Characterization of a Putative Fanconi Anemia D2 Protein Histone‐Binding Domain and Chromodomain

Abstract

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.