Nuclear Cytoskeleton in Virus Infection

The nuclear lamina is the main component of the nuclear cytoskeleton that maintains the integrity of the nucleus. However, it represents a natural barrier for viruses replicating in the cell nucleus. The lamina blocks viruses from being trafficked to the nucleus for replication, but it also impedes the nuclear egress of the progeny of viral particles. Thus, viruses have evolved mechanisms to overcome this obstacle. Large viruses induce the assembly of multiprotein complexes that are anchored to the inner nuclear membrane. Important components of these complexes are the viral and cellular kinases phosphorylating the lamina and promoting its disaggregation, therefore allowing virus egress. Small viruses also use cellular kinases to induce lamina phosphorylation and the subsequent disruption in order to facilitate the import of viral particles during the early stages of infection or during their nuclear egress. Another component of the nuclear cytoskeleton, nuclear actin, is exploited by viruses for the intranuclear movement of their particles from the replication sites to the nuclear periphery. This study focuses on exploitation of the nuclear cytoskeleton by viruses, although this is just the beginning for many viruses, and promises to reveal the mechanisms and dynamic of physiological and pathological processes in the nucleus.

Deformation of the nucleus by TGFβ1 via the remodeling of nuclear envelope and histone isoforms

The cause of nuclear shape abnormalities which are often seen in pre-neoplastic and malignant tissues is not clear. In this study we report that deformation of the nucleus can be induced by TGFβ1 stimulation in several cell lines including Huh7. In our results, the upregulated histone H3.3 expression downstream of SMAD signaling contributed to TGFβ1-induced nuclear deformation, a process of which requires incorporation of the nuclear envelope (NE) proteins lamin B1 and SUN1. During this process, the NE constitutively ruptured and reformed. Contrast to lamin B1 which was relatively stationary around the nucleus, the upregulated lamin A was highly mobile, clustering at the nuclear periphery and reintegrating into the nucleoplasm. The chromatin regions that lost NE coverage formed a supra-nucleosomal structure characterized by elevated histone H3K27me3 and histone H1, the formation of which depended on the presence of lamin A. These results provide evidence that shape of the nucleus can be modulated through TGFβ1-induced compositional changes in the chromatin and nuclear lamina.

Nuclear pore protein NUP210 depletion suppresses metastasis through heterochromatin-mediated disruption of tumor cell mechanical response

Mechanical signals from the extracellular microenvironment have been implicated in tumor and metastatic progression. Here, we identify nucleoporin NUP210 as a metastasis susceptibility gene for human estrogen receptor positive (ER+) breast cancer and a cellular mechanosensor. Nup210 depletion suppresses lung metastasis in mouse models of breast cancer. Mechanistically, NUP210 interacts with LINC complex protein SUN2 which connects the nucleus to the cytoskeleton. In addition, the NUP210/SUN2 complex interacts with chromatin via the short isoform of BRD4 and histone H3.1/H3.2 at the nuclear periphery. In Nup210 knockout cells, mechanosensitive genes accumulate H3K27me3 heterochromatin modification, mediated by the polycomb repressive complex 2 and differentially reposition within the nucleus. Transcriptional repression in Nup210 knockout cells results in defective mechanotransduction and focal adhesion necessary for their metastatic capacity. Our study provides an important role of nuclear pore protein in cellular mechanosensation and metastasis.

A novel enhancer that regulates Bdnf expression in developing neurons

Brain derived neurotrophic factor (BDNF) is a critical secreted peptide that promotes neuronal differentiation and survival, and its downregulation is implicated in many neurological disorders. Here, we investigated the regulation of the mouse Bdnf gene in cortical neurons and identified a novel enhancer that promotes the expression of many Bdnf transcript variants during differentiation, increasing total Bdnf mRNA levels. Enhancer activity contributes to Bdnf-mediated effects on neuronal clustering and activity-dependent dendritogenesis. During Bdnf activation, enhancer-promoter contacts increase, and the region moves away from the repressive nuclear periphery. Our findings suggest that changes in nuclear structure may contribute to the expression of essential growth factors during neuronal development.

Cell-type specialization is encoded by specific chromatin topologies

The three-dimensional (3D) structure of chromatin is intrinsically associated with gene regulation and cell function1–3. Methods based on chromatin conformation capture have mapped chromatin structures in neuronal systems such as in vitro differentiated neurons, neurons isolated through fluorescence-activated cell sorting from cortical tissues pooled from different animals and from dissociated whole hippocampi4–6. However, changes in chromatin organization captured by imaging, such as the relocation of Bdnf away from the nuclear periphery after activation7, are invisible with such approaches8. Here we developed immunoGAM, an extension of genome architecture mapping (GAM)2,9, to map 3D chromatin topology genome-wide in specific brain cell types, without tissue disruption, from single animals. GAM is a ligation-free technology that maps genome topology by sequencing the DNA content from thin (about 220 nm) nuclear cryosections. Chromatin interactions are identified from the increased probability of co-segregation of contacting loci across a collection of nuclear slices. ImmunoGAM expands the scope of GAM to enable the selection of specific cell types using low cell numbers (approximately 1,000 cells) within a complex tissue and avoids tissue dissociation2,10. We report cell-type specialized 3D chromatin structures at multiple genomic scales that relate to patterns of gene expression. We discover extensive ‘melting’ of long genes when they are highly expressed and/or have high chromatin accessibility. The contacts most specific of neuron subtypes contain genes associated with specialized processes, such as addiction and synaptic plasticity, which harbour putative binding sites for neuronal transcription factors within accessible chromatin regions. Moreover, sensory receptor genes are preferentially found in heterochromatic compartments in brain cells, which establish strong contacts across tens of megabases. Our results demonstrate that highly specific chromatin conformations in brain cells are tightly related to gene regulation mechanisms and specialized functions.

Histone N-terminal acetyltransferase NAA40 links one-carbon metabolism to chemoresistance

Aberrant function of epigenetic modifiers plays an important role not only in the progression of cancer but also the development of drug resistance. N-alpha-acetyltransferase 40 (NAA40) is a highly specific epigenetic enzyme catalyzing the transfer of an acetyl moiety at the N-terminal end of histones H4 and H2A. Recent studies have illustrated the essential oncogenic role of NAA40 in various cancer types but its role in chemoresistance remains unclear. Here, using transcriptomic followed by metabolomic analysis in colorectal cancer (CRC) cells, we demonstrate that NAA40 controls key one-carbon metabolic genes and corresponding metabolites. In particular, through its acetyltransferase activity NAA40 regulates the methionine cycle thereby affecting global histone methylation and CRC cell survival. Importantly, NAA40-mediated metabolic rewiring promotes resistance of CRC cells to antimetabolite chemotherapy in vitro and in xenograft models. Specifically, NAA40 stimulates transcription of the one-carbon metabolic gene thymidylate synthase (TYMS), whose product is targeted by 5-fluorouracil (5-FU) and accordingly in primary CRC tumours NAA40 expression associates with TYMS levels and poorer 5-FU response. Mechanistically, NAA40 activates TYMS by preventing enrichment of repressive H2A/H4S1ph at the nuclear periphery. Overall, these findings define a novel regulatory link between epigenetics and cellular metabolism mediated by NAA40, which is harnessed by cancer cells to evade chemotherapy.

Lamin C is required to establish genome organization after mitosis

Background The dynamic 3D organization of the genome is central to gene regulation and development. The nuclear lamina influences genome organization through the tethering of lamina-associated domains (LADs) to the nuclear periphery. Evidence suggests that lamins A and C are the predominant lamins involved in the peripheral association of LADs, potentially serving different roles. Results Here, we examine chromosome architecture in mouse cells in which lamin A or lamin C are downregulated. We find that lamin C, and not lamin A, is required for the 3D organization of LADs and overall chromosome organization. Striking differences in localization are present as cells exit mitosis and persist through early G1 and are linked to differential phosphorylation. Whereas lamin A associates with the nascent nuclear envelope (NE) during telophase, lamin C remains in the interior, surrounding globular LAD aggregates enriched on euchromatic regions. Lamin C association with the NE is delayed until several hours into G1 and correlates temporally and spatially with the post-mitotic NE association of LADs. Post-mitotic LAD association with the NE, and global 3D genome organization, is perturbed only in cells depleted of lamin C, and not lamin A. Conclusions Lamin C regulates LAD dynamics during exit from mitosis and is a key regulator of genome organization in mammalian cells. This reveals an unexpectedly central role for lamin C in genome organization, including inter-chromosomal LAD-LAD segregation and LAD scaffolding at the NE, raising intriguing questions about the individual and overlapping roles of lamin A/C in cellular function and disease.

A motor independent requirement for Dynein light chain in C. elegans meiotic synapsis

The dynein motor complex is thought to aid in homolog pairing in many organisms by moving chromosomes within the nuclear periphery to promote and test homologous interactions. This precedes synaptonemal complex (SC) formation during homolog synapsis, which stabilizes homolog proximity during recombination. We observed that depletion of the dynein light chain (DLC-1) in Caenorhabditis elegans irreversibly prevents synapsis, causing an increase in off-chromatin formation of SC protein foci with increasing temperature. This requirement for DLC-1 is independent of its function in dynein motors, as SYP protein foci do not form with depletion of other dynein motor components. In contrast to normal SC-related structures, foci formed with DLC-1 depletion are resistant to dissolution with 1,6-hexanediol, similar to aggregates of SC proteins formed in high growth temperatures. Dynein light chains have been shown to act as hub proteins that interact with other proteins through a conserved binding motif. We identified a similar DLC-1 binding motif in the C. elegans SC protein SYP-2, and mutation of the putative motif causes meiosis defects that are exacerbated by elevated temperatures. We propose that DLC-1 acts as a pre-synapsis chaperone-like factor for SYP proteins to help regulate their self-association prior to the signals for SC assembly, a role that is revealed by its increased essentiality at elevated temperatures.

Mad1’s ability to interact with Mad2 is essential to regulate and monitor meiotic synapsis in C. elegans

Meiotic homolog synapsis is essential to ensure accurate segregation of chromosomes during meiosis. In C. elegans, proper regulation of synapsis and a checkpoint that monitors synapsis relies on the spindle checkpoint components, Mad1 and Mad2, and Pairing Centers (PCs), cis-acting loci that interact with the nuclear envelope to mobilize chromosomes within the nucleus. Here, we test what specific functions of Mad1 and Mad2 are required to regulate and monitor synapsis. We find that a mutation that prevents Mad1’s localization to the nuclear periphery abolishes the synapsis checkpoint but has no effect on Mad2’s localization to the nuclear periphery or synapsis. By contrast, a mutation that prevents Mad1’s interaction with Mad2 abolishes the synapsis checkpoint, delays synapsis and fails to localize Mad2 to the nuclear periphery. These data indicate that Mad1’s primary role in regulating synapsis is through control of Mad2 and that Mad2 can bind other factors at the nuclear periphery. We also tested whether Mad2’s ability to adopt a specific conformation associated with its activity during spindle checkpoint function is required for its role in meiosis. A mutation that prevents Mad2 from adopting its active conformer fails to localize to the nuclear periphery, abolishes the synapsis checkpoint and exhibits substantial defects in meiotic synapsis. Thus, Mad2, and its regulation by Mad1, is an important regulator of meiotic synapsis in C. elegans.

Nup93 and CTCF modulate spatiotemporal dynamics and function of the HOXA gene locus during differentiation

Nucleoporins regulate nuclear transport and are also involved in DNA damage, repair, cell cycle, chromatin organization, and gene expression. Here, we studied the role of nucleoporin Nup93 and the chromatin organizer CTCF in regulating HOXA expression during differentiation. ChIP sequencing revealed a significant overlap between Nup93 and CTCF peaks. Interestingly, Nup93 and CTCF are associated with the 3' and 5′HOXA genes respectively. Depletions of Nup93 and CTCF antagonistically modulate expression levels of 3′and 5′HOXA genes in undifferentiated NT2/D1 cells. Nup93 also regulates the localization of the HOXA gene locus, which disengages from the nuclear periphery upon Nup93 but not CTCF depletion, consistent with its upregulation. The dynamic association of Nup93 and CTCF with the HOXA locus during differentiation correlates with its spatial positioning and expression. While Nup93 tethers the HOXA locus to the nuclear periphery, CTCF potentially regulates looping of the HOXA gene cluster in a temporal manner. In summary, Nup93 and CTCF complement one another in modulating the spatiotemporal dynamics and function of the HOXA gene locus during differentiation.