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

Negative Supercoiling Creates Single-Stranded Patches of DNA That Are Substrates for AID–Mediated Mutagenesis

Antibody diversification necessitates targeted mutation of regions within the immunoglobulin locus by activation-induced cytidine deaminase (AID). While AID is known to act on single-stranded DNA (ssDNA), the source, structure, and distribution of these substrates in vivo remain unclear. Using the technique of in situ bisulfite treatment, we characterized these substrates—which we found to be unique to actively transcribed genes—as short ssDNA regions, that are equally distributed on both DNA strands. We found that the frequencies of these ssDNA patches act as accurate predictors of AID activity at reporter genes in hypermutating and class switching B cells as well as in Escherichia coli. Importantly, these ssDNA patches rely on transcription, and we report that transcription-induced negative supercoiling enhances both ssDNA tract formation and AID mutagenesis. In addition, RNaseH1 expression does not impact the formation of these ssDNA tracts indicating that these structures are distinct from R-loops. These data emphasize the notion that these transcription-generated ssDNA tracts are one of many in vivo substrates for AID

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

R-Loop Formation, ssDNA Patches and Switching to IgA in WT and RNaseH1-Expressing CH12F3-2 Cells.

<p>A) Two CH12F3-2 control clones and two CH12F3-2 clones expressing RNaseH1 (hRH1) transfected with a hRH1 expression vector. Western blots were performed for RNaseH1 and β-actin. B) Switch region R-loop formation in control and RNaseH1-expressing CH12F3-2 cells. Extracted genomic DNA from <i>in situ</i> bisulfite treated-CH12F3-2 clones were subjected to PCR amplification using a standard forward primer and a reverse primer that preferentially binds to bisulfite-converted dCs on the top strand 5′ of the μ switch region to specifically amplify bisulfite converted products/R-loops (see <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002518#s4" target="_blank">Materials and Methods</a>). A hypothetical sequence in which all the dCs are converted to dUs is shown on the bottom for reference. The location of the reverse converted primer is depicted as a box. C) R-loop length as expressed as the mean length of contiguous converted dCs in control and RNaseH1-expressing CH12F3-2 cells. Data derived from B. Statistical analysis were conducted using the Student's two tailed t-test * = P = 0.016. D) ssDNA patch frequency, obtained from amplifying the switch region with unconverted primers, shown as ssDNA frequencies in control and RNaseH1-expressing CH12F3-2 cells before (Unstim) and after CSR-stimulation (Stim and hRH1) <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002518#pgen.1002518-Ramachandran1" target="_blank">[46]</a>. ssDNA frequency also analyzed in association with sequences that harbour R-loops (Cont with R-loop and hRH1 with R-loop). E) CSR to IgA in stimulated control and RNaseH1-expressing CH12F3-2 cells. ns = not significant. F) Mutation analysis in WT and hRH1 CH12F3-2 clones of the 5′Sμ region. Left Panel: Mutations are depicted at dC (opened triangle) or dA (closed triangle) along the 5′Sμ region. Middle Panel: mutation spectrum represented for WT and hRH1 samples. Right Panel: Mutation frequencies for WT and hRH1 samples (no significant difference observed).</p

ssDNA Frequencies at Non-Ig Sequences in LPS-Stimulated <i>Ex Vivo</i> Murine B Cells.

<p>A) ssDNA frequencies at the non-transcribed <i>CD4</i> gene, and other indicated genes that are transcribed in B cells. The genes are ordered from Spt5^hi (left) to Spt5^lo (right) and the number of αSpt5 TPM (tags per million sequences) obtained from Pavri <i>et al.</i> (see Table S3 in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002518#pgen.1002518-Pavri1" target="_blank">[3]</a>) is indicated above each gene, which correlates with Spt5 occupancy. B) ssDNA frequency plotted against the mutation frequency of each gene examined in (A). Mutation frequencies for each gene were obtained from Liu <i>et al... </i><a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002518#pgen.1002518-Liu1" target="_blank">[38]</a> (black symbols) and Pavri <i>et al... </i><a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002518#pgen.1002518-Pavri1" target="_blank">[3]</a> (grey symbols). C) Left Panel: ssDNA frequencies at the <i>Btg1</i> gene in pre-B cells, unstimulated B cell and LPS-stimulated B cells. Statistical analysis were performed using the Student's t-test (** = P = 0.0039 comparing ssDNA frequencies at the <i>Btg1</i> gene in pre-B cells and LPS-stimulated B cells). Right Panel: ssDNA patches depicted along the <i>Btg1</i> gene with unique AID-induced mutations. ssDNA patches observed in pre-B cells (open box), unstimulated mature B cells (grey box), and LPS-stimulated B cells (black box) are shown. Unique point mutations depicted at dC (open triangles) or dA (closed triangles) are shown and were obtained from Liu <i>et al... </i><a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002518#pgen.1002518-Liu1" target="_blank">[38]</a>. The location and strand distribution of WRC motifs are depicted as lines along the <i>Btg1</i> gene at the top of this panel.</p