The genome is constantly exposed to DNA damage from endogenous and exogenous sources. Fine modulation of DNA repair, chromatin remodeling, and transcription factors is necessary for protecting genome integrity, but the precise mechanisms are still largely unclear. We found that after ionizing radiation (IR), global trimethylation of histone H3 at lysine 4 (H3K4me3) was decreased at an early (5 min) post-IR phase but increased at an intermediate (180 min) post-IR phase in both human and mouse hematopoietic cells. We demonstrated that PTIP, a component of the MLL histone methyltransferase complex, is required for H3K4me3 upregulation in the intermediate post-IR phase and promotes cell cycle arrest by epigenetically inducing a cell cycle inhibitor, PRDM1. In addition, we found that PTIP expression is specifically downregulated in acute myeloid leukemia patients. These findings collectively suggest that the PTIP-PRDM1 axis plays an essential role in proper DNA damage response and its deregulation contributes to leukemogenesis.

暂无摘要。

Homologous-recombination deficiency due to breast cancer 1/2 (BRCA1/2) mutations or mimicking BRCA1/2 mutations confer synthetic lethality with poly-(ADP)-ribose polymerase 1/2 inhibitors. The chromatin regulator Pax2 transactivation domain interacting protein (PTIP) promotes stalled replication fork degradation in BRCA1-deficient cells, but the underlying mechanism by which PTIP regulates stalled replication fork stability is unclear. Here, we performed a series of in vitro analyses to dissect the function of UFMylation in regulating fork stabilization in BRCA1-deficient cells. By denaturing co-immunoprecipitation, we first found that replication stress can induce PTIP UFMylation. Interestingly, this post-translational modification promotes end resection and degradation of nascent DNA at stalled replication forks in BRCA1-deficient cells. By cell viability assay, we found that PTIP-depleted and UFL1-depleted BRCA1 knockdown cells are less sensitive to poly-(ADP)-ribose polymerase inhibitors than the siRNA targeting negative control BRCA1-deficient cells. These results identify a new mechanism by which PTIP UFMylation confers chemoresistance in BRCA1-deficient cells.

Esophageal squamous cell carcinoma (ESCC) is a highly aggressive malignancy and treatment failure is largely due to metastasis and invasion. Aberrant tumor cell adhesion is often associated with tumor progression and metastasis. However, the exact details of cell adhesion in ESCC progression have yet to be determined. In our study, the clinical relevance of Pax2 transactivation domain-interacting protein (PTIP/PAXIP1) was analyzed by immunohistochemistry of ESCC tissues. We found that low expression of PTIP was associated with lymph node metastasis in ESCC, and loss-of-function approaches showed that depletion of PTIP promoted ESCC cell migration and invasion both in vitro and in vivo. Analysis integrating RNA-seq and ChIP-seq data revealed that PTIP directly regulated ephrin type-A receptor 2 (EphA2) expression in ESCC cells. Moreover, PTIP inhibited EphA2 expression by competing with Fosl2, which attenuated the invasion ability of ESCC cells. These results collectively suggest that PTIP regulates ESCC invasion through modulation of EphA2 expression and hence presents a potential therapeutic target for its treatment.

Duplication of the genome poses one of the most significant threats to genetic integrity, cellular fitness, and organismal health. Therefore, numerous mechanisms have evolved that maintain replication fork stability in the face of DNA damage and allow faithful genome duplication. The fission yeast BRCT-domain-containing protein Brc1, and its budding yeast orthologue Rtt107, has emerged as a \"hub\" factor that integrates multiple replication fork protection mechanisms. Notably, the cofactors and pathways through which Brc1, Rtt107, and their human orthologue (PTIP) act have appeared largely distinct. This either represents true evolutionary functional divergence, or perhaps an incomplete genetic and biochemical analysis of each protein. In this regard, we recently showed that like Rtt107, Brc1 supports key functions of the Smc5-Smc6 complex, including its recruitment into DNA repair foci, chromatin association, and SUMO ligase activity. Furthermore, fission yeast cells lacking the Nse5-Nse6 genome stability factor were found to exhibit defects in Smc5-Smc6 function, similar to but more severe than those in cells lacking Brc1. Here, we place these findings in context with the known functions of Brc1, Rtt107, and Smc5-Smc6, present data suggesting a role for acetylation in Smc5-Smc6 chromatin loading, and discuss wider implications for genome stability.

DNA damage response mediator protein 53BP1 is a key regulator of non-homologous end-joining (NHEJ) repair. 53BP1 protects DNA broken ends from resection by recruiting two downstream factors, RIF1 (RAP1-interacting factor 1) and PTIP (Pax transactivation domain-interacting protein), to double-stranded breaks (DSBs) via ATM (ataxia telangiectasia mutated)-mediated 53BP1 phosphorylation, and competes with BRCA1-mediated homologous recombination (HR) repair in G1 phase. In contrast, BRCA1 antagonizes 53BP1-direct NHEJ repair in S/G2 phases. We and others have found that BRCA1 prevents the translocation of RIF1 to DSBs in S/G2 phases; however, the underlying mechanism remains unclear. Here we show that efficient ATM-dependent 53BP1 phosphorylation is restricted to the G1 phase of the cell cycle, as a consequence RIF1 and PTIP accumulation at DSB sites only occur in G1 phase. Mechanistically, both BRCT and RING domains of BRCA1 are required for the inhibition of 53BP1 phosphorylation in S and G2 phases. Thus, our findings reveal how BRCA1 antagonizes 53BP1 signaling to ensure that HR repair is the dominant repair pathway in S/G2 phases.

Deoxyribonucleic acid double-strand breaks (DSBs) are cytotoxic lesions that must be repaired either through homologous recombination (HR) or non-homologous end-joining (NHEJ) pathways. DSB repair is critical for genome integrity, cellular homeostasis and also constitutes the biological foundation for radiotherapy and the majority of chemotherapy. The choice between HR and NHEJ is a complex yet not completely understood process that will entail more future efforts. Herein we review our current understandings about how the choice is made over an antagonizing balance between p53-binding protein 1 and breast cancer 1 in the context of cell cycle stages, downstream effects, and distinct chromosomal histone marks. These exciting areas of research will surely bring more mechanistic insights about DSB repair and be utilized in the clinical settings.

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.

BACKGROUND: Kabuki syndrome (KS) is a complex multisystem developmental disorder associated with mutation of genes encoding histone-modifying proteins. In addition to craniofacial, intellectual, and cardiac defects, KS is also characterized by humoral immune deficiency and autoimmune disease, yet no detailed molecular characterization of the KS-associated immune phenotype has been reported.
OBJECTIVE: We sought to characterize the humoral immune defects found in patients with KS with lysine methyltransferase 2D (KMT2D) mutations.
METHODS: We comprehensively characterized B-cell function in a cohort (n = 13) of patients with KS (age, 4 months to 27 years).
RESULTS: Three quarters (77%) of the cohort had a detectable heterozygous KMT2D mutation (50% nonsense, 20% splice site, and 30% missense mutations), and 70% of the reported mutations are novel. Among the patients with KMT2D mutations (KMT2D(Mut/+)), hypogammaglobulinemia was detected in all but 1 patient, with IgA deficiency affecting 90% of patients and a deficiency in at least 1 other isoform seen in 40% of patients. Numbers of total memory (CD27(+)) and class-switched memory B cells (IgM(-)) were significantly reduced in patients with KMT2D(Mut/+) mutations compared with numbers in control subjects (P < .001). Patients with KMT2D(Mut/+) mutations also had significantly reduced rates of somatic hypermutation in IgG (P = .003) but not IgA or IgM heavy chain sequences. Impaired terminal differentiation was noted in primary B cells from patients with KMT2D(Mut/+) mutations. Autoimmune pathology was observed in patients with missense mutations affecting the SET domain and its adjacent domains.
CONCLUSIONS: In patients with KS, autosomal dominant KMT2D mutations are associated with dysregulation of terminal B-cell differentiation, leading to humoral immune deficiency and, in some cases, autoimmunity. All patients with KS should undergo serial clinical immune evaluations.

PARP inhibitors (PARPis) are being used in patients with BRCA1/2 mutations. However, doubly deficient BRCA1(-/-)53BP1(-/-) cells or tumors become resistant to PARPis. Since 53BP1 or its known downstream effectors, PTIP and RIF1 (RAP1-interacting factor 1 homolog), lack enzymatic activities directly implicated in DNA repair, we decided to further explore the 53BP1-dependent pathway. In this study, we uncovered a nuclease, Artemis, as a PTIP-binding protein. Loss of Artemis restores PARPi resistance in BRCA1-deficient cells. Collectively, our data demonstrate that Artemis is the major downstream effector of the 53BP1 pathway, which prevents end resection and promotes nonhomologous end-joining and therefore directly competes with the homologous recombination repair pathway.