Abstract LB-83: The hiIstone variant H3.3 deposition during p21 activation is regulated by HP1gamma

Abstract

Abstract The genome of eukaryotic cells is composed in nucleoprotein structure called ‘chromatin’. The basic repeating unit of chromatin is nucelosome which consists of two pairs of H2A-H2B dimer and one H3-H4 tetramer wrapped around 176 bp of double stranded DNA (dsDNA). Although, the structure of chromatin is necessary for hierarchical compaction of the entire genome, it is also an obstacle for some of the most important cellular processes like transcription, DAN repair, DNA replication, etc. In order to alter the structure between histones and DNA, eukaryotic cells imply three basic mechanisms: 1) Chromatin remodeling 2) Incorporation of histone variants 3) Post-translational modification. Unlike canonical histones, histone variants can be incorporated into the chromatin independent from DNA replication. Histone H2A and H3 variants have been introduced to the field of epigenetics for decades and numbers of different studies have shown the evidence of the histone variants involved in specific cellular processes causing different chromatin state. Histone H3.3 is one of H3 ‘replacement’ variants. Although, there are only 5 amino-acid sequence difference between human H3 and H3.3, the incorporation, localization, and the specification on chromatin states seem very distinct between two histones. For instance, H3.3 is significantly recognized in transcriptionally active chromatin states. However, it is not clear the mechanistic role of H3.3 such as whether H3.3 recognizes the active chromatin states or the activation state is initiated by H3.3. To study the specificity of H3.3, we first isolated H3 and H3.3 containing nucleosomes from the cell respectively after introducing ectopic H3 and H3.3 into HEK293T cells by transient transfection. The western blot using various antibodies that recognize specific histone modifications showed that acetylation, H3-K4-di/tri-methylation, and H4-K20-di/tri-methylation levels were relatively higher for H3.3 containing nucleosomes. This result verifies the known functions of H3.3 which dominantly involved in transcriptionally active regions of chromatin. Interestingly though, we also observed substantially higher level of HP1gamma binding for H3.3 containing nucleosomes. HP1 gamma is one of the subtypes of HP1 family originally known as heterochromatin binding protein. Previously, we have confirmed the high level of H3.3 incorporation into P21-RE is heavily increased upon DNA damage. Thereby, to study the effect of HP1gamma in H3.3 incorporation, we depleted endogenous HP1gamma in HEK293T cells using shRNA. As a result, we have observed that H3.3 incorporation was significantly depending on HP1gamma presence. In addition, the level of P21 mRNA was also decreased in HP1gamma depleted cell. Currently, we are now trying to discover the possible protein(s) which HP1gamma can recruit to aid H3.3/H3 exchange during the active transcription process using Mass. Spec analysis of HP1gamma specific binding protein complex purified from HP1gamma expressing stable cell line. Taken together, the incorporation of histone variant, H3.3 and its involvement in different cellular processes are still a great mystery in the field of epigenetics. Thereby, this research would give us a great opportunity to understand one of the mechanisms which can regulate the H3.3 incorporation during an important cellular process. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr LB-83.