This week we profile a recent publication in PNAS from Dr. Frederick Alt’s
lab at Boston Children’s Hospital and Dr. Richard Frock’s lab at Stanford.
Can you provide a brief overview of your lab’s current research focus?
This project was initiated in the Alt lab by Vipul Kumar who was then an M.D/ Ph.D. student as a collaboration with Richard Frock who was a postdoctoral fellow to extend their earlier published DNA end-joining study. The currently published study, which had moved to a very interesting point, was finished over the past few years by Zhuoyi (Johnny) Liang a current post doc in the Alt lab and Marie Le Bouteiller, a post doc in the Frock lab at Stanford. So, the final work resulted from a long-term collaboration among the various lead and senior authors at Boston Children’s and Stanford, who all collaborated on writing the final manuscript.
The Frock lab is interested in understanding how various cellular contexts affect the repair of DNA ends formed and how knowledge gained can be leveraged to direct repair outcomes for genome editing and cancer therapeutics. A specific area of interest is understanding how different DNA end joining pathways are selected for repair. To address these questions, the lab will continue to develop and apply new applications of the high-throughput genome-wide translocation sequencing (HTGTS) cloning platform that had been developed originally in the Alt lab.
The Alt lab continues to address the mechanisms by which DNA ends are joined during V(D)J recombination in G1-arrested progenitor B lymphocytes and during IgH class switch recombination in cycling mature B lymphocytes, as well as in developing neurons. Their work is currently focused on the contribution of genome architecture and, in particular, cohesin-mediated loop extrusion to bring together over long-ranges DNA target sequences for cleavage and then to also mediate appropriate joining of DNA DSB ends during these processes. Various new applications of the HTGTS methods are also being used to facilitate these studies and also to study the process of somatic hyper-mutation in antibody maturation.
What is the significance of the findings in this publication?
The joining of V(D)J recombination generated DSBs in G1-phase progenitor B lymphocytes (pro-B cells) has long been known to occur by the classical non-homologous DNA end-joining (C-NHEJ) pathway and to exclude alternative end-joining (A-EJ) pathways that can robustly join DSBs in cycling cells. Earlier studies indicated that the V(D)J recombination complex specifically guides V(D)J DSBs into the C-NHEJ pathway. The recently published project from the Frock and Alt labs focused on further elucidating mechanisms that might influence choice of C-NHEJ versus A-EJ pathways in G1-phase progenitor lymphocytes during joining of V(D)J recombination DSBs and to determine whether the C-NHEJ predominance also applied to the joining of other types of DSBs in G1 phase pro-B cells.
The current study exploited a new variation of the HTGTS platform, HTGTS-JoinT-seq, which additionally captures rejoined DNA ends arising from a solitary DSB at high resolution. HTGTS-JoinT-seq was used to study in great detail the joining of Cas9, ZFN, and RAG1/2 endonuclease-generated broken ends in G1-phase progenitor lymphocytes. The studies revealed that the Ku70/Ku80 “Ku” DNA DSB end-binding protein suppresses a DNA Ligase III dependent A-EJ pathway that can function on these various types of DNA ends, including V(D)J recombination DSB ends, in G1-phase pro-B cells when Ku is absent. This conclusion was supported by 1) the virtual absence of DNA end-joining G1-arrested pro-B lines in which DNA Ligase IV is deleted and 2) the restoration of substantial end-joining when Ku70 was deleted from Ligase IV-deficient pro-B lines in G1-arrested pro-B lines, and 3), the substantial A-EJ of cycling pro-B lines in the absence of Ligase IV. Thus, the Ku70/80 C-NHEJ DSB recognition complex, directs repair of various types of ectopic and physiological DSBs toward C-NHEJ by suppressing A-EJ in G1-phase cells. These findings indicate this Ku activity can reinforce the selectivity of RAG-endonuclease V(D)J recombination generated DSBs to the C-NHEJ pathway. The findings of this study also provide an explanation for how Ku-deficiency rescues the embryonic lethality and wide-spread neuronal apoptosis phenotypes of Ligase IV-deficient mice, namely by allowing sufficient A-EJ to rescue the wide-spread neuronal apoptosis phenotype due to lack of DSB end joining in newly generated postmitotic Ligase IV deficient neurons.
What are the next steps for this research?
A major unsolved question is how Ku activity suppresses A-EJ pathways in cycling cells. We have considered several possible mechanisms including increased resection of DSB ends and differential expression of other potentially relevant factors such as DNA-PKcs, Ligase I, or others. To elucidate mechanisms, we are developing various approaches to test processes and factors that could promote A-EJ in Ku sufficient G1-arrested pro-B lines.
This research was funded by:
NIH/NIAID/NCI and HHMI (Alt lab)
V Foundation for Cancer Research (Frock lab)
