Premium
Characterization of a Putative Fanconi Anemia D2 Protein Histone‐Binding Domain and Chromodomain
Author(s) -
Howlett Niall,
Azzinaro Paul,
Paquin Karissa
Publication year - 2015
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.29.1_supplement.878.3
Subject(s) - fancd2 , chromatin , chromodomain , biology , microbiology and biotechnology , fanconi anemia , fanca , genetics , dna repair , dna , gene , helicase , rna
Fanconi anemia (FA) is a genetic disease characterized by congenital defects, bone marrow failure and heightened cancer susceptibility. FA is caused by biallelic mutations in 16 genes. The FA proteins function cooperatively in the FA‐BRCA pathway to repair DNA interstrand crosslinks (ICLs). A central step in the activation of the FA‐BRCA pathway is the site‐specific monoubiquitination of the FANCD2 and FANCI proteins. Monoubiquitinated FANCD2 and FANCI localize to chromatin, where they recruit several structure‐specific endonucleases. However, how these proteins are targeted to chromatin remains undetermined. We recently uncovered sequence homology between FANCD2 and the histone H4 binding domain (HBD) of D. melanogaster p55, a component of the CAF1 and NuRD chromatin remodeling complexes. In silico analysis of this region uncovered a putative methyl‐lysine‐binding chromodomain (CD), with sequence homology to the chromodomains of HP1a and TIP60. In a histone peptide array screen, a FANCD2 fragment harboring the HBD and CD bound to unmodified, mono‐, di‐, and tri‐methylated H4K20. These findings were verified using in vitro peptide pull‐down assays. Guided by sequence conservation and molecular modeling of the crystal structure of the mouse Fancd2‐Fanci heterodimer, we engineered FANCD2 lentiviral vectors harboring missense mutations in several putative key HBD/CD residues, and stably transduced FA‐D2 ( FANCD2 ‐/‐ ) patient‐derived cells. As assessed by cytotoxicity assays, chromosome analysis, and G2‐M‐phase accumulation, mutation of the FANCD2 HBD/CD impairs FANCD2 function in ICL repair. Our studies describe an important chromatin reader domain in a FA protein and link this important human genetic disease to the regulation of chromatin plasticity.