Differential recognition of histone H10 by monoclonal antibodies during cell differentiation and the arrest of cell proliferation

Abstract

Individual anti-H10 monoclonal antibodies were screened in an immunolocalization assay to isolate clones able to recognize H10 in a differentiation-dependent manner using a murine erythroleukemia cell line. Two clones were selected, one recognizing H10 only in differentiating cells (clone 27 antibody), and the other recognizing the protein constitutively (clone 34 antibody). Both antibodies recognized a restricted region of the protein located at the N-terminal part of the globular domain. Amino acids 24-30, essential for the recognition of the protein by the clone 27 antibody, are extremely conserved in all known H10-like proteins from sea urchin to human. Within these residues, proline 26, responsible for a bend in this region, plays a particularly important role in the epitope recognition. The region involved in the protein recognition by clone 34 antibody is larger and encompasses amino acids 20-30. However, proline 26 does not play an essential role in the structure of this epitope. Detailed analysis of the differential recognition of H10 in chromatin during cell differentiation and proliferation suggests that the modification of chromatin structure as well as that of H10 conformation can account for this effect. Indeed, in vitro study of H10-four-way junction DNA interaction showed that the N-terminal tail domain of the protein can influence the recognition of H10 by these antibodies when the protein interacts with DNA. The two monoclonal antibodies described here therefore seem to be valuable tools for investigating fine modulations in chromatin structure and the concomitant changes occurring in the conformation of the protein.