A genetic screen reveals a role for the late G1-specific transcription factor Swi4p in diverse cellular functions including cytokinesis

1997 ◽  
Vol 110 (14) ◽  
pp. 1647-1654 ◽  
Author(s):  
J.C. Igual ◽  
W.M. Toone ◽  
L.H. Johnston
Keyword(s):  
Cell Cycle ◽  
Transcription Factor ◽  
Ectopic Expression ◽  
Single Copy ◽  
Restrictive Temperature ◽  
Cell Integrity ◽  
Cellular Functions ◽  
Synthetic Lethal ◽  
Cellular Processes ◽  
Set Up

The transcription factor Swi4p plays a crucial role in the control of the initiation of the cell cycle in budding yeast. To further understand Swi4p function, we set up a synthetic lethal screen for genes interacting with SWI4. Fourteen conditional mutations which resulted in lethality only in the absence of SWI4 have been isolated. Only two of them were suppressed by ectopic expression of CLN2, indicating that Swi4p is involved in diverse cellular processes in addition to its requirement for CLN1,2 regulation. In most of the mutants a cell cycle phenotype was observed, including defects in G1 progression, budding, the G2/M transition and cytokinesis. In addition, four of the mutations resulted in massive cell lysis at the restrictive temperature, indicating that Swi4p is involved in the maintenance of cell integrity. One of the mutants, rsf1 swi4delta, was characterized in detail and it is defective in cytokinesis at the restrictive temperature. Staining with Calcofluor revealed that the rsf1 swi4delta mutant is impaired in chitin biosynthesis. rsf1 is allelic to the AGM1 gene, coding for N-acetylglucosamine-phosphate mutase, an enzyme involved in the biosynthesis of chitin. A single copy of SWI4 suppressed the cytokinesis defect. The above data suggest that Swi4p has a role in cytokinesis and becomes essential in this process when chitin biosynthesis is compromised. As overexpression or ectopic expression of CLN did not suppress the rsf1 swi4delta mutant phenotype, Swi4p must control some other gene(s) involved in cytokinesis.

scholarly journals SUMO conjugation susceptibility of Akt/protein kinase B affects the expression of the pluripotency transcription factor Nanog in embryonic stem cells

PLoS ONE ◽  
2021 ◽  
Vol 16 (7) ◽  
pp. e0254447
Author(s):  
Marcos Francia ◽  
Martin Stortz ◽  
Camila Vazquez Echegaray ◽  
Camila Oses ◽  
Paula Verneri ◽  
...  
Keyword(s):  
Transcription Factor ◽  
Es Cells ◽  
Embryonic Stem ◽  
Dependent Manner ◽  
Cellular Functions ◽  
Cellular Processes ◽  
Sumo Conjugation ◽  
Heterologous System ◽  
Canonical Pathways ◽  
The Impact

Akt/PKB is a kinase involved in the regulation of a wide variety of cell processes. Its activity is modulated by diverse post-translational modifications (PTMs). Particularly, conjugation of the small ubiquitin-related modifier (SUMO) to this kinase impacts on multiple cellular functions, such as proliferation and splicing. In embryonic stem (ES) cells, this kinase is key for pluripotency maintenance. Among other functions, Akt is known to promote the expression of Nanog, a central pluripotency transcription factor (TF). However, the relevance of this specific PTM of Akt has not been previously analyzed in this context. In this work, we study the effect of Akt1 variants with differential SUMOylation susceptibility on the expression of Nanog. Our results demonstrate that both, the Akt1 capability of being modified by SUMO conjugation and a functional SUMO conjugase activity are required to induce Nanog gene expression. Likewise, we found that the common oncogenic E17K Akt1 mutant affected Nanog expression in ES cells also in a SUMOylatability dependent manner. Interestingly, this outcome takes places in ES cells but not in a non-pluripotent heterologous system, suggesting the presence of a crucial factor for this induction in ES cells. Remarkably, the two major candidate factors to mediate this induction, GSK3-β and Tbx3, are non-essential players of this effect, suggesting a complex mechanism probably involving non-canonical pathways. Furthermore, we found that Akt1 subcellular distribution does not depend on its SUMOylatability, indicating that Akt localization has no influence on the effect on Nanog, and that besides the membrane localization of E17K Akt mutant, SUMOylation is also required for its hyperactivity. Our results highlight the impact of SUMO conjugation in the function of a kinase relevant for a plethora of cellular processes, including the control of a key pluripotency TF.

scholarly journals Transcriptional Coregulation by the Cell Integrity Mitogen-Activated Protein Kinase Slt2 and the Cell Cycle Regulator Swi4

2001 ◽  
Vol 21 (19) ◽  
pp. 6515-6528 ◽  
Author(s):  
Kristin Baetz ◽  
Jason Moffat ◽  
Jennifer Haynes ◽  
Michael Chang ◽  
Brenda Andrews
Keyword(s):  
Gene Expression ◽  
Cell Cycle ◽  
Cell Wall ◽  
Transcription Factor ◽  
Protein Kinase ◽  
Mapk Pathway ◽  
Mitogen Activated Protein Kinase ◽  
Regulatory Subunit ◽  
Cell Integrity ◽  
Mitogen Activated Protein

ABSTRACT In Saccharomyces cerevisiae, the heterodimeric transcription factor SBF (for SCB binding factor) is composed of Swi4 and Swi6 and activates gene expression at the G1/S-phase transition of the mitotic cell cycle. Cell cycle commitment is associated not only with major alterations in gene expression but also with highly polarized cell growth; the mitogen-activated protein kinase (MAPK) Slt2 is required to maintain cell wall integrity during periods of polarized growth and cell wall stress. We describe experiments aimed at defining the regulatory pathway involving the cell cycle transcription factor SBF and Slt2-MAPK. Gene expression assays and chromatin immunoprecipitation experiments revealed Slt2-dependent recruitment of SBF to the promoters of the G1 cyclinsPCL1 and PCL2 after activation of the Slt2-MAPK pathway. We performed DNA microarray analysis and identified other genes whose expression was reduced in both SLT2and SWI4 deletion strains. Genes that are sensitive to both Slt2 and Swi4 appear to be uniquely regulated and reveal a role for Swi4, the DNA-binding component of SBF, which is independent of the regulatory subunit Swi6. Some of the Swi4- and Slt2-dependent genes do not require Swi6 for either their expression or for Swi4 localization to their promoters. Consistent with these results, we found a direct interaction between Swi4 and Slt2. Our results establish a new Slt2-dependent mode of Swi4 regulation and suggest roles for Swi4 beyond its prominent role in controlling cell cycle transcription.

scholarly journals Protein manipulation using single copies of short peptide tags in cultured cells and in Drosophila melanogaster

2020 ◽  
Author(s):  
M. Alessandra Vigano ◽  
Clara-Maria Ell ◽  
Manuela MM Kustermann ◽  
Gustavo Aguilar ◽  
Shinya Matsuda ◽  
...  
Keyword(s):  
Cultured Cells ◽  
Single Copy ◽  
Specific Protein ◽  
Cellular Functions ◽  
Cellular Processes ◽  
Peptide Tags ◽  
Genetic Technologies ◽  
Protein Binders ◽  
And Function

AbstractCellular development and specialized cellular functions are regulated processes which rely on highly dynamic molecular interactions among proteins, distributed in all cell compartments. Analysis of these interactions and their mechanisms of action has been one of the main topics in cellular and developmental research over the last fifty years. Studying and understanding the functions of proteins of interest (POIs) has been mostly achieved by their alteration at the genetic level and the analysis of the phenotypic changes generated by these alterations. Although genetic and reverse genetic technologies contributed to the vast majority of information and knowledge we have gathered so far, targeting specific interactions of POIs in a time- and space-controlled manner or analyzing the role of POIs in dynamic cellular processes such as cell migration or cell division would require more direct approaches. The recent development of specific protein binders, which can be expressed and function intracellularly, together with several improvements in synthetic biology techniques, have contributed to the creation of a new toolbox for direct protein manipulations. We selected a number of short tag epitopes for which protein binders from different scaffolds have been developed and tested whether these tags can be bound by the corresponding protein binders in living cells when they are inserted in a single copy in a POI. We indeed find that in all cases, a single copy of a short tag allows protein binding and manipulation. Using Drosophila, we also find that single short tags can be recognized and allow degradation and relocalization of POIs in vivo.

scholarly journals Protein Kinase SGK Mediates Survival Signals by Phosphorylating the Forkhead Transcription Factor FKHRL1 (FOXO3a)

2001 ◽  
Vol 21 (3) ◽  
pp. 952-965 ◽  
Author(s):  
Anne Brunet ◽  
Jongsun Park ◽  
Hien Tran ◽  
Linda S. Hu ◽  
Brian A. Hemmings ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Transcription Factor ◽  
Transcription Factors ◽  
Cell Survival ◽  
Cell Cycle Progression ◽  
Target Genes ◽  
Cellular Functions ◽  
Forkhead Transcription Factor ◽  
Pi3k Activation ◽  
Phosphoinositide 3 Kinase

ABSTRACT Serum- and glucocorticoid-inducible kinases (SGKs) form a novel family of serine/threonine kinases that are activated in response to a variety of extracellular stimuli. SGKs are related to Akt (also called PKB), a serine/threonine kinase that plays a crucial role in promoting cell survival. Like Akt, SGKs are activated by the phosphoinositide-3 kinase (PI3K) and translocate to the nucleus upon growth factor stimulation. However the physiological substrates and cellular functions of SGKs remained to be identified. We hypothesized that SGKs regulate cellular functions in concert with Akt by phosphorylating common targets within the nucleus. The best-characterized nuclear substrates of Akt are transcription factors of the Forkhead family. Akt phosphorylates Forkhead transcription factors such as FKHRL1, leading to FKHRL1's exit from the nucleus and the consequent shutoff of FKHRL1 target genes. We show here that SGK1, like Akt, promotes cell survival and that it does so in part by phosphorylating and inactivating FKHRL1. However, SGK and Akt display differences with respect to the efficacy with which they phosphorylate the three regulatory sites on FKHRL1. While both kinases can phosphorylate Thr-32, SGK displays a marked preference for Ser-315 whereas Akt favors Ser-253. These findings suggest that SGK and Akt may coordinately regulate the function of FKHRL1 by phosphorylating this transcription factor at distinct sites. The efficient phosphorylation of these three sites on FKHRL1 by SGK and Akt appears to be critical to the ability of growth factors to suppress FKHRL1-dependent transcription, thereby preventing FKHRL1 from inducing cell cycle arrest and apoptosis. These findings indicate that SGK acts in concert with Akt to propagate the effects of PI3K activation within the nucleus and to mediate the biological outputs of PI3K signaling, including cell survival and cell cycle progression.

scholarly journals MYC: a multipurpose oncogene with prognostic and therapeutic implications in blood malignancies

2021 ◽  
Vol 14 (1) ◽  
Author(s):  
Seyed Esmaeil Ahmadi ◽  
Samira Rahimi ◽  
Bahman Zarandi ◽  
Rouzbeh Chegeni ◽  
Majid Safa
Keyword(s):  
Cell Cycle ◽  
Transcription Factor ◽  
Dna Damage Response ◽  
Hematologic Malignancies ◽  
Cellular Functions ◽  
Therapeutic Implications ◽  
Myc Oncogene ◽  
Damage Response ◽  
Multiple Levels ◽  
Potential Inhibitors

AbstractMYC oncogene is a transcription factor with a wide array of functions affecting cellular activities such as cell cycle, apoptosis, DNA damage response, and hematopoiesis. Due to the multi-functionality of MYC, its expression is regulated at multiple levels. Deregulation of this oncogene can give rise to a variety of cancers. In this review, MYC regulation and the mechanisms by which MYC adjusts cellular functions and its implication in hematologic malignancies are summarized. Further, we also discuss potential inhibitors of MYC that could be beneficial for treating hematologic malignancies.

scholarly journals The FACT complex and cell cycle progression are essential to maintain asymmetric transcription factor partitioning during cell division

2016 ◽  
Author(s):  
Eva Herrero ◽  
Sonia Stinus ◽  
Eleanor Bellows ◽  
Peter H Thorpe
Keyword(s):  
Cell Cycle ◽  
Transcription Factor ◽  
Cell Division ◽  
Cell Cycle Progression ◽  
Asymmetric Cell Division ◽  
Cycle Progression ◽  
Cellular Processes ◽  
Daughter Cells ◽  
Cycle Arrest ◽  
Reverse Genetic Screen

AbstractThe polarized partitioning of proteins in cells underlies asymmetric cell division, which is an important driver of development and cellular diversity. Like most cells, the budding yeast Saccharomyces cerevisiae divides asymmetrically to give two distinct daughter cells. This asymmetry mimics that seen in metazoans and the key regulatory proteins are conserved from yeast to human. A well-known example of an asymmetric protein is the transcription factor Ace2, which localizes specifically to the daughter nucleus, where it drives a daughter-specific transcriptional network. We performed a reverse genetic screen to look for regulators of asymmetry based on the Ace2 localization phenotype. We screened a collection of essential genes in order to analyze the effect of core cellular processes in asymmetric cell division. This identified a large number of mutations that are known to affect progression through the cell cycle, suggesting that cell cycle delay is sufficient to disrupt Ace2 asymmetry. To test this model we blocked cells from progressing through mitosis and found that prolonged cell cycle arrest is sufficient to disrupt Ace2 asymmetry after release. We also demonstrate that members of the evolutionary conserved FACT chromatin-remodeling complex are required for both asymmetric and cell cycle-regulated localization of Ace2.

The Phox2b transcription factor coordinately regulates neuronal cell cycle exit and identity

Development ◽  
2000 ◽  
Vol 127 (23) ◽  
pp. 5191-5201 ◽  
Author(s):  
V. Dubreuil ◽  
M. Hirsch ◽  
A. Pattyn ◽  
J. Brunet ◽  
C. Goridis
Keyword(s):  
Cell Cycle ◽  
Transcription Factor ◽  
Cell Fate ◽  
Motor Neurons ◽  
Ventricular Zone ◽  
Ectopic Expression ◽  
Neuronal Cell ◽  
Cell Cycle Exit ◽  
Neuronal Precursors ◽  
Regulation Of Cell Cycle

In the vertebrate neural tube, cell cycle exit of neuronal progenitors is accompanied by the expression of transcription factors that define their generic and sub-type specific properties, but how the regulation of cell cycle withdrawal intersects with that of cell fate determination is poorly understood. Here we show by both loss- and gain-of-function experiments that the neuronal-subtype-specific homeodomain transcription factor Phox2b drives progenitor cells to become post-mitotic. In the absence of Phox2b, post-mitotic neuronal precursors are not generated in proper numbers. Conversely, forced expression of Phox2b in the embryonic chick spinal cord drives ventricular zone progenitors to become post-mitotic neurons and to relocate to the mantle layer. In the neurons thus generated, ectopic expression of Phox2b is sufficient to initiate a programme of motor neuronal differentiation characterised by expression of Islet1 and of the cholinergic transmitter phenotype, in line with our previous results showing that Phox2b is an essential determinant of cranial motor neurons. These results suggest that Phox2b coordinates quantitative and qualitative aspects of neurogenesis, thus ensuring that neurons of the correct phenotype are generated in proper numbers at the appropriate times and locations.

scholarly journals A Role for the START Gene–specific Transcription Factor Complex in the Inactivation of Cyclin B and Cut2 Destruction

2000 ◽  
Vol 11 (10) ◽  
pp. 3411-3424 ◽  
Author(s):  
Sylvie Tournier ◽  
Jonathan B.A. Millar
Keyword(s):  
Cell Cycle ◽  
Transcription Factor ◽  
Fission Yeast ◽  
Single Point ◽  
Cyclin B ◽  
Mitotic Catastrophe ◽  
Single Point Mutation ◽  
Restrictive Temperature ◽  
Specific Transcription Factor ◽  
Transcription Factor Complex

Hyperactivation of Cdc2 in fission yeast causes cells to undergo a lethal premature mitosis called mitotic catastrophe. This phenotype is observed in cdc2-3w wee1-50 cells at high temperature. Eleven of 17 mutants that suppress this phenotype define a single complementation group, mcs1. The mcs1-77mutant also suppresses lethal inactivation of the Wee1 and Mik1 tyrosine kinases and thus delays mitosis independently of Cdc2 tyrosine phosphorylation. We have cloned mcs1 by isolating suppressors of the cell cycle arrest phenotype of mcs1-77 cdc25-22 cells and found that it encodes Res2, a component of the START gene–specific transcription factor complex MBF (also known as DSC-1). The mcs1-77 mutant bears a single point mutation in the DNA-binding domain of Res2 that causes glycine 68 to be replaced by a serine residue. Importantly, two substrates of the anaphase-promoting complex (APC), the major B-type cyclin, Cdc13, and the anaphase inhibitor, Cut2, are unstable in G2-phasemcs1-77 cells. Consistent with this, we observe abnormal sister chromatid separation in mcs1-77 cdc25-22 cells at the restrictive temperature. Mutation of either Cdc10 or Res1 also deregulates MBF-dependent transcription and causes a G2 delay. We find that this cell cycle delay is abolished in the absence of the APC regulator Ste9/Srw1 and that the periodic expression of Ste9/Srw1 is controlled by the MBF complex. These data suggest that in fission yeast the MBF complex plays a key role in the inactivation of cyclin B and Cut2 destruction by controlling the periodic production of APC regulators.

scholarly journals Genetic and Physical Interactions Between Factors Involved in Both Cell Cycle Progression and Pre-mRNA Splicing inSaccharomyces cerevisiae

Genetics ◽  
2000 ◽  
Vol 156 (4) ◽  
pp. 1503-1517 ◽  
Author(s):  
Sigal Ben-Yehuda ◽  
Ian Dix ◽  
Caroline S Russell ◽  
Margaret McGarvey ◽  
Jean D Beggs ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Cell Cycle Progression ◽  
Synthetic Lethality ◽  
Mrna Splicing ◽  
Splicing Factors ◽  
Restrictive Temperature ◽  
Cycle Progression ◽  
Cellular Processes ◽  
Physical Interactions ◽  
Mutant Cells

AbstractThe PRP17/CDC40 gene of Saccharomyces cerevisiae functions in two different cellular processes: pre-mRNA splicing and cell cycle progression. The Prp17/Cdc40 protein participates in the second step of the splicing reaction and, in addition, prp17/cdc40 mutant cells held at the restrictive temperature arrest in the G2 phase of the cell cycle. Here we describe the identification of nine genes that, when mutated, show synthetic lethality with the prp17/cdc40Δ allele. Six of these encode known splicing factors: Prp8p, Slu7p, Prp16p, Prp22p, Slt11p, and U2 snRNA. The other three, SYF1, SYF2, and SYF3, represent genes also involved in cell cycle progression and in pre-mRNA splicing. Syf1p and Syf3p are highly conserved proteins containing several copies of a repeated motif, which we term RTPR. This newly defined motif is shared by proteins involved in RNA processing and represents a subfamily of the known TPR (tetratricopeptide repeat) motif. Using two-hybrid interaction screens and biochemical analysis, we show that the SYF gene products interact with each other and with four other proteins: Isy1p, Cef1p, Prp22p, and Ntc20p. We discuss the role played by these proteins in splicing and cell cycle progression.

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