Acetyltransferases GCN5 and PCAF Are Required for B Lymphocyte Maturation in Mice.

B lymphocyte development has two DNA recombination processes: V(D)J recombination of the immunoglobulin (Igh) gene variable region, and class switching of the Igh constant regions from IgM to IgG, IgA, or IgE. V(D)J recombination is required for the successful maturation of B cells from pro-B to pre-B to immature-B and then to mature B cells in the bone marrow. CSR occurs outside of the bone marrow when mature B cells migrate to peripheral lymphoid organs, such as spleen and lymph nodes. Both V(D)J recombination and CSR depend on an open chromatin state that makes DNA accessible to specific enzymes, recombination activating gene (RAG), and activation-induced cytidine deaminase (AID). Acetyltransferases GCN5 and PCAF possess redundant functions acetylating histone H3 lysine 9 (H3K9). Here, we generated a mouse model that lacked both GCN5 and PCAF in B cells. Double-deficient mice possessed low levels of mature B cells in the bone marrow and peripheral organs, an accumulation of pro-B cells in bone marrow, and reduced CSR levels. We concluded that both GCN5 and PCAF are required for B-cell development in vivo.

Site-specific characterization of endogenous SUMOylation across species and organs.

Small ubiquitin-like modifiers (SUMOs) are post-translational modifications that play crucial roles in most cellular processes. While methods exist to study exogenous SUMOylation, large-scale characterization of endogenous SUMO2/3 has remained technically daunting. Here, we describe a proteomics approach facilitating system-wide and in vivo identification of lysines modified by endogenous and native SUMO2. Using a peptide-level immunoprecipitation enrichment strategy, we identify 14,869 endogenous SUMO2/3 sites in human cells during heat stress and proteasomal inhibition, and quantitatively map 1963 SUMO sites across eight mouse tissues. Characterization of the SUMO equilibrium highlights striking differences in SUMO metabolism between cultured cancer cells and normal tissues. Targeting preferences of SUMO2/3 vary across different organ types, coinciding with markedly differential SUMOylation states of all enzymes involved in the SUMO conjugation cascade. Collectively, our systemic investigation details the SUMOylation architecture across species and organs and provides a resource of endogenous SUMOylation sites on factors important in organ-specific functions.

PTIP chromatin regulator controls development and activation of B cell subsets to license humoral immunity in mice.

B cell receptor signaling and downstream NF-κB activity are crucial for the maturation and functionality of all major B cell subsets, yet the molecular players in these signaling events are not fully understood. Here we use several genetically modified mouse models to demonstrate that expression of the multifunctional BRCT (BRCA1 C-terminal) domain-containing PTIP (Pax transactivation domain-interacting protein) chromatin regulator is controlled by B cell activation and potentiates steady-state and postimmune antibody production in vivo. By examining the effects of PTIP deficiency in mice at various ages during ontogeny, we demonstrate that PTIP promotes bone marrow B cell development as well as the neonatal establishment and subsequent long-term maintenance of self-reactive B-1 B cells. Furthermore, we find that PTIP is required for B cell receptor- and T:B interaction-induced proliferation, differentiation of follicular B cells during germinal center formation, and normal signaling through the classical NF-κB pathway. Together with the previously identified role for PTIP in promoting sterile transcription at the Igh locus, the present results establish PTIP as a licensing factor for humoral immunity that acts at several junctures of B lineage maturation and effector cell differentiation by controlling B cell activation.

Synthetic lethality between murine DNA repair factors XLF and DNA-PKcs is rescued by inactivation of Ku70.

DNA double-strand breaks (DSBs) are recognized and repaired by the Classical Non-Homologous End-Joining (C-NHEJ) and Homologous Recombination pathways. C-NHEJ includes the core Ku70 and Ku80 (or Ku86) heterodimer that binds DSBs and thus promotes recruitment of accessory downstream NHEJ factors XLF, PAXX, DNA-PKcs, Artemis and other core subunits, XRCC4 and DNA Ligase 4 (Lig4). In the absence of core C-NHEJ factors, DNA repair can be performed by Alternative End-Joining, which likely depends on DNA Ligase 1 and DNA Ligase 3. Genetic inactivation of C-NHEJ factors, such as Ku70, Ku80, XLF, PAXX and DNA-PKcs results in viable mice showing increased levels of genomic instability and sensitivity to DSBs. Knockouts of XRCC4 or Lig4, on the other hand, as well as combined inactivation of XLF and DNA-PKcs, or XLF and PAXX, result in late embryonic lethality in mice, which in most cases correlate with severe apoptosis in the central nervous system. Here, we demonstrate that inactivation of the Ku70 gene rescues the synthetic lethality between XLF and DNA-PKcs, resulting in triple knockout mice that are indistinguishable from Ku70-deficient littermates by size or levels of genomic instability. Moreover, we find that combined inactivation of Ku70 and XLF results in viable mice. Together, these findings suggest that Ku70 is epistatic with XLF and DNA-PKcs and support a model in which inactivation of Ku70 allows DNA lesions to become accessible to alternative DNA repair pathways.

DEK is required for homologous recombination repair of DNA breaks.

DEK is a highly conserved chromatin-bound protein whose upregulation across cancer types correlates with genotoxic therapy resistance. Loss of DEK induces genome instability and sensitizes cells to DNA double strand breaks (DSBs), suggesting defects in DNA repair. While these DEK-deficiency phenotypes were thought to arise from a moderate attenuation of non-homologous end joining (NHEJ) repair, the role of DEK in DNA repair remains incompletely understood. We present new evidence demonstrating the observed decrease in NHEJ is insufficient to impact immunoglobulin class switching in DEK knockout mice. Furthermore, DEK knockout cells were sensitive to apoptosis with NHEJ inhibition. Thus, we hypothesized DEK plays additional roles in homologous recombination (HR). Using episomal and integrated reporters, we demonstrate that HR repair of conventional DSBs is severely compromised in DEK-deficient cells. To define responsible mechanisms, we tested the role of DEK in the HR repair cascade. DEK-deficient cells were impaired for γH2AX phosphorylation and attenuated for RAD51 filament formation. Additionally, DEK formed a complex with RAD51, but not BRCA1, suggesting a potential role regarding RAD51 filament formation, stability, or function. These findings define DEK as an important and multifunctional mediator of HR, and establish a synthetic lethal relationship between DEK loss and NHEJ inhibition.

SCAI promotes DNA double-strand break repair in distinct chromosomal contexts.

DNA double-strand breaks (DSBs) are highly cytotoxic DNA lesions, whose accurate repair by non-homologous end-joining (NHEJ) or homologous recombination (HR) is crucial for genome integrity and is strongly influenced by the local chromatin environment. Here, we identify SCAI (suppressor of cancer cell invasion) as a 53BP1-interacting chromatin-associated protein that promotes the functionality of several DSB repair pathways in mammalian cells. SCAI undergoes prominent enrichment at DSB sites through dual mechanisms involving 53BP1-dependent recruitment to DSB-surrounding chromatin and 53BP1-independent accumulation at resected DSBs. Cells lacking SCAI display reduced DSB repair capacity, hypersensitivity to DSB-inflicting agents and genome instability. We demonstrate that SCAI is a mediator of 53BP1-dependent repair of heterochromatin-associated DSBs, facilitating ATM kinase signalling at DSBs in repressive chromatin environments. Moreover, we establish an important role of SCAI in meiotic recombination, as SCAI deficiency in mice leads to germ cell loss and subfertility associated with impaired retention of the DMC1 recombinase on meiotic chromosomes. Collectively, our findings uncover SCAI as a physiologically important component of both NHEJ- and HR-mediated pathways that potentiates DSB repair efficiency in specific chromatin contexts.

Proteome-wide analysis of arginine monomethylation reveals widespread occurrence in human cells.

The posttranslational modification of proteins by arginine methylation is functionally important, yet the breadth of this modification is not well characterized. Using high-resolution mass spectrometry, we identified 8030 arginine methylation sites within 3300 human proteins in human embryonic kidney 293 cells, indicating that the occurrence of this modification is comparable to phosphorylation and ubiquitylation. A site-level conservation analysis revealed that arginine methylation sites are less evolutionarily conserved compared to arginines that were not identified as modified by methylation. Through quantitative proteomics and RNA interference to examine arginine methylation stoichiometry, we unexpectedly found that the protein arginine methyltransferase (PRMT) family of arginine methyltransferases catalyzed methylation independently of arginine sequence context. In contrast to the frequency of somatic mutations at arginine methylation sites throughout the proteome, we observed that somatic mutations were common at arginine methylation sites in proteins involved in mRNA splicing. Furthermore, in HeLa and U2OS cells, we found that distinct arginine methyltransferases differentially regulated the functions of the pre-mRNA splicing factor SRSF2 (serine/arginine-rich splicing factor 2) and the RNA transport ribonucleoprotein HNRNPUL1 (heterogeneous nuclear ribonucleoprotein U-like 1). Knocking down PRMT5 impaired the RNA binding function of SRSF2, whereas knocking down PRMT4 [also known as coactivator-associated arginine methyltransferase 1 (CARM1)] or PRMT1 increased the RNA binding function of HNRNPUL1. High-content single-cell imaging additionally revealed that knocking down CARM1 promoted the nuclear accumulation of SRSF2, independent of cell cycle phase. Collectively, the presented human arginine methylome provides a missing piece in the global and integrative view of cellular physiology and protein regulation.

Cellular Barcoding Links B-1a B Cell Potential to a Fetal Hematopoietic Stem Cell State at the Single-Cell Level.

Hematopoietic stem cells (HSCs) undergo a functional switch in neonatal mice hallmarked by a decrease in self-renewing divisions and entry into quiescence. Here, we investigated whether the developmental attenuation of B-1a cell output is a consequence of a shift in stem cell state during ontogeny. Using cellular barcoding for in vivo single-cell fate analyses, we found that fetal liver definitive HSCs gave rise to both B-1a and B-2 cells. Whereas B-1a potential diminished in all HSCs with time, B-2 output was maintained. B-1a and B-2 plasticity could be reinitiated in a subset of adult HSCs by ectopic expression of the RNA binding protein LIN28B, a key regulator of fetal hematopoiesis, and this coincided with the clonal reversal to fetal-like elevated self-renewal and repopulation potential. These results anchor the attenuation of B-1a cell output to fetal HSC behavior and demonstrate that the developmental decline in regenerative potential represents a reversible HSC state.

Replication fork stability confers chemoresistance in BRCA-deficient cells.

Cells deficient in the Brca1 and Brca2 genes have reduced capacity to repair DNA double-strand breaks by homologous recombination and consequently are hypersensitive to DNA-damaging agents, including cisplatin and poly(ADP-ribose) polymerase (PARP) inhibitors. Here we show that loss of the MLL3/4 complex protein, PTIP, protects Brca1/2-deficient cells from DNA damage and rescues the lethality of Brca2-deficient embryonic stem cells. However, PTIP deficiency does not restore homologous recombination activity at double-strand breaks. Instead, its absence inhibits the recruitment of the MRE11 nuclease to stalled replication forks, which in turn protects nascent DNA strands from extensive degradation. More generally, acquisition of PARP inhibitors and cisplatin resistance is associated with replication fork protection in Brca2-deficient tumour cells that do not develop Brca2 reversion mutations. Disruption of multiple proteins, including PARP1 and CHD4, leads to the same end point of replication fork protection, highlighting the complexities by which tumour cells evade chemotherapeutic interventions and acquire drug resistance.

A PTIP-PA1 subcomplex promotes transcription for IgH class switching independently from the associated MLL3/MLL4 methyltransferase complex.

Class switch recombination (CSR) diversifies antibodies for productive immune responses while maintaining stability of the B-cell genome. Transcription at the immunoglobulin heavy chain (Igh) locus targets CSR-associated DNA damage and is promoted by the BRCT domain-containing PTIP (Pax transactivation domain-interacting protein). Although PTIP is a unique component of the mixed-lineage leukemia 3 (MLL3)/MLL4 chromatin-modifying complex, the mechanisms for how PTIP promotes transcription remain unclear. Here we dissected the minimal structural requirements of PTIP and its different protein complexes using quantitative proteomics in primary lymphocytes. We found that PTIP functions in transcription and CSR separately from its association with the MLL3/MLL4 complex and from its localization to sites of DNA damage. We identified a tandem BRCT domain of PTIP that is sufficient for CSR and identified PA1 as its main functional protein partner. Collectively, we provide genetic and biochemical evidence that a PTIP-PA1 subcomplex functions independently from the MLL3/MLL4 complex to mediate transcription during CSR. These results further our understanding of how multifunctional chromatin-modifying complexes are organized by subcomplexes that harbor unique and distinct activities.

Lysine succinylation is a frequently occurring modification in prokaryotes and eukaryotes and extensively overlaps with acetylation.

Recent studies have shown that lysines can be posttranslationally modified by various types of acylations. However, except for acetylation, very little is known about their scope and cellular distribution. We mapped thousands of succinylation sites in bacteria (E. coli), yeast (S. cerevisiae), human (HeLa) cells, and mouse liver tissue, demonstrating widespread succinylation in diverse organisms. A majority of succinylation sites in bacteria, yeast, and mouse liver were acetylated at the same position. Quantitative analysis of succinylation in yeast showed that succinylation was globally altered by growth conditions and mutations that affected succinyl-coenzyme A (succinyl-CoA) metabolism in the tricarboxylic acid cycle, indicating that succinylation levels are globally affected by succinyl-CoA concentration. We preferentially detected succinylation on abundant proteins, suggesting that succinylation occurs at a low level and that many succinylation sites remain unidentified. These data provide a systems-wide view of succinylation and its dynamic regulation and show its extensive overlap with acetylation.

Functional intersection of ATM and DNA-dependent protein kinase catalytic subunit in coding end joining during V(D)J recombination.

V(D)J recombination is initiated by the RAG endonuclease, which introduces DNA double-strand breaks (DSBs) at the border between two recombining gene segments, generating two hairpin-sealed coding ends and two blunt signal ends. ATM and DNA-dependent protein kinase catalytic subunit (DNA-PKcs) are serine-threonine kinases that orchestrate the cellular responses to DNA DSBs. During V(D)J recombination, ATM and DNA-PKcs have unique functions in the repair of coding DNA ends. ATM deficiency leads to instability of postcleavage complexes and the loss of coding ends from these complexes. DNA-PKcs deficiency leads to a nearly complete block in coding join formation, as DNA-PKcs is required to activate Artemis, the endonuclease that opens hairpin-sealed coding ends. In contrast to loss of DNA-PKcs protein, here we show that inhibition of DNA-PKcs kinase activity has no effect on coding join formation when ATM is present and its kinase activity is intact. The ability of ATM to compensate for DNA-PKcs kinase activity depends on the integrity of three threonines in DNA-PKcs that are phosphorylation targets of ATM, suggesting that ATM can modulate DNA-PKcs activity through direct phosphorylation of DNA-PKcs. Mutation of these threonine residues to alanine (DNA-PKcs(3A)) renders DNA-PKcs dependent on its intrinsic kinase activity during coding end joining, at a step downstream of opening hairpin-sealed coding ends. Thus, DNA-PKcs has critical functions in coding end joining beyond promoting Artemis endonuclease activity, and these functions can be regulated redundantly by the kinase activity of either ATM or DNA-PKcs.

53BP1 mediates productive and mutagenic DNA repair through distinct phosphoprotein interactions.

The DNA damage response (DDR) protein 53BP1 protects DNA ends from excessive resection in G1, and thereby favors repair by nonhomologous end-joining (NHEJ) as opposed to homologous recombination (HR). During S phase, BRCA1 antagonizes 53BP1 to promote HR. The pro-NHEJ and antirecombinase functions of 53BP1 are mediated in part by RIF1, the only known factor that requires 53BP1 phosphorylation for its recruitment to double-strand breaks (DSBs). Here, we show that a 53BP1 phosphomutant, 53BP18A, comprising alanine substitutions of the eight most N-terminal S/TQ phosphorylation sites, mimics 53BP1 deficiency by restoring genome stability in BRCA1-deficient cells yet behaves like wild-type 53BP1 with respect to immunoglobulin class switch recombination (CSR). 53BP18A recruits RIF1 but fails to recruit the DDR protein PTIP to DSBs, and disruption of PTIP phenocopies 53BP18A. We conclude that 53BP1 promotes productive CSR and suppresses mutagenic DNA repair through distinct phosphodependent interactions with RIF1 and PTIP.

The AID-induced DNA damage response in chromatin.

Chemical modifications to the DNA and histone protein components of chromatin can modulate gene expression and genome stability. Understanding the physiological impact of changes in chromatin structure remains an important question in biology. As one example, in order to generate antibody diversity with somatic hypermutation and class switch recombination, chromatin must be made accessible for activation-induced cytidine deaminase (AID)-mediated deamination of cytosines in DNA. These lesions are recognized and removed by various DNA repair pathways but, if not handled properly, can lead to formation of oncogenic chromosomal translocations. In this review, we focus the discussion on how chromatin-modifying activities and -binding proteins contribute to the native chromatin environment in which AID-induced DNA damage is targeted and repaired. Outstanding questions remain regarding the direct roles of histone posttranslational modifications and the significance of AID function outside of antibody diversity.

The DNA damage- and transcription-associated protein paxip1 controls thymocyte development and emigration.

Histone 3 lysine 4 trimethylation (H3K4me3) is associated with promoters of active genes and found at hot spots for DNA recombination. Here we have shown that PAXIP1 (also known as PTIP), a protein associated with MLL3 and MLL4 methyltransferase and the DNA damage response, regulates RAG-mediated cleavage and repair during V(D)J recombination in CD4(+) CD8(+) DP thymocytes. Loss of PAXIP1 in developing thymocytes diminished Jα H3K4me3 and germline transcription, suppressed double strand break formation at 3' Jα segments, but resulted in accumulation of unresolved T cell receptor α-chain gene (Tcra) breaks. Moreover, PAXIP1 was essential for release of mature single positive (SP) αß T cells from the thymus through transcriptional activation of sphingosine-1-phosphate receptor S1pr1 as well as for natural killer T cell development. Thus, in addition to maintaining genome integrity during Tcra rearrangements, PAXIP1 controls distinct transcriptional programs during DP differentiation necessary for Tcra locus accessibility, licensing mature thymocytes for trafficking and natural killer T cell development.

Loss of ATM kinase activity leads to embryonic lethality in mice.

Ataxia telangiectasia (A-T) mutated (ATM) is a key deoxyribonucleic acid (DNA) damage signaling kinase that regulates DNA repair, cell cycle checkpoints, and apoptosis. The majority of patients with A-T, a cancer-prone neurodegenerative disease, present with null mutations in Atm. To determine whether the functions of ATM are mediated solely by its kinase activity, we generated two mouse models containing single, catalytically inactivating point mutations in Atm. In this paper, we show that, in contrast to Atm-null mice, both D2899A and Q2740P mutations cause early embryonic lethality in mice, without displaying dominant-negative interfering activity. Using conditional deletion, we find that the D2899A mutation in adult mice behaves largely similar to Atm-null cells but shows greater deficiency in homologous recombination (HR) as measured by hypersensitivity to poly (adenosine diphosphate-ribose) polymerase inhibition and increased genomic instability. These results may explain why missense mutations with no detectable kinase activity are rarely found in patients with classical A-T. We propose that ATM kinase-inactive missense mutations, unless otherwise compensated for, interfere with HR during embryogenesis.

Roles for histone H3K4 methyltransferase activities during immunoglobulin class-switch recombination.

Germ-line transcription of an antigen receptor gene segment is an essential feature of the targeting mechanism for DNA double-strand break formation during physiological DNA rearrangements in lymphocytes. Alterations in chromatin structure have long been postulated to regulate accessibility of recombinase activities for lymphocytes to generate antibody diversity; however, whether or not germ-line transcripts are the cause or the effect of chromatin changes at antigen receptor loci is still not clear. Methylation of histone H3 at lysine 4 is one of the most well-studied histone post-translational modifications yet we have only recently begun to understand the significance of the MLL-like H3K4 methyltransferase activities in lymphocyte function. While it is clear during lymphocyte development that H3K4me3 plays a critical role in targeting and stimulating RAG1/2 recombinase activity for V(D)J recombination, recent work suggests roles for this histone mark and different MLL-like complexes in mature B cells during immunoglobulin class-switch recombination. In this review, we focus our discussion to advances on how MLL-like complexes and H3K4 methylation may function during the germ-line transcription and recombinase targeting steps of class-switch recombination. This article is part of a Special Issue entitled: Chromatin in time and space.

PTIP promotes chromatin changes critical for immunoglobulin class switch recombination.

Programmed genetic rearrangements in lymphocytes require transcription at antigen receptor genes to promote accessibility for initiating double-strand break (DSB) formation critical for DNA recombination and repair. Here, we showed that activated B cells deficient in the PTIP component of the MLL3 (mixed-lineage leukemia 3)-MLL4 complex display impaired trimethylation of histone 3 at lysine 4 (H3K4me3) and transcription initiation of downstream switch regions at the immunoglobulin heavy-chain (Igh) locus, leading to defective immunoglobulin class switching. We also showed that PTIP accumulation at DSBs contributes to class switch recombination (CSR) and genome stability independently of Igh switch transcription. These results demonstrate that PTIP promotes specific chromatin changes that control the accessibility of the Igh locus to CSR and suggest a nonredundant role for the MLL3-MLL4 complex in altering antibody effector function.

Multiple organ system defects and transcriptional dysregulation in the Nipbl(+/-) mouse, a model of Cornelia de Lange Syndrome.

Cornelia de Lange Syndrome (CdLS) is a multi-organ system birth defects disorder linked, in at least half of cases, to heterozygous mutations in the NIPBL gene. In animals and fungi, orthologs of NIPBL regulate cohesin, a complex of proteins that is essential for chromosome cohesion and is also implicated in DNA repair and transcriptional regulation. Mice heterozygous for a gene-trap mutation in Nipbl were produced and exhibited defects characteristic of CdLS, including small size, craniofacial anomalies, microbrachycephaly, heart defects, hearing abnormalities, delayed bone maturation, reduced body fat, behavioral disturbances, and high mortality (75-80%) during the first weeks of life. These phenotypes arose despite a decrease in Nipbl transcript levels of only approximately 30%, implying extreme sensitivity of development to small changes in Nipbl activity. Gene expression profiling demonstrated that Nipbl deficiency leads to modest but significant transcriptional dysregulation of many genes. Expression changes at the protocadherin beta (Pcdhb) locus, as well as at other loci, support the view that NIPBL influences long-range chromosomal regulatory interactions. In addition, evidence is presented that reduced expression of genes involved in adipogenic differentiation may underlie the low amounts of body fat observed both in Nipbl+/- mice and in individuals with CdLS.