FAM72A antagonizes UNG2 to promote mutagenic repair during antibody maturation.

Activation-induced cytidine deaminase (AID) catalyses the deamination of deoxycytidines to deoxyuracils within immunoglobulin genes to induce somatic hypermutation and class-switch recombination1,2. AID-generated deoxyuracils are recognized and processed by subverted base-excision and mismatch repair pathways that ensure a mutagenic outcome in B cells3-6. However, why these DNA repair pathways do not accurately repair AID-induced lesions remains unknown. Here, using a genome-wide CRISPR screen, we show that FAM72A is a major determinant for the error-prone processing of deoxyuracils. Fam72a-deficient CH12F3-2 B cells and primary B cells from Fam72a-/- mice exhibit reduced class-switch recombination and somatic hypermutation frequencies at immunoglobulin and Bcl6 genes, and reduced genome-wide deoxyuracils. The somatic hypermutation spectrum in B cells from Fam72a-/- mice is opposite to that observed in mice deficient in uracil DNA glycosylase 2 (UNG2)7, which suggests that UNG2 is hyperactive in FAM72A-deficient cells. Indeed, FAM72A binds to UNG2, resulting in reduced levels of UNG2 protein in the G1 phase of the cell cycle, coinciding with peak AID activity. FAM72A therefore causes U·G mispairs to persist into S phase, leading to error-prone processing by mismatch repair. By disabling the DNA repair pathways that normally efficiently remove deoxyuracils from DNA, FAM72A enables AID to exert its full effects on antibody maturation. This work has implications in cancer, as the overexpression of FAM72A that is observed in many cancers8 could promote mutagenesis.

SHLD2 promotes class switch recombination by preventing inactivating deletions within the Igh locus.

The newly identified shieldin complex, composed of SHLD1, SHLD2, SHLD3, and REV7, lies downstream of 53BP1 and acts to inhibit DNA resection and promote NHEJ. Here, we show that Shld2-/- mice have defective class switch recombination (CSR) and that loss of SHLD2 can suppress the embryonic lethality of a Brca1Δ11 mutation, highlighting its role as a key effector of 53BP1. Lymphocyte development and RAG1/2-mediated recombination were unaffected by SHLD2 deficiency. Interestingly, a significant fraction of Shld2-/- primary B-cells and 53BP1- and shieldin-deficient CH12F3-2 B-cells permanently lose expression of immunoglobulin upon induction of CSR; this population of Ig-negative cells is also seen in other NHEJ-deficient cells and to a much lesser extent in WT cells. This loss of Ig is due to recombination coupled with overactive resection and loss of coding exons in the downstream acceptor constant region. Collectively, these data show that SHLD2 is the key effector of 53BP1 and critical for CSR in vivo by suppressing large deletions within the Igh locus.

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.

Differences in the enzymatic efficiency of human and bony fish AID are mediated by a single residue in the C terminus modulating single-stranded DNA binding.

Activation-induced cytidine deaminase (AID) mediates antibody diversification by deaminating deoxycytidines to deoxyuridine within immunoglobulin genes. However, it also generates genome-wide DNA lesions, leading to transformation. Though the biochemical properties of AID have been described, its 3-dimensional structure has not been determined. Hence, to investigate the relationship between the primary structure and biochemical characteristics of AID, we compared the properties of human and bony fish AID, since these are most divergent in amino acid sequence. We show that AIDs of various species have different catalytic rates that are thermosensitive and optimal at native physiological temperatures. Zebrafish AID is severalfold more catalytically robust than human AID, while catfish AID is least active. This disparity is mediated by a single amino acid difference in the C terminus. Using functional assays supported by models of AID core and surface structure, we show that this residue modulates activity by affecting ssDNA binding. Furthermore, the cold-adapted catalytic rates of fish AID result from increased ssDNA binding affinity at lower temperatures. Our work suggests that AID may generate DNA damage with variable efficiencies in different organisms, identifies residues critical in regulating AID activity, and provides insights into the evolution of the APOBEC family of enzymes.

AID associates with single-stranded DNA with high affinity and a long complex half-life in a sequence-independent manner.

Activation-induced cytidine deaminase (AID) initiates secondary antibody diversification processes by deaminating cytidines on single-stranded DNA. AID preferentially mutates cytidines preceded by W(A/T)R(A/G) dinucleotides, a sequence specificity that is evolutionarily conserved from bony fish to humans. To uncover the biochemical mechanism of AID, we compared the catalytic and binding kinetics of AID on WRC (a hot-spot motif, where W equals A or T and R equals A or G) and non-WRC motifs. We show that although purified AID preferentially deaminates WRC over non-WRC motifs to the same degree observed in vivo, it exhibits similar binding affinities to either motif, indicating that its sequence specificity is not due to preferential binding of WRC motifs. AID preferentially deaminates bubble substrates of five to seven nucleotides rather than larger bubbles and preferentially binds to bubble-type rather than to single-stranded DNA substrates, suggesting that the natural targets of AID are either transcription bubbles or stem-loop structures. Importantly, AID displays remarkably high affinity for single-stranded DNA as indicated by the low dissociation constants and long half-life of complex dissociation that are typical of transcription factors and single-stranded DNA binding protein. These findings suggest that AID may persist on immunoglobulin and other target sequences after deamination, possibly acting as a scaffolding protein to recruit other factors.

The H2B deubiquitinase Usp22 promotes antibody class switch recombination by facilitating non-homologous end joining.

Class switch recombination (CSR) has a fundamental function during humoral immune response and involves the induction and subsequent repair of DNA breaks in the immunoglobulin (Ig) switch regions. Here we show the role of Usp22, the SAGA complex deubiquitinase that removes ubiquitin from H2B-K120, in the repair of programmed DNA breaks in vivo. Ablation of Usp22 in primary B cells results in defects in γH2AX and impairs the classical non-homologous end joining (c-NHEJ), affecting both V(D)J recombination and CSR. Surprisingly, Usp22 depletion causes defects in CSR to various Ig isotypes, but not IgA. We further demonstrate that IgG CSR primarily relies on c-NHEJ, whereas CSR to IgA is more reliant on the alternative end joining pathway, indicating that CSR to different isotypes involves distinct DNA repair pathways. Hence, Usp22 is the first deubiquitinase reported to regulate both V(D)J recombination and CSR in vivo by facilitating c-NHEJ.

A system for precise analysis of transcription-regulating elements of immunoglobulin genes.

BACKGROUND: Precise analysis of expression-regulating elements, such as enhancers and insulators, requires that they be tested under reproducible, isogenic conditions. The commonly used methods of transfecting DNA into cell lines and selecting for drug resistance lack the requisite precision, as they yield cell lines in which varying numbers of gene copies have inserted at varying and undefined sites. By contrast, recombination-mediated cassette exchange (RMCE), by which a site-specific recombinase is used to place a single copy of a transgene at a constant chromosomal site of a cell line, offers the necessary precision. Although RMCE is generally applicable, many regulatory elements of interest are tissue-specific in their function and so require cell lines in the appropriate ontogenetic state. RESULTS: As reported here, we have used RMCE in a mouse B hybridoma cell line to establish a system with several additional advantages. To avoid the non-physiological features of prokaryotic DNA, this system uses the immunoglobulin mu heavy chain (IgH) gene from the hybridoma as the reporter. Expression can be measured simply by bulk culture assays (ELISA, Northern blot) and single cell assays (flow cytometry). Expression of the IgH reporter gene varies only 1.5 fold among independent transfectants, and expression is greatly (> 50 fold) increased by inclusion of the IgH intronic enhancer. CONCLUSION: This system is suitable for precise analysis of the regulatory elements of the immunoglobulin loci.

The epigenetic stability of the locus control region-deficient IgH locus in mouse hybridoma cells is a clonally varying, heritable feature.

Cis-acting elements such as enhancers and locus control regions (LCRs) prevent silencing of gene expression. We have shown previously that targeted deletion of an LCR in the immunoglobulin heavy-chain (IgH) locus creates conditions in which the immunoglobulin micro heavy chain gene can exist in either of two epigenetically inherited states, one in which micro expression is positive and one in which micro expression is negative, and that the positive and negative states are maintained by a cis-acting mechanism. As described here, the stability of these states, i.e., the propensity of a cell to switch from one state to the other, varied among subclones and was an inherited, clonal feature. A similar variation in stability was seen for IgH loci that both lacked and retained the matrix attachment regions associated with the LCR. Our analysis of cell hybrids formed by fusing cells in which the micro expression had different stabilities indicated that stability was also determined by a cis-acting feature of the IgH locus. Our results thus show that a single-copy gene in the same chromosomal location and in the presence of the same transcription factors can exist in many different states of expression.

A weakened transcriptional enhancer yields variegated gene expression.

Identical genes in the same cellular environment are sometimes expressed differently. In some cases, including the immunoglobulin heavy chain (IgH) locus, this type of differential gene expression has been related to the absence of a transcriptional enhancer. To gain additional information on the role of the IgH enhancer, we examined expression driven by enhancers that were merely weakened, rather than fully deleted, using both mutations and insulators to impair enhancer activity. For this purpose we used a LoxP/Cre system to place a reporter gene at the same genomic site of a stable cell line. Whereas expression of the reporter gene was uniformly high in the presence of the normal, uninsulated enhancer and undetectable in its absence, weakened enhancers yielded variegated expression of the reporter gene; i.e., the average level of expression of the same gene differed in different clones, and expression varied significantly among cells within individual clones. These results indicate that the weakened enhancer allows the reporter gene to exist in at least two states. Subtle aspects of the variegation suggest that the IgH enhancer decreases the average duration (half-life) of the silent state. This analysis has also tested the conventional wisdom that enhancer activity is independent of distance and orientation. Thus, our analysis of mutant (truncated) forms of the IgH enhancer revealed that the 250 bp core enhancer was active in its normal position, approximately 1.4 kb 3' of the promoter, but inactive approximately 6 kb 3', indicating that the activity of the core enhancer was distance-dependent. A longer segment--the core enhancer plus approximately 1 kb of 3' flanking material, including the 3' matrix attachment region--was active, and the activity of this longer segment was orientation-dependent. Our data suggest that this 3' flank includes binding sites for at least two activators.

Positive and negative transcriptional states of a variegating immunoglobulin heavy chain (IgH) locus are maintained by a cis-acting epigenetic mechanism.

Analyses of transgene expression have defined essential components of a locus control region (LCR) in the J(H)-C(mu) intron of the IgH locus. Targeted deletion of this LCR from the endogenous IgH locus of hybridoma cells results in variegated expression, i.e., cells can exist in two epigenetically inherited states in which the Ig(mu) H chain gene is either active or silent; the active or silent state is typically transmitted to progeny cells through many cell divisions. In principle, cells in the two states might differ either in their content of specific transcription factors or in a cis-acting feature of the IgH locus. To distinguish between these mechanisms, we generated LCR-deficient, recombinant cell lines in which the Ig(mu) H chain genes were distinguished by a silent mutation and fused cells in which the mu gene was active with cells in which mu was silent. Our analysis showed that both parental active and silent transcriptional states were preserved in the hybrid cell, i.e., that two alleles of the same gene in the same nucleus can exist in two different states of expression through many cell divisions. These results indicate that the expression of the LCR-deficient IgH locus is not fully determined by the cellular complement of transcription factors, but is also subject to a cis-acting, self-propagating, epigenetic mark. The methylation inhibitor, 5-azacytidine, reactivated IgH in cells in which this gene was silent, suggesting that methylation is part of the epigenetic mark that distinguishes silent from active transcriptional states.

Efectos de la cocaína y adrenalina sobre la unión neuromuscular de la rata In Vitro: Ausencia de actividad antifatigante / Effects of the cocaine and adrenaline on the neuromuscular of rats in vitro:

Se evaluó el efecto antifatigante in vitro del clorhidrato de cocaína (Benzoilmetilecgonina), sobre la preparación neuromuscular nervio frénico-diafragma de la rata. La fatiga muscular fue inducida por estimulación del nervio frénico con una frecuencia de 3 pulsos/segundo en periodos de 5 minutos, y se tomó como índice de fatiga el decrecimiento de la tensión isométrica. Se encontró que la cocaína no afectó significativamente el proceso de fatiga en el rango de 20ng/ma a 200ug/ml. En esta preparación la interacción entre cocaína y adrenalina fue también nula sobre el proceso de fatiga muscular