SnoN in TGF-beta signaling and cancer biology.

The Transforming Growth Factor (TGF)- beta-Smad signaling pathway regulates diverse biological processes essential for normal development and homeostasis. The Smad-interacting transcriptional modulator SnoN and its related homologs have emerged as important modulators of TGF-beta signaling and responses. SnoN forms a physical complex with the TGF-beta-regulated Smad2/Smad3 and co-Smad4 proteins and either represses or stimulates TGF-beta-induced Smad-dependent transcription in a cell- and promoter-specific manner. In addition, the TGF-beta-activated Smads recruit several ubiquitin ligases to SnoN and thereby promote the ubiquitination and consequent degradation of SnoN. Additional modifications of SnoN, including sumoylation, may contribute to the regulation of SnoN function and its role in TGF-beta signaling. Collectively, these studies suggest that SnoN function is intimately linked to the TGF-beta-Smad pathway in cellular signaling. Although the mechanisms by which SnoN modulates signaling in the TGF-beta-Smad pathway are beginning to be characterized, the full range of SnoN functions and underlying mechanisms in normal development and disease processes remains to be elucidated.

Chl4p and iml3p are two new members of the budding yeast outer kinetochore.

Kinetochore proteins contribute to the fidelity of chromosome transmission by mediating the attachment of a specialized chromosomal region, the centromere, to the mitotic spindle during mitosis. In budding yeast, a subset of kinetochore proteins, referred to as the outer kinetochore, provides a link between centromere DNA-binding proteins of the inner kinetochore and microtubule-binding proteins. Using a combination of chromatin immunoprecipitation, in vivo localization, and protein coimmunoprecipitation, we have established that yeast Chl4p and Iml3p are outer kinetochore proteins that localize to the kinetochore in a Ctf19p-dependent manner. Chl4p interacts with the outer kinetochore proteins Ctf19p and Ctf3p, and Iml3p interacts with Chl4p and Ctf19p. In addition, Chl4p is required for the Ctf19p-Ctf3p and Ctf19p-Iml3p interactions, indicating that Chl4p is an important structural component of the outer kinetochore. These physical interaction dependencies provide insights into the molecular architecture and centromere DNA loading requirements of the outer kinetochore complex.

Identification of protein complexes required for efficient sister chromatid cohesion.

Ctf8p is a component of Ctf18-RFC, an alternative replication factor C-like complex required for efficient sister chromatid cohesion in Saccharomyces cerevisiae. We performed synthetic genetic array (SGA) analysis with a ctf8 deletion strain as a primary screen to identify other nonessential genes required for efficient sister chromatid cohesion. We then assessed proficiency of cohesion at three chromosomal loci in strains containing deletions of the genes identified in the ctf8 SGA screen. Deletion of seven genes (CHL1, CSM3, BIM1, KAR3, TOF1, CTF4, and VIK1) resulted in defective sister chromatid cohesion. Mass spectrometric analysis of immunoprecipitated complexes identified a physical association between Kar3p and Vik1p and an interaction between Csm3p and Tof1p that we confirmed by coimmunoprecipitation from cell extracts. These data indicate that synthetic genetic array analysis coupled with specific secondary screens can effectively identify protein complexes functionally related to a reference gene. Furthermore, we find that genes involved in mitotic spindle integrity and positioning have a previously unrecognized role in sister chromatid cohesion.

Identification of a Novel Link between the Protein Kinase NDR1 and TGFß Signaling in Epithelial Cells.

Transforming growth factor-beta (TGFß) is a secreted polypeptide that plays essential roles in cellular development and homeostasis. Although mechanisms of TGFß-induced responses have been characterized, our understanding of TGFß signaling remains incomplete. Here, we uncover a novel function for the protein kinase NDR1 (nuclear Dbf2-related 1) in TGFß responses. Using an immunopurification approach, we find that NDR1 associates with SnoN, a key component of TGFß signaling. Knockdown of NDR1 by RNA interference promotes the ability of TGFß to induce transcription and cell cycle arrest in NMuMG mammary epithelial cells. Conversely, expression of NDR1 represses TGFß-induced transcription and inhibits the ability of TGFß to induce cell cycle arrest in NMuMG cells. Mechanistically, we find that NDR1 acts in a kinase-dependent manner to suppress the ability of TGFß to induce the phosphorylation and consequent nuclear accumulation of Smad2, which is critical for TGFß-induced transcription and responses. Strikingly, we also find that TGFß reciprocally regulates NDR1, whereby TGFß triggers the degradation of NDR1 protein. Collectively, our findings define a novel and intimate link between the protein kinase NDR1 and TGFß signaling. NDR1 suppresses TGFß-induced transcription and cell cycle arrest, and counteracting NDR1's negative regulation, TGFß signaling induces the downregulation of NDR1 protein. These findings advance our understanding of TGFß signaling, with important implications in development and tumorigenesis.

ING2 as a novel mediator of transforming growth factor-beta-dependent responses in epithelial cells.

Members of the ING (inhibitor of growth) family of chromatin modifying proteins (ING1-ING5) have emerged as critical regulators of gene expression and cellular responses, suggesting that the ING proteins may impinge on specific signal transduction pathways and their mediated effects. Here, we demonstrate a role for the protein ING2 in mediating responses by the transforming growth factor (TGF)-beta-Smad signaling pathway. We show that ING2 promotes TGF-beta-induced transcription. Both gain-of-function and RNA interference-mediated knockdown of endogenous ING2 reveal that ING2 couples TGF-beta signals to the induction of transcription and cell cycle arrest. We also find that the Smad-interacting transcriptional modulator SnoN interacts with ING2 and promotes the assembly of a protein complex containing SnoN, ING2, and Smad2. Knockdown of endogenous SnoN blocks the ability of ING2 to promote TGF-beta-dependent transcription, and conversely expression of SnoN augments ING2 enhancement of the TGF-beta response. Collectively, our data suggest that ING2 collaborates with SnoN to mediate TGF-beta-induced Smad-dependent transcription and cellular responses.

Sumoylated SnoN represses transcription in a promoter-specific manner.

The transcriptional modulator SnoN controls a diverse set of biological processes, including cell proliferation and differentiation. The mechanisms by which SnoN regulates these processes remain incompletely understood. Recent studies have shown that SnoN exerts positive or negative regulatory effects on transcription. Because post-translational modification of proteins by small ubiquitin-like modifier (SUMO) represents an important mechanism in the control of the activity of transcriptional regulators, we asked if this modification regulates SnoN function. Here, we show that SnoN is sumoylated. Our data demonstrate that the SUMO-conjugating E2 enzyme Ubc9 is critical for SnoN sumoylation and that the SUMO E3 ligase PIAS1 selectively interacts with and enhances the sumoylation of SnoN. We identify lysine residues 50 and 383 as the SUMO acceptor sites in SnoN. Analyses of SUMO "loss-of-function" and "gain-of-function" SnoN mutants in transcriptional reporter assays reveal that sumoylation of SnoN contributes to the ability of SnoN to repress gene expression in a promoter-specific manner. Although this modification has little effect on SnoN repression of the plasminogen activator inhibitor-1 promoter and only modestly potentiates SnoN repression of the p21 promoter, SnoN sumoylation robustly augments the ability of SnoN to suppress transcription of the myogenesis master regulatory gene myogenin. In addition, we show that the SnoN SUMO E3 ligase, PIAS1, at its endogenous levels, suppresses myogenin transcription. Collectively, our findings suggest that SnoN is directly regulated by sumoylation leading to the enhancement of the ability of SnoN to repress transcription in a promoter-specific manner. Our study also points to a physiological role for SnoN sumoylation in the control of myogenin expression in differentiating muscle cells.

Spindle checkpoint maintenance requires Ame1 and Okp1.

Kinetochore proteins are required for high fidelity chromosome segregation and as a platform for checkpoint signaling. Ame1 is an essential component of the COMA (Ctf19, Okp1, Mcm21, Ame1) sub-complex of the central kinetochore of budding yeast. In this study, we describe the isolation and characterization of an Ame1 conditional mutant, ame1-4. ame1-4 cells exhibit chromosome segregation defects and Mad2-dependent cell cycle delay similar to okp1-5 cells. However, the viability of ame1-4 cells is markedly reduced relative to wild type and okp1-5 cells after three hours at restrictive temperature. To determine if ame1-4 cells enter anaphase with mis-segregated chromosomes, we monitored the localization of Bub3:VFP as a marker for anaphase onset. ame1-4 cells containing mis-segregated sister chromatids initially accumulate Bub3:VFP at kinetochores, indicating checkpoint activation and a metaphase arrest. Subsequently, Bub3:VFP de-localizes and cells reinitiate DNA duplication and budding without cytokinesis in the presence of un-segregated chromosomes. Overexpression of OKP1 in ame1-4 cells restores ame1-4 protein localization and a stable arrest. Based on our results, we propose that Ame1 and Okp1 are required for a sustained checkpoint arrest in the presence of mis-segregated chromosomes. Our results suggest that checkpoint response might be controlled not only at the level of activation but also via signals that ensure maintenance of the response.

The structural and dynamic basis of Ets-1 DNA binding autoinhibition.

The transcription factor Ets-1 is regulated by the allosteric coupling of DNA binding with the unfolding of an alpha-helix (HI-1) within an autoinhibitory module. To understand the structural and dynamic basis for this autoinhibition, we have used NMR spectroscopy to characterize Ets-1DeltaN301, a partially inhibited fragment of Ets-1. The NMR-derived Ets-1DeltaN301 structure reveals that the autoinhibitory module is formed predominantly by the hydrophobic packing of helices from the N-terminal (HI-1, HI-2) and C-terminal (H4, H5) inhibitory sequences, along with H1 of the intervening DNA binding ETS domain. The intramolecular interactions made by HI-1 in Ets-1DeltaN301 are similar to the intermolecular contacts observed in the crystal structure of an Ets-1DeltaN300 dimer, confirming that the latter represents a domain-swapped species. (15)N relaxation studies demonstrate that the backbone of the N-terminal inhibitory sequence is mobile on the nanosecond-picosecond and millisecond-microsecond time scales. Furthermore, hydrogen exchange measurements reveal that amide protons in helices HI-1 and HI-2 exchange with water at rates only approximately 15- and approximately 75-fold slower, respectively, than predicted for an unfolded polypeptide. These findings indicate that inhibitory helices are only marginally stable even in the absence of DNA. The energetic coupling of DNA binding with the facile unfolding of the labile HI-1 provides a mechanism for modulating Ets-1 DNA binding activity via protein partnerships, post-translational modifications, or mutations. Ets-1 autoinhibition illustrates how conformational equilibria within structural domains can regulate macromolecular interactions.

Systematic yeast synthetic lethal and synthetic dosage lethal screens identify genes required for chromosome segregation.

Accurate chromosome segregation requires the execution and coordination of many processes during mitosis, including DNA replication, sister chromatid cohesion, and attachment of chromosomes to spindle microtubules via the kinetochore complex. Additional pathways are likely involved because faithful chromosome segregation also requires proteins that are not physically associated with the chromosome. Using kinetochore mutants as a starting point, we have identified genes with roles in chromosome stability by performing genome-wide screens employing synthetic genetic array methodology. Two genetic approaches (a series of synthetic lethal and synthetic dosage lethal screens) isolated 211 nonessential deletion mutants that were unable to tolerate defects in kinetochore function. Although synthetic lethality and synthetic dosage lethality are thought to be based upon similar genetic principles, we found that the majority of interactions associated with these two screens were nonoverlapping. To functionally characterize genes isolated in our screens, a secondary screen was performed to assess defects in chromosome segregation. Genes identified in the secondary screen were enriched for genes with known roles in chromosome segregation. We also uncovered genes with diverse functions, such as RCS1, which encodes an iron transcription factor. RCS1 was one of a small group of genes identified in all three screens, and we used genetic and cell biological assays to confirm that it is required for chromosome stability. Our study shows that systematic genetic screens are a powerful means to discover roles for uncharacterized genes and genes with alternative functions in chromosome maintenance that may not be discovered by using proteomics approaches.

Ctf3p, the Mis6 budding yeast homolog, interacts with Mcm22p and Mcm16p at the yeast outer kinetochore.

The budding yeast kinetochore is composed of an inner and outer protein complex, which binds to centromere (CEN) DNA and attaches to microtubules. We performed a genetic synthetic dosage lethality screen to identify novel kinetochore proteins in a collection of chromosome transmission fidelity mutants. Our screen identified several new kinetochore-related proteins including YLR381Wp/Ctf3p, which is a member of a conserved family of centromere-binding proteins. Ctf3p interacts with Mcm22p, Mcm16p, and the outer kinetochore protein Ctf19p. We used chromatin immunoprecipitation to demonstrate that Ctf3p, Mcm22p, and Mcm16p bind to CEN DNA in a Ctf19p-dependent manner. In addition, Ctf3p, Mcm22p, and Mcm16p have a localization pattern similar to other kinetochore proteins. The fission yeast Ctf3p homolog, Mis6, is required for loading of a CENP-A centromere specific histone, Cnp1, onto centromere DNA. We find however that Ctf3p is not required for loading of the budding yeast CENP-A homolog, Cse4p, onto CEN DNA. In contrast, Ctf3p and Ctf19p fail to bind properly to the centromere in a cse4-1 mutant strain. We conclude that the requirements for CENP-A loading onto centromere DNA differ in fission versus budding yeast.