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

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

ARP2/3- and resection-coupled genome reorganization facilitates translocations [preprint]

DNA end-resection and nuclear actin-based movements orchestrate clustering of double-strand breaks (DSBs) into homology-directed repair (HDR) domains. Here, we analyze how actin nucleation by ARP2/3 affects damage-dependent and -independent 3D genome reorganization and facilitates pathologic repair. We observe that DNA damage, followed by ARP2/3-dependent establishment of repair domains enhances local chromatin insulation at a set of damage-proximal boundaries and affects compartment organization genome-wide. Nuclear actin polymerization also promotes interactions between DSBs, which in turn facilitates aberrant intra- and inter-chromosomal rearrangements. Notably, BRCA1 deficiency, which decreases end-resection, DSB mobility, and subsequent HDR, nearly abrogates recurrent translocations between AsiSI DSBs. In contrast, loss of functional BRCA1 yields unique translocations genome-wide, reflecting a critical role in preventing spontaneous genome instability and subsequent rearrangements. Our work establishes that the assembly of DSB repair domains is coordinated with multiscale alterations in genome architecture that enable HDR despite increased risk of translocations with pathologic potential

Building a nuclear envelope at the end of mitosis: coordinating membrane reorganization, nuclear pore complex assembly, and chromatin de-condensation

The metazoan nucleus is disassembled and re-built at every mitotic cell division. The nuclear envelope, including nuclear pore complexes, breaks down at the beginning of mitosis to accommodate the capture of massively condensed chromosomes by the spindle apparatus. At the end of mitosis, a nuclear envelope is newly formed around each set of segregating and de-condensing chromatin. We review the current understanding of the membrane restructuring events involved in the formation of the nuclear membrane sheets of the envelope, the mechanisms governing nuclear pore complex assembly and integration in the nascent nuclear membranes, and the regulated coordination of these events with chromatin de-condensation

RuvB-like ATPases Function in Chromatin Decondensation at the End of Mitosis

SummaryChromatin undergoes extensive structural changes during the cell cycle. Upon mitotic entry, metazoan chromatin undergoes tremendous condensation, creating mitotic chromosomes with 50-fold greater compaction relative to interphase chromosomes. At the end of mitosis, chromosomes reestablish functional interphase chromatin competent for replication and transcription through a decondensation process that is cytologically well described. However, the underlying molecular events and factors remain unidentified. We describe a cell-free system that recapitulates chromatin decondensation based on purified mitotic chromatin and Xenopus egg extracts. Using biochemical fractionation, we identify RuvB-like ATPases as chromatin decondensation factors and demonstrate that their ATPase activity is essential for decondensation. Our results show that decompaction of metaphase chromosomes is not merely an inactivation of known chromatin condensation factors but rather an active process requiring specific molecular machinery. Our cell-free system provides an important tool for further molecular characterization of chromatin decondensation and its coordination with concomitant processes

The nucleoporin Nup50 activates the Ran guanine nucleotide exchange factor RCC1 to promote NPC assembly at the end of mitosis

During mitotic exit, thousands of nuclear pore complexes (NPCs) assemble concomitant with the nuclear envelope to build a transport-competent nucleus. Here, we show that Nup50 plays a crucial role in NPC assembly independent of its well-established function in nuclear transport. RNAi-mediated downregulation in cells or immunodepletion of Nup50 protein in Xenopus egg extracts interferes with NPC assembly. We define a conserved central region of 46 residues in Nup50 that is crucial for Nup153 and MEL28/ELYS binding, and for NPC interaction. Surprisingly, neither NPC interaction nor binding of Nup50 to importin alpha/beta, the GTPase Ran, or chromatin is crucial for its function in the assembly process. Instead, an N-terminal fragment of Nup50 can stimulate the Ran GTPase guanine nucleotide exchange factor RCC1 and NPC assembly, indicating that Nup50 acts via the Ran system in NPC reformation at the end of mitosis. In support of this conclusion, Nup50 mutants defective in RCC1 binding and stimulation cannot replace the wild-type protein in in vitro NPC assembly assays, whereas excess RCC1 can compensate the loss of Nup50.ISSN:0261-4189ISSN:1460-207