Mitotic checkpoint protein Mad1 is required for early Nup153 recruitment to chromatin and nuclear envelope integrity.

Nucleoporin Nup153 is a multifunctional protein and a known binding partner of mitotic checkpoint protein Mad1 (also known as MAD1L1). The functional relevance of their interaction has remained elusive. Here, we have further dissected the interface and functional interplay of Nup153 and Mad1. Using in situ proximity ligation assays, we found that the presence of a nuclear envelope (NE) is a prerequisite for the Nup153-Mad1 association. Time-lapse microscopy revealed that depletion of Mad1 delayed recruitment of Nup153 to anaphase chromatin, which was often accompanied by a prolongation of anaphase. Furthermore, as seen by electron microscopic and three-dimensional structured illumination investigations, Nup153 and Mad1 depletion led to alterations in NE architecture, characterised by a change of membrane curvature at nuclear pore complexes (NPCs) and an expansion of the spacing between inner and outer nuclear membranes. Nup153 depletion, but not Mad1 depletion, caused defects in interphase NPC assembly, with partial displacement of cytoplasmic nucleoporins and a reduction in NPC density. Taken together, our results suggest that Nup153 has separable roles in NE and NPC formation: in post-mitotic NE re-formation in concert with Mad1 and in interphase NPC assembly, independent of Mad1.

Expression of Leukemia-Associated Nup98 Fusion Proteins Generates an Aberrant Nuclear Envelope Phenotype.

Chromosomal translocations involving the nucleoporin NUP98 have been described in several hematopoietic malignancies, in particular acute myeloid leukemia (AML). In the resulting chimeric proteins, Nup98's N-terminal region is fused to the C-terminal region of about 30 different partners, including homeodomain (HD) transcription factors. While transcriptional targets of distinct Nup98 chimeras related to immortalization are relatively well described, little is known about other potential cellular effects of these fusion proteins. By comparing the sub-nuclear localization of a large number of Nup98 fusions with HD and non-HD partners throughout the cell cycle we found that while all Nup98 chimeras were nuclear during interphase, only Nup98-HD fusion proteins exhibited a characteristic speckled appearance. During mitosis, only Nup98-HD fusions were concentrated on chromosomes. Despite the difference in localization, all tested Nup98 chimera provoked morphological alterations in the nuclear envelope (NE), in particular affecting the nuclear lamina and the lamina-associated polypeptide 2α (LAP2α). Importantly, such aberrations were not only observed in transiently transfected HeLa cells but also in mouse bone marrow cells immortalized by Nup98 fusions and in cells derived from leukemia patients harboring Nup98 fusions. Our findings unravel Nup98 fusion-associated NE alterations that may contribute to leukemogenesis.

Structural characterization of altered nucleoporin Nup153 expression in human cells by thin-section electron microscopy.

Nuclear pore complexes (NPCs) span the 2 membranes of the nuclear envelope (NE) and facilitate nucleocytoplasmic exchange of macromolecules. NPCs have a roughly tripartite structural organization with the so-called nuclear basket emanating from the NPC scaffold into the nucleoplasm. The nuclear basket is composed of the 3 nucleoporins Nup153, Nup50, and Tpr, but their specific role for the structural organization of this NPC substructure is, however, not well established. In this study, we have used thin-section transmission electron microscopy to determine the structural consequences of altering the expression of Nup153 in human cells. We show that the assembly and integrity of the nuclear basket is not affected by Nup153 depletion, whereas its integrity is perturbed in cells expressing high concentrations of the zinc-finger domain of Nup153. Moreover, even mild over-expression of Nup153 is coinciding with massive changes in nuclear organization and it is the excess of the zinc-finger domain of Nup153 that is sufficient to induce these rearrangements. Our data indicate a central function of Nup153 in the organization of the nucleus, not only at the periphery, but throughout the entire nuclear interior.

Dynamics and diverse functions of nuclear pore complex proteins.

Nuclear pore complexes (NPCs) are best known for their central role in controlling the molecular trafficking between the cytoplasm and the nucleus. NPCs are assembled from about 30 different proteins and a growing body of evidence suggests that these nucleoporins are not only acting in the context of NPCs, but also in the nucleoplasm and cytoplasm. In this context it is well accepted that a set of nucleoporins are important regulators of a variety of mitotic processes, including kinetochore assembly, spindle checkpoint control and cytokinesis, whereas others associate with chromatin and administer gene expression. However, the functional importance of nucleoporins go far beyond these roles and this review will provide an overview of the latest insights into the versatility of metazoan nucleoporins with an emphasis on their roles in cell migration, cellular signaling and tissue-specific activities.

Domain topology of nucleoporin Nup98 within the nuclear pore complex.

Nuclear pore complexes (NPCs) facilitate selective transport of macromolecules across the nuclear envelope in interphase eukaryotic cells. NPCs are composed of roughly 30 different proteins (nucleoporins) of which about one third are characterized by the presence of phenylalanine-glycine (FG) repeat domains that allow the association of soluble nuclear transport receptors with the NPC. Two types of FG (FG/FxFG and FG/GLFG) domains are found in nucleoporins and Nup98 is the sole vertebrate nucleoporin harboring the GLFG-type repeats. By immuno-electron microscopy using isolated nuclei from Xenopus oocytes we show here the localization of distinct domains of Nup98. We examined the localization of the C- and N-terminal domain of Nup98 by immunogold-labeling using domain-specific antibodies against Nup98 and by expressing epitope tagged versions of Nup98. Our studies revealed that anchorage of Nup98 to NPCs through its C-terminal autoproteolytic domain occurs in the center of the NPC, whereas its N-terminal GLFG domain is more flexible and is detected at multiple locations within the NPC. Additionally, we have confirmed the central localization of Nup98 within the NPC using super resolution structured illumination fluorescence microscopy (SIM) to position Nup98 domains relative to markers of cytoplasmic filaments and the nuclear basket. Our data support the notion that Nup98 is a major determinant of the permeability barrier of NPCs.

Nucleoporins: leaving the nuclear pore complex for a successful mitosis.

The nuclear envelope (NE) separates the cytoplasm and the cell nucleus of interphase eukaryotic cells and nuclear pore complexes (NPCs) mediate the macromolecular exchange between these two compartments. The NE and the NPCs of vertebrate cells disassemble during prophase and the nuclear pore proteins (nucleoporins) are distributed within the mitotic cytoplasm. For an increasing number of them active mitotic functions have been assigned over the past few years. Nucleoporins are participating in spindle assembly, kinetochore organisation, and the spindle assembly checkpoint, all processes that control chromosome segregation and are important for maintenance of genome integrity. But nucleoporins are also engaged in early and late mitotic events, such as centrosome positioning and cytokinesis. Here we will highlight recent progress in deciphering the roles for nucleoporins in the distinct steps of mitosis.

The basic helix-loop-helix transcription factor CrMYC2 controls the jasmonate-responsive expression of the ORCA genes that regulate alkaloid biosynthesis in Catharanthus roseus.

Jasmonates are plant signalling molecules that play key roles in defence against insects and certain pathogens, among others by controlling the biosynthesis of protective secondary metabolites. In Catharanthus roseus, the AP2/ERF-domain transcription factor ORCA3 controls the jasmonate-responsive expression of several genes encoding enzymes involved in terpenoid indole alkaloid biosynthesis. ORCA3 gene expression is itself induced by jasmonate. The ORCA3 promoter contains an autonomous jasmonate-responsive element (JRE) composed of a quantitative sequence responsible for the high level of expression and a qualitative sequence that acts as an on/off switch in response to methyl-jasmonate (MeJA). Here, we identify the basic helix-loop-helix (bHLH) transcription factor CrMYC2 as the major activator of MeJA-responsive ORCA3 gene expression. The CrMYC2 gene is an immediate-early jasmonate-responsive gene. CrMYC2 binds to the qualitative sequence in the ORCA3 JRE in vitro, and transactivates reporter gene expression via this sequence in transient assays. Knock-down of the CrMYC2 expression level via RNA interference caused a strong reduction in the level of MeJA-responsive ORCA3 mRNA accumulation. In addition, MeJA-responsive expression of the related transcription factor gene ORCA2 was significantly reduced. Our results show that MeJA-responsive expression of alkaloid biosynthesis genes in C. roseus is controlled by a transcription factor cascade consisting of the bHLH protein CrMYC2 regulating ORCA gene expression, and the AP2/ERF-domain transcription factors ORCA2 and ORCA3, which in turn regulate a subset of alkaloid biosynthesis genes.

Hedgehog signaling pathway is inactive in colorectal cancer cell lines.

The Hedgehog (Hh) signaling pathway plays an important role in human development. Abnormal activation of this pathway has been observed in several types of human cancers, such as the upper gastro-intestinal tract cancers. However, activation of the Hh pathway in colorectal cancers is controversial. We analyzed the expression of the main key members of the Hh pathway in 7 colon cancer cell lines in order to discover whether the pathway is constitutively active in these cells. We estimated the expression of SHH, IHH, PTCH, SMO, GLI1, GLI2, GLI3, SUFU and HHIP genes by RT-PCR. Moreover, Hh ligand, Gli3 and Sufu protein levels were quantified by western blotting. None of the cell lines expressed the complete set of Hh pathway members. The ligands were absent from Colo320 and HCT116 cells, Smo from Colo205, HT29 and WiDr. GLI1 gene was not expressed in SW480 cells nor were GLI2/GLI3 in Colo205 or Caco-2 cells. Furthermore the repressive form of Gli3, characteristic of an inactive pathway, was detected in SW480 and Colo320 cells. Finally treatment of colon cancer cells with cyclopamine, a specific inhibitor of the Hh pathway, did not downregulate PTCH and GLI1 genes expression in the colorectal cells, whereas it did so in PANC1 control cells. Taken together, these results indicate that the aberrant activation of the Hh signaling pathway is not common in colorectal cancer cell lines.

Distal ERBB2 promoter fragment displays specific transcriptional and nuclear binding activities in ERBB2 overexpressing breast cancer cells.

Overexpression of the ERBB2 gene occurs in 30% of human breast cancers and is correlated with poor prognosis. The deregulation is the consequence of an increased transcription level and gene amplification. Several laboratories, including our own, have identified, in the proximal promoter, enhancers implicated in the gene overexpression. However, our previous studies of a 6-kb ERBB2 promoter fragment revealed the presence of repressing fragments, which were able to overcome the effect of the proximal enhancers. These repressing elements were functional in all cell lines, regardless of their endogenous ERBB2 expression level. Here, we show that a distal ERBB2 promoter region restores high transcription rates specifically in ERBB2 overexpressing breast cancer cells. This distal promoter region thus contains enhancers essential for the overexpression of the gene. By EMSA, performed with nuclear extract of cells overexpressing (BT-474) or not (MDA-MB-231) the ERBB2 gene, we show that at least two sequences of the distal promoter region are bound exclusively by BT-474 extract. Further experiments reveal that AP-2 transcription factors contribute to this differential binding activity, by binding recognition sequences located 4500 bp and 4000 bp upstream of the transcription start site. These sites are occupied by AP2 in vivo, as demonstrated by ChIP assay. Inactivation of AP-2 proteins in ERBB2 overexpressing cells reduces the distal promoter activity up to 70%, indicating the AP-2 factors are implicated in the strong distal enhancing effect. Moreover, we identified a 54-bp fragment that is bound specifically by BT-474 nuclear extract. Further experiments did not lead to the identification of the protein responsible for this binding. Our results thus highlight the importance of ERBB2 distal promoter region and further implicate AP-2 in ERBB2 overexpression in breast cancer cells.

Zinc finger proteins act as transcriptional repressors of alkaloid biosynthesis genes in Catharanthus roseus.

In Catharanthus roseus cell suspensions, the expression of several terpenoid indole alkaloid biosynthetic genes, including two genes encoding strictosidine synthase (STR) and tryptophan decarboxylase (TDC), is coordinately induced by fungal elicitors such as yeast extract. To identify molecular mechanisms regulating the expression of these genes, a yeast one-hybrid screening was performed with an elicitor-responsive part of the TDC promoter. This screening identified three members of the Cys(2)/His(2)-type (transcription factor IIIA-type) zinc finger protein family from C. roseus, ZCT1, ZCT2, and ZCT3. These proteins bind in a sequence-specific manner to the TDC and STR promoters in vitro and repress the activity of these promoters in trans-activation assays. In addition, the ZCT proteins can repress the activating activity of APETALA2/ethylene response-factor domain transcription factors, the ORCAs, on the STR promoter. The expression of the ZCT genes is rapidly induced by yeast extract and methyljasmonate. These results suggest that the ZCT proteins act as repressors in the regulation of elicitor-induced secondary metabolism in C. roseus.

CrMYC1, a Catharanthus roseus elicitor- and jasmonate-responsive bHLH transcription factor that binds the G-box element of the strictosidine synthase gene promoter.

A cDNA encoding a bHLH transcription factor was isolated by the yeast one-hybrid system from a Catharanthus roseus cDNA library using the G-box element of the Strictosidine synthase gene promoter as bait. The corresponding protein (named CrMYC1) was shown to bind specifically to the G-box in yeast. In C. roseus suspension cells CrMYC1 mRNA levels are induced by fungal elicitor and jasmonate suggesting that CrMYC1 may be involved in the regulation of gene expression in response to these signals.