scholarly journals Histone titration against the genome sets the DNA-to-cytoplasm threshold for the Xenopus midblastula transition

2015 ◽  
Vol 112 (10) ◽  
pp. E1086-E1095 ◽  
Author(s):  
Amanda A. Amodeo ◽  
David Jukam ◽  
Aaron F. Straight ◽  
Jan M. Skotheim
Keyword(s):  
Cell Cycle ◽  
Transcriptional Activation ◽  
Cell Cycle Checkpoints ◽  
Midblastula Transition ◽  
Cell Cycles ◽  
Egg Extract ◽  
Activation Of Transcription ◽  
Maternal To Zygotic Transition ◽  
Synchronous Cell

During early development, animal embryos depend on maternally deposited RNA until zygotic genes become transcriptionally active. Before this maternal-to-zygotic transition, many species execute rapid and synchronous cell divisions without growth phases or cell cycle checkpoints. The coordinated onset of transcription, cell cycle lengthening, and cell cycle checkpoints comprise the midblastula transition (MBT). A long-standing model in the frog, Xenopus laevis, posits that MBT timing is controlled by a maternally loaded inhibitory factor that is titrated against the exponentially increasing amount of DNA. To identify MBT regulators, we developed an assay using Xenopus egg extract that recapitulates the activation of transcription only above the DNA-to-cytoplasm ratio found in embryos at the MBT. We used this system to biochemically purify factors responsible for inhibiting transcription below the threshold DNA-to-cytoplasm ratio. This unbiased approach identified histones H3 and H4 as concentration-dependent inhibitory factors. Addition or depletion of H3/H4 from the extract quantitatively shifted the amount of DNA required for transcriptional activation in vitro. Moreover, reduction of H3 protein in embryos induced premature transcriptional activation and cell cycle lengthening, and the addition of H3/H4 shortened post-MBT cell cycles. Our observations support a model for MBT regulation by DNA-based titration and suggest that depletion of free histones regulates the MBT. More broadly, our work shows how a constant concentration DNA binding molecule can effectively measure the amount of cytoplasm per genome to coordinate division, growth, and development.

scholarly journals Translation of Maternal TATA-Binding Protein mRNA Potentiates Basal but Not Activated Transcription in Xenopus Embryos at the Midblastula Transition

1999 ◽  
Vol 19 (12) ◽  
pp. 7972-7982 ◽  
Author(s):  
Gert Jan C. Veenstra ◽  
Olivier H. J. Destrée ◽  
Alan P. Wolffe
Keyword(s):  
Transcriptional Activation ◽  
Transcriptional Repression ◽  
Translational Regulation ◽  
Binding Protein ◽  
Cell Extract ◽  
Tata Binding Protein ◽  
Nucleosome Assembly ◽  
Midblastula Transition ◽  
Egg Extract

ABSTRACT Early embryonic development in Xenopus laevis is characterized by transcriptional repression which is relieved at the midblastula stage (MBT). Here we show that the relative abundance of TATA-binding protein (TBP) increases robustly at the MBT and that the mechanism underlying this increase is translation of maternally stored TBP RNA. We show that TBP is rate-limiting in egg extract under conditions that titrate nucleosome assembly. Precocious translation of TBP mRNA in Xenopus embryos facilitates transcription before the MBT, without requiring TBP to be prebound to the promoter before injection. This effect is transient in the absence of chromatin titration and is sustained when chromatin is titrated. These data show that translational regulation of TBP RNA contributes to limitations on the transcriptional capacity before the MBT. Second, we examined the ability of trans-acting factors to contribute to promoter activity before the MBT. Deletion of cis-acting elements does not affect histone H2B transcription in egg extract, a finding indicative of limited trans-activation. Moreover, in the context of the intact promoter, neither the transcriptional activator Oct-1, nor TBP, nor TFIID enable transcriptional activation in vitro. HeLa cell extract, however, reconstitutes activated transcription in mixed extracts. These data suggest a deficiency in egg extract cofactors required for activated transcription. We show that the capacity for activated H2B transcription is gradually acquired at the early gastrula transition. This transition occurs well after the blastula stage when the basal transcription machinery can first be complemented with TBP.

scholarly journals p53 upregulated by HIF-1α promotes hypoxia-induced G2/M arrest and renal fibrosis in vitro and in vivo

2018 ◽  
Vol 11 (5) ◽  
pp. 371-382 ◽  
Author(s):  
Limin Liu ◽  
Peng Zhang ◽  
Ming Bai ◽  
Lijie He ◽  
Lei Zhang ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Transcriptional Activation ◽  
Renal Fibrosis ◽  
Intraperitoneal Administration ◽  
Luciferase Reporter ◽  
Loss Of Function ◽  
P53 Expression ◽  
Tubular Cells

Abstract Hypoxia plays an important role in the genesis and progression of renal fibrosis. The underlying mechanisms, however, have not been sufficiently elucidated. We examined the role of p53 in hypoxia-induced renal fibrosis in cell culture (human and rat renal tubular epithelial cells) and a mouse unilateral ureteral obstruction (UUO) model. Cell cycle of tubular cells was determined by flow cytometry, and the expression of profibrogenic factors was determined by RT-PCR, immunohistochemistry, and western blotting. Chromatin immunoprecipitation and luciferase reporter experiments were performed to explore the effect of HIF-1α on p53 expression. We showed that, in hypoxic tubular cells, p53 upregulation suppressed the expression of CDK1 and cyclins B1 and D1, leading to cell cycle (G2/M) arrest (or delay) and higher expression of TGF-β, CTGF, collagens, and fibronectin. p53 suppression by siRNA or by a specific p53 inhibitor (PIF-α) triggered opposite effects preventing the G2/M arrest and profibrotic changes. In vivo experiments in the UUO model revealed similar antifibrotic results following intraperitoneal administration of PIF-α (2.2 mg/kg). Using gain-of-function, loss-of-function, and luciferase assays, we further identified an HRE3 region on the p53 promoter as the HIF-1α-binding site. The HIF-1α–HRE3 binding resulted in a sharp transcriptional activation of p53. Collectively, we show the presence of a hypoxia-activated, p53-responsive profibrogenic pathway in the kidney. During hypoxia, p53 upregulation induced by HIF-1α suppresses cell cycle progression, leading to the accumulation of G2/M cells, and activates profibrotic TGF-β and CTGF-mediated signaling pathways, causing extracellular matrix production and renal fibrosis.

RNA polymerase II cofactor PC2 facilitates activation of transcription by GAL4-AH in vitro

1994 ◽  
Vol 14 (6) ◽  
pp. 3927-3937
Author(s):  
M Kretzschmar ◽  
G Stelzer ◽  
R G Roeder ◽  
M Meisterernst
Keyword(s):  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Transcriptional Activation ◽  
Activation Domains ◽  
Positive Effects ◽  
Activation Of Transcription ◽  
Transcriptional Activation Domains ◽  
Necessary And Sufficient ◽  
Specific Pathway

We have isolated from a crude Hela cell cofactor fraction (USA) a novel positive cofactor that cooperates with the general transcription machinery to effect efficient stimulation of transcription by GAL4-AH, a derivative of the Saccharomyces cerevisiae regulatory factor GAL4. PC2 was shown to be a 500-kDa protein complex and to be functionally and biochemically distinct from native TFIID and previously identified cofactors. In the presence of native TFIID and other general factors, PC2 was necessary and sufficient for activation by GAL4-AH. Cofactor function was specific for transcriptional activation domains of GAL4-AH. The repressor histone H1 further potentiated but was not required for activation of transcription by GAL4-AH. On the basis of the observation that PC2 exerts entirely positive effects on transcription, we propose a model in which PC2 increases the activity of the preinitiation complex in the presence of an activator, thereby establishing a specific pathway during activation of RNA polymerase II.

190 CELL CYCLE REGULATION IN RHESUS MONKEY OOCYTES AND EMBRYOS OF DIFFERENT DEVELOPMENTAL POTENTIAL

2009 ◽  
Vol 21 (1) ◽  
pp. 194
Author(s):  
N. Mtango ◽  
K. Latham
Keyword(s):  
Cell Cycle ◽  
Rhesus Monkey ◽  
Transcriptional Activation ◽  
Cell Cycle Regulation ◽  
Technical Assistance ◽  
Regulatory Gene ◽  
Developmental Potential ◽  
Cycle Regulation

After fertilization, cell division is required for development during the transition from a zygote to an embryo. Degradation of oocyte transcripts, transcriptional activation of the nucleus, and chromatin remodeling occur during early cleavage divisions. Defects in cell cycle regulation decrease the ability of embryo to grow and can be detrimental. In the rhesus monkey, embryos derived by fertilization of oocytes from prepubertal females or oocytes collected during the non-breeding season undergo cleavage arrest (Schramm and Bavister 1994; Zheng et al. 2001). We employed the Primate Embryo Gene Expression Resource (PREGER; www.Preger.org) to examine the expression pattern of 70 mRNAs involved in cell cycle regulation in rhesus monkey oocytes and embryos derived from different stimulation protocols (non-stimulated, FSH stimulated-in vitro matured, and FSH and hCG stimulated-in vivo matured; Mtango and Latham 2007, 2008; Zheng et al. 2005). The resource encompasses a large, biologically rich set of more than 170 samples with 1 to 4 oocytes or embryos which were constructed using the quantitative amplification and dot blotting method. This method entails the direct lysis of small numbers of oocytes or embryos in a reverse transcription buffer supplemented with nonionic detergent, thereby avoiding RNA losses associated with organic extractions (Brady and Iscove 1993). We find that aberrant regulation of cell cycle regulatory gene mRNAs is a prominent feature of oocytes and embryos of compromised developmental potential (FSH stimulated-moderate reduced potential and NS-severely compromised potential). Of the 56 mRNAs for which expression was detected, there was significant aberrations related to oocyte and embryo quality in the expression of more than half (n = 30), P < 0.05), 26 of 30 display significant differences in metaphase II stage oocytes, 20 being altered in FSH stimulated females and 24 of 30 being altered in NS females. The comparison between monkey and previously reported mouse array expression data (Zeng et al. 2004) revealed striking differences between 2 species. These data provide novel information about disruptions in the expression of genes controlling the cell cycle in oocytes and embryos of compromised developmental potential. We thank Bela Patel, Malgorzata McMenamin, and Ann Marie Paprocki for their technical assistance. We also thank R. Dee Schramm for his contribution to the development of the PREGER resource. This work was supported by National Centers for Research Resources Grant RR-15253.

scholarly journals The atypical cyclin-like protein Spy1 overrides p53-mediated tumour suppression and promotes susceptibility to breast tumourigenesis

2019 ◽  
Vol 21 (1) ◽  
Author(s):  
Bre-Anne Fifield ◽  
Ingrid Qemo ◽  
Evie Kirou ◽  
Robert D. Cardiff ◽  
Lisa Ann Porter
Keyword(s):  
Cell Cycle ◽  
Mouse Model ◽  
Lobular Carcinoma ◽  
Critical Role ◽  
Mammary Development ◽  
Cell Cycle Checkpoints ◽  
Checkpoint Control ◽  
Protein Activity ◽  
Tumour Suppression

Abstract Background Breast cancer is the most common cancer to affect women and one of the leading causes of cancer-related deaths. Proper regulation of cell cycle checkpoints plays a critical role in preventing the accumulation of deleterious mutations. Perturbations in the expression or activity of mediators of cell cycle progression or checkpoint activation represent important events that may increase susceptibility to the onset of carcinogenesis. The atypical cyclin-like protein Spy1 was isolated in a screen for novel genes that could bypass the DNA damage response. Clinical data demonstrates that protein levels of Spy1 are significantly elevated in ductal and lobular carcinoma of the breast. We hypothesized that elevated Spy1 would override protective cell cycle checkpoints and support the onset of mammary tumourigenesis. Methods We generated a transgenic mouse model driving expression of Spy1 in the mammary epithelium. Mammary development, growth characteristics and susceptibility to tumourigenesis were studied. In vitro studies were conducted to investigate the relationship between Spy1 and p53. Results We found that in the presence of wild-type p53, Spy1 protein is held ‘in check’ via protein degradation, representing a novel endogenous mechanism to ensure protected checkpoint control. Regulation of Spy1 by p53 is at the protein level and is mediated in part by Nedd4. Mutation or abrogation of p53 is sufficient to allow for accumulation of Spy1 levels resulting in mammary hyperplasia. Sustained elevation of Spy1 results in elevated proliferation of the mammary gland and susceptibility to tumourigenesis. Conclusions This mouse model demonstrates for the first time that degradation of the cyclin-like protein Spy1 is an essential component of p53-mediated tumour suppression. Targeting cyclin-like protein activity may therefore represent a mechanism of re-sensitizing cells to important cell cycle checkpoints in a therapeutic setting.

scholarly journals AMF1 (GPS2) Modulates p53 Transactivation

2001 ◽  
Vol 21 (17) ◽  
pp. 5913-5924 ◽  
Author(s):  
Yu-Cai Peng ◽  
Felix Kuo ◽  
David E. Breiding ◽  
Yu-Fang Wang ◽  
Claire P. Mansur ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Cell Death ◽  
Transcriptional Activation ◽  
Nuclear Factor ◽  
P53 Response ◽  
U2os Cells ◽  
Stress Signals ◽  
Cellular Transcription

ABSTRACT We have reported that the papillomavirus E2 protein binds the nuclear factor AMF1 (also called G-protein pathway suppressor 2 or GPS2) and that their interaction is necessary for transcriptional activation by E2. It has also been shown that AMF1 can influence the activity of cellular transcription factors. These observations led us to test whether AMF1 regulates the functions of p53, a critical transcriptional activator that integrates stress signals and regulates cell cycle and programmed cell death. We report that AMF1 associates with p53 in vivo and in vitro and facilitates the p53 response by augmenting p53-dependent transcription. Overexpression of AMF1 in U2OS cells increases basal level p21WAF1/CIP1 expression and causes a G1 arrest. U2OS cells stably overexpressing AMF1 show increased apoptosis upon exposure to UV irradiation. These data demonstrate that AMF1 modulates p53 activities.

scholarly journals Modulation of liver-specific transcription by interactions between hepatocyte nuclear factor 3 and nuclear factor 1 binding DNA in close apposition.

1993 ◽  
Vol 13 (4) ◽  
pp. 2401-2410 ◽  
Author(s):  
D A Jackson ◽  
K E Rowader ◽  
K Stevens ◽  
C Jiang ◽  
P Milos ◽  
...  
Keyword(s):  
Transcriptional Activation ◽  
Mutational Analysis ◽  
Regulatory Element ◽  
Nuclear Factor ◽  
Hepatocyte Nuclear Factor ◽  
In Vitro Differentiation ◽  
Enhancer Activity ◽  
Activation Of Transcription ◽  
Nuclear Factor 1

The liver-specific enhancer of the serum albumin gene contains an essential segment, designated eH, which binds the hepatocyte nuclear factor 3 alpha (HNF3 alpha) and ubiquitous nuclear factor 1/CCAAT transcription factor (NF1/CTF) proteins in tight apposition. We previously showed that activation of transcription by the eH site was correlated with an increase in intracellular HNF3 alpha levels during the in vitro differentiation of the hepatic cell line H2.35. We now show that transfection of an HNF3 alpha cDNA expression vector into dedifferentiated H2.35 cells is sufficient to induce transcription from the eH site. Mutational analysis of the enhancer demonstrates that NF1/CTF cooperates with HNF3 alpha to induce enhancer activity. However, when the eH site is removed from the context of the enhancer, NF1/CTF can inhibit transcriptional activation by HNF3 alpha. We conclude that the ternary complex of HNF3 alpha, NF1/CTF, and the eH site forms a novel, composite regulatory element that is sensitive to the local DNA sequence environment and suggest that the transcriptional stimulatory activity of NF1/CTF depends on its higher-order interactions with other proteins during hepatocyte differentiation.

scholarly journals Excess histone H3 is a Chk1 inhibitor that controls embryonic cell cycle progression

2020 ◽  
Author(s):  
Yuki Shindo ◽  
Amanda A. Amodeo
Keyword(s):  
Cell Cycle ◽  
Cell Fate ◽  
Cell Cycle Progression ◽  
Histone H3 ◽  
Embryonic Cell ◽  
Cycle Progression ◽  
Cell Cycles ◽  
Embryonic Cell Cycle

AbstractThe early embryos of many species undergo a switch from rapid, reductive cleavage divisions to slower, cell fate-specific division patterns at the Mid-Blastula Transition (MBT). The maternally loaded histone pool is used to measure the increasing ratio of nuclei to cytoplasm (N/C ratio) to control MBT onset, but the molecular mechanism of how histones regulate the cell cycle has remained elusive. Here, we show that excess histone H3 inhibits the DNA damage checkpoint kinase Chk1 to promote cell cycle progression in the Drosophila embryo. We find that excess H3-tail that cannot be incorporated into chromatin is sufficient to shorten the embryonic cell cycle and reduce the activity of Chk1 in vitro and in vivo. Removal of the Chk1 phosphosite in H3 abolishes its ability to regulate the cell cycle. Mathematical modeling quantitatively supports a mechanism where changes in H3 nuclear concentrations over the final cell cycles leading up to the MBT regulate Chk1-dependent cell cycle slowing. We provide a novel mechanism for Chk1 regulation by H3, which is crucial for proper cell cycle remodeling during early embryogenesis.

The protein phosphatase inhibitor okadaic acid induces defects in cytokinesis and organellar genome segregation in Trypanosoma brucei

1994 ◽  
Vol 107 (12) ◽  
pp. 3477-3483 ◽  
Author(s):  
A. Das ◽  
M. Gale ◽  
V. Carter ◽  
M. Parsons
Keyword(s):  
Cell Cycle ◽  
Mitochondrial Dna ◽  
Okadaic Acid ◽  
Protein Phosphatase ◽  
Specific Inhibitor ◽  
Eukaryotic Cell ◽  
Cell Cycles ◽  
African Trypanosomes ◽  
20 Nm

Mitosis and cytokinesis are events that are highly coordinated in most eukaryotic cell cycles. African trypanosomes possess a single mitochondrion and must additionally coordinate the organellar division cycle. Here we report that okadaic acid, a potent and specific inhibitor of protein phosphatases PP1and PP2A, uncouples these cycles in living trypanosomes. Cell cycle analysis of treated cells revealed elevated DNA content. Microscopic examination indicated that okadaic acid treatment yielded multinucleate cells with a single mitochondrial network indicating these cells have undergone mitosis but failed to complete cytokinesis. Immunofluorescence analysis of 5-bromo-2-deoxyuridine incorporation demonstrated that the mitochondrial DNA was replicated but did not segregate. The dose response curve for inhibition of the normal cell cycle paralleled that for the in vitro inhibition of protein phosphatase activities with IC50s of approximately 20 nM okadaic acid. These results suggest the involvement of a PP1/PP2A-like activity in coordinating mitosis, mitochondrial DNA division and cytokinesis in trypanosomes.

The zebrafish midblastula transition

Development ◽  
1993 ◽  
Vol 119 (2) ◽  
pp. 447-456 ◽  
Author(s):  
D.A. Kane ◽  
C.B. Kimmel
Keyword(s):  
Cell Cycle ◽  
Cell Motility ◽  
Cycle Length ◽  
Cell Cycle Regulation ◽  
Midblastula Transition ◽  
Timing Mechanism ◽  
Cell Synchrony ◽  
Activation Of Transcription ◽  
Blastomere Volume ◽  
Cycle Regulation

The zebrafish midblastula transition (MBT) begins at cycle 10. It is characterized by cell cycle lengthening, loss of cell synchrony, activation of transcription and appearance of cell motility. Superceding a 15 minute oscillator that controls the first nine cycles, the nucleocytoplasmic ratio appears to govern the MBT. This timing mechanism operates cell autonomously: clones of labeled cells initiate cell cycle lengthening independently of neighbors but dependent on immediate lineal ancestors. Unequal divisions, when they occur, produce asymmetric cell cycle lengthening based on the volume of each daughter. During the several cycles after the MBT begins, cycle length is correlated with the reciprocal of the blastomere volume, suggesting a continuation of cell cycle regulation by the nucleocytoplasmic ratio during an interval that we term the ‘MBT period’.

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