Recent studies have revealed a structural role for DNA ligase 4 (Lig4) in the maintenance of a repair complex during non-homologous end joining (NHEJ) of DNA double-strand breaks. In cultured cell lines, catalytically inactive Lig4 can partially alleviate the severe DNA repair phenotypes observed in cells lacking Lig4. To study the structural role of Lig4 in vivo, a mouse strain harboring a point mutation to Lig4's catalytic site was generated. In contrast to the ablation of Lig4, catalytically inactive Lig4 mice are born alive. These mice display marked growth retardation and have clear deficits in lymphocyte development. We considered that the milder phenotype results from inactive Lig4 help to recruit another ligase to the repair complex. We next generated a mouse strain deficient for nuclear Lig3. Nuclear Lig3-deficient mice are moderately smaller and have elevated incidences of cerebral ventricle dilation but otherwise appear normal. Strikingly, in experiments crossing these two strains, mice lacking nuclear Lig3 and expressing inactive Lig4 were not obtained. Timed mating revealed that fetuses harboring both mutations underwent resorption, establishing an embryonic lethal genetic interaction. These data suggest that Lig3 is recruited to NHEJ complexes to facilitate end joining in the presence (but not activity) of Lig4.
© The Author(s) 2024. Published by Oxford University Press on behalf of Nucleic Acids Research.

MRL/lpr mice develop systemic lupus erythematosus-like autoimmunity due to defective FAS-mediated apoptosis. We generated Fas lpr mice deficient in EAF2, a transcription elongation-associated factor known to promote apoptosis in germinal center (GC) B cells and crucial for preventing autoimmunity. Contrary to expectations, EAF2 deficiency significantly reduced lymphadenopathy and splenomegaly, extended lifespan, and alleviated nephritis by decreasing renal immune complex deposition. Additionally, EAF2 deficiency markedly reduced accumulation of activated B cells, GC B cells, plasma cells, and the abnormal B220+CD3+ T cells in Fas lpr mice. Further analysis revealed that Eaf2 -/- Fas lpr B cells showed hyperactivation upon various stimulations, followed by increased death. RNA sequencing of the B220+CD3+ cells revealed a downregulation in survival-promoting genes such as Bcl-2 and Akt and an upregulation of proapoptotic genes. We conclude that the combined deficiency in FAS- and EAF2-mediated apoptotic pathways leads to B cell hyperactivation and subsequent death, thereby ameliorating systemic autoimmunity in this model.
© 2024 The Author(s).

Understanding the molecular regulation of arterial and hemogenic specification during early embryonic vascular development is crucial for guiding vascular and hematopoietic regeneration. Accumulating evidence emphasizes the role of long non-coding RNAs (lncRNAs) in cell fate decision. However, the dynamic expression and the potential roles of lncRNAs in early vascular development are still unknown. Here, we first constructed a single-cell landscape of lncRNA expression based on the deeply sequenced tag-based single-cell transcriptome data of early embryonic vascular endothelial cells (VECs). We revealed the contribution of lncRNAs to VEC heterogeneity and identified 295 lncRNAs with specific expression in eight VEC populations. Furthermore, we identified a series of lncRNAs potentially involved in regulating the two waves of arterial specification and hemogenic specification. We uncovered a transient downregulation of H19 in the hemogenic endothelial population during endothelial-to-hematopoietic transition. Additionally, we constructed a transcription factor regulatory network composed of 287 regulons for early VEC development. We further revealed differential activation patterns of regulons and modules in the eight VEC populations, and predicted potential lncRNA-regulon regulatory network. Moreover, unsupervised analysis of the lncRNA expression profile revealed novel VEC subpopulations strongly associated with the maturation of VECs, suggesting the prominent roles of lncRNAs in endothelial maturation. In summary, our study fills the gap in understanding of lncRNA regulatory networks in early vascular development and provides insights into the fields of vascular and hematopoietic regeneration research.

To produce a diverse antibody repertoire, immunoglobulin heavy-chain (Igh) loci undergo large-scale alterations in structure to facilitate juxtaposition and recombination of spatially separated variable (VH), diversity (DH), and joining (JH) genes. These chromosomal alterations are poorly understood. Uncovering their patterns shows how chromosome dynamics underpins antibody diversity. Using tiled Capture Hi-C, we produce a comprehensive map of chromatin interactions throughout the 2.8-Mb Igh locus in progenitor B cells. We find that the Igh locus folds into semi-rigid subdomains and undergoes flexible looping of the VH genes to its 3' end, reconciling two views of locus organization. Deconvolution of single Igh locus conformations using polymer simulations identifies thousands of different structures. This heterogeneity may underpin the diversity of V(D)J recombination events. All three immunoglobulin loci also participate in a highly specific, developmentally regulated network of interchromosomal interactions with genes encoding B cell-lineage factors. This suggests a model of interchromosomal coordination of B cell development.
Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.

The nonhomologous end-joining (NHEJ) pathway is a major DNA double-strand break repair pathway in mammals and is essential for lymphocyte development. Ku70 and Ku80 heterodimer (KU) initiates NHEJ, thereby recruiting and activating the catalytic subunit of DNA-dependent protein kinase (DNA-PKcs). While DNA-PKcs deletion only moderately impairs end-ligation, the expression of kinase-dead DNA-PKcs completely abrogates NHEJ. Active DNA-PK phosphorylates DNA-PKcs at two clusters-PQR around S2056 (S2053 in mouse) and ABCDE around T2609. Alanine substitution at the S2056 cluster moderately compromises end-ligation on plasmid-based assays. But, mice carrying alanine substitution at all five serine residues within the S2056 cluster (DNA-PKcsPQR/PQR) display no defect in lymphocyte development, leaving the physiological significance of S2056 cluster phosphorylation elusive. Xlf is a nonessential NHEJ factor. Xlf -/- mice have substantial peripheral lymphocytes that are completely abolished by the loss of DNA-PKcs, the related ATM kinases, other chromatin-associated DNA damage response factors (e.g., 53BP1, MDC1, H2AX, and MRI), or RAG2-C-terminal regions, suggesting functional redundancy. While ATM inhibition does not further compromise end-ligation, here we show that in XLF-deficient background, DNA-PKcs S2056 cluster phosphorylation is critical for normal lymphocyte development. Chromosomal V(D)J recombination from DNA-PKcsPQR/PQRXlf -/- B cells is efficient but often has large deletions that jeopardize lymphocyte development. Class-switch recombination junctions from DNA-PKcsPQR/PQRXlf -/- mice are less efficient and the residual junctions display decreased fidelity and increased deletion. These findings establish a role for DNA-PKcs S2056 cluster phosphorylation in physiological chromosomal NHEJ, implying that S2056 cluster phosphorylation contributes to the synergy between XLF and DNA-PKcs in end-ligation.