A Novel FANCD2-Chromatin Bound Complex in Replication Stress Response and HSC Maintenance
Abstract
DNA replication is essential for the propagation of life. However, DNA replication is also a highly risky process, as it can lead to genome instability. Conditions challenging DNA replication, termed as replication stress, can halt replication fork progression. Stalled replication forks are unstable, and mechanisms are urgently needed to counteract fork collapse and promote an efficient restart of DNA replication. The Fanconi anemia (FA) pathway is a major player in the control of DNA replication integrity in response to replication stress. Germline defect in the pathway results in the FA syndrome characterized by developmental abnormalities, bone marrow (BM) failure, and genome instability which greatly elevates the incidence of cancers. During the activation of the FA DNA repair pathway, FANCD2 and FANCI (ID2) are coupled to an ubiquitin moiety by the FA core complex, which includes eight proteins (FANCA–FANCG, FANCL, and FAAP100) and UBE2T/FANCT. This monoubiquitination event enables the recruitment of the ID2 complex to chromatin and nuclear foci at sites of DNA damage. This posttranslational modification of ID2 complex is central to the collective function of the FA pathway. The molecular mechanisms regarding the cellular DNA repair function of the ID2 complex remain poorly understood and are subjected to intense studies. Our preliminary proteomic (RIME) studies have now identified (by mass-spectrometry) and validated (by co-IPs) several novel FANCD2 interacting proteins that are involved in various cellular activities such as transcription, ubiquitin ligases, structure-specific endonuclease, and several other DNA repair factors. Built on these novel findings, we hypothesize that FANCD2, through interaction with the novel replication stress factors, maintains genome stability and function of hematopoietic stem/progenitor cells (HSPCs) under replication stress. The goals of the project are (1) to verify and characterize novel FANCD2 binding partners in response to replication stress and examine the mechanistic insights into regulation of the FANCD2 complex recruitment to stalled/collapsed replication forks on newly replicated DNA, and (2) and defining the molecular and functional collaboration between the FA pathway and its interacting partners at stalled/collapsed replication forks in the context of HSC maintenance. The proposal will employ many innovative techniques such as RIME, iPOND, unique 3XFLAG-HA Fancd2 knock-in mouse, ES cells, and innovative FA xenotransplant model using primary FA BM samples and the "humanized" NSGS mice. Successful completion of the proposed study will uncover new molecular details of the DNA replication stress response, thereby improving our understanding of mechanistic underpinnings of the FA pathway, and be applicable toward the prevention and treatment of bone marrow failure and of childhood leukemia.
Document Details
- Document Type
- DoD Grant Award
- Publication Date
- Jul 16, 2019
- Source ID
- W81XWH1910262
Entities
People
- Ruhikanta Meetei
Organizations
- United States Army