Structural and functional analysis of an HDAC3-H1.3 complex and its role in mitotic progression and microtubule dynamics
Histone deacetylase 3 (HDAC3) and linker histone H1 are both involved in regulation of chromatin compaction and mitotic progression. A novel HDAC3-H1.3 complex has recently been found in our lab. This complex was found to be increasingly abundant in late-G2 and mitosis. The complexed-HDAC3 was found to be specifically active in mitosis, and in vitro assays suggested that CK2 could be responsible for this activation by phosphorylating HDAC3-S424. Silver staining of the complex demonstrated that at least five other proteins of the complex remain unidentified. Interestingly, the complex was found to localize to polar microtubules and spindle poles in mitosis, suggesting that this complex could function to regulate microtubule dynamics in mitosis. The research in this dissertation seeks to further clarify the structure of the HDAC3-H1.3 complex and determine its mitotic function. Structurally, this work demonstrated that the association between HDAC3 and H1.3 is direct, and that HDAC3 and H1.3 are the only HDAC and linker histone subtype present in the complex. Mass spectrometry and immunoprecipitation followed by Western blotting analysis of the complex identified silencing mediator for retinoid or thyroid hormone receptor (SMRT), nuclear receptor co-repressor 1 (NCoR), myosin phosphatase Rho-interacting protein (M-RIP), and unconventional myosin 18A (MYO18A) as members of the HDAC3-H1.3 complex. Functionally, this work has demonstrated that CK2 phosphorylates HDAC3-S424 in mitosis in cell culture. Microtubule regrowth assays showed a role of HDAC3 and H1.3 in promoting microtubule growth in mitosis, possibly through the deacetylation of β-tubulin-K252. Additionally, in this dissertation we identify a potential inhibitory role for the linker histone H1.3 on spindle length and integrity, as siRNA knockdown of H1.3 was able to recover the shortened mitotic spindle and aberrant metaphase spindle phenotype exhibited in HDAC3 knockdowns. The identification of M-RIP and MYO18A as members of the HDAC3-H1.3 complex suggests that the complex may serve a role in telophase and cytokinesis. In this dissertation we propose a model for the HDAC3-H1.3 complex in multiple mitotic functions: promotion of microtubule polymerization, regulation of kinetochore-microtubule attachment, and the regulation of membrane ingression and abscission in telophase and cytokinesis through M-RIP and potentially MYO18A.