The BBAP E3 Ligase Monoubiquitylates Histone H4 at Lysine 91 and Selectively Modulates the DNA Damage Response in Chemotherapy-Resistant Lymphomas.

Abstract 3958

Poster Board III-894

Although the BBAP E3 ligase and its binding partner, BAL, are overexpressed in chemotherapy-resistant diffuse large B-cell lymphomas (DLBCLs), the role of these proteins in response to DNA damage remains undefined. The BBAP and BAL1 genes are located on chromosome 3q21 in a head-to-head orientation and regulated by the same gIFN-responsive bidirectional promoter. These findings are of note because BBAP and BAL1 are most abundant in DLBCLs with a prominent, but ineffective, immune/inflammatory infiltrate and increased gIFN production.

Since BAL proteins modulate promoter-coupled transcription and contain structural motifs associated with chromatin remodeling and DNA repair, we reasoned that the BBAP E3 ligase might target nucleosomal proteins. The nucleosome consists of eight core histone proteins (two each of H2A, H2B, H3 and H4) encircled by a genomic DNA segment. Individual histone proteins undergo extensive post-translational modifications that are essential for proper chromatin assembly, regulation of chromatin structure and DNA damage repair. We found that the BBAP E3 ligase selectively monoubiquitylated histone H4 lysine 91, interacted with the chromatin-associated histone and protected cells exposed to DNA damaging agents.

For these reasons, we visually assessed the role of BBAP in the DNA damage response (DDR) pathway. DNA damage generates double-strand DNA breaks and the relocalization of DDR factors to damaged chromatin with phosphorylated ATM, g-H2AX, and MDC-1 accumulating early in the response, followed by 53BP-1 and BRCA1. BBAP-depletion did not affect the accumulation of the early response factors, ATM, g-H2AX or MDC, to sites of DNA damage. In marked contrast, BBAP-depletion significantly altered the DDR response to the checkpoint mediator, 53BP1. At 1 - 4 hrs following induction of DNA damage, there were significantly fewer 53BP1 foci in BBAP-depleted cells than in controls.

Since 53BP1 is recruited to histones via the specific interaction of its tandem tudor domains with mono- and dimethyl H4K20, we next compared BBAP expression, H4 monoubiquitylation and H4K20 methylation in control and BBAP-depleted cells at serial timepoints following DNA damage. In parental or control siRNA-treated Hela cells, DNA damage increased the abundance of BBAP and the respective histone H4 modifications - monoubiquitylated histone H4 and mono- and dimethylated H4K20 – with similar kinetics. However, when BBAP was depleted prior to DNA damage, there was a significant decrease in monoubiquitylated histone H4 and mono- and dimethylated histone H4K20. Taken together, these data directly associate BBAP-mediated monoubiquitylation of histone H4 with the additional histone H4 modifications, H4K20 mono- and dimethylation, necessary for 53BP1 recruitment.

PR-Set7/Set8 catalyses the monomethylation of histone H4K20, which is a prerequisite for H4K20 dimethylation. Since histone H4K20 mono- and dimethylation were significantly reduced in BBAP-depleted cells, we next assessed PR-Set7/Set8 levels following BBAP knockdown. BBAP depletion significantly decreased the abundance of chromatin-associated PR-Set7/Set8, providing a likely mechanism for BBAP-associated changes in histone H4K20 methylation and 53BP1 foci formation.

BBAP-mediated monoubiquitylation of H4K91 and modulation of an associated DNA damage response may represent a mechanism of gIFN-mediated “immunoediting”, limiting the host reponse against lymphoma. More generally, BBAP's role in decreasing the efficacy of chemotherapy-induced DNA damage responses has implications for the design of more effective treatment regimens.