Leah Vardy

Fission yeast ch‐TOG/XMAP215 homologue Alp14 connects mitotic spindles with the kinetochore and is a component of the Mad2‐dependent spindle checkpoint

The TOG/XMAP215‐related proteins play a role in microtubule dynamics at its plus end. Fission yeast Alp14, a newly identified TOG/XMAP215 family protein, is essential for proper chromosome segregation in concert with a second homologue Dis1. We show that the alp14 mutant fails to progress towards normal bipolar spindle formation. Intriguingly, Alp14 itself is a component of the Mad2‐dependent spindle checkpoint cascade, as upon addition of microtubule‐destabilizing drugs the alp14 mutant is incapable of maintaining high H1 kinase activity, which results in securin destruction and premature chromosome separation. Live imaging of Alp14–green fluorescent protein shows that during mitosis, Alp14 is associated with the peripheral region of the kinetochores as well as with the spindle poles. This is supported by ChIP (chromatin immunoprecipitation) and overlapping localization with the kinetochore marker Mis6. An intact spindle is required for Alp14 localization to the kinetochore periphery, but not to the poles. These results indicate that the TOG/XMAP215 family may play a central role as a bridge between the kinetochores and the plus end of pole to chromosome microtubules.

The fission yeast γ-tubulin complex is required in G1 phase and is a component of the spindle assembly checkpoint

Microtubule polymerization is initiated from the microtubule organizing centre (MTOC), which contains the γ‐tubulin complex. We have identified fission yeast Alp4 and Alp6, which are homologues of the γ‐tubulin‐interacting proteins Sc.Spc97/Hs.Gcp2 and Sc.Spc98/Hs.Gcp3, respectively. The size of the fission yeast γ‐tubulin complex is large (>2000 kDa), comparable to that in metazoans. Both Alp4 and Alp6 localize to the spindle pole body (SPB) and also to the equatorial MTOC. Temperature‐sensitive (ts) alp4 and alp6 mutants show two types of microtubular defects. First, monopolar mitotic spindles form. Secondly, abnormally long cytoplasmic microtubules appear that do not stop at the cell tips and are still associated with the SPB. Alp4 function is required in G1 phase and ts mutants become lethal before S‐phase. alp4 and alp6 mutants are hypersensitive to the microtubule‐ destabilizing drug thiabendazole (TBZ) and show a lethal ‘cut’ phenotype in its presence. Furthermore, alp4mad2 double mutants show an exaggerated multiple septation phenotype in TBZ. These results indicate that Alp4 and Alp6 may play a crucial role in the spindle pole‐mediated checkpoint pathway.

Activin/Nodal signaling controls divergent transcriptional networks in human embryonic stem cells and in endoderm progenitors.

Activin/Nodal signaling is necessary to maintain pluripotency of human embryonic stem cells (hESCs) and to induce their differentiation toward endoderm. However, the mechanisms by which Activin/Nodal signaling achieves these opposite functions remain unclear. To unravel these mechanisms, we examined the transcriptional network controlled in hESCs by Smad2 and Smad3, which represent the direct effectors of Activin/Nodal signaling. These analyses reveal that Smad2/3 participate in the control of the core transcriptional network characterizing pluripotency, which includes Oct‐4, Nanog, FoxD3, Dppa4, Tert, Myc, and UTF1. In addition, similar experiments performed on endoderm cells confirm that a broad part of the transcriptional network directing differentiation is downstream of Smad2/3. Therefore, Activin/Nodal signaling appears to control divergent transcriptional networks in hESCs and in endoderm. Importantly, we observed an overlap between the transcriptional network downstream of Nanog and Smad2/3 in hESCs; whereas, functional studies showed that both factors cooperate to control the expression of pluripotency genes. Therefore, the effect of Activin/Nodal signaling on pluripotency and differentiation could be dictated by tissue specific Smad2/3 partners such as Nanog, explaining the mechanisms by which signaling pathways can orchestrate divergent cell fate decisions. STEM CELLS 2011;29:1176–1185

Transcriptional consequences of genomic structural aberrations in breast cancer

Using a long-span, paired-end deep sequencing strategy, we have comprehensively identified cancer genome rearrangements in eight breast cancer genomes. Herein, we show that 40%-54% of these structural genomic rearrangements result in different forms of fusion transcripts and that 44% are potentially translated. We find that single segmental tandem duplication spanning several genes is a major source of the fusion gene transcripts in both cell lines and primary tumors involving adjacent genes placed in the reverse-order position by the duplication event. Certain other structural mutations, however, tend to attenuate gene expression. From these candidate gene fusions, we have found a fusion transcript (RPS6KB1-VMP1) recurrently expressed in ∼30% of breast cancers associated with potential clinical consequences. This gene fusion is caused by tandem duplication on 17q23 and appears to be an indicator of local genomic instability altering the expression of oncogenic components such as MIR21 and RPS6KB1.

A conserved small GTP-binding protein Alp41 is essential for the cofactor-dependent biogenesis of microtubules in fission yeast

The proper folding of tubulins and their incorporation into microtubules consist of a series of reactions, in which evolutionarily conserved proteins, cofactors A to E, play a vital role. We have cloned a fission yeast gene (alp41 +) which encodes a highly conserved small GTP‐binding protein homologous to budding yeast CIN4 and human ARF‐like Arl2. alp41 + is essential, disruption of which results in microtubule dysfunction and growth polarity defects. Genetic analysis indicates that Alp41 plays a crucial role in the cofactor‐dependent pathway, in which it functions upstream of the cofactor D homologue Alp1D and possibly in concert with Alp21E.

AMD1 is essential for ESC self-renewal and is translationally down-regulated on differentiation to neural precursor cells

The gene expression networks governing embryonic stem cell (ESC) pluripotency are complex and finely regulated during differentiation toward specific lineages. We describe a new role for Amd1 (adenosyl methionine decarboxylase), a key enzyme in the polyamine synthesis pathway, in regulating both ESC self-renewal and differentiation to the neural lineage. Amd1 is highly expressed in ESCs and is translationally down-regulated by the neural precursor cell (NPC)-enriched microRNA miR-762 during NPC differentiation. Overexpression of Amd1 or addition of the polyamine spermine blocks ESC-to-NPC conversion, suggesting Amd1 must be down-regulated to decrease the levels of inhibitory spermine during differentiation. In addition, we demonstrate that high levels of Amd1 are required for maintenance of the ESC state. We show that forced overexpression of Amd1 in ESCs results in maintenance of high Myc levels and a delay in differentiation on removal of LIF. We propose that Amd1 is a major regulator of ESC self-renewal and that its essential role lies in its regulation of Myc levels within the cell.

Regulation of Cyclin A protein in meiosis and early embryogenesis

In contrast to the extensive analysis of the regulation of Cyclin B protein levels during developmental progression through meiosis in oogenesis, little is known about Cyclin A. Repression of cyclin A translation early in prophase I in Drosophila is important to maintain the oocyte in meiosis, and this has been shown to be mediated by deadenylation of the mRNA and inhibition by the Bruno repressor. We find that at oocyte maturation as meiosis resumes, Cyclin A protein reappears, coincident with polyadenylation of the mRNA and loss of Bruno repressor. Cyclin A is multiphosphorylated in a pattern consistent with autophosphorylation, and this form accumulates aberrantly in metaphase I if the Cortex form of the Anaphase Promoting Complex/Cyclosome is inactive. The PAN GU (PNG) kinase positively promotes translation of Cyclin A, beginning in oogenesis, an earlier onset than previously recognized. After egg activation and the completion of meiosis, PNG promotes further polyadenylation of cyclin A mRNA and appears to antagonize repression of translation by the PUMILIO inhibitor. Epistasis studies with png; apc mutants indicate that PNG acts solely to promote translation, rather than having a parallel function to inhibit degradation. These studies reveal multiple levels of posttranscriptional regulation of Cyclin A protein by translational and proteolytic control during oocyte maturation and the onset of embryogenesis.

Deletion of Mia1/Alp7 activates Mad2-dependent spindle assembly checkpoint in fission yeast

Deletion of Mia1/Alp7 activates Mad2-dependent spindle assembly checkpoint in fission yeast

Contrasting expression patterns of coding and noncoding parts of the human genome upon oxidative stress

Oxidative stress (OS) is caused by an imbalance between pro- and anti-oxidant reactions leading to accumulation of reactive oxygen species within cells. We here investigate the effect of OS on the transcriptome of human fibroblasts. OS causes a rapid and transient global induction of transcription characterized by pausing of RNA polymerase II (PolII) in both directions, at specific promoters, within 30 minutes of the OS response. In contrast to protein-coding genes, which are commonly down-regulated, this novel divergent, PolII pausing-phenomenon leads to the generation of thousands of long noncoding RNAs (lncRNAs) with promoter-associated antisense lncRNAs transcripts (si-paancRNAs) representing the major group of stress-induced transcripts. OS causes transient dynamics of si-lncRNAs in nucleus and cytosol, leading to their accumulation at polysomes, in contrast to mRNAs, which get depleted from polysomes. We propose that si-lncRNAs represent a novel component of the transcriptional stress that is known to determine the outcome of immediate-early and later cellular stress responses and we provide insights on the fate of those novel mature lncRNA transcripts by showing that their association with polysomal complexes is significantly increased in OS.

Translatome analysis of CHO cells to identify key growth genes

We report the first investigation of translational efficiency on a global scale, also known as translatome, of a Chinese hamster ovary (CHO) DG44 cell line producing monoclonal antibodies (mAb). The translatome data was generated via combined use of high resolution and streamlined polysome profiling technology and proprietary Nimblegen microarrays probing for more than 13K annotated CHO-specific genes. The distribution of ribosome loading during the exponential growth phase revealed the translational activity corresponding to the maximal growth rate, thus allowing us to identify stably and highly translated genes encoding heterogeneous nuclear ribonucleoproteins (Hnrnpc and Hnrnpa2b1), protein regulator of cytokinesis 1 (Prc1), glucose-6-phosphate dehydrogenase (G6pdh), UTP6 small subunit processome (Utp6) and RuvB-like protein 1 (Ruvbl1) as potential key players for cellular growth. Moreover, correlation analysis between transcriptome and translatome data sets showed that transcript level and translation efficiency were uncoupled for 95% of investigated genes, suggesting the implication of translational control mechanisms such as the mTOR pathway. Thus, the current translatome analysis platform offers new insights into gene expression in CHO cell cultures by bridging the gap between transcriptome and proteome data, which will enable researchers of the bioprocessing field to prioritize in high-potential candidate genes and to devise optimal strategies for cell engineering toward improving culture performance.