IntroductionA fundamental hallmark of humoral immunity is the ability to produce class-switched antibodies that have enhanced affinity for antigen. This process, termed affinity maturation, allows clonally selected B cells to refine their response to theoretically target any antigen with high specificity. After activation, B cells mutate the immunoglobulin (Ig) locus in a process known as somatic hypermutation (SHM). 1 Mutated clones that have acquired increased affinity for antigen preferentially expand over their low-affinity counterparts. The selection process occurs in the germinal center (GC), a transient microenvironment that forms in secondary lymphoid tissue shortly after antigen exposure. 2 The GC provides a competitive setting for B cells whereby ineffectual clones are actively cleared from the system via apoptosis, although the processes that govern this selection still remain vague.SHM and class switch recombination (CSR) are initiated by the enzyme activation-induced cytidine deaminase (AID), 3,4 which deaminates cytidines to uridines specifically at the Ig locus. 5-10 AID-generated uridines are then engaged by various DNA repair pathways that either lead to the generation of point mutations in the antibody variable region or recombinogenic events that lead to CSR. AID Ϫ/Ϫ mice lack mutations at their Ig locus and are incapable of producing class-switched antibodies. 4 Interestingly, these mice were previously shown to harbor an abnormally high proportion of splenic GC B cells. 11 This phenotype is also recapitulated in humans with AID deficiencies. 3 Although a link between reduced gut immunity (resulting from an inability to produce mucosal IgA) and peripheral GC formation was hypothesized to account for these abnormalities, this remains to be conclusively proven, and the possibility of a B cell-intrinsic effect that could explain the profound expansion of GC B cells has not been examined.GC B cells represent a unique lymphoid compartment of actively proliferating cells where numerous apoptotic factors must synergize to induce the elimination of nonproductive clones. Factors contributing to the intrinsic pathway of apoptosis, such as Bcl-2, Bcl-x L , and Bim, 12-17 and the extrinsic pathway, such as Fas, [18][19][20][21][22] have been implicated in GC selection. The unique physiology of these cells makes them highly susceptible to disease progression, often serving as etiologic sites for autoimmune and malignant B cells. 13,[23][24][25][26] Recent evidence has implicated AID as a fundamental contributor to the genetic aberrations that lead to these disease phenotypes. 24,[27][28][29] Given the importance of apoptosis as a parameter for both GC B-cell selection and lymphomagenesis, we investigated the relationship between AID-induced DNA mutation and cell death to understand how this enzyme may impinge on survival and death within the GC niche. Here we report that many of the potentially harmful AID-induced genetic alterations may indeed lead to the death of these cells within the GC. Methods Mice...

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.

In , transcripts derived from the pericentromeric and repeats promote heterochromatin formation via RNAi as well as an RNAi-independent mechanism involving the RNA polymerase II (RNAPII)-associated RNA-binding protein Seb1 and RNA processing activities. We show that Seb1 promotes long-lived RNAPII pauses at pericentromeric repeat regions and that their presence correlates with the heterochromatin-triggering activities of the corresponding and DNA fragments. Globally increasing RNAPII stalling by other means induces the formation of novel large ectopic heterochromatin domains. Such ectopic heterochromatin occurs even in cells lacking RNAi. These results uncover Seb1-mediated polymerase stalling as a signal necessary for heterochromatin nucleation.

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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.

Antibody diversification processes play a major role in protecting humans from pathogens. Somatic hypermutation and gene conversion increase the affinity of pathogen-specific antibodies by changing the sequence within antibody variable genes, while the class switch recombination (CSR) process changes the antibody's effector function by replacing the constant region of the antibody gene with a different constant region. Activation-induced cytidine deaminase (AID) initiates each of these three processes by deaminating cytidines within antibody genes, while a host of other DNA transacting factors are involved in either creating new mutations or repairing DNA lesions introduced during these processes. This review will discuss the main features of antibody diversification and their role in lymphomagenesis, highlight outstanding issues and questions that remain in the field, and discuss our contributions to this field.

peer-reviewed) is the author/funder. All rights reserved. No reuse allowed without permission.The copyright holder for this preprint (which was not . http://dx.doi.org/10.1101/217968 doi: bioRxiv preprint first posted online Nov. 11, 2017; 3 To understand how Seb1 interfaces with the transcription of dg and dh repeats to promote heterochromatin, we employed a previously identified viable, heterochromatindefective allele, seb1-1 15 . When combined with mutants in the RNAi machinery, seb1-1 eliminates pericentromeric heterochromatin, while the corresponding single mutants decrease H3K9me, indicative of partially redundant pathways 15 . We examined transcription of heterochromatin at single-nucleotide resolution and tested the impact of the seb1-1 allele using Nascent Elongating Transcript Sequencing (NET-Seq) 20 . To analyze the intrinsic transcriptional properties of heterochromatic sequences prior to the establishment of heterochromatin assembly, we used the clr4∆ mutant, which lacksH3K9me and displays full derepression of most silenced chromatin regions. We compared this strain to a clr4∆ seb1-1 double mutant to assess the impact of seb1-1.We first examined the effect of seb1-1 on transcription of non-heterochromatic regions peer-reviewed) is the author/funder. All rights reserved. No reuse allowed without permission.The copyright holder for this preprint (which was not . http://dx.doi.org/10.1101/217968 doi: bioRxiv preprint first posted online Nov. 11, 2017; 4 Extended Data Figure 1). The seb1-1 mutation causes a reduced median 5' traveling ratio and an increased median 3' traveling ratio for both clusters ( for p values). These data indicate that the seb1-1 allele leads to decreased RNAPII pausing at gene 5' ends with an associated increased 3' signal; the latter may be due to polymerase release from upstream pauses. peer-reviewed) is the author/funder. All rights reserved. No reuse allowed without permission.The copyright holder for this preprint (which was not . http://dx.doi.org/10.1101/217968 doi: bioRxiv preprint first posted online Nov. 11, 2017; 5 To compare the binding of Seb1 across transcript classes, we computed the fraction of RNA covered by Seb1 PAR-CLIP read clusters. We observed a ~12-fold higher PAR-CLIP cluster coverage for pericentromeric repeat intervals than for coding gene intervals (Figure 2d and Extended Data Figure 4a). Non-coding RNAs display the highest coverage at a mean level ~100-fold higher than that of coding-genes (Extended Data Figure 4b).We next examined the NET-Seq profiles of pericentromeric heterochromatin sequences of clr4∆ and clr4∆ seb1-1 strains (replicate experiments were conducted). The copyright holder for this preprint (which was not . http://dx.doi.org/10.1101/217968 doi: bioRxiv preprint first posted online Nov. 11, 2017; 6 Figure 4d).These data reveal detectable Seb1-dependent RNAPII pauses in pericentromeric sequences. (Figure 2g). Silencing of ura4 + was determined using YS-FOA plates, which selects for ura4+ repression. The insert of Fragment ...

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