Thomas MacCarthy

Ubiquitylated PCNA plays a role in somatic hypermutation and class-switch recombination and is required for meiotic progression

Somatic hypermutation (SHM) and class-switch recombination (CSR) of Ig genes are dependent upon activation-induced cytidine deaminase (AID)-induced mutations. The scaffolding properties of proliferating cell nuclear antigen (PCNA) and ubiquitylation of its residue K164 have been suggested to play an important role organizing the error-prone repair events that contribute to the AID-induced diversification of the Ig locus. We generated knockout mice for PCNA (Pcna−/−), which were embryonic lethal. Expression of PCNA with the K164R mutation rescued the lethal phenotype, but the mice (Pcna−/−tgK164R) displayed a meiotic defect in early pachynema and were sterile. B cells proliferated normally in Pcna−/−tgK164R mice, but a PCNA-K164R mutation resulted in impaired ex vivo CSR to IgG1 and IgG3, which was associated with reduced mutation frequency at the switch regions and a bias toward blunt junctions. Analysis of the heavy chain V186.2 region after NP-immunization showed in Pcna−/−tgK164R mice a significant reduction in the mutation frequency of A:T residues in WA motifs preferred by polymerase-η (Polη), and a strand-biased increase in the mutation frequency of G residues, preferentially in the context of AID-targeted GYW motifs. The phenotype of Pcna−/−tgK164R mice supports the idea that ubiquitylation of PCNA participates directly in the meiotic process and the diversification of the Ig locus through class-switch recombination (CSR) and somatic hypermutation (SHM).

A core erythroid transcriptional network is repressed by a master regulator of myelo-lymphoid differentiation

Two mechanisms that play important roles in cell fate decisions are control of a “core transcriptional network” and repression of alternative transcriptional programs by antagonizing transcription factors. Whether these two mechanisms operate together is not known. Here we report that GATA-1, SCL, and Klf1 form an erythroid core transcriptional network by co-occupying >300 genes. Importantly, we find that PU.1, a negative regulator of terminal erythroid differentiation, is a highly integrated component of this network. GATA-1, SCL, and Klf1 act to promote, whereas PU.1 represses expression of many of the core network genes. PU.1 also represses the genes encoding GATA-1, SCL, Klf1, and important GATA-1 cofactors. Conversely, in addition to repressing PU.1 expression, GATA-1 also binds to and represses >100 PU.1 myelo-lymphoid gene targets in erythroid progenitors. Mathematical modeling further supports that this dual mechanism of repressing both the opposing upstream activator and its downstream targets provides a synergistic, robust mechanism for lineage specification. Taken together, these results amalgamate two key developmental principles, namely, regulation of a core transcriptional network and repression of an alternative transcriptional program, thereby enhancing our understanding of the mechanisms that establish cellular identity.

Coevolution of robustness, epistasis, and recombination favors asexual reproduction

The prevalence of sexual reproduction remains one of the most perplexing phenomena in evolutionary biology. The deterministic mutation hypothesis postulates that sexual reproduction will be advantageous under synergistic epistasis, a condition in which mutations cause a greater reduction in fitness when combined than would be expected from their individual effects. The inverse condition, antagonistic epistasis, correspondingly is predicted to favor asexual reproduction. To assess this hypothesis, we introduce a finite population evolutionary process that combines a recombination modifier formalism with a gene-regulatory network model. We demonstrate that when reproductive mode and epistasis are allowed to coevolve, asexual reproduction outcompetes sexual reproduction. In addition, no correlation is found between the level of synergistic epistasis and the fixation time of the asexual mode. However, a significant correlation is found between the level of antagonistic epistasis and asexual mode fixation time. This asymmetry can be explained by the greater reduction in fitness imposed by sexual reproduction as compared with asexual reproduction. Our findings present evidence and suggest plausible explanations that challenge both the deterministic mutation hypothesis and recent arguments asserting the importance of emergent synergistic epistasis in the maintenance of sexual reproduction.

The limits of subfunctionalization

BackgroundThe duplication-degeneration-complementation (DDC) model has been proposed as an explanation for the unexpectedly high retention of duplicate genes. The hypothesis proposes that, following gene duplication, the two gene copies degenerate to perform complementary functions that jointly match that of the single ancestral gene, a process also known as subfunctionalization. We distinguish between subfunctionalization at the regulatory level and at the product level (e.g within temporal or spatial expression domains).ResultsIn contrast to what is expected under the DDC model, we use in silico modeling to show that regulatory subfunctionalization is expected to peak and then decrease significantly. At the same time, neofunctionalization (recruitment of novel interactions) increases monotonically, eventually affecting the regulatory elements of the majority of genes. Furthermore, since this process occurs under conditions of stabilizing selection, there is no need to invoke positive selection. At the product level, the frequency of subfunctionalization is no higher than would be expected by chance, a finding that was corroborated using yeast microarray time-course data. We also find that product subfunctionalization is not necessarily caused by regulatory subfunctionalization.ConclusionOur results suggest a more complex picture of post-duplication evolution in which subfunctionalization plays only a partial role in conjunction with redundancy and neofunctionalization. We argue that this behavior is a consequence of the high evolutionary plasticity in gene networks.

IGHV-unmutated and IGHV-mutated chronic lymphocytic leukemia cells produce activation-induced deaminase protein with a full range of biologic functions

Clonal evolution occurs during the course of chronic lymphocytic leukemia (CLL) and activation-induced deaminase (AID) could influence this process. However, this possibility has been questioned in CLL because the number of circulating AID mRNA(+) cells is exceedingly low; synthesis of AID protein by blood CLL cells has not been demonstrated; the full range of AID functions is lacking in unmutated CLL (U-CLL), and no prospective analysis linking AID expression and disease severity has been reported. The results of the present study show that circulating CLL cells and those within secondary lymphoid tissues can make AID mRNA and protein. This production is related to cell division because more AID mRNA was detected in recently divided cells and AID protein was limited to the dividing fraction and was up-regulated on induction of cell division. AID protein was functional because AID(+) dividing cells exhibited more double-stranded DNA breaks, IGH class switching, and new IGHV-D-J mutations. Each of these actions was documented in U-CLL and mutated CLL (M-CLL). Furthermore, AID protein was associated with worse patient outcome and adverse cytogenetics. We conclude that the production of fully functional AID protein by U-CLL and M-CLL cells could be involved in clonal evolution of the disease.

SHMTool: a webserver for comparative analysis of somatic hypermutation datasets.

The somatic hypermutation (SHM) of Immunoglobulin variable (V) regions is a key process in the generation of antibody diversity. The growing number of datasets of point mutations that occur during SHM in mice and humans often include comparisons between wild-type and individuals or strains genetically defective in the repair mechanisms that contribute to SHM. However, it has been difficult to compare the results of different studies because the analyses have not been standardized for criteria such as correction for base composition and the inclusion of unique mutations. If many mutations are involved, the analysis can also be time consuming. To overcome these problems and facilitate a standardized analysis and display of similar data, we present a webserver (SHMTool) for comparing SHM datasets, available at http://scb.aecom.yu.edu/shmtool.

V-region mutation in vitro, in vivo, and in silico reveal the importance of the enzymatic properties of AID and the sequence environment

The somatic hypermutation of Ig variable regions requires the activity of activation-induced cytidine deaminase (AID) which has previously been shown to preferentially deaminate WRC (W = A/T, R = A/G) motif hot spots in in vivo and in vitro assays. We compared mutation profiles of in vitro assays for the 3′ flanking intron of VhJ558-Jh4 region to previously reported in vivo profiles for the same region in the Msh2−/−Ung−/− mice that lack base excision and mismatch repair. We found that the in vitro and in vivo mutation profiles were highly correlated for the top (nontranscribed) strand, while for the bottom (transcribed) strand the correlation is far lower. We used an in silico model of AID activity to elucidate the relative importance of motif targeting in vivo. We found that the mutation process entails substantial complexity beyond motif targeting, a large part of which is captured in vitro. To elucidate the contribution of the sequence environment to the observed differences between the top and bottom strands, we analyzed intermutational distances. The bottom strand shows an approximately exponential distribution of distances in vivo and in vitro, as expected from a null model. However, the top strand deviates strongly from this distribution in that mutations approximately 50 nucleotides apart are greatly reduced, again both in vivo and in vitro, illustrating an important strand asymmetry. While we have confirmed that AID targeting of hot and cold spots is a key part of the mutation process, our results suggest that the sequence environment plays an equally important role.

Overlapping hotspots in CDRs are critical sites for V region diversification.

Significance The somatic hypermutation of immunoglobulin (Ig) variable (V) regions is required to produce high-affinity protective antibodies. Activation-induced deaminase (AID) initiates the accumulation of mutations in the V regions at frequencies that are a million times higher than the normal mutation rates in non-Ig genes. On the basis of the in vivo pattern of mutations in the highly used IGHV3-23*01 human V region and the manipulation of DNA sequence motifs in that V region in a mutating B-cell line and biochemically, we have concluded that particular DNA sequence motifs focus and influence AID activity on those parts of the V region that affect antigen binding and should be considered in vaccine strategies. Activation-induced deaminase (AID) mediates the somatic hypermutation (SHM) of Ig variable (V) regions that is required for the affinity maturation of the antibody response. An intensive analysis of a published database of somatic hypermutations that arose in the IGHV3-23*01 human V region expressed in vivo by human memory B cells revealed that the focus of mutations in complementary determining region (CDR)1 and CDR2 coincided with a combination of overlapping AGCT hotspots, the absence of AID cold spots, and an abundance of polymerase eta hotspots. If the overlapping hotspots in the CDR1 or CDR2 did not undergo mutation, the frequency of mutations throughout the V region was reduced. To model this result, we examined the mutation of the human IGHV3-23*01 biochemically and in the endogenous heavy chain locus of Ramos B cells. Deep sequencing revealed that IGHV3-23*01 in Ramos cells accumulates AID-induced mutations primarily in the AGCT in CDR2, which was also the most frequent site of mutation in vivo. Replacing the overlapping hotspots in CDR1 and CDR2 with neutral or cold motifs resulted in a reduction in mutations within the modified motifs and, to some degree, throughout the V region. In addition, some of the overlapping hotspots in the CDRs were at sites in which replacement mutations could change the structure of the CDR loops. Our analysis suggests that the local sequence environment of the V region, and especially of the CDR1 and CDR2, is highly evolved to recruit mutations to key residues in the CDRs of the IgV region.

Using large-scale perturbations in gene network reconstruction

BackgroundRecent analysis of the yeast gene network shows that most genes have few inputs, indicating that enumerative gene reconstruction methods are both useful and computationally feasible. A simple enumerative reconstruction method based on a discrete dynamical system model is used to study how microarray experiments involving modulated global perturbations can be designed to obtain reasonably accurate reconstructions. The method is tested on artificial gene networks with biologically realistic in/out degree characteristics.ResultsIt was found that a relatively small number of perturbations significantly improve inference accuracy, particularly for low-order inputs of one or two genes. The perturbations themselves should alter the expression level of approximately 50–60% of the genes in the network.ConclusionsTime-series obtained from perturbations are a common form of expression data. This study illustrates how gene networks can be significantly reconstructed from such time-series while requiring only a relatively small number of calibrated perturbations, even for large networks, thus reducing experimental costs.

The evolutionary potential of the Drosophila sex determination gene network.

The evolution of sex determination mechanisms is known to be relatively rapid, though recent evidence indicates that certain parts of the mechanism may be more highly conserved. These characteristics establish the sex determination mechanism as a good candidate for the theoretical study of gene network evolution, particularly of networks involved in development. We investigate the short-term evolutionary potential of the sex determination mechanism in Drosophila melanogaster with the aid of a synchronous logical model. We introduce general theoretical concepts such as a network-specific form of mutation, and a notion of functional equivalence between networks. We apply this theoretical framework to the sex determination mechanism and compare it to a population of random networks, enabling us to find features both general to sex determination networks, and particular to the Drosophila network. In general, sex determination networks exist within large sets of functionally equivalent networks all of which satisfy the sex determination task. These large sets are in turn composed of subsets which are mutationally related, suggesting a high degree of flexibility is available without compromising the core functionality. Two particular characteristics of the Drosophila network are found: (a) a parsimonious use of gene interactions, and (b) the network structure can produce a relatively large number of dynamical pattern variations through single network mutations.