A natural histone H2A variant lacking the Bub1 phosphorylation site and regulated depletion of centromeric histone CENP-A foster evolvability in Candida albicans.

Eukaryotes have evolved elaborate mechanisms to ensure that chromosomes segregate with high fidelity during mitosis and meiosis, and yet specific aneuploidies can be adaptive during environmental stress. Here, we identify a chromatin-based system required for inducible aneuploidy in a human pathogen. Candida albicans utilizes chromosome missegregation to acquire tolerance to antifungal drugs and for nonmeiotic ploidy reduction after mating. We discovered that the ancestor of C. albicans and 2 related pathogens evolved a variant of histone 2A (H2A) that lacks the conserved phosphorylation site for kinetochore-associated Bub1 kinase, a key regulator of chromosome segregation. Using engineered strains, we show that the relative gene dosage of this variant versus canonical H2A controls the fidelity of chromosome segregation and the rate of acquisition of tolerance to antifungal drugs via aneuploidy. Furthermore, whole-genome chromatin precipitation analysis reveals that Centromere Protein A/ Centromeric Histone H3-like Protein (CENP-A/Cse4), a centromeric histone H3 variant that forms the platform of the eukaryotic kinetochore, is depleted from tetraploid-mating products relative to diploid parents and is virtually eliminated from cells exposed to aneuploidy-promoting cues. We conclude that genetically programmed and environmentally induced changes in chromatin can confer the capacity for enhanced evolvability via chromosome missegregation

Investigations into the Targeting and Substrate Specificity of Activation-induced Deaminase

The processes of secondary antibody diversification are initiated by the mutagenic, B cell specific enzyme, Activation-Induced Deaminase (AID). AID deaminates deoxycytosine (dC) that is located in single-stranded DNA (ssDNA) in actively transcribed DNA to initiate the processes of somatic hypermutation (SHM), gene conversion (GCV) and class switch recombination (CSR) at the antibody gene loci. These processes lead to high affinity antibodies and antibodies of various effector functions that are required to efficiently neutralize invading pathogens. It is currently unclear how the antibody genes are specifically targeted by AID over other genes. I found that AID is able to mutate a non-immunoglobulin (Ig) transgene independent of its chromosomal integration site at rates that were above background mutation rates, but were ~10-fold lower than at the antibody variable (V) region. This result suggests that AID can mutate non-Ig genes at low rates, which may explain AID’s role in oncogenesis, but nevertheless shows that AID preferentially mutates the Ig locus over other loci. While it is understood that AID specifically deaminates dC bases in ssDNA, the size, distribution and origin of these ssDNA substrates is unknown. By utilizing a unique in situ sodium bisulfite assay to detect regions of ssDNA in intact nuclei, I characterized ssDNA regions and found that they are accurate predictors of AID activity during the processes of SHM and CSR in mammalian B cells and E.coli. Importantly, with the use of E.coli models, I show that these ssDNA substrates are the product of transcription-induced negative-supercoiled DNA that correlates strongly with the mutagenic activity of AID. While several underlying mechanisms exist to prevent the mistargeting of AID, my findings suggest that by simply gaining access to ssDNA that is produced by transcription-induced negative supercoiling, AID has the potential to mutate non-Ig genes, albeit at lower rates than the antibody V-region.Ph

A natural histone H2A variant lacking the Bub1 phosphorylation site and regulated depletion of centromeric histone CENP-A foster evolvability in Candida albicans.

Eukaryotes have evolved elaborate mechanisms to ensure that chromosomes segregate with high fidelity during mitosis and meiosis, and yet specific aneuploidies can be adaptive during environmental stress. Here, we identify a chromatin-based system required for inducible aneuploidy in a human pathogen. Candida albicans utilizes chromosome missegregation to acquire tolerance to antifungal drugs and for nonmeiotic ploidy reduction after mating. We discovered that the ancestor of C. albicans and 2 related pathogens evolved a variant of histone 2A (H2A) that lacks the conserved phosphorylation site for kinetochore-associated Bub1 kinase, a key regulator of chromosome segregation. Using engineered strains, we show that the relative gene dosage of this variant versus canonical H2A controls the fidelity of chromosome segregation and the rate of acquisition of tolerance to antifungal drugs via aneuploidy. Furthermore, whole-genome chromatin precipitation analysis reveals that Centromere Protein A/ Centromeric Histone H3-like Protein (CENP-A/Cse4), a centromeric histone H3 variant that forms the platform of the eukaryotic kinetochore, is depleted from tetraploid-mating products relative to diploid parents and is virtually eliminated from cells exposed to aneuploidy-promoting cues. We conclude that genetically programmed and environmentally induced changes in chromatin can confer the capacity for enhanced evolvability via chromosome missegregation

Phospho-site mutants of the RNA Polymerase II C-terminal domain alter subtelomeric gene expression and chromatin modification state in fission yeast

Eukaryotic gene expression requires that RNA Polymerase II (RNAP II) gain access to DNA in the context of chromatin. The C-terminal domain (CTD) of RNAP II recruits chromatin modifying enzymes to promoters, allowing for transcription initiation or repression. Specific CTD phosphorylation marks facilitate recruitment of chromatin modifiers, transcriptional regulators, and RNA processing factors during the transcription cycle. However, the readable code for recruiting such factors is still not fully defined and how CTD modifications affect related families of genes or regional gene expression is not well understood. Here, we examine the effects of manipulating the Y S P T S P S heptapeptide repeat of the CTD of RNAP II in Schizosaccharomyces pombe by substituting non-phosphorylatable alanines for Ser2 and/or Ser7 and the phosphomimetic glutamic acid for Ser7. Global gene expression analyses were conducted using splicing-sensitive microarrays and validated via RT-qPCR. The CTD mutations did not affect pre-mRNA splicing or snRNA levels. Rather, the data revealed upregulation of subtelomeric genes and alteration of the repressive histone H3 lysine 9 methylation (H3K9me) landscape. The data further indicate that H3K9me and expression status are not fully correlated, suggestive of CTD-dependent subtelomeric repression mechansims that act independently of H3K9me levels. 1 2 3 4 5 6

ssDNA Frequencies during Induction of CSR in <i>Ex Vivo</i> Murine B Cells.

<p>A) ssDNA patches depicted 3′ of the JH4-region and 5′ of the μ switch region in <i>ex vivo</i> mouse B cells. ssDNA patches observed in unstimulated B cells (grey box), and LPS-stimulated B cells for 48 hours (black box) are shown. Patches are depicted as bars and are distributed to depict top- or bottom-strandedness. Wagon-wheels depict the number of ssDNA patches per sequence for both regions examined under unstimulated or LPS stimulated conditions. B) ssDNA patch lengths of the mouse V-region and 5′μ switch region. Median patch lengths depicted by a black line. C) ssDNA frequencies observed within murine <i>CD4</i> and the 3′JH4 region (left panel) and 5′Sμ (right panel) in unstimulated (open bar), LPS-stimulated (grey bar), and IgM and α-CD40 stimulated (dark grey bars) <i>ex vivo</i> mouse B cells. Statistical analyses were performed using the Student's t-test (Left Panel: ** = P = 0.002 comparing ssDNA frequencies at the CD4 gene and unstimulated 3′JH4 region; * = P = 0.021 comparing ssDNA frequencies at the V-region between unstimulated and LPS-stimulated B cells; Right Panel: * = P = 0.039 comparing ssDNA frequencies at the CD4 gene and the unstimulated 5′Sμ * = P = 0.036 comparing ssDNA frequencies at the 5′Sμ region between unstimulated and LPS-stimulated B cells). D) ssDNA frequencies at the 5′μ switch in CH12F3-2 cells and primary mouse B cells that were untreated or treated with Camptothecin (Campt) or Actinomycin D (Act-D) for 24 hrs. Since no ssDNA patches were observed in the Campt-treated CH12F3-2 cells, the data is presented as if one dC were converted to dT Statistical analysis were performed using the Student's t-test (* = P = 0.023 comparing CH12F3-2 no drug and Act-D LD50; * = P = 0.026 comparing Primary Mouse B cells LPS stim and LPS CPT LD50).</p

ssDNA Patches in the V-Region and GFP Transgene in Ramos Cells.

<p>A) Location and lengths of patches identified in the V-region and GFP transgene. Patches are depicted as black bars and distributed to depict top- or bottom-strandedness. Wagon-wheels depict the number of ssDNA patches per sequence for each gene. B) Cluster plot of patch lengths of ssDNA identified at the V region and GFP genes. Black line depicts the median patch size. C) Strand bias as an expression of ssDNA frequencies on the top strand divided by total ssDNA frequencies for each Ramos clone at the V-region and GFP transgene. A value of 0.5 suggests no strand bias. D) Mutation frequencies at the V-region and GFP genes for individual clones are reported. Statistical analysis was performed using the Mann-Whitney test (** = P = 0.0061). Values for V-region mutation frequencies were obtained from 2 Ramos clones for this study, as well as 5 Ramos clones from previous analyses <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002518#pgen.1002518-Zhang1" target="_blank">[28]</a>, <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002518#pgen.1002518-Parsa1" target="_blank">[65]</a>. E) ssDNA frequencies at the V-region and GFP genes for individual clones. Statistical analyses were performed using the Mann-Whitney test (** = P = 0.0013). F) Linear regression analysis depicting ssDNA frequencies plotted against mutation frequencies for the GFP gene for 12 individual clones (GFP = open circles; VPS GFP = light grey circles; VPL GFP = dark grey circles; see <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002518#pgen.1002518.s002" target="_blank">Figure S2C</a>). X and Y axes are plotted as linear values. G) Quantitative RT-PCR analyses for GFP expression was performed on the clones in which the GFP gene mutated at high rates (<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002518#pgen.1002518.s002" target="_blank">Figure S2C</a>; VPL-6) or low rates (<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002518#pgen.1002518.s002" target="_blank">Figure S2C</a>; GFP-14). See also <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002518#pgen-1002518-g001" target="_blank">Figure 1F</a>. The expression of GFP was plotted relative to GAPDH expression. The relative GFP expression levels of one replicate of clone GFP-14 was set to 1. Statistical analysis was performed using the Student's t-test (*** = P = 0.0009). H) ssDNA frequencies at the V-region in Ramos cells treated with the transcription elongation inhibitor, actinomycin D, or the topoisomerase I inhibitor, camptothecin, for 24 hrs with varying concentrations of inhibitors (LD25 and LD50). Act-D = Actinomycin D; Campt = Camptothecin. Statistical analysis was performed using the Student's t-test (* = P = 0.0115 comparing ssDNA frequencies in Act-D and untreated cells; * = P = 0.0108 comparing ssDNA frequencies in Campt and untreated cells).</p