Assembly of the nuclear envelope (NE) in telophase is essential for higher eukaryotic cells to re-establish a functional nucleus. Time-lapse, FRAP and FRET analyses in human cells showed that barrier-to-autointegration factor (BAF), a DNA-binding protein, assembled first at the distinct `core' region of the telophase chromosome and formed an immobile complex by directly binding with other core-localizing NE proteins, such as lamin A and emerin. Correlative light and electron microscopy after live cell imaging, further showed that BAF formed an electron-dense structure on the chromosome surface of the core, close to spindle microtubules (MTs) prior to the attachment of precursor NE membranes, suggesting that MTs may mediate core assembly of BAF. Disruption of the spindle MTs consistently abolished BAF accumulation at the core. In addition, RNAi of BAF eliminated the core assembly of lamin A and emerin, caused abnormal cytoplasmic accumulation of precursor nuclear membranes and resulted in a significant delay of NE assembly. These results suggest that the MT-mediated BAF accumulation at the core facilitates NE assembly at the end of mitosis.

Loss of functional emerin, a nuclear membrane protein, causes X‐linked recessive Emery–Dreifuss muscular dystrophy. In a yeast two‐hybrid screen, we found that emerin interacts with Btf, a death‐promoting transcriptional repressor, which is expressed at high levels in skeletal muscle. Biochemical analysis showed that emerin binds Btf with an equilibrium affinity (KD) of 100 nm. Using a collection of 21 clustered alanine‐substitution mutations in emerin, the residues required for binding to Btf mapped to two regions of emerin that flank its lamin‐binding domain. Two disease‐causing mutations in emerin, S54F and Δ95–99, disrupted binding to Btf. The Δ95–99 mutation was relatively uninformative, as this mutation also disrupts emerin binding to lamin A and a different transcription repressor named germ cell‐less (GCL). In striking contrast, emerin mutant S54F, which binds normally to barrier‐to‐autointegration factor, lamin A and GCL, selectively disrupted emerin binding to Btf. We localized endogenous Btf in HeLa cells by indirect immunoflurorescence using affinity‐purified antibodies against Btf. In nonapoptotic HeLa cells Btf was found in dot‐like structures throughout the nuclear interior. However, within 3 h after treating cells with Fas antibody to induce apoptosis, the distribution of Btf changed, and Btf concentrated in a distinct zone near the nuclear envelope. These results suggest that Btf localization is regulated by apoptotic signals, and that loss of emerin binding to Btf may be relevant to muscle wasting in Emery–Dreifuss muscular dystrophy.

It is generally believed that silent chromatin is condensed and transcriptionally active chromatin is decondensed. However, little is known about the relationship between the condensation levels and gene expression. Here we report the condensation levels of interphase chromatin in the fission yeast Schizosaccharomyces pombe examined by super-resolution fluorescence microscopy. Unexpectedly, silent chromatin is less condensed than the euchromatin. Furthermore, the telomeric silent regions are flanked by highly condensed chromatin bodies, or ‘knobs'. Knob regions span ∼50 kb of sequence devoid of methylated histones. Knob condensation is independent of HP1 homologue Swi6 and other gene silencing factors. Disruption of methylation at lysine 36 of histone H3 (H3K36) eliminates knob formation and gene repression at the subtelomeric and adjacent knob regions. Thus, epigenetic marks at H3K36 play crucial roles in the formation of a unique chromatin structure and in gene regulation at those regions in S. pombe.

Inner nuclear membrane proteins interact with chromosomes in the nucleus and are important for chromosome activity. Lem2 and Man1 are conserved members of the LEM-domain nuclear membrane protein family. Mutations of LEM-domain proteins are associated with laminopathy, but their cellular functions remain unclear. Here, we report that Lem2 maintains genome stability in the fission yeast Schizosaccharomyces pombe. S. pombe cells disrupted for the lem2 + gene (lem2Δ) showed slow growth and increased rate of the minichromosome loss. These phenotypes were prominent in the rich culture medium, but not in the minimum medium. Centromeric heterochromatin formation was augmented upon transfer to the rich medium in wild-type cells. This augmentation of heterochromatin formation was impaired in lem2Δ cells. Notably, lem2Δ cells occasionally exhibited spontaneous duplication of genome sequences flanked by the long-terminal repeats of retrotransposons. The resulting duplication of the lnp1 + gene, which encodes an endoplasmic reticulum membrane protein, suppressed lem2Δ phenotypes, whereas the lem2Δ lnp1Δ double mutant showed a severe growth defect. A combination of mutations in Lem2 and Bqt4, which encodes a nuclear membrane protein that anchors telomeres to the nuclear membrane, caused synthetic lethality. These genetic interactions imply that Lem2 cooperates with the nuclear membrane protein network to regulate genome stability.

Ciliated protozoa have two functionally distinct nuclei, a micronucleus (MIC) and a macronucleus (MAC) [1]. These two nuclei are distinct in size, transcriptional activity, and division cycle control, proceeding with cycles of DNA replication and nuclear division at different times within the same cell [2, 3]. The structural basis generating functionally distinct nuclei remains unknown. Here, we show that, in Tetrahymena thermophila, the nuclear pore complexes (NPCs) of MIC and MAC are composed of different sets of nucleoporins. Among the 13 nucleoporins identified, Nup98 homologs were of interest because two out of the four homologs were localized exclusively in the MAC and the other two were localized exclusively in the MIC. The two MAC-localizing Nup98s contain repeats of GLFG [4]. In contrast, the two MIC-localizing Nup98s lack the GLFG repeats and instead contain a novel repeat signature of NIFN. Ectopic expression of a chimeric MIC-localizing Nup98 homolog bearing GLFG repeats obstructed the nuclear accumulation of MIC-specific nuclear proteins, and expression of a chimeric MAC-localizing Nup98 homolog bearing NIFN repeats obstructed the nuclear accumulation of MAC-specific nuclear proteins. These results suggest that Nup98s act as a barrier to misdirected localization of nucleus-specific proteins. Our findings provide the first evidence that the NPC contributes to nucleus-selective transport in ciliates.