Appointment period: 3/1/2007 to 2/29/2008

Award: NIH Minority Supplement Training Award, 2008-2009

Project

The Role of K8 Alpha K-bZIP in Chromatin Remodeling of the Kaposi’s Sarcoma-Associated Herpes virus in Transformed B-lymphoma Cells

The research focused on:

1. Assessment of the role of K-bZIP in the transcriptional regulation and chromatin remodeling of the KSHS genome.
2. Assessment of the role of posttranslational modifications of K-bZIP chromatin binding sites in the KSHS genome.

Dr. Fitzgerald focused on determining how K-bZIP globally represses transcription in the host system. K-bZIP is an early gene expressed in the KSHS genome upon lytic activation. It is a strong transcriptional repressor and a moderate transcriptional activator. In the absence of other viral proteins, K-bZIP is a general repressor of host genes and an effective inducer of H3K9 trimethylation. It is known that sumoylation and histone H3K9 methylation are hallmarks of heterochromatin and silenced genes. Therefore she wanted to determine if sumoylation as well as histone methylation served as a mechanism by which K-bZIP could globally repress the host genome. To date her data shows that K-bZIP is sumoylated at lysine 158, when this site is mutated )-bZIP no longer serves as a global repressor. K-bZIP also represses transcription through its interaction with transcriptional machinery, such as JMJD2a, a histone demethylase. She was able to demonstrate that K-bZIP interacts with JMJD2a using immunoflourescence as well as co-immunoprecipation assays. She has mapped the domain of K-bZIP that interacts with JMJD2a. It appears that upon deletion of the N-terminus (aa 1-121) or deletion of the C-terminus (aa 121-237), both mutants bind to JMJD2a, however it appears that the deletion of the C-terminus binds less to JMJD2a when compared to deletion of the N-terminus. She also tested whether the interaction between K-bZIP affected JMJD2a demethylase activity using mass spectrophotometry. The findings demonstrate that when K-bZIP and JMJD2a were incubated with H3K9me3 peptide, JMJD2a was no longer able to demethylate H3)9me3 when compared to that of JMJD2a alone, suggesting that indeed K-bZIP serves as an inhibitor of JMJD2a activity. Dr.Fitzgerald also determined which region of K-bZIP is necessary for inhibiting JMJD2a demethylase activity.

K-bZIP is a SUMO-binding protein and behaves like a SUMO-ligase/adaptor, so we wanted to determine if JMJD2A is sumoylated, since it interacts with K-bZIP. Our results show that JMJD2A is sumoylated and colocalized with SUMO1 foci. In addition, JMJD2A is transcriptionally down-modulated by K-bZIP. Thus, K-bZIP utilizes multiple ways to shut off JMJD2A which was observed that over expression of JMJD2A in BCBL-1 increases the level of reactivation of latent genome. These findings suggest that K-bZIP plays a pivotal role in the switch from latency to lytic reactivation through interaction with JMJD2a and modulating the chromatin structure of the infecting genome.