The lysine demethylase LSD1 regulates nuclear envelope formation at the end of mitosis

Abstract

The eukaryotic genome is compartmentalized and organized within the two membranes of the nuclear envelope. Integrated at pores spanning the envelope are nuclear pore complexes, which mediate the regulated exchange of macromolecules between the nuclear compartment and the cell cytoplasm. During mitosis, the metazoan nucleus is disassembled so that the mitotic spindle can access the highly condensed chromosomes and mediate their faithful segregation. The nuclear envelope and nuclear pore complexes must therefore be rebuilt on the de-condensing chromatin at the end of every mitotic cell division. In recent years, advances have been made towards elucidating the molecular composition and structural features of the nuclear envelope and nuclear pore complexes. Additionally, the importance and several determinants of three dimensional chromatin organisation within the boundaries of the nuclear envelope have begun to come to light. However, although many essential factors have been identified, the molecular mechanisms governing the establishment of nuclear architecture at the end of mitosis remain poorly defined. It is particularly unclear how the assembly of the nuclear envelope and pore complexes is coordinated with the changing chromatin landscape at the end of mitosis. The work presented in this thesis aimed to identify regulatory landmarks of nuclear envelope formation. A cell-free nuclear reconstitution system based on Xenopus laevis extracts was employed to screen various chemical inhibitors for nuclear assembly defects. A group of inhibitors targeting Lysine (K) Specific Demethylase 1 (A) (LSD1/KDM1A) blocked the formation of a closed nuclear envelope and nuclear pore complex assembly in vitro. LSD1 catalyzes the demethylation of mono- and di-methylated lysines of histone H3 tails. Immunodepletion of LSD1 and rescue experiments using recombinant proteins confirmed that LSD1-dependent demethylation is specifically required for cell-free nuclear assembly. Accordingly RNAi-mediated depletion of LSD1 in human cells significantly extended the length of telophase, during which the nuclear envelope is formed, based on live cell imaging experiments and the automated tracking and annotation of chromatin features during cell division. A modified version of the cell-free nuclear reconstitution assay that employs mitotic chromatin clusters was developed and used to specifically assay the role of LSD1 in post-mitotic chromatin decondensation. Although the nuclear and chromatin-occupied volume was consistently smaller both in vitro and in cultured cells in the absence of LSD1 activity, LSD1 did not seem to be required for the initial steps of chromatin decondensation. Nonetheless, additional biochemical experiments indicated that LSD1 activity was essential for the recruitment of early-associating nuclear envelope and nuclear pore complex proteins to chromatin. The data presented here demonstrate that LSD1 regulates the recruitment and assembly of a functional nuclear envelope on post-mitotic chromatin. Although non-histone protein targets cannot be excluded, the identification of the histone demethylase LSD1 as an essential regulator of nuclear assembly represents one of the first descriptions of a factor linking nuclear envelope formation with the changing chromatin landscape at the end of mitosis.