Elucidating Mechanisms of Histone H2B Dynamic Modification in Mammalian Apoptosis

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DESCRIPTION (provided by applicant): Chromatin comprised of DNA and associated histone proteins is the physiological template of our genome. An increasing body of evidence suggests that Chromatin can undergo dynamic changes, including covalent histone modifications, remodeling by ATP-dependent complexes, or utilization of histone variants during different biological processes, such as apoptosis. Apoptosis is a conserved pathway essential in all metazoans, characterized by morphological hallmarks including Chromatin condensation, and DNA fragmentation. Acquired resistance toward apoptosis is a major characteristic in nearly all types of cancer. Previous studies in our laboratory have demonstrated a dynamic phosphorylation of serine 14 in core histone H2B (hereafter H2BS14) in mammalian cells and of serine 10 (H2BS10) in S. cerevisiae in response to apoptotic stimuli. This phosphorylation is a key regulatory step of programmed cell death in yeast as replacement of yeast histone H2B with a non-phosphorylable form results in resistance to apoptosis. Recently, in S. cerevisiae, deacetylation of lysine 11 in H2B (H2BK11) by Hbs 3, a histone deacetylase (HDAC), has been shown to be a prerequisite for H2BS10 phosphorylation to occur, thus forming a unidirectional "cross-talk" between the two histone H2B marks. Based on these preliminary data in yeast, I propose to investigate the regulatory mechanisms of dynamic modifications of histone H2B in mammalian apoptosis. I propose to identify the "signature profiles" of H2B covalent modifications during cell growth and apoptosis using biochemical assays and mass spectrometry; I propose to examine the "crosstalk" regulation in mammalian cells using both in vitro kinase assays and in vivo functional cellular assays during apoptosis. I further propose to identify and characterize the "effectors" downstream of phosphorylated H2BS14 in mammalian apoptosis, using in vitro peptide pull-down assays, and in vivo mononucleosomal co-IPs. These studies will shed light on the mechanisms of dynamic histone H2B modifications involved in the regulation of mammalian apoptosis. Advances made in gaining insights into these fundamental processes will lead to therapeutic strategies that impact on human biology and human diseases, notably cancer.