Project description:We perform RNA-seq in CH12F3 and Lig4-/- CH12F3 cells. The results show the deficiency of Lig4 triggers RIG-I-like receptor, NOD-like receptor, IL-17 signaling, Cytosolic DNA-sensing pathway, and Toll-like receptor signaling pathways. As these pathways play impotant roles in immune resposne, the RNA-seq results suggest Lig4 may regulate the immune repsonse in CH12F3 cells.
Project description:We characterized the DNA sequences spanning telomere fusion junctions focusing on XpYp:17p inter-chromosomal events in HCT116 LIG3-/-:mL3, HCT116 LIG4-/-, WT HCT116 and MRC5HPVE6E7 cells.
Project description:Hematopoietic stem cells (HSCs) maintain the blood system during steady state and in response to stress. As a result, the accumulation of DNA damage has been proposed as a principal factor that contributes to the functional decline in HSC renewal during aging and stress. However, how the HSCs are sustained in the face of increased DNA damage remains unknown. Here, we address this question by studying the role of non-homologous end joining (NHEJ) in HSC. Using a mouse model of a naturally occurring human hypomorphic Lig4 mutation, we show a definitive effect on the steady state of activated HSCs. This population is severely diminished in the Lig4 mutant mice from the loss of selfrenewing HSCs. Our data suggests that defective NHEJ resulting from this Lig4 mutation depletes only the cycling HSCs but not the dormant HSCs, demonstrating that Lig4 is required for maintaining HSC by preserving their cycling pool.
Project description:Classical nonhomologous end-joining (C-NHEJ) repairs DNA double-stranded breaks (DSBs) throughout interphase but is thought to predominate in G1-phase when homologous recombination is unavailable. Complexes containing the Ku70/80 ("Ku") and XRCC4/Ligase IV (Lig4) core C-NHEJ factors are required, respectively, for sensing and joining DSBs. While such factors are exclusively required for joining RAG1/2-initiated DSBs during V(D)J recombination in G1-phase lymphocyte progenitors, cycling cells deficient for core C-NHEJ factors join chromosomal DSBs by alternative end-joining (A-EJ) pathways. Restriction of V(D)J recombination to C-NHEJ has been attributed to RAG-mediated exclusion of A-EJ; however, it remains unclear whether A-EJ is similarly excluded from more general DSBs in G1. Here, we report that Ku actively and robustly suppresses A-EJ of RAG1/2 and two classes of engineered endonuclease-mediated DSBs in G1-arrested progenitor B cell lines. Thus, while targeted DSBs remain as free broken ends in Lig4-deficient G1-arrested progenitor B cells, deletion of Ku70 in Lig4-deficient cells restores DSB rejoining and translocation to levels observed in Ku70-deficient counterparts. Correspondingly, while V(D)J recombination is abrogated in Ligase4-deficient lines, V(D)J-like joining occurs in Ku70-deficient and Ku70/Lig4 double-deficient lines through a translocation-based A-EJ mechanism. We conclude that in G1, Lig4-deficient progenitor B cells are functionally end-joining deficient due to a near complete Ku-dependent block in A-EJ. Thus, the differential impacts of Ku deficiency versus XRCC4/Ligase IV deficiency on V(D)J recombination, severity of neuronal apoptosis, and embryonic development, including Ku-deficiency rescuing Lig4-deficient embryonic lethality, may be explained by Ku-mediated inhibition of A-EJ in the G1 cell cycle phase.