Fanconi Anemia (FA) is a multi-genic disorder that results in progressive bone marrow failure and a strong predisposition to myelodysplastic syndrome and leukemia. FA proteins are involved in the cellular response to DNA damage by a largely unknown mechanism. A nuclear complex of at least 8 FA proteins is required for the monoubiquitination of FANCD2. Monoubiquitination of FANCD2 is required for resistance to the crosslinker mitomycin C. The ATR checkpoint kinase also regulates FANCD2 monoubiquitination and potentially links FANCD2 to the detection of DNA damage at the replication fork. The goal of this study is to understand the regulation and function of FANCD2 monoubiquitination, and thereby to better understand the collective role of FA proteins in promoting normal hematopoiesis. Certain FA proteins are mutated in spontaneous cancers in the general population, so these studies are also relevant to understanding how cancer develops.
The specific aims of the study are as follows: 1) Delineate mechanisms of the ATR-dependent regulation of FANCD2 monoubiquitination. How, and whether, MCM7 and Rad17, along with Rad9, cooperate to couple FANCD2 monoubiquitination to the detection of DNA damage at the replication fork will be determined. ShRNA-mediated suppression of these proteins in primary human CD34+ cells, and expression of mutants in an immortalized myeloid precursor line, will be utilized. Whether these DNA damage sensors prevent chromosomal instability and apoptosis in response to MMC will also be determined. 2) Determine whether defects in FANCD2 monoubiquitination and the assembly of FANCD2 foci results in chromosomal instability and sensitivity to MMC in CD34+ hematopoietic stem/progenitor cells. FANCD2 monoubiquitination will be inhibited by shRNA-mediated suppression of FANCA. Also, mechanisms involved in the recruitment of FANCD2 to blocked replication forks will be elucidated using real-time microscopy in cells reconstituted with EGFP- FANCD2. PROJECT NARRATIVE It is increasingly clear that alterations in some Fanconi anemia pathway proteins are involved in some spontanous cancers seen in the general population. Thus the studies described in this application may shed new light on both normal and abnormal stem cell function and on cancer formation.

Agency
National Institute of Health (NIH)
Institute
National Heart, Lung, and Blood Institute (NHLBI)
Type
Research Project (R01)
Project #
5R01HL085587-05
Application #
8296565
Study Section
Hematopoiesis Study Section (HP)
Program Officer
Qasba, Pankaj
Project Start
2008-07-08
Project End
2014-06-30
Budget Start
2012-07-01
Budget End
2014-06-30
Support Year
5
Fiscal Year
2012
Total Cost
$334,125
Indirect Cost
$111,375
Name
Cincinnati Children's Hospital Medical Center
Department
Type
DUNS #
071284913
City
Cincinnati
State
OH
Country
United States
Zip Code
45229
Park, Jung-Young; Virts, Elizabeth L; Jankowska, Anna et al. (2016) Complementation of hypersensitivity to DNA interstrand crosslinking agents demonstrates that XRCC2 is a Fanconi anaemia gene. J Med Genet 53:672-80
Alavattam, Kris G; Kato, Yasuko; Sin, Ho-Su et al. (2016) Elucidation of the Fanconi Anemia Protein Network in Meiosis and Its Function in the Regulation of Histone Modifications. Cell Rep 17:1141-1157
Bick, Gregory; Zhang, Fan; Meetei, A Ruhikanta et al. (2016) Coordination of the recruitment of the FANCD2 and PALB2 Fanconi anemia proteins by an ubiquitin signaling network. Chromosoma :
Kato, Yasuko; Alavattam, Kris G; Sin, Ho-Su et al. (2015) FANCB is essential in the male germline and regulates H3K9 methylation on the sex chromosomes during meiosis. Hum Mol Genet 24:5234-49
Hasegawa, Kazuteru; Sin, Ho-Su; Maezawa, So et al. (2015) SCML2 establishes the male germline epigenome through regulation of histone H2A ubiquitination. Dev Cell 32:574-88
Broering, Tyler J; Alavattam, Kris G; Sadreyev, Ruslan I et al. (2014) BRCA1 establishes DNA damage signaling and pericentric heterochromatin of the X chromosome in male meiosis. J Cell Biol 205:663-75
Park, J-Y; Singh, T R; Nassar, N et al. (2014) Breast cancer-associated missense mutants of the PALB2 WD40 domain, which directly binds RAD51C, RAD51 and BRCA2, disrupt DNA repair. Oncogene 33:4803-12
Park, Jung-Young; Zhang, Fan; Andreassen, Paul R (2014) PALB2: the hub of a network of tumor suppressors involved in DNA damage responses. Biochim Biophys Acta 1846:263-75
Zhang, Fan; Bick, Gregory; Park, Jung-Young et al. (2012) MDC1 and RNF8 function in a pathway that directs BRCA1-dependent localization of PALB2 required for homologous recombination. J Cell Sci 125:6049-57
Sin, Ho-Su; Barski, Artem; Zhang, Fan et al. (2012) RNF8 regulates active epigenetic modifications and escape gene activation from inactive sex chromosomes in post-meiotic spermatids. Genes Dev 26:2737-48

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