NuMA interaction with chromatin is vital for proper chromosome decondensation at the mitotic exit

Abstract

NuMA is an abundant long coiled-coil protein that plays a prominent role in spindle organization during mitosis. In interphase, NuMA is localized to the nucleus and hypothesized to control gene expression and chromatin organization. However, because of the prominent mitotic phenotype upon NuMA loss, its precise function in the interphase nucleus remains elusive. Here, we report that NuMA is associated with chromatin in interphase and prophase but released upon nuclear envelope breakdown (NEBD) by the action of Cdk1. We uncover that NuMA directly interacts with DNA via evolutionarily conserved sequences in its C-terminus. Notably, the expression of the DNA-binding deficient mutant of NuMA affects chromatin decondensation at the mitotic exit, and nuclear shape in interphase. We show that the nuclear shape defects observed upon mutant NuMA expression are due to its potential to polymerize into higher-order fibrillar structures. Overall, this work establishes the spindle independent function of NuMA in choreographing proper chromatin decompaction and nuclear shape by directly associating with the DNA. [Media: see text] [Media: see text] [Media: see text] [Media: see text] [Media: see text] [Media: see text] [Media: see text] [Media: see text] [Media: see text] [Media: see text] [Media: see text]

Membrane compartmentalization of Ect2/Cyk4/Mklp1 and NuMA/dynein/dynactin is essential for cleavage furrow formation during anaphase

“…To track the spatiotemporal localization of NuMA, we performed live-cell imaging in monoclonal stable HeLa Kyoto cells coexpressing AcGFP (Aequora coerulescens GFP) and a mono FLAG epitope-tagged NuMA and mCherry-H2B (Rajeevan et al, 2020). Analogous to endogenous protein, AcGFP-NuMA localizes to the equatorial membrane in cells transfected with Ect2 siRNA, compared to control cells (Figure 1R-1T).…”

“…siRNA resistant Ect2 full-length and Ect2 r Δmem was cloned into a pIRES-AcGFP-FLAG plasmid (a gift from Mark Petronczki) using Age1 and EcoR1 sites. NuMA full-length was cloned into the pIRES-AcGFP-FLAG plasmid using Age1 and EcoR1 sites as reported in Rajeevan et al, 2020. All the clones were confirmed by sequencing analysis.…”

The Nuclear Mitotic Apparatus (NuMA) Protein: A Key Player for Nuclear Formation, Spindle Assembly, and Spindle Positioning

“…Previously, human NuMA was reported to interact with defined DNA sequences called matrix attached regions (MARs) in vitro (Luderus et al, 1994). Recently, two studies demonstrated that the C-terminal region of human NuMA interacts with DNA in vitro and chromatin in cells (Rajeevan et al, 2020;Serra-Marques et al, 2020). Rajeevan et al (2020) showed that the C-terminus NuMA 2058−2115 fragment is sufficient to bind DNA in vitro, and the basic amino acids within the region are critical for its interaction with chromatin in cells (Figure 2C).…”

“…Recently, two studies demonstrated that the C-terminal region of human NuMA interacts with DNA in vitro and chromatin in cells (Rajeevan et al, 2020;Serra-Marques et al, 2020). Rajeevan et al (2020) showed that the C-terminus NuMA 2058−2115 fragment is sufficient to bind DNA in vitro, and the basic amino acids within the region are critical for its interaction with chromatin in cells (Figure 2C). When endogenous NuMA was replaced with mutated versions lacking its DNA-binding ability, cells showed improper chromosome decondensation during mitotic exit and an abnormal nuclear shape (Rajeevan et al, 2020), suggesting that NuMA-DNA interactions are critical for proper regulation of chromosome decondensation during nuclear reformation.…”

“…In fact, the well conserved threonine at 2055 (T2055) is phosphorylated by CDK during metaphase (Compton and Luo, 1995;Kotak et al, 2013;Seldin et al, 2013), which inhibits NuMA's cortical association and thus promotes microtubule-binding. CDK-based phosphorylation of NuMA is also critical for releasing NuMA from the chromosomes at mitotic entry (Rajeevan et al, 2020). During anaphase, NuMA is dephosphorylated by the PPP2CA-B55gamma-PPP2R1B complex (Keshri et al, 2020), which dissociates NuMA from the spindle poles (Gehmlich et al, 2004) and promotes its cortical association.…”