scholarly journals YAP controls retinal stem cell DNA replication timing and genomic stability

eLife ◽  
2015 ◽  
Vol 4 ◽  
Author(s):  
Pauline Cabochette ◽  
Guillermo Vega-Lopez ◽  
Juliette Bitard ◽  
Karine Parain ◽  
Romain Chemouny ◽  
...  
Keyword(s):  
Stem Cells ◽  
Dna Replication ◽  
Hippo Pathway ◽  
Genomic Stability ◽  
Replication Timing ◽  
S Phase ◽  
Downstream Effector ◽  
Frog Retina ◽  
Temporal Firing ◽  
The Hippo Pathway

The adult frog retina retains a reservoir of active neural stem cells that contribute to continuous eye growth throughout life. We found that Yap, a downstream effector of the Hippo pathway, is specifically expressed in these stem cells. Yap knock-down leads to an accelerated S-phase and an abnormal progression of DNA replication, a phenotype likely mediated by upregulation of c-Myc. This is associated with an increased occurrence of DNA damage and eventually p53-p21 pathway-mediated cell death. Finally, we identified PKNOX1, a transcription factor involved in the maintenance of genomic stability, as a functional and physical interactant of YAP. Altogether, we propose that YAP is required in adult retinal stem cells to regulate the temporal firing of replication origins and quality control of replicated DNA. Our data reinforce the view that specific mechanisms dedicated to S-phase control are at work in stem cells to protect them from genomic instability.

scholarly journals A non-transcriptional function of Yap orchestrates the DNA replication program

2021 ◽  
Author(s):  
Rodrigo Meléndez García ◽  
Olivier Haccard ◽  
Albert Chesneau ◽  
Hemalatha Narassimprakash ◽  
Jerome E Roger ◽  
...  
Keyword(s):  
Stem Cells ◽  
Dna Replication ◽  
Embryonic Stem ◽  
Replication Timing ◽  
S Phase ◽  
Hippo Signaling ◽  
Downstream Effector ◽  
Egg Extracts ◽  
Replication Foci ◽  
Eukaryotic Organisms

In multicellular eukaryotic organisms, the initiation of DNA replication occurs asynchronously throughout S-phase according to a regulated replication timing program. Here, using Xenopus egg extracts, we showed that Yap (Yes-associated protein 1), a downstream effector of the Hippo signaling pathway, is required for the control of DNA replication dynamics. We found that Yap is recruited to chromatin at the start of DNA replication and that Yap depletion accelerates DNA replication dynamics by increasing the number of activated replication origins. Furthermore, we identified Rif1, a major regulator of the DNA replication timing program, as a novel Yap binding protein. In Xenopus embryos, using a Trim-Away approach during cleavage stages devoid of transcription, we found that both Yap and Rif1 depletion trigger an acceleration of cell divisions, suggesting a shorter S-phase by alterations of the replication program. Finally, our data show that Rif1 knockdown leads to defects in the partitioning of early versus late replication foci in retinal stem cells, as we previously showed for Yap. Altogether, our findings unveil a non-transcriptional role for Yap in regulating replication dynamics. We propose that Yap and Rif1 function as breaks to control the DNA replication program in early embryos and post-embryonic stem cells.

scholarly journals High-throughput analysis of DNA replication in single human cells reveals constrained variability in the location and timing of replication initiation

2021 ◽  
Author(s):  
Dashiell J Massey ◽  
Amnon Koren
Keyword(s):  
Dna Replication ◽  
Replication Origin ◽  
Replication Timing ◽  
S Phase ◽  
Human Cells ◽  
Replication Initiation ◽  
High Throughput Analysis ◽  
Timing Variability ◽  
Fixed Set ◽  
Fixed Order

DNA replication occurs throughout the S phase of the cell cycle, initiating from replication origin loci that fire at different times. Debate remains about whether origins are a fixed set of loci used across all cells or a loose agglomeration of potential origins used stochastically in individual cells, and about how consistent their firing time during S phase is across cells. Here, we develop an approach for profiling DNA replication in single human cells and apply it to 2,305 replicating cells spanning the entire S phase. The resolution and scale of the data enabled us to specifically analyze initiation sites and show that these sites have confined locations that are consistently used among individual cells. Further, we find that initiation sites are activated in a similar, albeit not fixed, order across cells. Taken together, our results suggest that replication timing variability is constrained both spatially and temporally, and that the degree of variation is consistent across human cell lines.

scholarly journals Genomic Analysis of the DNA Replication Timing Program during Mitotic S Phase in Maize (Zea mays) Root Tips

2017 ◽  
Vol 29 (9) ◽  
pp. 2126-2149 ◽  
Author(s):  
Emily E. Wear ◽  
Jawon Song ◽  
Gregory J. Zynda ◽  
Chantal LeBlanc ◽  
Tae-Jin Lee ◽  
...  
Keyword(s):  
Zea Mays ◽  
Dna Replication ◽  
Genomic Analysis ◽  
Replication Timing ◽  
S Phase ◽  
Root Tips ◽  
Dna Replication Timing

scholarly journals A83 INTESTINAL EPITHELIAL CELL STEMNESS AND DIFFERENTIATION ARE REGULATED BY THE HIPPO PATHWAY EFFECTOR YAP1

2020 ◽  
Vol 3 (Supplement_1) ◽  
pp. 97-98
Author(s):  
S Fallah ◽  
J Beaulieu
Keyword(s):  
Colorectal Cancer ◽  
Stem Cells ◽  
Transcription Factors ◽  
Cancer Stem Cells ◽  
Hippo Pathway ◽  
High Rate ◽  
Intestinal Cell ◽  
Intestinal Differentiation ◽  
Ht29 Cells ◽  
The Hippo Pathway

Abstract Background The high rate of cell turnover in the intestinal epithelium is supported by the LGR5+ crypt base columnar (CBC) stem cells, which are located at the lower part of the gland. Among of the various factors and signals like Wnt and Notch, YAP1 (yes associated protein) also plays an important role in stemness of CBC stem cells. YAP1 is the effector of the Hippo pathway. Hippo Pathway restricts the cells proliferation, tissues overgrowth and cancer formation through the phosphorylation and inactivation of the YAP1 protein. When active, YAP1 transfers into nucleus, forms the complex with TEADs transcription factors and promotes the transcription of genes involved in cell growth and proliferation. Aims In the present study, we investigated the role of the YAP1 in the colorectal cancer multipotent HT29 cell line, which contain cancer stem cells (CSC). Methods For approaching to this goal, YAP1 expression was knocked down using shRNAs in HT29 cells. Then stem cells and intestinal cell lineages (secretory goblet, Paneth and enteroendocrine and absorptive) markers expression was analyzed using qPCR and Western blot. Results The results showed the reduction of the expression of stem cells markers including LGR5 in YAP1 knockdown HT29 cells compare with control. Expression of the goblet cells markers (MUC2 and trefoil factor 3) and absorptive cells markers (sucrase-isomaltase and dipeptidylpeptidase IV) were significantly increased in YAP1 knockdown cells but Paneth (DEFA5 and lysozyme) and enteroendocrine (CHGA) were not detected. Finally, examination of the main transcription factors for intestinal differentiation revealed an increase in CDX2 expression. Conclusions These results suggest that YAP1 is involved in the maintenance of colorectal cancer stem cells while preventing intestinal differentiation in both secretory and absorptive lineages through the repression of CDX2. Funding Agencies CIHR

scholarly journals RASSF effectors couple diverse RAS subfamily GTPases to the Hippo pathway

2020 ◽  
Vol 13 (653) ◽  
pp. eabb4778 ◽  
Author(s):  
Thillaivillalan Dhanaraman ◽  
Swati Singh ◽  
Ryan C. Killoran ◽  
Anamika Singh ◽  
Xingjian Xu ◽  
...  
Keyword(s):  
Hippo Pathway ◽  
Effector Proteins ◽  
Domain Family ◽  
Ras Gtpases ◽  
Downstream Effector ◽  
Ras Superfamily ◽  
Ras Family ◽  
Key Residues ◽  
The Hippo Pathway ◽  
Apoptotic Induction

Small guanosine triphosphatases (GTPases) of the RAS superfamily signal by directly binding to multiple downstream effector proteins. Effectors are defined by a folded RAS-association (RA) domain that binds exclusively to GTP-loaded (activated) RAS, but the binding specificities of most RA domains toward more than 160 RAS superfamily GTPases have not been characterized. Ten RA domain family (RASSF) proteins comprise the largest group of related effectors and are proposed to couple RAS to the proapoptotic Hippo pathway. Here, we showed that RASSF1-6 formed complexes with the Hippo kinase ortholog MST1, whereas RASSF7-10 formed oligomers with the p53-regulating effectors ASPP1 and ASPP2. Moreover, only RASSF5 bound directly to activated HRAS and KRAS, and RASSFs did not augment apoptotic induction downstream of RAS oncoproteins. Structural modeling revealed that expansion of the RASSF effector family in vertebrates included amino acid substitutions to key residues that direct GTPase-binding specificity. We demonstrated that the tumor suppressor RASSF1A formed complexes with the RAS-related GTPases GEM, REM1, REM2, and the enigmatic RASL12. Furthermore, interactions between RASSFs and RAS GTPases blocked YAP1 nuclear localization. Thus, these simple scaffolds link the activation of diverse RAS family small G proteins to Hippo or p53 regulation.

scholarly journals Loss of Mob1a/b impairs the differentiation of mouse embryonic stem cells into the three germ layer lineages

2019 ◽  
Vol 51 (11) ◽  
pp. 1-12 ◽  
Author(s):  
June Sung Bae ◽  
Sun Mi Kim ◽  
Yoon Jeon ◽  
Juyeon Sim ◽  
Ji Yun Jang ◽  
...  
Keyword(s):  
Stem Cells ◽  
Stem Cell ◽  
Hippo Pathway ◽  
Critical Role ◽  
Embryonic Stem ◽  
Mouse Escs ◽  
Germ Layers ◽  
Stem Cell Maintenance ◽  
The Hippo Pathway ◽  
Cell Maintenance

AbstractThe Hippo pathway plays a crucial role in cell proliferation and apoptosis and can regulate stem cell maintenance and embryonic development. MOB kinase activators 1A and 1B (Mob1a/b) are key components of the Hippo pathway, whose homozygous deletion in mice causes early embryonic lethality at the preimplantation stage. To investigate the role of Mob1a/b in stem cell maintenance and differentiation, an embryonic stem cell (ESC) clone in which Mob1a/b could be conditionally depleted was generated and characterized. Although Mob1a/b depletion did not affect the stemness or proliferation of mouse ESCs, this depletion caused defects in differentiation into the three germ layers. Yap knockdown rescued the in vitro and in vivo defects in differentiation caused by Mob1a/b depletion, suggesting that differentiation defects caused by Mob1a/b depletion were Yap-dependent. In teratoma experiments, Yap knockdown in Mob1a/b-depleted ESCs partially restored defects in differentiation, indicating that hyperactivation of Taz, another effector of the Hippo pathway, inhibited differentiation into the three germ layers. Taken together, these results suggest that Mob1a/b or Hippo signaling plays a critical role in the differentiation of mouse ESCs into the three germ layers, which is dependent on Yap. These close relationship of the Hippo pathway with the differentiation of stem cells supports its potential as a therapeutic target in regenerative medicine.

scholarly journals The Hippo Pathway Effector YAP1 Regulates Intestinal Epithelial Cell Differentiation

Cells ◽  
2020 ◽  
Vol 9 (8) ◽  
pp. 1895 ◽  
Author(s):  
Sepideh Fallah ◽  
Jean-François Beaulieu
Keyword(s):  
Stem Cells ◽  
Cell Differentiation ◽  
Epithelial Cells ◽  
Cell Line ◽  
Intestinal Epithelial Cells ◽  
Hippo Pathway ◽  
Intestinal Cell ◽  
Intestinal Epithelial ◽  
The Hippo Pathway

The human intestine is covered by epithelium, which is continuously replaced by new cells provided by stem cells located at the bottom of the glands. The maintenance of intestinal stem cells is supported by a niche which is composed of several signaling proteins including the Hippo pathway effectors YAP1/TAZ. The role of YAP1/TAZ in cell proliferation and regeneration is well documented but their involvement on the differentiation of intestinal epithelial cells is unclear. In the present study, the role of YAP1/TAZ on the differentiation of intestinal epithelial cells was investigated using the HT29 cell line, the only multipotent intestinal cell line available, with a combination of knockdown approaches. The expression of intestinal differentiation cell markers was tested by qPCR, Western blot, indirect immunofluorescence and electron microscopy analyses. The results show that TAZ is not expressed while the abolition of YAP1 expression led to a sharp increase in goblet and absorptive cell differentiation and reduction of some stem cell markers. Further studies using double knockdown experiments revealed that most of these effects resulting from YAP1 abolition are mediated by CDX2, a key intestinal cell transcription factor. In conclusion, our results indicate that YAP1/TAZ negatively regulate the differentiation of intestinal epithelial cells through the inhibition of CDX2 expression.

scholarly journals Regulation of Epstein-Barr Virus Origin of Plasmid Replication (OriP) by the S-Phase Checkpoint Kinase Chk2

2010 ◽  
Vol 84 (10) ◽  
pp. 4979-4987 ◽  
Author(s):  
Jing Zhou ◽  
Zhong Deng ◽  
Julie Norseen ◽  
Paul M. Lieberman
Keyword(s):  
Dna Replication ◽  
Epstein Barr Virus ◽  
Replication Timing ◽  
S Phase ◽  
Plasmid Replication ◽  
Acceptor Site ◽  
Plasmid Maintenance ◽  
Barr Virus ◽  
Amino Terminal ◽  
Epstein Barr

ABSTRACT The Epstein-Barr virus (EBV) origin of plasmid replication (OriP) is required for episome stability during latent infection. Telomere repeat factor 2 (TRF2) binds directly to OriP and facilitates DNA replication and plasmid maintenance. Recent studies have found that TRF2 interacts with the DNA damage checkpoint protein Chk2. We show here that Chk2 plays an important role in regulating OriP plasmid stability, chromatin modifications, and replication timing. The depletion of Chk2 by small interfering RNA (siRNA) leads to a reduction in DNA replication efficiency and a loss of OriP-dependent plasmid maintenance. This corresponds to a change in OriP replication timing and an increase in constitutive histone H3 acetylation. We show that Chk2 interacts with TRF2 in the early G1/S phase of the cell cycle. We also show that Chk2 can phosphorylate TRF2 in vitro at a consensus acceptor site in the amino-terminal basic domain of TRF2. TRF2 mutants with a serine-to-aspartic acid phosphomimetic substitution mutation were reduced in their ability to recruit the origin recognition complex (ORC) and stimulate OriP replication. We suggest that the Chk2 phosphorylation of TRF2 is important for coordinating ORC binding with chromatin remodeling during the early S phase and that a failure to execute these events leads to replication defects and plasmid instability.

scholarly journals Shavenbaby and Yorkie mediate Hippo signaling to protect adult stem cells from apoptosis

2017 ◽  
Author(s):  
Jérôme Bohère ◽  
Alexandra Mancheno-Ferris ◽  
Kohsuke Akino ◽  
Yuya Yamabe ◽  
Sachi Inagaki ◽  
...  
Keyword(s):  
Stem Cells ◽  
Cell Death ◽  
Transcription Factor ◽  
Adult Stem Cells ◽  
Hippo Pathway ◽  
Inhibitor Of Apoptosis ◽  
Hippo Signaling ◽  
Polished Rice ◽  
Organ Regeneration ◽  
The Hippo Pathway

AbstractTo compensate for accumulating damages and cell death, adult homeostasis (e.g., body fluids and secretion) requires organ regeneration, operated by long-lived stem cells. How stem cells can survive throughout the animal life yet remains poorly understood. Here we show that the transcription factor Shavenbaby (Svb, OvoL in vertebrates) is expressed in renal/nephric stem cells (RNSCs) ofDrosophilaand required for their maintenance during adulthood. As recently shown in embryos, Svb function in adult RNSCs further needs a post-translational processing mediated by Polished rice (Pri) smORF peptides and impairing Svb function leads to RNSC apoptosis. We show that Svb interacts both genetically and physically with Yorkie (YAP/TAZ in vertebrates), a nuclear effector of the Hippo pathway, to activate the expression of the inhibitor of apoptosisDIAP1. These data therefore identify Svb as a novel nuclear effector in the Hippo pathway, critical for the survival of adult somatic stem cells.

scholarly journals αCGRP Affects BMSCs’ Migration and Osteogenesis via the Hippo-YAP Pathway

2019 ◽  
Vol 28 (11) ◽  
pp. 1420-1431 ◽  
Author(s):  
Bin Wang ◽  
Jie Lin ◽  
Qin Zhang ◽  
Xinyuan Zhang ◽  
Hui Yu ◽  
...  
Keyword(s):  
Hippo Pathway ◽  
In Vitro Tests ◽  
Downstream Effector ◽  
Intrinsic Mechanism ◽  
Pathophysiological Role ◽  
Enhanced Expression ◽  
The Hippo Pathway ◽  
The Impact

Alpha-calcitonin gene-related peptide (αCGRP) plays a significant pathophysiological role in the regulation of bone metabolism. Our previous research indicated that αCGRP might have a potential application in enhancing osseointegration in vivo. To further uncover the intrinsic mechanism of its networks in bone regeneration, here we investigate the impact of αCGRP on osteogenic differentiation in bone marrow-derived mesenchymal stem cells (BMSCs) from both wild-type and αCGRP-/- mice. Considering the half-life of αCGRP in plasma is only 10 min, we applied αCGRP lentivirus and stably transfected it into BMSCs, followed by transfection identification and cell cycle assay. We further conducted a series of in vitro tests, and the results revealed that biological functions including migratory ability and osteogenicity exhibited positive correlation with BMSCs’ αCGRP expression. Meanwhile, this phenomenon was associated with an enhanced expression of YAP (Yes-associated protein), the key downstream effector of the Hippo pathway. To sum up, our data together with previous in vivo observations is likely to elucidate the intrinsic mechanism of αCGRP in bone remodeling, and αCGRP would appear to be a novel treatment to promote bone wound healing.

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