, when sister chromatids can serve as donors for faithful repair [8,9]. Homologous recombination can also have a mutagenic outcome by promoting recombination between nonallelic repeated sequences, leading to genomic instability, or by templating repair from a homolog rather than a sister chromatid, leading to loss of heterozygosity (LOH). In a living cell, multiple pathways compete to repair the same kinds of damage. For example, double-strand breaks (DSBs) can be repaired by nonhomologous end-joining, which may be accompanied by sequence loss or translocation [5,10,11]. Nicks can be efficiently repaired in situ, or by short- or long-patch repair pathways that use the complementary strand as a template [12,13].
Chromatin structure plays an important role in repair at the site of DNA damage. A critical signal for DSB repair is C-terminal phosphorylation of the variant histone H2AX by ATM and ATR, to generate γ-H2AX [14–16]. γ-H2AX is recruited to the break and extends over a large region surrounding the break site, creating a boundary of modified chromatin, and recruits the cohesin complex to sites of damage, to promote DSB repair using the sister chromatid as a template [17,18]. γ-H2AX alerts DNA damage checkpoints, and is recognized by histone acetyltransferases and chromatin remodeling complexes [19–22]. Changes in chromatin structure also facilitate synapsis of severed DNA ends for nonhomologous end-joining [23].
Homologous recombination involves two DNA molecules, the recipient, which is the site of the DNA lesion, and the donor. Two lines of evidence suggest that donor chromatin structure may contribute to homologous recombination. At the yeast mating type locus, changes in histone acetylation occur at the donor locus that are distinct from those at or near the DNA break [24]. In human cells, transcription of a donor promotes its use in gene conversion [25]. However, the role of donor chromatin structure in regulating recombination has not been directly tested in vertebrate cells.
Immunoglobulin (Ig) gene diversification in chicken B cells provides a powerful model for studying homologous recombination. Chickens have a limited number of functional heavy and light chain variable (V) regions, which undergo V(D)J recombination early in B cell development [26,27]. The rearranged V genes then undergo sequence diversification by gene conversion, using an array of homeologous upstream pseudo-V (ψV) regions as donors (Figure 1A). The ψV regions are nonfunctional, as they lack promoters and cannot be transcribed. The mechanism of Ig gene conversion is readily studied in the DT40 cell line, which derives from a bursal lymphoma and constitutively diversifies its Ig heavy and Ig light chain (Igλ) genes by gene conversion [28–30]. DT40 also supports very high levels of homologous gene targeting, thought to reflect elevated expression or activity of factors that promote recombinational repair [31–33].
(A) Schematic diagram of the rearranged chicken Igλ locus, showing the 25 ψVλ regions and the rearranged VλR gene. Cλ, constant region; L, leader; VλJλ, variable region.
(B) Summary of a representative chromatin immunoprecipitation experiment, assaying N-terminal acetylation of histones H3 and H4 (AcH3 and AcH4). Sites interrogated were as follows: a region approximately 1 kb upstream of the ψVλ array (flank), ψVλ25, ψVλ24, ψVλ13, ψVλ5, ψVλ1, the region between ψVλ1 and Vλ, and the rearranged VλR and unrearranged VλU alleles. See Materials and Methods for details. Bars indicate standard deviation.
Ig gene conversion in DT40 depends upon ubiquitous and conserved factors. The substrate for repair is a nick produced by successive action of three factors. The B-cell-specific enzyme activation-induced deaminase (AID) [34–37] deaminates cytosine to uracil in transcribed and targeted genes [38–41]; uracil DNA glycosylase excises the uracil produced by AID to generate an abasic site [42–46]; and the MRE11 abasic lyase, functioning within the MRE11/RAD50/NBS1 complex, nicks at the abasic site [47,48]. Strand transfer and new DNA synthesis are carried out by ubiquitous DNA repair factors including the RAD51 paralogs, BRCA2, FANCC, FANCD2, and polη [49–55]. Deficiencies in some of these factors, particularly the RAD51 paralogs, or targeted deletion of some or all of the ψV donors [56], impair gene conversion and can contribute to a shift in the processing of AID-initiated breaks so that templated repair is accompanied or even supplanted by nontemplated mutagenesis. ψV regions preferentially used as donors are in opposite orientation to the functional V region, suggesting that local chromosomal architecture may guide templated repair [57]. However, nothing is known about how epigenetic features of the donors affect recombination.
To understand how donor chromatin structure affects gene conversion in particular, and homologous recombination more generally, we characterized and experimentally manipulated chromatin structure at the Igλ locus in the chicken B cell line DT40. We found that the ψVλ donors contain acetylated histones, consistent with an open chromatin structure. To test whether this reflects requirements of gene conversion, we tethered HP1 (Drosophila melanogaster Su[var]205) to the ψVλ array in a DT40 derivative in which polymerized lactose operator (PolyLacO) has been inserted into that chromosomal region. HP1 is known to promote heterochromatic gene silencing [58–60]. Tethered HP1 caused a local transition of the donor sequences from an open to a nonpermissive state, and a switch from templated to nontemplated diversification, evident as point mutations. These observations demonstrate that permissive chromatin structure at the donor is a key regulator of gene conversion, and that nonpermissive chromatin structure can prevent homologous recombination and result in point mutagenesis. These results have implications for our understanding of homologous recombination and of the mechanisms that promote LOH, leading to tumorigenesis and nonallelic recombination between repeats. These results should also inform design of donor constructs for targeted gene therapy.
Permissive Chromatin Structure at Vλ and ψVλ Donor Templates
In DT40 B cells, the Vλ gene is rearranged and expressed at one Igλ allele, but it is unrearranged at the other allele. We characterized chromatin structure at the rearranged (VλR) and unrearranged (VλU) alleles and the ψVλ array by chromatin immunoprecipitation (ChIP). ChIP was carried out with antibodies specific for lysine acetylation at the N-termini of histones H3 and H4. Recovered DNA was amplified in duplex PCR reactions; recovery was normalized to an amplicon from the ovalbumin (Ova) gene, which is not expressed in B cells; and enrichment was normalized to a total DNA input control (see Materials and Methods for details). The distinct genomic structure of VλR and VλU permit them to be distinguished by PCR with specific primers. ChIP demonstrated considerable enrichment of acetylated histones H3 and H4 (AcH3 and AcH4) at the rearranged VλR gene. In a typical experiment, AcH3 was enriched more than 80-fold at VλR, and AcH4 more than 30-fold (Figure 1B). In contrast, at the VλU allele, the levels of AcH3 and AcH4 were much lower than at VλR (16-fold and 7-fold lower, respectively), and only a few fold enriched relative to input DNA.
Chromatin structure within the ψVλ array was assayed by amplification with primers that interrogated seven sites, including a region between ψVλ1 and the Vλ gene, ψVλ1, ψVλ5, ψVλ13, ψVλ18, ψVλ24, ψVλ25, and the upstream flanking region. (Because of a paucity of polymorphisms, the ψVλ arrays at the two Igλ alleles in DT40 cannot be readily distinguished by PCR.) Strikingly, we observed considerable enrichment of AcH3 and AcH4 throughout the ψVλ array (Figure 1B). Enrichment was not proportional to distance from the transcribed VλR gene, as sites distant from VλR did not consistently display lower levels of enrichment than proximal sites (Figure 1B). Thus, enrichment of acetylated histones within the ψVλ array does not simply represent a graded spreading of chromatin modification from the transcribed VλR gene to sites upstream. The nonuniform chromatin structure of the locus suggests the presence of cis-elements that regulate chromatin structure at the ψVλ array.
Reversible Tethering of Lactose Repressor Fusion Proteins to the ψVλ Array in DT40 PolyLacO-λR
Local modification of chromatin structure can be achieved by tethering regulators to DNA binding sites as appropriate fusion proteins. This strategy has, for example, been used to show that the heterochromatin protein HP1, expressed as a fusion with Escherichia coli lactose repressor (LacI-HP1), promotes a closed chromatin structure and inactivation of reporter genes neighboring a LacO repeat in Drosophila [61,62], and to show that tethering of the vertebrate G9a histone methyltransferase to a GAL4 binding site within V(D)J minigene reporter impairs nonhomologous-mediated recombination of that construct [63]. Our laboratory has recently constructed a cell line, DT40 PolyLacO-λR, that is a DT40 derivative in which PolyLacO has been inserted by homologous gene targeting between ψVλ17 and ψVλ20, 17 kb upstream of the transcribed VλR (Figure 2A; M. Yabuki, E. C. Ordinario, W. J. Cummings, R. P. Larson, M. M. Fujii, et al., unpublished data). The PolyLacO insert is 3.8 kb in length and composed of approximately 100 copies of a 20-mer operator [64]. Using this cell line, it is possible to assay the effects of tethered regulatory factors on homologous recombination in a physiological process within an endogenous locus, avoiding the need for a transgene reporter. Control experiments have shown that the PolyLacO tag does not affect cell proliferation, cell cycle, or Ig gene diversification (M. Yabuki, E. C. Ordinario, W. J. Cummings, R. P. Larson, M. M. Fujii, et al., unpublished data).
(A) Schematic diagram of the rearranged chicken Igλ locus in DT40, with PolyLacO inserted between ψVλ17 and ψVλ20. Notations as in Figure 1.
(B) Fluorescent images of DT40 GFP-LacI transfectants and DT40 PolyLacO-VλR GFP-LacI transfectants cultured in the absence of IPTG (center) or in the presence of 100 μM IPTG overnight (right).
In DT40 PolyLacO-λR GFP-LacI cells, which stably express enhanced green fluorescent protein (GFP) fused to LacI (GFP-LacI), the tagged λR allele can be directly imaged by fluorescence microscopy and appears as a distinct dot in each cell (Figure 2B, center). Tethering is reversible, as bright dots are not evident following overnight culture with 100 μM isopropyl-β-D-thiogalactoside (IPTG), which prevents LacI from binding to PolyLacO (Figure 2B, right).
Tethered HP1 Diminishes Modifications Characteristic of Active Chromatin at ψVλ
To manipulate chromatin structure at the ψVλ array, we generated stable transfectants of DT40 PolyLacO-λR that express the D. melanogaster HP1 protein fused to LacI (LacI-HP1). HP1 is a nonhistone heterochromatin protein that functions in heterochromatic gene silencing, the spreading of heterochromatin, and histone deacetylation [58–60]. Tethered HP1 has been shown to promote a closed chromatin structure at adjacent genes [61,62,65–67]. Staining DT40 PolyLacO-λR LacI-HP1 transfectants with anti-LacI antibodies showed that LacI-HP1 colocalized with DAPI-dense regions corresponding to pericentric heterochromatin (Figure 3A), behaving as a functional marker of heterochromatin [68].
(A) Representative fluorescent images of single DT40 PolyLacO-VλR LacI-HP1 transfectants, stained with anti-LacI antibodies (left), DAPI (center), or merged image (right).
(B) Enrichment of AcH3 and AcH4 at ψVλ17R in DT40 PolyLacO-VλR GFP-LacI and DT40 PolyLacO-VλR LacI-HP1 transfectants. Following ChIP, duplex PCR was carried out with Ova and ψVλ17R primers. Enrichment is expressed relative to the total DNA input control ± standard deviation of four separate amplifications of increasing amounts of template DNA.
(C) Histogram showing enrichment of AcH3 and AcH4 at ψVλ17R (from [B]) and the polε promoter in DT40 PolyLacO-VλR GFP-LacI and DT40 PolyLacO-VλR LacI-HP1 transfectants. Bars indicate standard deviation.
To ask if tethered LacI-HP1 altered chromatin structure, we assayed chromatin modifications at ψVλ17. This is the only site in the ψVλ array at which the rearranged and unrearranged alleles could be readily distinguished by use of specific PCR primers. Following ChIP, DNA was amplified with PCR primers specific for the targeted rearranged allele (ψVλ17R). Enrichment of ψVλ17R was compared to the nonexpressed Ova gene as an internal control, and normalized to the ψVλ17R:Ova enrichment ratio in total input DNA (see Material and Methods). AcH3 and AcH4 were enriched 2.2-fold and 5.9-fold, respectively, at ψVλ17R in DT40 PolyLacO-λR GFP-LacI controls (Figure 3B and 3C). These levels of enrichment are comparable to those documented in DT40 (Figure 1B). (Note that analysis of modification at ψVλ in the survey of the parental DT40 line necessarily included both alleles, which may underestimate activating modifications at the rearranged allele. In contrast, analysis of modifications at ψVλ17R interrogates only the active allele.) AcH3 and AcH4 were not enriched at ψVλ17R in DT40 PolyLacO-λR LacI-HP1 transfectants (0.6- and 1.0-fold, respectively; Figure 3B and 3C), consistent with HP1-mediated silencing. HP1 can effect silencing by recruitment of a histone methyltransferase that modifies lysine 9 of histone H3 [65–67], but may also promote silencing independently of this modification [61]. ChIP using antibodies against either di- and trimethylated H3 (lysine 9) did not reveal clear enrichment of the H3 lysine 9 methylation modification (data not shown). Dimethylation of lysine 4 of histone H3 (diMeK4[H3]) is associated with transcription and generally exhibits an overlapping distribution with acetylation [69,70]. Assays of diMeK4(H3) at ψVλ17R demonstrated that this modification was 18.9-fold enriched in DT40 PolyLacO-λR GFP-LacI cells, but at background levels in DT40 PolyLacO-λR LacI-HP1 cells (Figure 3B and 3C).
HP1 promotes maintenance and spreading of heterochromatin [65]. To verify that changes in chromatin structure promoted by tethered HP1 did not spread throughout the chromosome, we examined another site near the Igλ locus on Chromosome 15, the gene encoding the catalytic subunit of DNA polε. DNA polε is ubiquitously expressed and essential for chromosomal replication in eukaryotes [71], and it is encoded by a gene mapping approximately 2.1 Mb from Igλ. We found no difference in enrichment of AcH3 at the polε promoter region in the DT40 PolyLacO-λR LacI-HP1 transfectants relative to DT40 PolyLacO-λR GFP-LacI controls (polε/Ova enrichment 8.5-fold and 8.4-fold, respectively; Figure 3C). Similarly, there was no difference in AcH4 at the polε promoter in the DT40 PolyLacO-λR LacI-HP1 transfectants relative to DT40 PolyLacO-λR GFP-LacI controls (polε/Ova enrichment 1.9-fold and 1.7-fold, respectively; Figure 3C). Thus, tethering of LacI-HP1 at ψVλ caused local modifications in chromatin structure, diminishing the AcH3, AcH4, and diMeK4(H3) modifications characteristic of open chromatin at ψVλ17R, and causing chromatin to adopt a less permissive state.
Tethered HP1 Does Not Affect Vλ Gene Expression
We asked how tethered HP1 affected AcH3 and AcH4 levels at the expressed VλR by comparing these modifications in DT40 PolyLacO-λR LacI-HP1 cells and the DT40 PolyLacO-λR GFP-LacI control transfectants (Figure 4A). Tethered HP1 diminished AcH3 and AcH4 levels to approximately 40% and 20% of the control levels, respectively. To ask if this affected gene expression, we assayed both surface IgM (sIgM) expression and Vλ transcript levels. Staining cells with mouse anti-chicken IgM showed that sIgM expression was comparable in DT40 PolyLacO-λR GFP-LacI and DT40 PolyLacO-λR LacI-HP1 lines, cultured in either the presence or absence of IPTG (Figure 4B). Vλ transcript levels were assayed in RNA harvested from DT40 PolyLacO-λR GFP-LacI and DT40 PolyLacO-λR LacI-HP1 cells, and normalized to β-actin as a control (Figure 4C). No significant difference was observed between Vλ transcript levels in the two cell lines, demonstrating that transcription is not affected by tethering of HP1 within the ψVλ array. Thus tethered LacI-HP1 did not affect expression of the downstream Ig gene, although it did diminish AcH3 and AcH4 levels at VλR. The very high AcH3 and AcH4 levels characteristic of Vλ (Figures 1B and 4A) are therefore not essential to maintain high levels of gene expression.
(A) Relative enrichment of AcH3 and AcH4 at VλR in DT40 PolyLacO-VλR GFP-LacI and DT40 PolyLacO-VλR LacI-HP1 transfectants. Enrichment values were normalized to the DT40 PolyLacO-VλR GFP-LacI control. Bars indicate standard deviation of four separate amplifications of increasing amounts of template DNA.
(B) Relative intensity of sIgM expression in DT40 PolyLacO-λR GFP-LacI and DT40 PolyLacO-λR LacI-HP1 transfectants cultured in the presence or absence of 250 μM IPTG. sIgM levels were quantitated by measuring intensity of staining with mouse anti-chicken IgM antibody, and normalized to the level in DT40 PolyLacO-λR GFP-LacI transfectants. Details as in (A).
(C) Relative levels of VλR transcripts in DT40 PolyLacO-λR GFP-LacI and DT40 PolyLacO-λR LacI-HP1 transfectants. Transcript levels were quantitated by reverse transcriptase–PCR and normalized to the level in DT40 PolyLacO-λR GFP-LacI transfectants. Details as in (A).
Tethered HP1 Alters Local Chromatin Structure
To assess how extensive the chromatin effects of LacI-HP1 were, we examined AcH3 and AcH4 levels throughout the Igλ locus at the same amplicons examined in Figure 1, including one in the flank, six in the ψVλ array, and one at the expressed Vλ. Levels of modification were determined by comparing ψVλ17R:Ova ratios of immunoprecipitated and input conditions, as in Figure 3B. AcH3 modifications at the sites surveyed ranged from 24% to 63% of the levels at the same sites in the controls (Figure 5A, dark bars), and the average level of H3 acetylation across all of the sites was 38% of the DT40 PolyLacO-λR GFP-LacI control. Culture of DT40 PolyLacO-λR LacI-HP1 transfectants for 3 d with 250 μM IPTG increased acetylation of H3 at all eight sites surveyed (Figure 5A, compare dark and light bars). The effects of IPTG culture were somewhat variable, but at most sites IPTG culture restored levels of AcH3 to at least 45% of the level in the DT40 PolyLacO-λR GFP-LacI control cells, with an average of over 80%. Thus, the chromatin modifications at ψVλ17R in DT40 PolyLacO-λR LacI-HP1 cells resulted directly from tethered LacI-HP1, and were largely reversible.
(A) Summary of a ChIP experiment, assaying N-terminal acetylation of histone H3 in chromatin from the DT40 PolyLacO-VλR LacI-HP1 cell line cultured for 3 d in the presence or absence of 250 μM IPTG. ChIP enrichment values (see Material and Methods) were normalized to values obtained from a parallel analysis of chromatin from DT40 PolyLacO-VλR GFP-LacI cells. Bars indicate standard deviation of four separate amplifications of increasing amounts of template DNA.
(B) Summary of a ChIP experiment, assaying N-terminal acetylation of histone H4 in chromatin from the DT40 PolyLacO-VλR LacI-HP1 cell line cultured for 3 d in the presence or absence of 250 μM IPTG. Details as in (A).
H4 acetylation was surveyed at the same eight sites (Figure 5B, dark bars). AcH4 modifications were found to range from 18% to 42% of control levels, and the average level was 29% of that of the control cell line. Culture with IPTG for 3 d increased acetylation of H4 at all eight sites surveyed (Figure 5B, compare dark and light bars), restoring H4 acetylation to at least 57% of the level in the DT40 PolyLacO-λR GFP-LacI control cells, with an average of over 80%. Moreover, IPTG can at least partially reverse the effects of LacI-HP1.
These results show that the observed chromatin modifications in the ψVλ array are due to tethering of HP1. Moreover, the fact that these modifications are reversible shows that an active mechanism reverses histone modifications imposed by tethering chromatin modification factors at ψVλ.
Tethered HP1 Impairs Templated Mutagenesis
The ability to manipulate chromatin structure at ψVλ by tethering LacI-HP1 (Figures 3–5) enabled us to directly ask whether and how chromatin structure influences Ig gene conversion. We used the sIgM loss variant assay to determine if tethered LacI-HP1 affected the clonal rate of sequence diversification of the rearranged VλR gene. This fluctuation assay measures the fraction of variant cells that no longer express structurally intact sIgM, and thus scores mutation events resulting from either gene conversion or point mutagenesis [47,50]. Independent clonal derivatives of DT40 PolyLacO-λR GFP-LacI and DT40 PolyLacO-λR LacI-HP1 were established by limiting dilution cloning of sIgM+ cells; the fraction of sIgM− cells in each population was determined by flow cytometry of cells cultured for 4 wk and then stained with anti-IgM antibody. The median sIgM loss rate was 0.5% for DT40 PolyLacO-λR GFP-LacI cells and 2.8% for DT40 PolyLacO-λR LacI-HP1 cells (Figure 6A). This corresponds to a 5.6-fold acceleration of clonal diversification rates in LacI-HP1 transfectants relative to GFP-LacI controls.
(A) sIgM loss fluctuation assay of panels of independent DT40 PolyLacO-VλR GFP-LacI (n = 27) and DT40 PolyLacO-VλR LacI-HP1 (n = 16). The figure shows combined data from at least two independent transfectants for each fusion construct. Median diversification rates are shown below.
(B) Summary of sequence analysis of Vλ regions carrying unique mutations from DT40 PolyLacO-VλR GFP-LacI (n = 71) and DT40 PolyLacO-VλR LacI-HP1 (n = 36) transfectants, as analyzed by single-cell PCR. Sequences were pooled from two independent transfectants.
Ig gene diversification in chicken B cells occurs predominantly by gene conversion (templated mutation), but if gene conversion is impaired, for example by the absence of essential factors, repair can create a significant fraction of nontemplated mutations [50–55]. This is typically accompanied by an increase in the clonal diversification rate, because the ψVλ templates for gene conversion are about 80% identical to the rearranged gene, and a significant fraction of DNA lesions that are repaired by gene conversion do not undergo any alteration of sequence; in contrast, repair by a mutagenic polymerase is more likely to alter DNA sequence. To determine how tethering of HP1 accelerated diversification, we sorted single sIgM− cells from the DT40 PolyLacO-λR GFP-LacI and DT40 PolyLacO-λR LacI-HP1 transfectants, amplified expressed Vλ regions by single-cell PCR, and sequenced these regions. Sequence changes were categorized as templated if they were within a tract containing two or more base changes and the tract was an exact match to at least 9 bp of a donor ψVλ sequence, and as ambiguous if they consisted of only a single base change while matching at least 9 bp of a donor ψVλ sequence. Nontemplated events, consisting of point mutations, deletions, and insertions, were also scored. In the control DT40 PolyLacO-λR GFP-LacI transfectants, 55 templated events and two ambiguous events were documented among 71 mutations; thus, most events (77%) were templated, and a small fraction of events (20%) were point mutations (Figure 6B, left; Figure S1A). Strikingly, in DT40 PolyLacO-λR LacI-HP1 cells, point mutations predominated (58%), accompanied by deletions (8%) and insertions (14%), while only one clearly templated event and six ambiguous events were documented among 36 mutations (Figure 6B, right; Figure S1B). Thus, only 3% of mutations were clearly templated, and even including the ambiguous class of potentially templated mutations, templating could account for no more than 19% of mutation. Statistical comparisons showed that the difference between the fraction of clearly templated mutations in DT40 PolyLacO-λR GFP-LacI control cells and DT40 PolyLacO-λR LacI-HP1 transfectants (77% compared to 3%) was highly significant (p = 7.5 × 10−7, Fisher's exact test). The difference in the fraction of ambiguous, potentially templated mutations in the control cells (3%) and HP1 transfectants (17%) is also significant (p = 0.05, Fisher's exact test). This suggests that some mutations in this category may arise as a result of limitations on the length of a gene conversion tract imposed by nonpermissive donor chromatin. Thus, tethering of HP1 accelerated clonal rates of mutagenesis by impairing templated mutation.
Gene conversion at the chicken Ig loci uses an array of upstream ψV donors as templates for homology-directed repair of lesions targeted to the rearranged and transcribed V genes. We have shown that in chicken B cells carrying out active Ig gene conversion, chromatin within the donor ψVλ array is characterized by enrichment of AcH3 and AcH4, modifications that correlate with an open chromatin structure. We directly demonstrated the importance of permissive chromatin structure for Ig gene conversion by showing that tethering the heterochromatin protein HP1 to the ψVλ donor array caused local changes in chromatin structure, diminishing the AcH3, AcH4, and diMeK4(H3) modifications characteristic of open chromatin. Although these changes were not accompanied by the lysine 9 methylation (H3) modification characteristic of closed chromatin, they caused the region to adopt a state less permissive for gene conversion. Tethering of HP1 was accompanied by a dramatic shift in the Ig Vλ mutation spectrum, so that templated mutations were in the minority and point mutations predominated. Importantly, this effect on mutagenesis was correlated with a change in chromatin structure and not changes in expression of the locus. Thus, chromatin structure can dictate whether gene conversion occurs at an endogenously generated DNA lesion.
The Mechanism of Gene Conversion within a Complex Chromatin Landscape
Gene conversion at Vλ results from priming of new DNA synthesis at the 3′ end of a break using a ψVλ region as template. Gene conversion requires synapsis between the donor and recipient DNA, as well as access to the donor by factors that carry out homology-directed repair. The elevated levels of H3 and H4 acetylation characteristic of the ψVλ array in wild-type DT40 are evidence of a relaxed chromatin structure, which would increase the accessibility of the ψVλ genes to trans-acting factors and also create a three-dimensional architecture that is favorable for sequence synapsis.
HP1 tethered within the ψVλ donor array impaired gene conversion at the rearranged VλR, without affecting Vλ gene expression. Chromatin changes caused by tethered HP1 may impair gene conversion by impeding access of repair factors and the invading strand to the donor template. Tethered HP1 may also contribute to larger chromosomal architecture that affects the mechanics of DNA repair pathways, such as looping necessary to juxtapose donor and recipient sequences. The point mutations that accumulated in LacI-HP1 transfectants are typical of thwarted recombinational repair, and are characteristic of cells lacking either trans-acting factors essential for recombination [49–55] or some or all of the ψV donor array [56]. HP1 regulates chromatin structure and heterochromatic gene silencing in two ways, by partnering with a histone methyltransferase [65] and by recruiting histone deacetylases [60]. Tethered HP1 caused modification changes characteristic of a nonpermissive chromatin structure within ψVλ.
Histone acetylation has been documented at actively transcribed mammalian Ig genes undergoing somatic hypermutation and class switch recombination, but whether hyperacetylation contributes to targeting of diversification has yet to be resolved [72–76]. A connection between histone acetylation and gene conversion was suggested by experiments showing that treatment of DT40 cells with the histone deacetylase inhibitor trichostatin A promotes genome-wide histone deacetylation accompanied by increased gene conversion at VλR [77]. However, the interpretation of those results is complicated by the fact that the effects of trichostatin A are genome-wide, and not specific. The DT40 PolyLacO-λR cell line permits local manipulation of chromatin structure, avoiding that complication. Moreover, we were able to demonstrate that the effects of tethering a LacI-HP1 fusion protein were largely reversed upon culture with IPTG, so an active mechanism must determine chromatin modification at ψVλ. For studies of homologous recombination, the DT40 PolyLacO-λR B cell line has the further advantage that Ig gene conversion is a physiological process within an endogenous locus, avoiding the need for a transgene reporter.
Chromatin Structure, Genome Stability, Aging, and Gene Therapy
The importance of chromatin structure to the outcome of homologous recombination has implications for understanding the mechanisms that normally maintain genomic stability. There are vast numbers of repetitive elements distributed throughout the vertebrate genome, and recombination between these elements can lead to genomic instability [78]. In the human genome, there are approximately one million Alu elements, and recombination between Alu elements can cause duplications leading to tumorigenesis and genetic disease [79,80]. Histones carrying repressive modifications are enriched at repetitive elements [81]. These modifications undoubtedly maintain transcriptional repression; our results suggest they may also contribute to suppression of recombination.
LOH occurs as a result of unequal mitotic recombination between homologs at allelic sites. The mechanism of LOH is of particular interest, because it contributes to loss of tumor suppressor gene function, leading to tumorigenesis [82]. Recent experiments have demonstrated an age-dependent increase in LOH in Saccharomyces cerevisiae [83] and in reporter genes in Drosophila germ cells [84], and an increase in homologous recombination in mouse pancreatic cells [85]. Mechanisms proposed to explain age-associated LOH include elevated rates of DNA damage, changes in the cell cycle distribution, and inactivation of homology-independent repair pathways with aging. Our results suggest another possibility, that relaxation of chromatin structure may accompany aging and promote a genome-wide increase in homologous recombination in aging cells. This possibility is supported by recent analysis of Drosophila [86], as well as by recent evidence that the mutant form lamin A polypeptide (product of the human LMNA gene) responsible for Hutchinson-Gilford progeria syndrome leads to a genome-wide loss of H3 methylation [87].
The finding that chromatin structure regulates homologous recombination also has practical ramifications. Considerable current effort is directed toward developing strategies that harness a cell's capacity for homology-dependent repair to promote gene therapy, by providing an intact donor gene to replace a deficient target gene [88]. Our results suggest that permissive structure at the donor will be an important design parameter in developing donor genes for therapeutic applications.
Materials and Methods
Chromatin immunoprecipitation.
ChIP was carried out as previously described [48,89]. For all experiments at least two chromatin preparations from at least two independent stably transfected lines were analyzed. Figures present one representative experiment in which results from analysis of four separate amplifications were used to calculate a standard deviation. Enrichment of the experimental amplicon was normalized to enrichment of an internal control amplicon from the Ova gene, amplified in the same tube by duplex PCR, and enrichment upon ChIP with specific antibodies was normalized to parallel experiments in which ChIP was carried out with total input DNA controls. Inclusion of the Ova internal control amplicon enabled us to normalize for immunoprecipitation efficiency, background carryover, and differences in gel loading. Enrichment equaled [(ψVλ/Ova)Ab]/[(ψVλ/Ova)Input]. As an additional control, the ratio of the experimental and control amplicons in the total input control was compared to a control ChIP with polyspecific IgG; in all cases, enrichment in input and IgG controls were essentially equal.
Antibodies used were as follows: anti-AcH3 (06–599), anti-AcH4 (06–866), and diMeK4(H3) (07–030) from Upstate ( http://www.upstate.com/). PCR primers for ChIP were as follows: VλR, 5′-GCCGTCACTGATTGCCGTTTTCTCCCCTC-3′ and 5′-CGAGACGAGGTCAGCGACTCACCTAGGAC-3′; region between ψVλ1 and Vλ, 5′-CTGTGGCCTGTCAGTGCTTA-3′ and 5′-GCAGGGAACCACAAGAACAT-3′; ψVλ1, 5′-GGGACTTGTGTCACCAGGAT-3′ and 5′-CGCAGTCACATGTGGAATATC-3′; ψVλ5, 5′-GAGCCCCATTTTCTCTCCTC-3′ and 5′-GAGATGTGCAGCAACAAGGA-3′; ψVλ13, 5′-CCCTCTCCCTATGCAGGTTC-3′ and 5′-CCCCTATCACCATACCAGGA-3′; ψVλ18, 5′-CCATTTTCTCCCCTCTCTCC-3′ and 5′-TCACCCTACAGCTTCAGTGC-3′; ψVλ24, 5′-CCATTTTCTCCCCTCTCTCC-3′ and 5′-CAGCCCATCACTCCCTCTTA-3′; ψVλ25, 5′-TCTGTTGGTTTCAGCACAGC-3′ and 5′-GCAGTTCTGTGGGATGAGGT-3′; ψVλ upstream flank, 5′-GGCTCCTGTAGCTGATCCTG-3′ and 5′-GTTCTTTGCTCTTCGGTTGC-3′; ψVλ17 at the PolyLacO-targeted allele, 5′-TAGATAGGGATAACAGGGTAATAGC-3′ and 5′-AGGGCTGTACCTCAGTTTCAC-3′; Ova, 5′-ATTGCGCATTGTTATCCACA-3′ and 5′-TAAGCCCTGCCAGTTCTCAT-3′; and polε, 5′-GGGCTGGCTCATCAACAT-3′ and 5′-CTGGGTGGCCACATAGAAGT-3′.
Constructs, transfection, and cell culture.
The LacI-HP1 expression plasmid was created by substituting LacI-HP1 from a construct provided by L. Wallrath (University of Iowa) for AID in pAIDPuro (from H. Arakawa, Munich, Germany), to position LacI-HP1 downstream of the chicken β-actin promoter. The GFP-LacI expression plasmid (p3′ss-EGFP-LacI) was provided by A. Belmont (University of Illinois). Cell culture and transfection were carried out as previously described [47]. DT40 PolyLacO-λR was generated by homologous gene targeting, using a construct carrying approximately 3.8 kb of PolyLacO flanked by arms designed for targeting the region between ψVλ17 and ψVλ20, 17 kb upstream of the transcribed VλR (M. Yabuki, E. C. Ordinario, W. J. Cummings, R. P. Larson, M. M. Fujii, et al., unpublished data). In brief, homologous integrants were identified by PCR, and the selectable marker deleted by Cre expression. The DT40 bursal lymphoma derives from B cells in which only one Igλ allele is rearranged, and in which the two parental chromosomes are distinguished by a polymorphism near ψVλ17. This enabled us to determine whether the rearranged or unrearranged allele had been targeted by PCR. Control experiments established that cell cycle distribution was comparable in DT40 PolyLacO-λR, DT40 PolyLacO-λR GFP-LacI, and DT40 PolyLacO-λR LacI-HP1 cells, and that culture of cells with up to 500 μM IPTG for 7 d did not affect proliferation rate or chromatin modifications at ψVλ17R in DT40 PolyLacO-λR GFP-LacI control cells. Oligonucleotides for Vλ sequence analysis have been described [47].
Fluorescence imaging.
For fluorescence imaging, cells (2 × 105) were cytospun onto glass slides and fixed with 2% paraformaldehyde for 20 min, permeabilized with 0.1% NP-40 for 15 min, and stained as previously described [90]. Primary staining was with an anti-LacI monoclonal antibody (1:500 dilution; Upstate), and the secondary antibody was donkey anti-mouse IgG Alexa Fluor 594 (1:2,000; Molecular Probes, http://probes.invitrogen.com/). To visualize the nucleus, cells were stained with DAPI (Sigma-Aldrich, http://www.sigmaaldrich.com/). Fluorescent images were acquired using the DeltaVision microscopy system (Applied Precision, http://www.appliedprecision.com/) and processed with softWoRx software (Applied Precision).
Reverse transcriptase–PCR.
RNA was harvested from cells using TRIzol Reagent (Invitrogen, http://www.invitrogen.com/), purified with a PreAnalytiX column (Qiagen, http://www1.qiagen.com/), and subject to one round of reverse transcription prior to PCR. Vλ transcripts were PCR-amplified following dilution of the template (1:1,300), and β-actin was PCR-amplified from an undiluted sample. The primers for amplification of Vλ were 5′-GTCAGCAAACCCAGGAGAAAC-3′ and 5′-AATCCACAGTCACTGGGCTG-3′. The primers for amplification of β-actin have been described [36].
Quantitation of sIgM loss variants and sequence analysis.
The sIgM loss variant assay, which measures the accumulated sIgM loss variants resulting from frameshift or nonsense mutations in mutated V regions, was used to quantitate Ig V region diversification [47,50]. In brief, sIgM+ cells were isolated by flow cytometry followed by limiting dilution cloning, and expanded for 4 wk. To quantitate the fraction of sIgM− cells, approximately 1 × 106 cells were stained with anti-chicken IgM-RPE (SouthernBiotech, http://www.southernbiotech.com/) and analyzed on a FACScan with CellQuest software (BD Biosciences, http://www.bdbiosciences.com/).
Single-cell PCR and sequence analysis were performed as described [47]. In brief, sIgM− cells were sorted and aliquoted to single wells, Vλ regions were amplified and sequenced, and their sequences were compared to those of the ψVλ donors to determine if mutations were templated or nontemplated. The criterion for a templated mutation was that nine consecutive bases must be an exact match in donor and recipient. Sequences were derived from two independently transfected lines. Only unique sequences were included for classification of the mutations.
Supporting Information
Figure S1. Sequence Alignment of Mutated DT40 PolyLacO-λR GFP-LacI and DT40 PolyLacO-λR LacI-HP1 Clones
Sequences of unique, mutated Vλ regions from diversified (A) DT40 PolyLacO-λR GFP-LacI and (B) DT40 PolyLacO-λR LacI-HP1 cells. Clear blue boxes outline gene conversion tracts; red circles denote point mutations; black dotted boxes indicate nontemplated insertions; orange triangles denote deletions; blue-shaded boxes indicate ambiguous changes.
We thank Andrew Belmont (University of Illinois) for providing p3′ss-EGFP, Lori Wallrath (University of Iowa) for providing the LacI-HP1 construct, and Masayuki Sekimata and all members of the Maizels laboratory for discussions.
Author Contributions
WJC conceived and designed the experiments. WJC, MY, ECO, DWB, and SQ performed the experiments. WJC, MY, and NM contributed reagents/materials/analysis tools. WJC and NM analyzed the data and wrote the paper.
- 1. West SC (2003) Molecular views of recombination proteins and their control. Nat Rev Mol Cell Biol 4: 435–445.
- 2. Lee GS, Neiditch MB, Salus SS, Roth DB (2004) RAG proteins shepherd double-strand breaks to a specific pathway, suppressing error-prone repair, but RAG nicking initiates homologous recombination. Cell 117: 171–184.
- 3. Essers J, Houtsmuller AB, Kanaar R (2006) Analysis of DNA recombination and repair proteins in living cells by photobleaching microscopy. Methods Enzymol 408: 463–485.
- 4. Wyman C, Kanaar R (2006) DNA double-strand break repair: All's well that ends well. Annu Rev Genet 40: 363–383.
- 5. Weinstock DM, Richardson CA, Elliott B, Jasin M (2006) Modeling oncogenic translocations: Distinct roles for double-strand break repair pathways in translocation formation in mammalian cells. DNA Repair (Amst) 5: 1065–1074.
- 6. Sugawara N, Haber JE (2006) Repair of DNA double strand breaks: In vivo biochemistry. Methods Enzymol 408: 416–429.
- 7. Brugmans L, Kanaar R, Essers J (2007) Analysis of DNA double-strand break repair pathways in mice. Mutat Res 614: 95–108.
- 8. Garber PM, Vidanes GM, Toczyski DP (2005) Damage in transition. Trends Biochem Sci 30: 63–66.
- 9. Rodrigue A, Lafrance M, Gauthier MC, McDonald D, Hendzel M, et al. (2006) Interplay between human DNA repair proteins at a unique double-strand break in vivo. EMBO J 25: 222–231.
- 10. Haber JE (2000) Partners and pathways repairing a double-strand break. Trends Genet 16: 259–264.
- 11. Rooney S, Chaudhuri J, Alt FW (2004) The role of the non-homologous end-joining pathway in lymphocyte development. Immunol Rev 200: 115–131.
- 12. Caldecott KW (2003) XRCC1 and DNA strand break repair. DNA Repair (Amst) 2: 955–969.
- 13. Sung JS, Demple B (2006) Roles of base excision repair subpathways in correcting oxidized abasic sites in DNA. FEBS J 273: 1620–1629.
- 14. Rogakou EP, Pilch DR, Orr AH, Ivanova VS, Bonner WM (1998) DNA double-stranded breaks induce histone H2AX phosphorylation on serine 139. J Biol Chem 273: 5858–5868.
- 15. Burma S, Chen BP, Murphy M, Kurimasa A, Chen DJ (2001) ATM phosphorylates histone H2AX in response to DNA double-strand breaks. J Biol Chem 276: 42462–42467.
- 16. Ward IM, Chen J (2001) Histone H2AX is phosphorylated in an ATR-dependent manner in response to replicational stress. J Biol Chem 276: 47759–47762.
- 17. Rogakou EP, Boon C, Redon C, Bonner WM (1999) Megabase chromatin domains involved in DNA double-strand breaks in vivo. J Cell Biol 146: 905–916.
- 18. Unal E, Arbel-Eden A, Sattler U, Shroff R, Lichten M, et al. (2004) DNA damage response pathway uses histone modification to assemble a double-strand break-specific cohesin domain. Mol Cell 16: 991–1002.
- 19. Paull TT, Rogakou EP, Yamazaki V, Kirchgessner CU, Gellert M, et al. (2000) A critical role for histone H2AX in recruitment of repair factors to nuclear foci after DNA damage. Curr Biol 10: 886–895.
- 20. van Attikum H, Fritsch O, Hohn B, Gasser SM (2004) Recruitment of the INO80 complex by H2A phosphorylation links ATP-dependent chromatin remodeling with DNA double-strand break repair. Cell 119: 777–788.
- 21. Morrison AJ, Highland J, Krogan NJ, Arbel-Eden A, Greenblatt JF, et al. (2004) INO80 and gamma-H2AX interaction links ATP-dependent chromatin remodeling to DNA damage repair. Cell 119: 767–775.
- 22. Downs JA, Allard S, Jobin-Robitaille O, Javaheri A, Auger A, et al. (2004) Binding of chromatin-modifying activities to phosphorylated histone H2A at DNA damage sites. Mol Cell 16: 979–990.
- 23. Jazayeri A, McAinsh AD, Jackson SP (2004) Saccharomyces cerevisiae Sin3p facilitates DNA double-strand break repair. Proc Natl Acad Sci U S A 101: 1644–1649.
- 24. Tamburini BA, Tyler JK (2005) Localized histone acetylation and deacetylation triggered by the homologous recombination pathway of double-strand DNA repair. Mol Cell Biol 25: 4903–4913.
- 25. Schildkraut E, Miller CA, Nickoloff JA (2006) Transcription of a donor enhances its use during double-strand break-induced gene conversion in human cells. Mol Cell Biol 26: 3098–3105.
- 26. Arakawa H, Buerstedde JM (2004) Immunoglobulin gene conversion: Insights from bursal B cells and the DT40 cell line. Dev Dyn 229: 458–464.
- 27. Maizels N (2005) Immunoglobulin gene diversification. Annu Rev Genet 39: 23–46.
- 28. Thompson CB, Neiman PE (1987) Somatic diversification of the chicken immunoglobulin light chain gene is limited to the rearranged variable gene segment. Cell 48: 369–378.
- 29. Reynaud CA, Anquez V, Grimal H, Weill JC (1987) A hyperconversion mechanism generates the chicken light chain preimmune repertoire. Cell 48: 379–388.
- 30. Reynaud CA, Dahan A, Anquez V, Weill JC (1989) Somatic hyperconversion diversifies the single VH gene of the chicken with a high incidence in the D region. Cell 59: 171–183.
- 31. Buerstedde JM, Arakawa H, Watahiki A, Carninci PP, Hayashizaki YY, et al. (2002) The DT40 web site: Sampling and connecting the genes of a B cell line. Nucleic Acids Res 30: 230–231.
- 32. Yamazoe M, Sonoda E, Hochegger H, Takeda S (2004) Reverse genetic studies of the DNA damage response in the chicken B lymphocyte line DT40. DNA Repair (Amst) 3: 1175–1185.
- 33. Sale JE (2004) Immunoglobulin diversification in DT40: A model for vertebrate DNA damage tolerance. DNA Repair (Amst) 3: 693–702.
- 34. Muramatsu M, Kinoshita K, Fagarasan S, Yamada S, Shinkai Y, et al. (2000) Class switch recombination and hypermutation require activation-induced cytidine deaminase (AID), a potential RNA editing enzyme. Cell 102: 553–563.
- 35. Revy P, Muto T, Levy Y, Geissmann F, Plebani A, et al. (2000) Activation-induced cytidine deaminase (AID) deficiency causes the autosomal recessive form of the Hyper-IgM syndrome (HIGM2). Cell 102: 565–575.
- 36. Arakawa H, Hauschild J, Buerstedde JM (2002) Requirement of the activation-induced deaminase (AID) gene for immunoglobulin gene conversion. Science 295: 1301–1306.
- 37. Harris RS, Sale JE, Petersen-Mahrt SK, Neuberger MS (2002) AID is essential for immunoglobulin V gene conversion in a cultured B cell line. Curr Biol 12: 435–438.
- 38. Petersen-Mahrt SK, Harris RS, Neuberger MS (2002) AID mutates E. coli suggesting a DNA deamination mechanism for antibody diversification. Nature 418: 99–103.
- 39. Bransteitter R, Pham P, Scharff MD, Goodman MF (2003) Activation-induced cytidine deaminase deaminates deoxycytidine on single-stranded DNA but requires the action of RNase. Proc Natl Acad Sci U S A 100: 4102–4107.
- 40. Chaudhuri J, Tian M, Khuong C, Chua K, Pinaud E, et al. (2003) Transcription-targeted DNA deamination by the AID antibody diversification enzyme. Nature 422: 726–730.
- 41. Ramiro AR, Stavropoulos P, Jankovic M, Nussenzweig MC (2003) Transcription enhances AID-mediated cytidine deamination by exposing single-stranded DNA on the nontemplate strand. Nat Immunol 4: 452–456.
- 42. Di Noia J, Neuberger MS (2002) Altering the pathway of immunoglobulin hypermutation by inhibiting uracil-DNA glycosylase. Nature 419: 43–48.
- 43. Rada C, Williams GT, Nilsen H, Barnes DE, Lindahl T, et al. (2002) Immunoglobuin isotype switching is inhibited and somatic hypermutation perturbed in UNG-deficient mice. Curr Biol 12: 1748–1755.
- 44. Imai K, Slupphaug G, Lee WI, Revy P, Nonoyama S, et al. (2003) Human uracil-DNA glycosylase deficiency associated with profoundly impaired immunoglobulin class-switch recombination. Nat Immunol 4: 1023–1028.
- 45. Di Noia JM, Neuberger MS (2004) Immunoglobulin gene conversion in chicken DT40 cells largely proceeds through an abasic site intermediate generated by excision of the uracil produced by AID-mediated deoxycytidine deamination. Eur J Immunol 34: 504–508.
- 46. Saribasak H, Saribasak N, Ipek F, Ellwart J, Arakawa H, et al. (2006) Uracil DNA glycosylase disruption blocks Ig gene conversion and induces transition mutations. J Immunol 176: 365–371.
- 47. Yabuki M, Fujii MM, Maizels N (2005) The MRE11-RAD50-NBS1 complex accelerates somatic hypermutation and gene conversion of immunoglobulin variable regions. Nat Immunol 6: 730–736.
- 48. Larson ED, Cummings WJ, Bednarski DW, Maizels N (2005) MRE11/RAD50 cleaves DNA in the AID/UNG-dependent pathway of immunoglobulin gene diversification. Mol Cell 20: 367–375.
- 49. Takata M, Sasaki MS, Sonoda E, Fukushima T, Morrison C, et al. (2000) The Rad51 paralog Rad51B promotes homologous recombinational repair. Mol Cell Biol 20: 6476–6482.
- 50. Sale JE, Calandrini DM, Takata M, Takeda S, Neuberger MS (2001) Ablation of XRCC2/3 transforms immunoglobulin V gene conversion into somatic hypermutation. Nature 412: 921–926.
- 51. Niedzwiedz W, Mosedale G, Johnson M, Ong CY, Pace P, et al. (2004) The Fanconi anaemia gene FANCC promotes homologous recombination and error-prone DNA repair. Mol Cell 15: 607–620.
- 52. Hatanaka A, Yamazoe M, Sale JE, Takata M, Yamamoto K, et al. (2005) Similar effects of Brca2 truncation and Rad51 paralog deficiency on immunoglobulin V gene diversification in DT40 cells support an early role for Rad51 paralogs in homologous recombination. Mol Cell Biol 25: 1124–1134.
- 53. Yamamoto K, Hirano S, Ishiai M, Morishima K, Kitao H, et al. (2005) Fanconi anemia protein FANCD2 promotes immunoglobulin gene conversion and DNA repair through a mechanism related to homologous recombination. Mol Cell Biol 25: 34–43.
- 54. McIlwraith M, Mcllwraith M, Vaisman A, Liu Y, Fanning E, et al. (2005) Human DNA polymerase eta promotes DNA synthesis from strand invasion intermediates of homologous recombination. Mol Cell 20: 783–792.
- 55. Kawamoto T, Araki K, Sonoda E, Yamashita YM, Harada K, et al. (2005) Dual roles for DNA polymerase eta in homologous DNA recombination and translesion DNA synthesis. Mol Cell 20: 793–799.
- 56. Arakawa H, Saribasak H, Buerstedde JM (2004) Activation-induced cytidine deaminase initiates immunoglobulin gene conversion and hypermutation by a common intermediate. PLoS Biol 2: e179..
- 57. McCormack WT, Thompson CB (1990) Chicken IgL variable region gene conversions display pseudogene donor preference and 5′ to 3′ polarity. Genes Dev 4: 548–558.
- 58. James TC, Eissenberg JC, Craig C, Dietrich V, Hobson A, et al. (1989) Distribution patterns of HP1, a heterochromatin-associated nonhistone chromosomal protein of Drosophila. Eur J Cell Biol 50: 170–180.
- 59. Eissenberg JC, James TC, Foster-Hartnett DM, Hartnett T, Ngan V, et al. (1990) Mutation in a heterochromatin-specific chromosomal protein is associated with suppression of position-effect variegation in Drosophila melanogaster. Proc Natl Acad Sci U S A 87: 9923–9927.
- 60. Nielsen AL, Ortiz JA, You J, Oulad-Abdelghani M, Khechumian R, et al. (1999) Interaction with members of the heterochromatin protein 1 (HP1) family and histone deacetylation are differentially involved in transcriptional silencing by members of the TIF1 family. EMBO J 18: 6385–6395.
- 61. Li Y, Danzer JR, Alvarez P, Belmont AS, Wallrath LL (2003) Effects of tethering HP1 to euchromatic regions of the Drosophila genome. Development 130: 1817–1824.
- 62. Danzer JR, Wallrath LL (2004) Mechanisms of HP1-mediated gene silencing in Drosophila. Development 131: 3571–3580.
- 63. Osipovich O, Milley R, Meade A, Tachibana M, Shinkai Y, et al. (2004) Targeted inhibition of V(D)J recombination by a histone methyltransferase. Nat Immunol 5: 309–316.
- 64. Robinett CC, Straight A, Li G, Willhelm C, Sudlow G, et al. (1996) In vivo localization of DNA sequences and visualization of large-scale chromatin organization using lac operator/repressor recognition. J Cell Biol 135: 1685–1700.
- 65. Bannister AJ, Zegerman P, Partridge JF, Miska EA, Thomas JO, et al. (2001) Selective recognition of methylated lysine 9 on histone H3 by the HP1 chromo domain. Nature 410: 120–124.
- 66. Jacobs SA, Taverna SD, Zhang Y, Briggs SD, Li J, et al. (2001) Specificity of the HP1 chromo domain for the methylated N-terminus of histone H3. EMBO J 20: 5232–5241.
- 67. Lachner M, O'Carroll D, Rea S, Mechtler K, Jenuwein T (2001) Methylation of histone H3 lysine 9 creates a binding site for HP1 proteins. Nature 410: 116–120.
- 68. Verschure PJ, van der Kraan I, de Leeuw W, van der Vlag J, Carpenter AE, et al. (2005) In vivo HP1 targeting causes large-scale chromatin condensation and enhanced histone lysine methylation. Mol Cell Biol 25: 4552–4564.
- 69. Strahl BD, Ohba R, Cook RG, Allis CD (1999) Methylation of histone H3 at lysine 4 is highly conserved and correlates with transcriptionally active nuclei in Tetrahymena. Proc Natl Acad Sci U S A 96: 14967–14972.
- 70. Litt MD, Simpson M, Gaszner M, Allis CD, Felsenfeld G (2001) Correlation between histone lysine methylation and developmental changes at the chicken beta-globin locus. Science 293: 2453–2455.
- 71. Araki H, Ropp PA, Johnson AL, Johnston LH, Morrison A, et al. (1992) DNA polymerase II, the probable homolog of mammalian DNA polymerase epsilon, replicates chromosomal DNA in the yeast Saccharomyces cerevisiae. EMBO J 11: 733–740.
- 72. Nambu Y, Sugai M, Gonda H, Lee CG, Katakai T, et al. (2003) Transcription-coupled events associating with immunoglobulin switch region chromatin. Science 302: 2137–2140.
- 73. Woo CJ, Martin A, Scharff MD (2003) Induction of somatic hypermutation is associated with modifications in immunoglobulin variable region chromatin. Immunity 19: 479–489.
- 74. Li Z, Luo Z, Scharff MD (2004) Differential regulation of histone acetylation and generation of mutations in switch regions is associated with Ig class switching. Proc Natl Acad Sci U S A 101: 15428–15433.
- 75. Odegard VH, Kim ST, Anderson SM, Shlomchik MJ, Schatz DG (2005) Histone modifications associated with somatic hypermutation. Immunity 23: 101–110.
- 76. Wang L, Whang N, Wuerffel R, Kenter A (2006) AID-dependent histone acetylation is detected in immunoglobulin S regions. J Exp Med 203: 215–226.
- 77. Seo H, Masuoka M, Murofushi H, Takeda S, Shibata T, et al. (2005) Rapid generation of specific antibodies by enhanced homologous recombination. Nat Biotechnol 23: 731–735.
- 78. Stankiewicz P, Lupski JR (2002) Genome architecture, rearrangements and genomic disorders. Trends Genet 18: 74–82.
- 79. Batzer MA, Deininger PL (2002) Alu repeats and human genomic diversity. Nat Rev Genet 3: 370–379.
- 80. Aplan PD (2006) Causes of oncogenic chromosomal translocation. Trends Genet 22: 46–55.
- 81. Martens JH, O'Sullivan RJ, Braunschweig U, Opravil S, Radolf M, et al. (2005) The profile of repeat-associated histone lysine methylation states in the mouse epigenome. EMBO J 24: 800–812.
- 82. Tycko B (2003) Genetic and epigenetic mosaicism in cancer precursor tissues. Ann N Y Acad Sci 983: 43–54.
- 83. McMurray MA, Gottschling DE (2003) An age-induced switch to a hyper-recombinational state. Science 301: 1908–1911.
- 84. Preston CR, Flores C, Engels WR (2006) Age-dependent usage of double-strand-break repair pathways. Curr Biol 16: 2009–2015.
- 85. Wiktor-Brown DM, Hendricks CA, Olipitz W, Engelward BP (2006) Age-dependent accumulation of recombinant cells in the mouse pancreas revealed by in situ fluorescence imaging. Proc Natl Acad Sci U S A 103: 11862–11867.
- 86. Peng JC, Karpen GH (2007) H3K9 methylation and RNA interference regulate nucleolar organization and repeated DNA stability. Nat Cell Biol 9: 25–35.
- 87. Shumaker DK, Dechat T, Kohlmaier A, Adam SA, Bozovsky MR, et al. (2006) Mutant nuclear lamin A leads to progressive alterations of epigenetic control in premature aging. Proc Natl Acad Sci U S A 103: 8703–8708.
- 88. Porteus MH, Connelly JP, Pruett SM (2006) A look to future directions in gene therapy research for monogenic diseases. PLoS Genet 2: e133..
- 89. Larson ED, Duquette ML, Cummings WJ, Streiff RJ, Maizels N (2005) MutSalpha binds to and promotes synapsis of transcriptionally activated immunoglobulin switch regions. Curr Biol 15: 470–474.
- 90. Liu Y, Maizels N (2000) Coordinated response of mammalian Rad51 and Rad52 to DNA damage. EMBO Rep 1: 85–90.


text:
This is my guess.
1) Matt Hasselbeck
2) Josh Johnson
3) Pat Devlin
I'm not wild about Hasselbeck, but I think the Niners will look for a vet QB to groom an eventual Starter of the future. The choices of vet QBs that are versed in the WCO are pretty limited. Hasselbeck and McNabb are the only ones I see, and I don't see McNabb meshing with Harbaugh.
With Hasselbeck in the fold, the Niners draft a Devlin and trade for Johnson and see which one can be the starter in the future.

subset: commoncrawl


text:
Hello everyone,
Recently I've installed a plugin (polylan) for multilingual purpose and using two languages (English & Persian/فارسی). everything is translated. but the only thing which is not translated in Persian is "Continue reading". If any one help me how to add translation of this link as well?
Note: my theme is twenty eleven.
Thanks in advance


text:
The story of 11-year old (apparent) Youtube.com phenom (I guess) Jessi Slaughter has once again lead me down the path of old man internet crotchety-ness. To sum it up quickly "Jessi" posted videos spouting her mouth off and internet cesspool message board 4chan.com took the hint and posted questionable pics of Jessi, her address, her phone number and other tidbits of information. This lead to prank phone calls, death threats, and the "Slaughter" family being placed under police protective custody. I know it sounds f-ing nuts.
So that first paragraph is a lot to digest and before we break it down I'll do my best to bring it home for us gamers. Blizzard recently tried to launch the surprisingly forward thinking and horribly myopic "Real ID." "Real ID" would have used your actual name for message board posts. The idea being without the cloak of anonymity (+2 to charisma) people would be less inclined to be a-holes to each other. It didn't go over well.
Back to Jenni then? So in the six-hundred and sixty-six ring circus that is the internet Jenni was a Mad Max Star Mel Gibson star one day and Crazy Mel Gibson the day after. This "Jenni Slaughter" story (or Jenni-nomicon) brings to light one of the problems with our online world. People are idiots (which isn't new) and the internet is filled with them at their most idiotic. So who is the idiot here? and what does this have to do with video gaming.
- Jenni forgot the first rule of stardom - don't believe the hype also ignore your critics
- Jenni's parents forgot to simply monitor what she was doing on the internet. A simple check of the real world information Jenni was letting out would have revealed a few warning signs.
- 4chan.com forgot that picking on an 11 year old is never a good thing. No matter how much Jenni preens and postures she is still an 11 year old girl.
So everyone's a fault. If the internet were a city there is a dark alley in that city (probably behind a porn shop) with a dumpster behind it, the sludge clinging to the underside of that dumpster is 4chan.com. 4chan.com is famous for the vile wretchedness that spills forth from its posters mouths. Why do people feel the need to go on to 4chan.com and unleash such horribly under-thought floods of hate? I'll never know but on 4chan.com they can do it without giving out their identity. The irony of a bunch of anonymous posters releasing an 11-year old's real world identity is apparently lost on 4chan's denizens.
Bringing it back home to gaming. There is a lot of hate fling back and forth on the internet. People with the emotional age of an under-matured 11 year old, spouting their mouths off. If we took a step back and realized that we wouldn't say this in real life. We could get along a lot better. Posters on this site often enter into flame wars that amount to nothing more than a "you suck..." and act like there isn't a problem with it. There is a problem with it and the problem is if you had to be responsible and actually own up to what you said you wouldn't say it. That is the lesson Jenni is learning right now; eventually your actions will come back to haunt you. Everyone has to responsible for what they've done eventually. (insert Lindsey Lohan joke) Blizzard's "Real ID" program was a try at speeding up the "eventually" part.
Also don't pick on 11 year olds, they are 11-friggin-years old, come on.
Oh and if you an 11-year old get off the internet. Go play stick ball or Barbie.


text: The The End of the Battle wiki last edited by pikahyper on 08/11/13 04:13AM View full history It's down to the 11th hour as Yugi and Jonouchi fight to save their friend Anzu's soul! Bandit Keith, the ruthless American duelist, challenges Jonouchi to a rematch using his own God Card, the "Wicked Eraser"! Meanwhile, Yugi faces Yako Tenma, Pegasus's favorite pupil, in a battle which pits god against god!Final Volume


text:
Did you enjoy your fake 10% rally? LOLOLOLOL - NEW 52 weeks lows - I love it ..yum yum yum lolololol
If it makes you shorts happy I took a small loss and lightened up on my BOIL position @ 11.31 just to be on the safe side. I grabbed shares @ 11.15 to bring my average down but didn't take me back to break even so I decided to unload 1/2 my position for a 10 cents loss which is really nothing when you consider the big picture and the gains I made in the last few days. Short may want to get some momentum so I need to keep my powder dry. Congrats to the shorts they win today. ;)
I am long, what at $2.45. I mean it.
...Your not alone...I'm long too again as of yesterday...bought more today...natural gas has not made new lows...lot's of fear mongering going on today...the future is bright...not going to fall another 90% from here...
Everyday is a new day. I closed my UNG position yesterday and sold most of my BOIL and grabbed more BOIL @ 11.15 now averaged in @ 11.41. I am only down a dime so not too scared. I think the shorts could get hurt just like the longs have been at some point and I want to be there when it happens. I only day trade NG or hold for a few days max unless I am sitting fat and a small position where I am willing to add lower. With that said I am prepared to stop out shares for a small loss / gain if I sense more weakness intra-day and will add lower. It takes some serious finesse trading to be long on NG so I appreciate your sentiment but no need to kick people when they are down. It all depends on your time frame.
These message boards crack me up. I banked on shorting silver yesterday with ZSL but didn't have the desire to go the SLV board and gloat about it and tell the longs how much of an idiot they are. I am holding plenty of physical silver and bullish long term still on silver but the opportunity to do a short short term trade was there and I grabbed it and was rewarded accordingly. I feel the same way about NG. I congrat the shorts for giving me an opportunity to go long lower and hopefully they made some money too.
YUM YUM YUM! alot of retail investors got burnt on that headfake.
so whats the point of this post?oh ya i know know its so i can use that ignore button:)


text: I'm trying to run a Windows 98 program in a Windows 98 virtual machine in Windows Virtual PC that requires a USB device (more specifically LEGO Mindstorms Robotics Invention System 2.0 and its USB IR tower that is used to connect with the RCX) and that does not run properly on NT-based computers (the program's readme states that it is "not designed to run on Windows NT/2000"). I know that Windows Virtual PC can redirect USB devices to virtual guests. However, the only menus that I see in the VM window are Action and Tools. The USB menu which shows up in Windows XP Mode that allows me to redirect USB devices does not show up in Windows 98. Why does it not show up?


text:
There are several different ways you can get in touch. However, before you do, you might find the answer to your enquiry in our advice or services sections.
You should always call 999 if you or someone else is in immediate danger, or if the crime is in progress; otherwise please call us on 101.
The Police and Crime Commissioner (PCC) has responsibility for ensuring that our Constabulary is efficient, effective and fair.
You can visit the Police and Crime Commissioner's Website to find out more about about the role of the PCC, the latest news from the PCC's office and how to get in touch.


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New rules would offer you more protection against bill collectors who go over the line. Read more
If you're tired of being hounded by creditors, here's a plan to help you break free.Read more
Deferred student loan debt now counts against you when applying for an FHA loan.Read more
See if walking away from a Veterans Affairs-backed mortgage jeopardizes benefits.Read more
Don't let student loans haunt you after college. Try these tips to right-size your debt.Read more
For some homeowners, it could make sense to refinance with a home equity loan.Read more
Debt consolidation is supposed to help you fix your credit. But much of what is said about the process is bogus.Read more
Bankruptcy will cloud your credit, but you can recover. It takes time and discipline, but it isn't unthinkable.Read more
Consider these three factors before making any consequential decisions, a debt counselor advises.Read more
Dear Debt Adviser, I have about $50,000 of debt on credit cards. My credit rating is still high. I am thinking about a debt consolidation loan. Will that adversely affect my credit rating? -- DT Dear DT, The cliche... Read more


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By Carmen Popwell, Human Resources CommandFebruary 23, 2011
FORT KNOX, Ky. Aca,!" Active duty and active Guard-Reserve lieutenant colonels and majors interested in becoming professors of military science must have their master's or higher degree by May 31 to be considered for positions in the August board at U.S. Army Human Resources Command.
The U.S. Army Cadet Command Professor of Military Science centralized selection board will convene at HRC Aug. 23 and 24. Selected officers can expect to report for duty before the start of the 2012-2013 academic year.
Interested officers must also have completed Intermediate Level Education or Command and General Staff College, have recently deployed in support of Operations Enduring or Iraqi Freedom or New Dawn, have broad experience in command and staff positions, demonstrate moral attributes and personal traits to serve in a position of prestige in an academic environment, meet Army standards of military bearing and appearance, and be able to serve a three-year tour.
Each volunteer must be eligible for a permanent change of station for summer 2012. Officers should update their Officer Record Brief, DA photo, and ensure their undergraduate and graduate transcripts are in their Official Military Personnel File no later than Aug. 1, 2011.
The Professor of Military Science selection board has averaged between 250 to 350 professors a year for four years, according to Phil Ayer, acting chief, Personnel Management Division, Cadet Command, Fort Knox.
Officers meeting these qualifications must provide their branch assignment officers a preference sheet by June 15 to begin the Professor of Military Science board process. Officers may download the Fiscal Year
2012 Professor of Military Science Board preference sheet from the Cadet Command website at:
http://www.rotc.usaac.army.mil/pms-board.htmm, which also lists projected summer 2012 PMS vacancies. Any questions may be directed to the email address: email@example.com.
-- Master Sgt. Christina Steiner contributing


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| By Scott Dunn
Windows users face the greatest security risks today not from flaws in Windows itself but from unpatched media players.
That's because many Windows Secrets readers, according to an online test we sponsored, are running versions of Flash, Java, and QuickTime that are unpatched against the latest security threats.
Readers' systems are rife with outdated add-ons
In two of our recent issues, subscribers to the paid version of the Windows Secrets Newsletter were asked to scan their computers using the Software Inspector, a service of Secunia.com. The scan reveals versions of Windows and builds of applications that have security flaws for which a vendor patch is available.
Contributing editor Ryan Russell, whose columns appeared in the July 26 and Aug. 9 issues of the newsletter, described how we affiliated with Secunia.com, a respected security firm that conducts the tests. We've found that Secunia's service provides such important information that we want all of our free subscribers to take the test as well. A link to the test is provided near the end of this article.
The tests of our paid subscribers showed which applications are the most likely to be installed but unpatched on users' PCs. In the following list, number 1 represents the unpatched application that was found on the greatest number of readers' machines, with higher numbers representing fewer machines:
1. Adobe Flash Player 9.x
2. Sun Java JRE 1.6.x/6.x
3. Macromedia Flash Player 6.x
4. Macromedia Flash Player 8.x
5. Macromedia Flash Player 7.x
6. Apple QuickTime 7.x
7. Macromedia Flash Player 5.x
8. Mozilla Firefox 2.0.x
9. Macromedia Flash Player 4.x
10. Adobe Reader 7.x
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Suzuki Verona Repair and Maintenance
A guide to problems, costs, maintenance and repair for your Suzuki Verona
Verified by mechanics, reported by drivers like you
Ask a mechanic in our community
Never overpay. Check prices with our estimator
Our experts translate recalls into plain English
Suzuki Verona ProblemsTCM software upgrade to improve shift quality
Poor shift quality of the automatic transmission may be improved by upgrading the software in the transmission control module (TCM).
The airbag light may illuminate due to damaged wires under the drivers and/or passenger front seat. Damaged wires should be repaired and properly secured to correct this issue.
Powertrain control module (PCM) software updates are available to correct false fault codes related to the oxygen (O2) sensors and evaporative control (EVAP) system.
Suzuki Verona QuestionsWhat is causes heater blower/fan to make loud growling sound when in use? (1 answer)
Recently whenever the blower or fan is turned on at any of the speed selections for heat or A/C it makes a loud growling or grinding sound. What causes this and how costly is the repair?
There was oil in the spark plug casing
Today when I was driving to work I notice the temperature gauge in my car was all the way up to hot. The car was not over heating at all and it drove fine.
Suzuki Verona RecallsPotential Fire From Instrument Panel
In the affected vehicles excess heat could be generated within the daytime running lights module located in the instrument panel. As a result the module could melt, increasing the risk of a fire. Dealers will replace the daytime running lights module to correct this concern.
The power steering pump pulley may separate from the pump due to inadequate wall thickness of the pulley. This could cause the drive belt to come off, resulting in heavier than normal steering and potential engine overheating and/or stalling. Dealers will inspect and replace the power steering pulley as necessary. This recall is scheduled to begin on December 1, 2005. The Suzuki recall number is KJ.
The brake light switch may be knocked out of adjustment if an upward/rearward force contacts the brake pedal. If this happens, the lights would remain on whether or not the brake pedal is applied, and will remain illuminated even when the key is switched off. This condition could drain the battery if left, and increase the risk of a rear end crash since drivers will not know when the brakes are applied. This condition would also disable the automatic transmission torque converter clutch, cruise control, and the traction control system. Dealers will replace the brake light switch. The recall began March 16, 2005. The Suzuki recall number is KF.


text:
76th Precinct
Carroll Gardens
Cobble Hill–Red Hook
Old gold
A thief stole a 20-year-old Jeep off Degraw Street overnight on June 15 — but then was arrested after the car's owner saw the perp driving around in his wheels!
The victim told police that he had parked the 1990 relic between Clinton and Court streets at around 11 am, but when he returned to it at 9 am the next day, it was gone.
But later in the day, the victim was on Third Avenue near 15th Street when he noticed his rusty wheels in the possession of the thief. He called 911 and the thug was quickly wrangled.
"He caught his own crook!" said a source at the 76th Precinct.
iPhone mugging
A thief punched out a man as he left the Smith-Ninth Street subway station early on June 19, knocking him to the ground and stealing his iPhone.
The victim told cops that he was near Hamilton Avenue at around 1:10 am when a thug approached, punched him and stole his fancy cellular device.
Night moves
A thief broke into a Richards Street home after removing the air-conditioner and stole $530 from a nightstand on June 15.
The victim said that the crime inside her apartment, at Dikeman Street, must have occurred between 4:30 pm and midnight.
Jewel time
A thug got thousands in jewelry and electronics from a Pacific Street apartment on June 16.
The victim told cops that he was not home between 9 am and 11:15 pm, when the burglar entered and stole a TV, a laptop and $2,000 in jewelry from the unit, which is between Henry and Clinton streets.
Lapped up
A couple lost their two laptops from their Kane Street apartment when a burglar broke in on June 18.
No one was home between 8:50 am and 8:15 pm, when the burglar plied his pernicious trade, swiping the computers and a cufflink-filled jewelry box. The house is at Cheever Place.
Summer bummer
In what has become a regular occurance in our police blotter, thugs stole two people's belongings while they frolicked in Red Hook Park last week.
• A man's wallet and cellphone were swiped while he played a hot handball game on June 14. He told cops that he had put his stuff down near Columbia and Bay streets at around 4 pm, but they were all gone an hour later.
• Another man said that he lost his stuff during a soccer game in the park on June 19 from 6:30-8 pm.
— Gersh Kuntzman
©2010 Community News Group
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Michael Kohl
Generally known as citizen428 online.
Main programming language:
Some other languages I enjoy/am interested in:
Swift, Clojure, Common Lisp, Elixir, Erlang, Haskell, Lua, Ocaml, Rust, Smalltalk.
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J, Scheme, Factor, R, 6502 assembler, Python, Forth, Potion and everything else I can get my hands on.
Contributed bug fixes to:
JRuby (x2), Potion
Languages I'm glad I don't have to touch anymore:
Cobol, PL/I, PHP
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our mailing list. It's so wizard.
by Pizzo
10 February, 2012@1:33 pm
1 comments
10 February, 2012@1:33 pm
1 comments
Another leak off that HNIC 3 mixtape, hitting soon.
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Ready in 10 minutes
Simply delicious.
26 people want to try | 31 have favorited
Mix cream cheese with powdered sugar; beat until fluffy, and then fold in half of the whipped cream.
Spread this mixture over the pie crust.
Beat pudding mix with milk for about 2 minutes;
now for the 2nd layer, pour this mixture over the first layer.
Top with remaining whipped topping, and chill.
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Ports in the U.S. and Canadian Pacific Northwest are planning feverishly for the larger cargo surges they know will occur in the coming years from big ships operated by powerful carrier alliances.
United States
Press Releases
26 Jul 2016
US trucking industry pioneer built one of the largest LTL carriers, now UPS Freight.
26 Jul 2016
DP World expects its modest growth in the first half of the year to speed up.
26 Jul 2016
Automating the freight booking process may be a little closer with the launching of an online marketplace for China-U.S. shipments.
25 Jul 2016
Plans for the second of six planned inland ports are moving ahead in Georgia, part of an effort to expand the port of Savannah's intermodal reach into the fast-growing U.S. Southeast market.
25 Jul 2016
The immediate roadblocks, stalemates and impasses the group confronted at the July 15 highlight the deep divisions among the members of the congressional working group and the challenges they'll face in the months ahead.
25 Jul 2016
TransForce's package division increased sales 6 percent, but its trucking business struggled in the second quarter.
25 Jul 2016
Port touts its proximity to distribution centers along Florida's I-4 corridor.
23 Jul 2016
U.S. exports continue to struggle amid low freight rates and the strong dollar.
22 Jul 2016
The fifth-largest U.S. truckload carrier is cutting some shippers loose as they seek deep rate cuts in a soft market.
22 Jul 2016
Direct ChassisLink's acquisition this week of a technology company will broaden the intermodal equipment manager's ability to manage assets.


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WOAH! Keisha has a ridiculous booty on her doesn't she? Those titties and nice, big and natural too! I love the way she sucks my fucking cock... deepthroating it, all the the way to the back. Watching her eyes tear up, yet I know she loves every second of it. Don't even get me started on that pussy either! Goddamn... so tight and warm as an oven. Best part is that she knows I'm a scumbag and I fuck other girls on the side, but stays loyal to me anyhow. I love banging up her sweet cunt and feeling that nice juicy ass bounce up and down on my prick. If only all women were like her! Hell yeah!
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We're back with a big booty white girl! Keisha Grey is a sexy babe with a whole lot of ass. Flawless I tell you! We caught Keisha checking herself out in the mirror before starting the scene. Which was an absolute turn on. Rico loved every second of it. He couldn't wait to stuff his big dick in her pussy. Keisha sucked his dick then hopped on it and went for a long ride. Watching that juicy ass getting pounded is an incredible sight to see. ENjoy!
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Keisha Grey is 20 years old and has one of the juiciest ass in the porn biz. I'm talking about an ass that will make you come within seconds. It's flawless I tell you! In this update of AssParade you're going to see that ass bounce and jiggle all over the cock. By the looks of it, Mike Adreiano seemed to be loving this ass. I know I would. Cum and watch. Enjoy!
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Track using Order ID
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- Track individual Orders
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|Paper Size
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|100 Sheets
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|Gloss,Micro Porous Coating
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|260 gsm
|400 gm
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Vikram Shekhar
Nov 15, 2012
glossy gloss papersHere comes my first flipkart review. Its been wonderful to shop with flipkart and love it everytime and I am always looking to shop for more from flipkart.


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We've had our second nor'easter blow through here in a matter of weeks, and–while the water is great for my newly planted sod–I know that our guests are not thrilled when there's rain on their vacation. The good news is … Continue reading
Category Archives: shopping
Uptown Girl
Looking for something to do tonight? Join this uptown girl as I do the San Marco Crawl, part of Uptown Saturday Nights in St. Augustine. From 5 til 9 tonight, all the art galleries, antique stores, and boutiques along San … Continue reading


text:
Melania Trump Thinks Her Husband Is the 'Sexiest Man in the World'
The Donald's wife, and jewelry designer, answers our usual 21 questions.
Name: Melania Trump
Age: 39
Neighborhood: Upper East Side
Occupation: Mom, wife, entrepreneur. Her new jewelry line debuts tonight on QVC.
Who's your favorite New Yorker, living or dead, real or fictional?
Donald J. Trump.
What's the best meal you've eaten in New York?
One thing I love most about New York is the variety of amazing foods you can eat. My all-time favorite is the Chicken Parmigianino at Jean Georges.
In one sentence, what do you actually do all day in your job?
Being a mommy first, businesswoman, designing my jewelry for QVC, and being a loving, supporting wife.
Would you live here on a $35,000 salary?
This is one of the most expensive cities in the world, so that would very difficult!
What's the last thing you saw on Broadway?
American Idiot, the new Green Day musical. My husband and I went to the premiere and it was great!
Do you give money to panhandlers?
Sometimes when I am out and about. My husband and I are big givers to charity and we are teaching our son Barron all about giving his old toys away to children who might not have any.
What's your drink?
Diet Coke from the classic glass bottles.
How often do you prepare your own meals?
Every morning.
What's your favorite medication?
Sleep and hearing Barron's laugh.
What's hanging above your sofa?
A beautiful portrait of my son Barron.
How much is too much to spend on a haircut?
Sometimes it's fun to splurge on a great haircut or treatment. For just your regular cut above $200.
When's bedtime?
Every day is a different time.
Which do you prefer, the old Times Square or the new Times Square?
The new Times Square. It is much easier to navigate, cleaner, and more people-friendly.
What do you think of Donald Trump?
Sexiest man in the world!!!
What do you hate most about living in New York?
Seeing trash on the streets and parks! With so many people in a small space, we should all be taking extra care to keep our city clean.
Who is your mortal enemy?
When's the last time you drove a car?
Last month in Palm Beach..
How has the Wall Street crash affected you?
I have not felt a huge impact from it, but I know that everyone — the whole world — has been affected by it.
Times, Post, or Daily News?
I read all three of them. They all report the news a little bit differently.
Where do you go to be alone?
My bedroom.
What makes someone a New Yorker?
Living here, loving it, and having the energy to keep up with the city's tempo.


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She was granted the marital home, a vehicle, and about 80% of the assets even though the judge made it look like a 50/50 split. The balance on the house was $50k when it was taken away from me. She refinanced the house when prices went sky high for over $275,000 and then abandoned the house when prices plummeted and kept the money. I have the ability to pay the alimony but she does not have the need for it. She makes over $30,000/year and she chose to lower her standard of living by moving into an apt when she voluntarily walked away from the house in order to keep all the money she had received from the bank. Three lawyers have told me that because i make more money now......leave it alone because I could end up paying her more. Why? She has more than me.
Criminal Defense Attorney
You have already spoken with 3 attorneys about this, and you want another opinion? I guess that is because none of them would tell you what you wanted to hear. If there has been a substantial change in circumstances since the entrance of the final judgment you can file a supplemental petition for modification. But if you are making more now, it might backfire on you.
R. Jason de Groot, Esq., 386-337-8239
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President Bush stood "in the round" Thursday night, virtually surrounded by his Republican Party admirers.
Was it symbolic of what the president hopes occurs Nov. 2 when voters cast their ballots to determine whether he gets re-elected for another four years? Will the country surround him with its support?
Time will tell.
But now the campaign begins as Bush, Vice President Cheney, the thousands of devoted delegates and the army of Republican political operatives leave New York for the campaign trail.
Bush sought to do something a bit different by talking from a stage set up close to the admirers packed into Madison Square Garden. It wasn't overly hokey, which is how some observers described Elizabeth Dole's Oprah-like stroll through the audience at the 1996 GOP convention that nominated her husband, Bob Dole, as its presidential candidate.
As for the speech itself, the president did what he needed to do - spell out how he intends to lead the nation in a second term. He made some important points, such as: The country cannot let up for one moment against international terror; we must simplify the tax code to allow people to continue to buy homes, invest in business ventures, establish tax-free retirement accounts and develop job skills.
"We seek to provide not just a government program," Bush said, "but a path - a path to greater opportunity, more freedom and more control over your life."
This election appears headed for a close result, although it seems highly unlikely it will be a repeat of the 2000 photo finish.
Bush has laid down some important markers. The country will learn in less than two months whether his speech in the round foretold a grand victory or was just so much show biz.
Amarillo Globe-News © 2016. All Rights Reserved. Terms of Service | Privacy Policy / About Our Ads


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November by funandcakeWatch
©2012-2016 funandcake
Just another drawing ....
Im in love with the jacket *___*
Perfect, just like all your other drawings!
wow pretty! ♥
This is really cute I love the blazer!
can't believe it!
it looks really fantastic!
just great!
Keep it up!
can't believe it!
it looks really fantastic!
just great!
Keep it up!
Ooooh, this is beautiful! What did u use Doing this?
Are u a topmodel.biz-Member?
Are u a topmodel.biz-Member?
Thank you! I used colored pencils, regular markers and some gel pen for outlines.
Yes I am a memebr of English and Swedish TM.biz
Yes I am a memebr of English and Swedish TM.biz
Thank you!!!!!!!!! once again
thats so nice i like her hair so beautiful
ow my gosh!!! beautiful!!!♥
Really stylish I was wondering whether you ever have designs that you don't like and don't upload (I doubt it because you're so good!)
haha...of course I have! I sometimes have ideas that I can not execute from first attempt. So I do the same drawing twice.
and thank you
and thank you
Beautiful I love how you draw the hair, and the eyes!! They're incredible :> Well done
Btw, can you maybe do a video where you draw the eyes? I won't copy, thats for sure(!!!!), but I'm just so curious about how you do it
I am so happy when I see people copying my style. This is why I do tutorials. I want to share whatever I have learned with others. Besides I am not creating anything new. I am also trying to copy from other designers But no matter how hard I try I never can do an exact copy instead I develop my own style. It sounds kinda confusing but I hope you understand what I mean..
Nice work!
I love it!!! It's looks cute and vintage


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In this interview I caught up with Dana Simerkoff of HiSoftware to talk about SharePoint, accessibility and how partners can use HiSoftware's expertise to ensure their SharePoint implementations are accessible by design and going forward too.
HiSoftware developed the Accessiblity Kit for SharePoint (AKS) for Microsoft so they know a thing or two about the subject.
Embed this video in your blog:
<iframe src="http://silverlight.services.live.com/invoke/14377/PTV%20HiSoftware/iframe.html" scrolling="no" frameborder="0" style="width:500px; height:400px"></iframe>
Download the video here: