Stage-specific regulation of DNA methylation by TET enzymes during human cardiac differentiation.

Changes in DNA methylation are associated with normal cardiogenesis, whereas altered methylation patterns can occur in congenital heart disease. Ten-eleven translocation (TET) enzymes oxidize 5-methylcytosine (5mC) and promote locus-specific DNA demethylation. Here, we characterize stage-specific methylation dynamics and the function of TETs during human cardiomyocyte differentiation. Human embryonic stem cells (hESCs) in which all three TET genes are inactivated fail to generate cardiomyocytes (CMs), with altered mesoderm patterning and defective cardiac progenitor specification. Genome-wide methylation analysis shows TET knockout causes promoter hypermethylation of genes encoding WNT inhibitors, leading to hyperactivated WNT signaling and defects in cardiac mesoderm patterning. TET activity is also needed to maintain hypomethylated status and expression of NKX2-5 for subsequent cardiac progenitor specification. Finally, loss of TETs causes a set of cardiac structural genes to fail to be demethylated at the cardiac progenitor stage. Our data demonstrate key roles for TET proteins in controlling methylation dynamics at sequential steps during human cardiac development.

Blastocyst-like structures generated from human pluripotent stem cells.

Limited access to embryos has hampered the study of human embryogenesis and disorders that occur during early pregnancy. Human pluripotent stem cells provide an alternative means to study human development in a dish1-7. Recent advances in partial embryo models derived from human pluripotent stem cells have enabled human development to be examined at early post-implantation stages8-14. However, models of the pre-implantation human blastocyst are lacking. Starting from naive human pluripotent stem cells, here we developed an effective three-dimensional culture strategy with successive lineage differentiation and self-organization to generate blastocyst-like structures in vitro. These structures-which we term 'human blastoids'-resemble human blastocysts in terms of their morphology, size, cell number, and composition and allocation of different cell lineages. Single-cell RNA-sequencing analyses also reveal the transcriptomic similarity of blastoids to blastocysts. Human blastoids are amenable to embryonic and extra-embryonic stem cell derivation and can further develop into peri-implantation embryo-like structures in vitro. Using chemical perturbations, we show that specific isozymes of protein kinase C have a critical function in the formation of the blastoid cavity. Human blastoids provide a readily accessible, scalable, versatile and perturbable alternative to blastocysts for studying early human development, understanding early pregnancy loss and gaining insights into early developmental defects.

MMP inhibitors attenuate doxorubicin cardiotoxicity by preventing intracellular and extracellular matrix remodelling.

AIMS: Heart failure is a major complication in cancer treatment due to the cardiotoxic effects of anticancer drugs, especially from the anthracyclines such as doxorubicin (DXR). DXR enhances oxidative stress and stimulates matrix metalloproteinase-2 (MMP-2) in cardiomyocytes. We investigated whether MMP inhibitors protect against DXR cardiotoxicity given the role of MMP-2 in proteolyzing sarcomeric proteins in the heart and remodelling the extracellular matrix. METHODS AND RESULTS: Eight-week-old male C57BL/6J mice were treated with DXR weekly with or without MMP inhibitors doxycycline or ONO-4817 by daily oral gavage for 4 weeks. Echocardiography was used to determine cardiac function and left ventricular remodelling before and after treatment. MMP inhibitors ameliorated DXR-induced systolic and diastolic dysfunction by reducing the loss in left ventricular ejection fraction, fractional shortening, and E'/A'. MMP inhibitors attenuated adverse left ventricular remodelling, reduced cardiomyocyte dropout, and prevented myocardial fibrosis. DXR increased myocardial MMP-2 activity in part also by upregulating N-terminal truncated MMP-2. Immunogold transmission electron microscopy showed that DXR elevated MMP-2 levels within the sarcomere and mitochondria which were associated with myofilament lysis, mitochondrial degeneration, and T-tubule distention. DXR-induced myofilament lysis was associated with increased titin proteolysis in the heart which was prevented by ONO-4817. DXR also increased the level and activity of MMP-2 in human embryonic stem cell-derived cardiomyocytes, which was reduced by ONO-4817. CONCLUSIONS: MMP-2 activation is an early event in DXR cardiotoxicity and contributes to myofilament lysis by proteolyzing cardiac titin. Two orally available MMP inhibitors ameliorated DXR cardiotoxicity by attenuating intracellular and extracellular matrix remodelling, suggesting their use may be a potential prophylactic strategy to prevent heart injury during chemotherapy.

Baicalin inhibits Salmonella typhimurium-induced inflammation and mediates autophagy through TLR4/MAPK/NF-κB signalling pathway.

Baicalin has been reported to protect mice against Salmonella typhimurium (S. typhimurium) infection, while its molecular mechanisms are unclear. In this study, multiplicity of infection (MOI) and observation time were measured. Cell viability and LDH levels were examined in RAW264.7 cells and H9 cells. RAW264.7 cells were stimulated with S typhimurium in the presence or absence of Baicalin, and the levels of pro-inflammatory cytokines were detected by enzyme-linked immunosorbent assay (ELISA). The changes in reactive oxygen species (ROS) production were determined by fluorescence microscopy and ELISA. The autophagy and TLR4/MAPK/NF-κB signalling pathway were examined by immunofluorescence microscopy, quantitative reverse transcription-polymerase chain reaction and Western blotting. The results indicated that MOI of 30 and duration of autophagy evident at 5 h were applicable to this study. Baicalin prevented death of macrophages, promoted bactericidal activity, decreased the levels of pro-inflammatory cytokines and ROS and reduced the changes of key biomarkers in autophagy and TLR4/MAPK/NF-κB signalling pathway infected by S typhimurium. TLR4-overexpressed cells, autophagy and TLR4/MAPK/NF-κB signalling pathway were activated by S typhimurium, which was suppressed by Baicalin. Our findings indicated that Baicalin exerts anti-inflammatory and cell-protective effects, and it mediates autophagy by down-regulating the activity of TLR4 infected by S typhimurium.

TBK1 regulates regeneration of pancreatic ß-cells.

Small-molecule inhibitors of non-canonical IκB kinases TANK-binding kinase 1 (TBK1) and IκB kinase ε (IKKε) have shown to stimulate ß-cell regeneration in multiple species. Here we demonstrate that TBK1 is predominantly expressed in ß-cells in mammalian islets. Proteomic and transcriptome analyses revealed that genetic silencing of TBK1 increased expression of proteins and genes essential for cell proliferation in INS-1 832/13 rat ß-cells. Conversely, TBK1 overexpression decreased sensitivity of ß-cells to the elevation of cyclic AMP (cAMP) levels and reduced proliferation of ß-cells in a manner dependent on the activity of cAMP-hydrolyzing phosphodiesterase 3 (PDE3). While the mitogenic effect of (E)3-(3-phenylbenzo[c]isoxazol-5-yl)acrylic acid (PIAA) is derived from inhibition of TBK1, PIAA augmented glucose-stimulated insulin secretion (GSIS) and expression of ß-cell differentiation and proliferation markers in human embryonic stem cell (hESC)-derived ß-cells and human islets. TBK1 expression was increased in ß-cells upon diabetogenic insults, including in human type 2 diabetic islets. PIAA enhanced expression of cell cycle control molecules and ß-cell differentiation markers upon diabetogenic challenges, and accelerated restoration of functional ß-cells in streptozotocin (STZ)-induced diabetic mice. Altogether, these data suggest the critical function of TBK1 as a ß-cell autonomous replication barrier and present PIAA as a valid therapeutic strategy augmenting functional ß-cells.

DNMT3B deficiency alters mitochondrial biogenesis and α-ketoglutarate levels in human embryonic stem cells.

Embryonic stem cell renewal and differentiation is regulated by metabolites that serve as cofactors for epigenetic enzymes. An increase of α-ketoglutarate (α-KG), a cofactor for histone and DNA demethylases, triggers multilineage differentiation in human embryonic stem cells (hESCs). To gain further insight into how the metabolic fluxes in pluripotent stem cells can be influenced by inactivating mutations in epigenetic enzymes, we generated hESCs deficient for de novo DNA methyltransferases (DNMTs) 3A and 3B. Our data reveal a bidirectional dependence between DNMT3B and α-KG levels: a-KG is significantly upregulated in cells deficient for DNMT3B, while DNMT3B expression is downregulated in hESCs treated with α-KG. In addition, DNMT3B null hESCs exhibit a disturbed mitochondrial fission and fusion balance and a switch from glycolysis to oxidative phosphorylation. Taken together, our data reveal a novel link between DNMT3B and the metabolic flux of hESCs.

Global Landscape and Dynamics of Parkin and USP30-Dependent Ubiquitylomes in iNeurons during Mitophagic Signaling.

The ubiquitin ligase Parkin, protein kinase PINK1, USP30 deubiquitylase, and p97 segregase function together to regulate turnover of damaged mitochondria via mitophagy, but our mechanistic understanding in neurons is limited. Here, we combine induced neurons (iNeurons) derived from embryonic stem cells with quantitative proteomics to reveal the dynamics and specificity of Parkin-dependent ubiquitylation under endogenous expression conditions. Targets showing elevated ubiquitylation in USP30-/- iNeurons are concentrated in components of the mitochondrial translocon, and the ubiquitylation kinetics of the vast majority of Parkin targets are unaffected, correlating with a modest kinetic acceleration in accumulation of pS65-Ub and mitophagic flux upon mitochondrial depolarization without USP30. Basally, ubiquitylated translocon import substrates accumulate, suggesting a quality control function for USP30. p97 was dispensable for Parkin ligase activity in iNeurons. This work provides an unprecedented quantitative landscape of the Parkin-modified ubiquitylome in iNeurons and reveals the underlying specificity of central regulatory elements in the pathway.

Single cell transcriptome dynamics from pluripotency to FLK1+ mesoderm.

Hemangiogenic progenitors generating blood and endothelial cells are specified from FLK1-expressing (FLK1+) mesoderm by the transcription factor ETV2. FLK1+ mesoderm also contributes to smooth muscle and cardiomyocytes. However, the developmental process of FLK1+ mesoderm generation and its allocation to various cell fates remain obscure. Recent single cell RNA-sequencing studies of early embryos or in vitro-differentiated human embryonic stem (ES) cells have provided unprecedented information on the spatiotemporal resolution of cells in embryogenesis. These snapshots, however, lack information on continuous dynamic developmental processes. Here, we performed single cell RNA sequencing of in vitro-differentiated mouse ES cells to capture the continuous developmental process leading to hemangiogenesis. We found that hemangiogenic progenitors from ES cells develop through intermediate gastrulation stages, which are gradually specified by 'relay'-like highly overlapping transcription factor modules. Moreover, the transcriptional program of the Flk1+ mesoderm was maintained in the smooth muscle lineage, suggesting that smooth muscle is the default fate of Flk1+ mesoderm. We also identified the SRC kinase contributing to ETV2-mediated activation of the hemangiogenic program. This continuous transcriptome map will facilitate both basic and applied studies of mesoderm development.

HMGB2 is a negative regulator of telomerase activity in human embryonic stem and progenitor cells.

High-mobility group box (HMGB)1 and HMGB2 proteins are the subject of intensive research because of their involvement in DNA replication, repair, transcription, differentiation, proliferation, cell signaling, inflammation, and tumor migration. Using inducible, stably transfected human embryonic stem cells (hESCs) capable of the short hairpin RNA-mediated knockdown (KD) of HMGB1 and HMGB2, we provide evidence that deregulation of HMGB1 or HMGB2 expression in hESCs and their differentiated derivatives (neuroectodermal cells) results in distinct modulation of telomere homeostasis. Whereas HMGB1 enhances telomerase activity, HMGB2 acts as a negative regulator of telomerase activity in the cell. Stimulation of telomerase activity in the HMGB2-deficient cells may be related to activation of the PI3K/protein kinase B/ glycogen synthase kinase-3ß/ß-catenin signaling pathways by HMGB1, augmented TERT/telomerase RNA subunit transcription, and possibly also because of changes in telomeric repeat-containing RNA (TERRA) and TERRA-polyA+ transcription. The impact of HMGB1/2 KD on telomerase transcriptional regulation observed in neuroectodermal cells is partially masked in hESCs by their pluripotent state. Our findings on differential roles of HMGB1 and HMGB2 proteins in regulation of telomerase activity may suggest another possible outcome of HMGB1 targeting in cells, which is currently a promising approach aiming at increasing the anticancer activity of cytotoxic agents.-Kucírek, M., Bagherpoor, A. J., Jaros, J., Hampl, A., Stros, M. HMGB2 is a negative regulator of telomerase activity in human embryonic stem and progenitor cells.

The role of selenium-mediated redox signaling by selenophosphate synthetase 1 (SEPHS1) in hESCs.

Selenium (Se) plays a vital role in reactive oxygen species (ROS) homeostasis and redox regulation in intracellular signaling via selenocysteine (Sec), known as the 21st proteinogenic amino acid, but its specific biological functions in development and disease remain undiscovered. In this study, we explored the role of selenophosphate synthetase 1 (SEPHS1) in the pluripotency maintenance and reprogramming. We found that high level of SEPHS1 is retained in undifferentiated embryonic stem cells (ESCs), which is decreased during their differentiation. SEPHS1 knockdown significantly reduced reprogramming efficiency, proving that SEPHS1 is required for acquisition of pluripotency. However, SEPHS1 knockdown did not affect the expression of significant pluripotency genes, suggesting that SEPHS1 may be involved in the survival of pluripotent stem cells rather than in the regulation of pluripotency genes. Transcriptome analysis revealed altered expression of the gene set related to the ROS pathway and apoptosis in SEPHS1-knockdown cells. We also demonstrated the role of SEPHS1 in human ESC clonogenicity, and we found improved single-cell survival of hESCs by selenium treatment in a concentration-dependent manner. Our study implies that hSEPHS1 is a regulator of selenium-mediated redox-signaling in human pluripotent stem cells and plays a role in their survival.

Sarco/endoplasmic reticulum Ca2+-ATPase is a more effective calcium remover than sodium-calcium exchanger in human embryonic stem cell-derived cardiomyocytes.

Human pluripotent stem cell (hPSCs)-derived ventricular (V) cardiomyocytes (CMs) display immature Ca2+-handing properties with smaller transient amplitudes and slower kinetics due to such differences in crucial Ca2+-handling proteins as the poor sarco/endoplasmic reticulum Ca2+-ATPase (SERCA) pump but robust Na+-Ca2+ exchanger (NCX) activities in human embryonic stem cell (ESC)-derived VCMs compared with adult. Despite their fundamental importance in excitation-contraction coupling, the relative contribution of SERCA and NCX to Ca2+-handling of hPSC-VCMs remains unexplored. We systematically altered the activities of SERCA and NCX in human embryonic stem cell-derived ventricular cardiomyocytes (hESC-VCMs) and their engineered microtissues, followed by examining the resultant phenotypic consequences. SERCA overexpression in hESC-VCMs shortened the decay of Ca2+ transient at low frequencies (0.5 Hz) without affecting the amplitude, SR Ca2+ content and Ca2+ baseline. Interestingly, short hairpin RNA-based NCX suppression did not prolong the transient decay, indicating a compensatory response for Ca2+ removal. Although hESC-VCMs and their derived microtissues exhibited negative frequency-transient/force responses, SERCA overexpression rendered them less negative at high frequencies (>2 Hz) by accelerating Ca2+ sequestration. We conclude that for hESC-VCMs and their microtissues, SERCA, rather than NCX, is the main Ca2+ remover during diastole; poor SERCA expression is the leading cause for immature negative-frequency/force responses, which can be partially reverted by forced expression. Combinatorial approach to mature calcium handling in hESC-VCMs may help shed further mechanistic insights.NEW & NOTEWORTHY In this study of human pluripotent stem cell-derived cardiomyocytes, we studied the role of sarco/endoplasmic reticulum Ca2+-ATPase (SERCA) and Na+-Ca2+ exchanger (NCX) in Ca2+ handling. Our data support the notion that SERCA is more effective in cytosolic calcium removal than the NCX.

Insulin Stimulates PI3K/AKT and Cell Adhesion to Promote the Survival of Individualized Human Embryonic Stem Cells.

Insulin is present in most maintenance media for human embryonic stem cells (hESCs), but little is known about its essential role in the cell survival of individualized cells during passage. In this article, we show that insulin suppresses caspase cleavage and apoptosis after dissociation. Insulin activates insulin-like growth factor (IGF) receptor and PI3K/AKT cascade to promote cell survival and its function is independent of rho-associated protein kinase regulation. During niche reformation after passaging, insulin activates integrin that is essential for cell survival. IGF receptor colocalizes with focal adhesion complex and stimulates protein phosphorylation involved in focal adhesion formation. Insulin promotes cell spreading on matrigel-coated surfaces and suppresses myosin light chain phosphorylation. Further study showed that insulin is also required for the cell survival on E-cadherin coated surface and in suspension, indicating its essential role in cell-cell adhesion. This work highlights insulin's complex roles in signal transduction and niche re-establishment in hESCs. Stem Cells 2019;37:1030-1041.

Rare mutations of ADAM17 from TOFs induce hypertrophy in human embryonic stem cell-derived cardiomyocytes via HB-EGF signaling.

Tetralogy of Fallot (TOF) is the most common cyanotic form of congenital heart defects (CHDs). The right ventricular hypertrophy is associated with the survival rate of patients with repaired TOF. However, very little is known concerning its genetic etiology. Based on mouse model studies, a disintergrin and metalloprotease 10/17 (ADAM10 and ADAM17) are the key enzymes for the NOTCH and ErbB pathways, which are critical pathways for heart development. Mutations in these two genes have not been previously reported in human TOF patients. In this study, we sequenced ADAM10 and ADAM17 in a Han Chinese CHD cohort comprised of 80 TOF patients, 286 other CHD patients, and 480 matched healthy controls. Three missense variants of ADAM17 were only identified in 80 TOF patients, two of which (Y42D and L659P) are novel and not found in the Exome Aggregation Consortium (ExAC) database. Point mutation knock-in (KI) and ADAM17 knock-out (KO) human embryonic stem cells (hESCs) were generated by CRISPR/Cas9 and programmed to differentiate into cardiomyocytes (CMs). Y42D or L659P KI cells or complete KO cells all developed hypertrophy with disorganized sarcomeres. RNA-seq results showed that phosphatidylinositide 3-kinases/protein kinase B (PI3K/Akt), which is downstream of epidermal growth factor receptor (EGFR) signaling, was affected in both ADAM17 KO and KI hESC-CMs. In vitro experiments showed that these two mutations are loss-of-function mutations in shedding heparin-binding EGF-like growth factor (HB-EGF) but not NOTCH signaling. Our results revealed that CM hypertrophy in TOF could be the result of mutations in ADAM17 which affects HB-EGF/ErbB signaling.

Fluorescent Ti3C2 MXene quantum dots for an alkaline phosphatase assay and embryonic stem cell identification based on the inner filter effect.

As an emerging two-dimensional material, MXenes have attracted much attention due to their unique physicochemical properties, but their application in biosensing has been lagging far behind because of their poor salt tolerance. Herein, a titanium carbide MXene quantum dot (Ti3C2 QD)-based fluorescent probe for the alkaline phosphatase (ALP) activity assay and embryonic stem cell (ESC) identification was developed by taking advantage of the inner filter effect (IFE). Ti3C2 QDs with ∼4.2 nm in diameter were prepared from Ti3C2 MXenes by hydrothermal treatment, exhibiting excellent salt tolerance, anti-photobleaching and dispersion stability in aqueous solution. Owing to the remarkable overlap between the absorption spectrum of p-nitrophenol (p-NP) and the excitation and emission spectrum of Ti3C2 QDs, p-NP generated from ALP-catalyzed dephosphorylation of the substrate, p-nitrophenyl phosphate (p-NPP), can effectively quench the fluorescence of Ti3C2 QDs through the IFE. As a result, sensitive fluorometric analysis of ALP activity was achieved without complicating the conventional colorimetric ALP detection system. The proposed assay was successfully applied to determine ALP activity with a low limit of detection (0.02 U L-1) as well as monitoring the enzyme activity in real time. Finally, accurate analysis of ALP, the biomarker of ESC, in ESC lysates was also achieved using this IFE-based method, affording an alternative method for ESC identification.

A Practical Protocol for the Conditional Depletion of Rho Isoforms in Human Embryonic Stem Cells.

Human pluripotent stem cells indefinitely proliferate and survive in culture while retaining genomic integrity, providing a unique opportunity to study human molecular biology. Here, we introduced an RNA interference-based protocol of inducible gene silencing in human embryonic stem cells, which has several advantages in handling simplicity/convenience, cost/time performance, and applicability. Using this method, we had succeeded to elucidate the isoform-unique roles of Rho-family small GTPases in human embryonic stem cells.

Dynamics of PARKIN-Dependent Mitochondrial Ubiquitylation in Induced Neurons and Model Systems Revealed by Digital Snapshot Proteomics.

Flux through kinase and ubiquitin-driven signaling systems depends on the modification kinetics, stoichiometry, primary site specificity, and target abundance within the pathway, yet we rarely understand these parameters and their spatial organization within cells. Here we develop temporal digital snapshots of ubiquitin signaling on the mitochondrial outer membrane in embryonic stem cell-derived neurons, and we model HeLa cell systems upon activation of the PINK1 kinase and PARKIN ubiquitin ligase by proteomic counting of ubiquitylation and phosphorylation events. We define the kinetics and site specificity of PARKIN-dependent target ubiquitylation, and we demonstrate the power of this approach to quantify pathway modulators and to mechanistically define the role of PARKIN UBL phosphorylation in pathway activation in induced neurons. Finally, through modulation of pS65-Ub on mitochondria, we demonstrate that Ub hyper-phosphorylation is inhibitory to mitophagy receptor recruitment, indicating that pS65-Ub stoichiometry in vivo is optimized to coordinate PARKIN recruitment via pS65-Ub and mitophagy receptors via unphosphorylated chains.

Insights into the ubiquitin-proteasome system of human embryonic stem cells.

Human embryonic stem cells (hESCs) exhibit high levels of proteasome activity, an intrinsic characteristic required for their self-renewal, pluripotency and differentiation. However, the mechanisms by which enhanced proteasome activity maintains hESC identity are only partially understood. Besides its essential role for the ability of hESCs to suppress misfolded protein aggregation, we hypothesize that enhanced proteasome activity could also be important to degrade endogenous regulatory factors. Since E3 ubiquitin ligases are responsible for substrate selection, we first define which E3 enzymes are increased in hESCs compared with their differentiated counterparts. Among them, we find HECT-domain E3 ligases such as HERC2 and UBE3A as well as several RING-domain E3s, including UBR7 and RNF181. Systematic characterization of their interactome suggests a link with hESC identity. Moreover, loss of distinct up-regulated E3s triggers significant changes at the transcriptome and proteome level of hESCs. However, these alterations do not dysregulate pluripotency markers and differentiation ability. On the contrary, global proteasome inhibition impairs diverse processes required for hESC identity, including protein synthesis, rRNA maturation, telomere maintenance and glycolytic metabolism. Thus, our data indicate that high proteasome activity is coupled with other determinant biological processes of hESC identity.

CHAC2 is essential for self-renewal and glutathione maintenance in human embryonic stem cells.

Glutathione (GSH), the major non-enzymatic antioxidant, plays a critical role in cellular reactive oxygen species (ROS) neutralization. Moreover, GSH is required for the self-renewal maintenance of human embryonic stem cells (hESCs), and is highly accumulated in undifferentiated cells. Among 8 GSH biosynthesis-related enzymes, we found CHAC2 is highly enriched in undifferentiated hESCs. CHAC2 downregulation in hESCs efficiently decreased the levels of GSH and blocked self-renewal. The self-renewal of sh-CHAC2 cells can be rescued by GSH supplement. CHAC2 downregulation promoted mesoderm differentiation and hampered both teratoma formation and the expression of Nrf2 and glutamate-cysteine ligase (GCL). Notably, CHAC1 knockdown restored the self-renewability of CHAC2-downregulated cells. Although both CHAC1 and CHAC2 purified protein alone showed the catalytic activities to GSH, our data extraordinarily revealed that CHAC2 prevented CHAC1-mediated GSH degradation, which suggests that CHAC2 competes with CHAC1 to maintain GSH homeostasis. This is the first report to demonstrate that CHAC2 is critical for GSH maintenance and the novel roles of the CHAC family in hESC renewal.

Nuclear insulin-like growth factor 1 receptor phosphorylates proliferating cell nuclear antigen and rescues stalled replication forks after DNA damage.

We have previously shown that the insulin-like growth factor 1 receptor (IGF-1R) translocates to the cell nucleus, where it binds to enhancer-like regions and increases gene transcription. Further studies have demonstrated that nuclear IGF-1R (nIGF-1R) physically and functionally interacts with some nuclear proteins, i.e. the lymphoid enhancer-binding factor 1 (Lef1), histone H3, and Brahma-related gene-1 proteins. In this study, we identified the proliferating cell nuclear antigen (PCNA) as a nIGF-1R-binding partner. PCNA is a pivotal component of the replication fork machinery and a main regulator of the DNA damage tolerance (DDT) pathway. We found that IGF-1R interacts with and phosphorylates PCNA in human embryonic stem cells and other cell lines. In vitro MS analysis of PCNA co-incubated with the IGF-1R kinase indicated tyrosine residues 60, 133, and 250 in PCNA as IGF-1R targets, and PCNA phosphorylation was followed by mono- and polyubiquitination. Co-immunoprecipitation experiments suggested that these ubiquitination events may be mediated by DDT-dependent E2/E3 ligases (e.g. RAD18 and SHPRH/HLTF). Absence of IGF-1R or mutation of Tyr-60, Tyr-133, or Tyr-250 in PCNA abrogated its ubiquitination. Unlike in cells expressing IGF-1R, externally induced DNA damage in IGF-1R-negative cells caused G1 cell cycle arrest and S phase fork stalling. Taken together, our results suggest a role of IGF-1R in DDT.

Generation of a homozygous GBA deletion human embryonic stem cell line.

We describe the generation of a biallelic GBA deletion human embryonic stem cell line using zinc finger nuclease-mediated gene targeting. The homozygous targeting of exon 4 of the GBA locus leads to a complete loss of glucocerebrosidase (GCase) protein expression.