Regulation Of Cell Fate Determination Research Articles

VGLL1 cooperates with TEAD4 to control human trophectoderm lineage specification

In contrast to rodents, the mechanisms underlying human trophectoderm and early placenta specification are understudied due to ethical barriers and the scarcity of embryos. Recent reports have shown that human pluripotent stem cells (PSCs) can differentiate into trophectoderm (TE)-like cells (TELCs) and trophoblast stem cells (TSCs), offering a valuable in vitro model to study early placenta specification. Here, we demonstrate that the VGLL1 (vestigial-like family member 1), which is highly expressed during human and non-human primate TE specification in vivo but is negligibly expressed in mouse, is a critical regulator of cell fate determination and self-renewal in human TELCs and TSCs derived from naïve PSCs. Mechanistically, VGLL1 partners with the transcription factor TEAD4 (TEA domain transcription factor 4) to regulate chromatin accessibility at target gene loci through histone acetylation and acts in cooperation with GATA3 and TFAP2C. Our work is relevant to understand primate early embryogenesis and how it differs from other mammalian species.

Range of chromatin accessibility configurations are permissive of GABAergic fate acquisition in developing mouse brain

In recent single-cell -omics studies, both the differential activity of transcription factors regulating cell fate determination and differential genome activation have been tested for utility as descriptors of cell types. Naturally, genome accessibility and gene expression are interlinked. To understand the variability in genomic feature activation in the GABAergic neurons of different spatial origins, we have mapped accessible chromatin regions and mRNA expression in single cells derived from the developing mouse central nervous system (CNS). We first defined a reference set of open chromatin regions for scATAC-seq read quantitation across samples, allowing comparison of chromatin accessibility between brain regions and cell types directly. Second, we integrated the scATAC-seq and scRNA-seq data to form a unified resource of transcriptome and chromatin accessibility landscape for the cell types in di- and telencephalon, midbrain and anterior hindbrain of E14.5 mouse embryo. Importantly, we implemented resolution optimization at the clustering, and automatized the cell typing step. We show high level of concordance between the cell clustering based on the chromatin accessibility and the transcriptome in analyzed neuronal lineages, indicating that both genome and transcriptome features can be used for cell type definition. Hierarchical clustering by the similarity in accessible chromatin reveals that the genomic feature activation correlates with neurotransmitter phenotype, selector gene expression, cell differentiation stage and neuromere origins.

Notch Signaling in Insect Development: A Simple Pathway with Diverse Functions.

Notch signaling is an evolutionarily conserved pathway which functions between adjacent cells to establish their distinct identities. Despite operating in a simple mechanism, Notch signaling plays remarkably diverse roles in development to regulate cell fate determination, organ growth and tissue patterning. While initially discovered and characterized in the model insect Drosophila melanogaster, recent studies across various insect species have revealed the broad involvement of Notch signaling in shaping insect tissues. This review focuses on providing a comprehensive picture regarding the roles of the Notch pathway in insect development. The roles of Notch in the formation and patterning of the insect embryo, wing, leg, ovary and several specific structures, as well as in physiological responses, are summarized. These results are discussed within the developmental context, aiming to deepen our understanding of the diversified functions of the Notch signaling pathway in different insect species.

Single-cell transcriptome atlas reveals spatiotemporal developmental trajectories in the basal roots of moso bamboo (Phyllostachys edulis).

Roots are essential for plant growth and development. Bamboo is a large Poaceae perennial with 1642 species worldwide. However, little is known about the transcriptional atlas that underpins root cell-type differentiation. Here, we set up a modified protocol for protoplast preparation and report single-cell transcriptomes of 14 279 filtered single cells derived from the basal root tips of moso bamboo. We identified four cell types and defined new cell-type-specific marker genes for the basal root. We reconstructed the developmental trajectories of the root cap, epidermis, and ground tissues and elucidated critical factors regulating cell fate determination. According to in situ hybridization and pseudotime trajectory analysis, the root cap and epidermis originated from a common initial cell lineage, revealing the particularity of bamboo basal root development. We further identified key regulatory factors for the differentiation of these cells and indicated divergent root developmental pathways between moso bamboo and rice. Additionally, PheWOX13a and PheWOX13b ectopically expressed in Arabidopsis inhibited primary root and lateral root growth and regulated the growth and development of the root cap, which was different from WOX13 orthologs in Arabidopsis. Taken together, our results offer an important resource for investigating the mechanism of root cell differentiation and root system architecture in perennial woody species of Bambusoideae.

Translational landscape in human early neural fate determination.

Gene expression regulation in eukaryotes is a multi-level process, including transcription, mRNA translation, and protein turnover. Many studies have reported the sophisticated transcriptional regulations during neural development, but the global translational dynamics are still ambiguous. Here, we differentiate human embryonic stem cells (ESCs) into neural progenitor cells (NPCs) with high efficiency and perform ribosome sequencing and RNA sequencing on both ESCs and NPCs. Data analysis reveals that translational controls engage in many critical pathways and contribute significantly to neural fate determination regulation. Furthermore, we show that the sequence characteristics in the untranslated region (UTR) might regulate translation efficiency. Specifically, genes with short 5'UTR and intense Kozak sequence are associated with high translation efficiency in human ESCs, while genes with long 3'UTR are related to high translation efficiency in NPCs. In addition, we have identified four biasedly-used codons (GAC, GAT, AGA, and AGG) and dozens of short open reading frames during neural progenitor differentiation. Thus, our study reveals the translational landscape during early human neural differentiation and provides insights into the regulation of cell fate determination at the translational level.

Stomatal regulators are co-opted for seta development in the astomatous liverwort Marchantia polymorpha.

The evolution of special types of cells requires the acquisition of new gene regulatory networks controlled by transcription factors (TFs). In stomatous plants, a TF module formed by subfamilies Ia and IIIb basic helix-loop-helix TFs (Ia-IIIb bHLH) regulates stomatal formation; however, how this module evolved during land plant diversification remains unclear. Here we show that, in the astomatous liverwort Marchantia polymorpha, a Ia-IIIb bHLH module regulates the development of a unique sporophyte tissue, the seta, which is found in mosses and liverworts. The sole Ia bHLH gene, MpSETA, and a IIIb bHLH gene, MpICE2, regulate the cell division and/or differentiation of seta lineage cells. MpSETA can partially replace the stomatal function of Ia bHLH TFs in Arabidopsis thaliana, suggesting that a common regulatory mechanism underlies setal and stomatal formation. Our findings reveal the co-option of a Ia-IIIb bHLH TF module for regulating cell fate determination and/or cell division of distinct types of cells during land plant evolution.

LncRNA-Smad7 mediates cross-talk between Nodal/TGF-β and BMP signaling to regulate cell fate determination of pluripotent and multipotent cells.

Transforming growth factor β (TGF-β) superfamily proteins are potent regulators of cellular development and differentiation. Nodal/Activin/TGF-β and BMP ligands are both present in the intra- and extracellular milieu during early development, and cross-talk between these two branches of developmental signaling is currently the subject of intense research focus. Here, we show that the Nodal induced lncRNA-Smad7 regulates cell fate determination via repression of BMP signaling in mouse embryonic stem cells (mESCs). Depletion of lncRNA-Smad7 dramatically impairs cardiomyocyte differentiation in mESCs. Moreover, lncRNA-Smad7 represses Bmp2 expression through binding with the Bmp2 promoter region via (CA)12-repeats that forms an R-loop. Importantly, Bmp2 knockdown rescues defects in cardiomyocyte differentiation induced by lncRNA-Smad7 knockdown. Hence, lncRNA-Smad7 antagonizes BMP signaling in mESCs, and similarly regulates cell fate determination between osteocyte and myocyte formation in C2C12 mouse myoblasts. Moreover, lncRNA-Smad7 associates with hnRNPK in mESCs and hnRNPK binds at the Bmp2 promoter, potentially contributing to Bmp2 expression repression. The antagonistic effects between Nodal/TGF-β and BMP signaling via lncRNA-Smad7 described in this work provides a framework for understanding cell fate determination in early development.

Assemblies of JAG1 and JAG2 determine tracheobronchial cell fate in mucosecretory lung disease.

Mucosecretory lung disease compromises airway epithelial function and is characterized by goblet cell hyperplasia and ciliated cell hypoplasia. Goblet and ciliated cell types are derived from tracheobronchial stem/progenitor cells via a Notch-dependent mechanism. Although specific arrays of Notch receptors regulate cell fate determination, the function of the ligands Jagged1 (JAG1) and JAG2 is unclear. This study examined JAG1 and JAG2 function using human air-liquid-interface cultures that were treated with γ-secretase complex (GSC) inhibitors, neutralizing peptides/antibodies, or WNT/β-catenin pathway antagonists/agonists. These experiments revealed that JAG1 and JAG2 regulated cell fate determination in the tracheobronchial epithelium; however, their roles did not adhere to simple necessity and sufficiency rules. Biochemical studies indicated that JAG1 and JAG2 underwent posttranslational modifications that resulted in generation of a JAG1 C-terminal peptide and regulated the abundance of full-length JAG2 on the cell surface. GSC and glycogen synthase kinase 3 were implicated in these posttranslational events, but WNT agonist/antagonist studies and RNA-Seq indicated a WNT-independent mechanism. Collectively, these data suggest that posttranslational modifications create distinct assemblies of JAG1 and JAG2, which regulate Notch signal strength and determine the fate of tracheobronchial stem/progenitor cells.

Trp53 controls chondrogenesis and endochondral ossification by negative regulation of TAZ activity and stability via β-TrCP-mediated ubiquitination

Transformation-related protein 53 (Trp53) is a critical regulator of cell fate determination by controlling cell proliferation and differentiation. Ablation of Trp53 signaling in osteoblast lineages significantly promotes osteogenesis, bone formation, and bone remodeling. However, how Trp53 regulates chondrogenesis and endochondral bone formation is undefined. In this study, we found that Trp53 expression gradually decreased in tibia growth plates during embryonic development in vivo and during chondrogenesis in vitro. By deleting Trp53 in chondrocyte lineage using Col2-Cre transgenic line, we found that loss of Trp53 in chondrocytes significantly increased growth plate growth and bone formation by increasing chondrocyte proliferation, matrix production and maturation, and bone dynamic formation rate. Mechanistically, our data revealed loss of Trp53 significantly promoted TAZ transcriptional activity through inhibition of TAZ phosphorylation and nuclear translocation, whereas its activity was pronouncedly inhibited after forced expression of Trp53. Furthermore, Co-IP data demonstrated that Trp53 associated with TAZ. Moreover, Trp53 decreased the stability of TAZ protein and promoted its degradation through β-TrCP-mediated ubiquitination. Ablation of TAZ in Col2-Cre;Trp53f/f mice rescued the phenotypes of enhanced chondrogenesis and bone formation caused by Trp53 deletion. Collectively, this study revealed that Trp53 modulates chondrogenesis and endochondral ossification through negative regulation of TAZ activity and stability, suggesting that targeting Trp53 signaling may be a potential strategy for fracture healing, heterotopic ossification, arthritis, and other bone diseases.

Isolation, culture, and characterisation of bovine ovarian fetal fibroblasts and gonadal ridge epithelial-like cells and comparison to their adult counterparts.

During ovarian development, gonadal ridge epithelial-like (GREL) cells arise from the epithelial cells of the ventral surface of the mesonephros. They ultimately develop into follicular granulosa cells or into ovarian surface epithelial cells. Stromal fibroblasts arise from the mesonephros and penetrate the ovary. We developed methods for isolating and culturing fetal ovarian GREL cells and ovarian fibroblasts by expansion of colonies without passage. In culture, these two cell types were morphologically different. We examined the expression profile of 34 genes by qRT-PCR, of which 24 genes had previously been studied in whole fetal ovaries. Expression of nine of the 10 newly-examined genes in fetal ovaries correlated with gestational age (MUC1, PKP2, CCNE1 and CCNE2 negatively; STAR, COL4A1, GJA1, LAMB2 and HSD17B1 positively). Comparison between GREL cells and fetal fibroblasts revealed higher expression of KRT19, PKP2, OCLN, MUC1, ESR1 and LGR5 and lower expression of GJA1, FOXL2, NR2F2, FBN1, COL1A1, NR5A1, CCND2, CCNE1 and ALDH1A1. Expression of CCND2, CCNE1, CCNE2, ESR2 and TGFBR1 was higher in the fetal fibroblasts than in adult fibroblasts; FBN1 was lower. Expression of OCLN, MUC1, LAMB2, NR5A1, ESR1, ESR2, and TGFBR3 was lower in GREL cells than ovarian surface epithelial cells. Expression of KRT19, DSG2, PKP2, OCLN, MUC1, FBN1, COL1A1, COL3A1, STAR and TGFBR2 was higher and GJA1, CTNNB1, LAMB2, NR5A1, CYP11A1, HSD3B1, CYP19A1, HSD17B1, FOXL2, ESR1, ESR2, TGFBR3 and CCND2 was lower in GREL cells compared to granulosa cells. TGFβ1 altered the expression of COL1A1, COL3A1 and FBN1 in fetal fibroblasts and epidermal growth factor altered the expression of FBN1 and COL1A1. In summary, the two major somatic cell types of the developing ovary have distinct gene expression profiles. They, especially GREL cells, also differ from the cells they ultimately differentiate in to. The regulation of cell fate determination, particularly of the bi-potential GREL cells, remains to be elucidated.

Metabolic sensor O-GlcNAcylation regulates erythroid differentiation and globin production via BCL11A

BackgroundHuman erythropoiesis is a tightly regulated, multistep process encompassing the differentiation of hematopoietic stem cells (HSCs) toward mature erythrocytes. Cellular metabolism is an important regulator of cell fate determination during the differentiation of HSCs. However, how O-GlcNAcylation, a posttranslational modification of proteins that is an ideal metabolic sensor, contributes to the commitment of HSCs to the erythroid lineage and to the terminal erythroid differentiation has not been addressed.MethodsCellular O-GlcNAcylation was manipulated using small molecule inhibition or CRISPR/Cas9 manipulation of catalyzing enzyme O-GlcNAc transferase (OGT) and removing enzyme O-GlcNAcase (OGA) in two cell models of erythroid differentiation, starting from: (i) human umbilical cord blood-derived CD34+ hematopoietic stem/progenitor cells (HSPCs) to investigate the erythroid lineage specification and differentiation; and (ii) human-derived erythroblastic leukemia K562 cells to investigate the terminal differentiation. The functional and regulatory roles of O-GlcNAcylation in erythroid differentiation, maturation, and globin production were investigated, and downstream signaling was delineated.ResultsFirst, we observed that two-step inhibition of OGT and OGA, which were established from the observed dynamics of O-GlcNAc level along the course of differentiation, promotes HSPCs toward erythroid differentiation and enucleation, in agreement with an upregulation of a multitude of erythroid-associated genes. Further studies in the efficient K562 model of erythroid differentiation confirmed that OGA inhibition and subsequent hyper-O-GlcNAcylation enhance terminal erythroid differentiation and affect globin production. Mechanistically, we found that BCL11A is a key mediator of O-GlcNAc-driven erythroid differentiation and β- and α-globin production herein. Additionally, analysis of biochemical contents using synchrotron-based Fourier transform infrared (FTIR) spectroscopy showed unique metabolic fingerprints upon OGA inhibition during erythroid differentiation, supporting that metabolic reprogramming plays a part in this process.ConclusionsThe evidence presented here demonstrated the novel regulatory role of O-GlcNAc/BCL11A axis in erythroid differentiation, maturation, and globin production that could be important in understanding erythropoiesis and hematologic disorders whose etiology is related to impaired erythroid differentiation and hemoglobinopathies. Our findings may lay the groundwork for future clinical applications toward an ex vivo production of functional human reticulocytes for transfusion from renewable cell sources, i.e., HSPCs and pluripotent stem cells.

Comparative analysis of protein-protein interaction networks in neural differentiation mechanisms

Neural differentiation as a major process during neural cell therapy is one of the main issues that is not fully characterized. This study focuses on the major deconstruction of the transcriptional networks that regulate cell fate determination during neural differentiation under the influence of RA signalling. In our studies, we used four different microarray datasets containing a total of 15,660 genes to determine which genes were differentially expressed during neural differentiation from pluripotent stem cells (P19), among the 17 samples from four different datasets that were integrated via meta-analysis approaches. Of the 15,660 gene expression in our data integration, 443 DEGs are induced during neural differentiation. Upstream dissection of these 443 DEGs revealed a network of protein-protein interactions (PPIs) from TFs and kinases, as well as intermediate proteins between them, which are indicated by three (POU51, NANOG, and FOXO1) down-expression genes and one PAX6 up-expression gene playing roles in up-stream of these 443 induced DEGs during neural differentiation. The constructed network from the PPIs database revealed that four novel sub-networks play major roles in neuron differentiation in cluster 3, retinol metabolism in cluster 4, Rap1 signalling pathways in cluster 2, and axonogenesis in cluster 6. These four clusters have revealed very useful information about how neural characterization will be created from pluripotent stem cells.This research reveals a plethora of information on the neural differentiation process, including cell commitment and neural differentiation, and lays the groundwork for future research into particular pathways involving protein-protein interactions in neurogenesis.

MiR-130a-3p regulates neural stem cell differentiation in vitro by targeting Acsl4.

In the adult mammalian brain, neural stem cells (NSCs) are the precursor cells of neurons that contribute to nervous system development, regeneration, and repair. MicroRNAs (miRNAs) are small non‐coding RNAs that regulate cell fate determination and differentiation by negatively regulating gene expression. Here, we identified a post‐transcriptional mechanism, centred around miR‐130a‐3p that regulated NSC differentiation. Importantly, overexpressing miR‐130a‐3p promoted NSC differentiation into neurons, whereas inhibiting miR‐130a‐3p function reduced the number of neurons. Then, the quantitative PCR, Western blot and dual‐luciferase reporter assays showed that miR‐130a‐3p negatively regulated acyl‐CoA synthetase long‐chain family member 4 (Acsl4) expression. Additionally, inhibition of Acsl4 promoted NSC differentiation into neurons, whereas silencing miR‐130a‐3p partially suppressed the neuronal differentiation induced by inhibiting Acsl4. Furthermore, overexpressing miR‐130a‐3p or inhibiting Acsl4 increased the levels of p‐AKT, p‐GSK‐3β and PI3K. In conclusion, our results suggested that miR‐130a‐3p targeted Acsl4 to promote neuronal differentiation of NSCs via regulating the Akt/PI3K pathway. These findings may help to develop strategies for stem cell‐mediated treatment for central nervous system diseases.

Mitochondrial homeostasis regulates definitive endoderm differentiation of human pluripotent stem cells

Cellular organelles play fundamental roles in almost all cell behaviors. Mitochondria have been reported to be functionally linked to various biological processes, including reprogramming and pluripotency maintenance. However, very little about the role of mitochondria has been revealed in human early development and lineage specification. Here, we reported the characteristics and function of mitochondria during human definitive endoderm differentiation. Using a well-established differentiation system, we first investigated the change of mitochondrial morphology by comparing undifferentiated pluripotent stem cells, the intermediate mesendoderm cells, and differentiated endoderm cells, and found that mitochondria were gradually elongated and matured along differentiation. We further analyzed the expression pattern of mitochondria-related genes by RNA-seq, indicating that mitochondria became active during differentiation. Supporting this notion, the production of adenosine triphosphate (ATP) and reactive oxygen species (ROS) was increased as well. Functionally, we utilized chemicals and genome editing techniques, which could interfere with mitochondrial homeostasis, to determine the role of mitochondria in human endoderm differentiation. Treatment with mitochondrial inhibitors, or genetic depletion of mitochondrial transcription factor A (TFAM), significantly reduced the differentiation efficiency of definitive endoderm. In addition, the defect in endoderm differentiation due to dysfunctional mitochondria could be restored to some extent by the addition of ATP. Moreover, the clearance of excessive ROS due to dysfunctional mitochondria by N-acetylcysteine (NAC) improved the differentiation as well. We further found that ATP and NAC could partially replace the growth factor activin A for definitive endoderm differentiation. Our study illustrates the essential role of mitochondria during human endoderm differentiation through providing ATP and regulating ROS levels, which may provide new insight for metabolic regulation of cell fate determination.

The Carboxyl-Terminus of TRANSPARENT TESTA GLABRA1 Is Critical for Its Functions in Arabidopsis.

The Arabidopsis WD40 repeat protein TRANSPARENT TESTA GLABRA1 (TTG1) regulates cell fate determination, including trichome initiation and root hair formation, as well as secondary metabolism such as flavonoid biosynthesis and seed coat mucilage production. TTG1 regulates different processes via regulating the expression of its downstream target genes by forming MYB-bHLH-WD40 (MBW) activator complexes with different R2R3 MYB and bHLH transcription factors. Here, we report the identification of the carboxyl (C)-terminus as a critical domain for TTG1′s functions in Arabidopsis. We found that the ttg1Δ15aa mutant shows pleiotropic phenotypes identical to a TTG1 loss-of-function mutant. Gene sequencing indicates that a single nucleotide substitution in TTG1 led to a premature stop at the W327 residue, leading to the production of a truncated TTG1 protein with a deletion of the last 15 C-terminal amino acids. The expression of TTG1 under the control of its native promoter fully restored the ttg1Δ15aa mutant phenotypes. Consistent with these observations, the expression levels of TTG1 downstream genes such as GLABRA2 (GL2) and CAPRICE (CPC) were reduced in the ttg1Δ15aa mutant. Assays in Arabidopsis protoplast show that TTG1Δ15aa failed to interact with the bHLH transcription factor GL3, and the deletion of the last 3 C-terminal amino acids or the 339L amino acid alone fully abolished the interaction of TTG1 with GL3. Furthermore, the expression of TTG1Δ3aa under the control of TTG1 native promoter failed to restore the ttg1Δ15aa mutant phenotypes. Taken together, our results suggest that the C-terminal domain of TTG1 is required for its proper function in Arabidopsis.

Notch Signaling in the Dynamics of Perivascular Stem Cells and their Niches

The Notch signaling pathway is a fundamental regulator of cell fate determination in homeostasis and regeneration. In this work, we aimed to determine how Notch signaling mediates the interactions between perivascular stem cells and their niches in human dental mesenchymal tissues, both in homeostatic and regenerative conditions. By single cell RNA sequencing analysis, we showed that perivascular cells across the dental pulp and periodontal human tissues all express NOTCH3, and that these cells are important for the response to traumatic injuries in vivo in a transgenic mouse model. We further showed that the behavior of perivascular NOTCH3‐expressing stem cells could be modulated by cellular and molecular cues deriving from their microenvironments. Taken together, the present studies, reinforced by single‐cell analysis, reveal the pivotal importance of Notch signaling in the crosstalk between perivascular stem cells and their niches in tissue homeostasis and regeneration.

LSD1-mediated demethylation of OCT4 safeguards pluripotent stem cells by maintaining the transcription of PORE-motif-containing genes

Reversible lysine methylation is essential for regulating histones and emerges to critically regulate non-histone proteins as well. Here we show that the master transcription factor OCT4 in pluripotent stem cells (PSCs) was methylated at multiple lysine residues. LSD1 that is highly expressed in PSCs can directly interact with and demethylate OCT4 at lysine 222 (K222) in the flexible linker region. Reduced LSD1 activity led to the methylation of OCT4-K222 that diminished the differentiation potential of PSCs while facilitating proteasome-independent degradation of OCT4 proteins. Furthermore, site-specifically replacing K222 with phenylalanine to mimic the constitutively methylated lysine promoted the ‘locked-in’ mode engagement of the OCT4 PORE-homodimers that tightly bind to and block the transcription of multiple PORE-motif-containing target genes regulating cell fate determination and cell junction organization, and thereby reducing the pluripotency of PSCs. Thus, LSD1-mediated demethylation of OCT4 plays a crucial role in restricting the ‘locked-in’ mode binding of OCT4 PORE-homodimers to the PORE-motif-containing genes and thereby maintaining their transcription to safeguard the pluripotency of PSCs.

Epigenetic regulation and intercellular communication during male gametophyte development

The male gametophyte of angiosperms has long been recognized as an ideal system for the study of the molecular mechanisms regulating cell fate determination. Recent findings on histone variants in two cell lineages, vegetative-cell-derived small interfering RNA and transposable element expression provide new power for relevant investigations.

Beyond PROX1: transcriptional, epigenetic, and noncoding RNA regulation of lymphatic identity and function.

The lymphatic vascular system acts as the major transportation highway of tissue fluids, and its activation or impairment is associated with a wide range of diseases. There has been increasing interest in understanding the mechanisms that control lymphatic vessel formation (lymphangiogenesis) and function in development and disease. Here, we discuss recent insights into new players whose identification has contributed to deciphering the lymphatic regulatory code. We reveal how lymphatic endothelial cells, the building blocks of lymphatic vessels, utilize their transcriptional, post-transcriptional, and epigenetic portfolio to commit to and maintain their vascular lineage identity and function, with a particular focus on development.

Rucaparib Treatment Alters p53 Oscillations in Single Cells to Enhance DNA-Double-Strand-Break-Induced Cell Cycle Arrest

DNA double strand breaks induce oscillatory expression of the transcription factor p53 that is dependent on ataxia telangiectasia mutated (ATM) activity and the rate of double strand break resolution. Although p53 dynamics are known to play a role in the regulation of cell fate determination, the consequences of the variability in dynamics associated with differences in repair rates and utilized repair pathways are unknown. Using single-cell time-lapse microscopy, we found that disruption of specific repair pathways has distinct impacts on p53 dynamics. The small-molecule rucaparib, an inhibitor of the alternative end-joining-associated protein poly (ADP-ribose) polymerase (PARP), increased p53 pulse duration, altering the temporal expression of multiple p53 target genes. As a result, combination treatments of the radiomimetic drug neocarzinostatin with rucaparib drove prolonged growth arrest beyond that of DNA damage alone. This study highlights how pharmacological manipulation of DNA repair pathways may be used to alter p53 dynamics to enhance therapeutic regimens.