Codanin-1, the Product of the Gene Mutated In Congenital Dyserythropoietic Anemia Type I (CDA I), Binds to Histone Chaperone Asf1a and Inhibits Its Nucleosome Assembly Activity

Abstract 1004

Congenital dyserythropoietic anemia type I (CDA1) is an inherited recessive macrocytic anemia associated with ineffective erythropoiesis. The disorder is characterized by the accumulation of erythroid precursors containing spongy heterochromatin and internuclear chromatin bridges. The mutated gene (CDAN1) encodes an ubiquitously expressed protein (codanin-1) of unknown function. We have previously shown that codanin-1 is a direct transcriptional target of the E2F1 transcription factor and that the levels of codanin-1 increase during S-phase and decrease during mitosis. In an attempt to further define the role of codanin-1, we conducted a yeast two-hybrid screen using a human bone marrow library and found that codanin-1 binds to Asf1a. Asf1 (anti silencing function) is a H3/H4 histone chaperone involved in the chromatin structure dynamics by its role in nucleosome assembly and disassembly. Using coimmunoprecipitation experiments we confirmed that histone chaperone Asf1a is a direct binding partner of codanin-1. Minimal 100 amino acids domain of codanin-1, involved in binding Asf1, was identified and defined as the Asf1-binding domain. We found that codanin-1 binds to the conserved N-terminal core of Asf1a, where histones and other histone chaperones also bind. FLAG-tagged codanin-1 or its Asf1-binding domain immunoprecipitated from transfected Hela cells and subsequently coimmunoprecipitated histone H3 and Asf1a simultaneously. A pull-down assay of purified Asf1-binding domain in the presence of core histones showed, however, no direct binding of this domain of codanin-1 to H3/H4 histones. By using the replication-independent nucleosome formation assay we noticed that the nucleosome assembly activity of GST-Asf1a was severely decreased by the addition of the purified Asf1-binding domain of codanin-1, a phenotype similar to the one observed in Asf1 depletion. One possible explanation is that binding of codanin-1 inhibits dissociation of histones from Asf1a, which therefore cannot be deposited onto DNA. It will be of interest to determine if codanin-1 is involved in modulating Asf1a activity in vivo and also in response to DNA replication or damage and to determine its role in erythroid heterochromatin formation.