TATA box-binding protein-related factor 3 drives the mesendoderm specification of human embryonic stem cells by globally interacting with the TATA box of key mesendodermal genes.

BACKGROUND: Mesendodermal formation during early gastrulation requires the expression of lineage-specific genes, while the regulatory mechanisms during this process have not yet been fully illustrated. TATA box-binding protein (TBP) and TBP-like factors are general transcription factors responsible for the transcription initiation by recruiting the preinitiation complex to promoter regions. However, the role of TBP family members in the regulation of mesendodermal specification remains largely unknown. METHODS: We used an in vitro mesendodermal differentiation system of human embryonic stem cells (hESCs), combining with the microarray and quantitative polymerase chain reaction (qRT-PCR) analysis, loss of function and gain of function to determine the function of the TBP family member TBP-related factor 3 (TRF3) during mesendodermal differentiation of hESCs. The chromatin immunoprecipitation (ChIP) and biochemistry analysis were used to determine the binding of TRF3 to the promoter region of key mesendodermal genes. RESULTS: The mesendodermal differentiation of hESCs was confirmed by the microarray gene expression profile, qRT-PCR, and immunocytochemical staining. The expression of TRF3 mRNA was enhanced during mesendodermal differentiation of hESCs. The TRF3 deficiency did not affect the pluripotent marker expression, alkaline phosphatase activity, and cell cycle distribution of undifferentiated hESCs or the expression of early neuroectodermal genes during neuroectodermal differentiation. During the mesendodermal differentiation, the expression of pluripotency markers decreased in both wild-type and TRF3 knockout (TRF3-/-) cells, while the TRF3 deficiency crippled the expression of the mesendodermal markers. The reintroduction of TRF3 into the TRF3-/- hESCs rescued inhibited mesendodermal differentiation. Mechanistically, the TRF3 binding profile was significantly shifted to the mesendodermal specification during mesendodermal differentiation of hESCs based on the ChIP-seq data. Moreover, ChIP and ChIP-qPCR analysis showed that TRF3 was enriched at core promoter regions of mesendodermal developmental genes, EOMESODERMIN, BRACHYURY, mix paired-like homeobox, and GOOSECOID homeobox, during mesendodermal differentiation of hESCs. CONCLUSIONS: These results reveal that the TBP family member TRF3 is dispensable in the undifferentiated hESCs and the early neuroectodermal differentiation. However, it directs mesendodermal lineage commitment of hESCs via specifically promoting the transcription of key mesendodermal transcription factors. These findings provide new insights into the function and mechanisms of the TBP family member in hESC early lineage specification.

SMYD2 Drives Mesendodermal Differentiation of Human Embryonic Stem Cells Through Mediating the Transcriptional Activation of Key Mesendodermal Genes.

Histone methyltransferases play a critical role in early human development, whereas their roles and precise mechanisms are less understood. SET and MYND domain-containing protein 2 (SMYD2) is a histone lysine methyltransferase induced during early differentiation of human embryonic stem cells (hESCs), but little is known about its function in undifferentiated hESCs and in their early lineage fate decision as well as underlying mechanisms. Here, we explored the role of SMYD2 in the self-renewal and mesendodermal lineage commitment of hESCs. We demonstrated that the expression of SMYD2 was significantly enhanced during mesendodermal but not neuroectodermal differentiation of hESCs. SMYD2 knockout (SMYD2-/- ) did not affect self-renewal and early neuroectodermal differentiation of hESCs, whereas it blocked the mesendodermal lineage commitment. This phenotype was rescued by reintroduction of SMYD2 into the SMYD2-/- hESCs. Mechanistically, the bindings of SMYD2 at the promoter regions of critical mesendodermal transcription factor genes, namely, brachyury (T), eomesodermin (EOMES), mix paired-like homeobox (MIXL1), and goosecoid homeobox (GSC) were significantly enhanced during mesendodermal differentiation of SMYD2+/+ hESCs but totally suppressed in SMYD2-/- ones. Concomitantly, such a suppression was associated with the remarkable reduction of methylation at histone 3 lysine 4 and lysine 36 but not at histone 4 lysine 20 globally and specifically on the promoter regions of mesendodermal genes, namely, T, EOMES, MIXL1, and GSC. These results reveal that the histone methyltransferase SMYD2 is dispensable in the undifferentiated hESCs and the early neuroectodermal differentiation, but it promotes the mesendodermal differentiation of hESCs through the epigenetic control of critical genes to mesendodermal lineage commitment. Stem Cells 2019;37:1401-1415.

Functional expression of the Ca2+ signaling machinery in human embryonic stem cells.

Emerging evidence suggests that Ca2+ signals are important for the self-renewal and differentiation of human embryonic stem cells (hESCs). However, little is known about the physiological and pharmacological properties of the Ca2+-handling machinery in hESCs. In this study we used RT-PCR and Western blotting to analyze the expression profiles of genes encoding Ca2+-handling proteins; we also used confocal Ca2+ imaging and pharmacological approaches to determine the contribution of the Ca2+-handling machinery to the regulation of Ca2+ signaling in hESCs. We revealed that hESCs expressed pluripotent markers and various Ca2+-handling-related genes. ATP-induced Ca2+ transients in almost all hESCs were inhibited by the inositol-1,4,5-triphosphate receptor (IP3R) blocker 2-APB or xestospongin C. In addition, Ca2+ transients were induced by a ryanodine receptor (RyR) activator, caffeine, in 10%-15% of hESCs and were blocked by ryanodine, whereas caffeine and ATP did not have additive effects. Moreover, store-operated Ca2+ entry (SOCE) but not voltage-operated Ca2+ channel-mediated Ca2+ entry was observed. Inhibition of sarco/endoplasmic reticulum (ER) Ca2+-ATPase (SERCA) by thapsigargin induced a significant increase in the cytosolic free Ca2+ concentration ([Ca2+]i). For the Ca2+ extrusion pathway, inhibition of plasma membrane Ca2+ pumps (PMCAs) by carboxyeosin induced a slow increase in [Ca2+]i, whereas the Na+/Ca2+ exchanger (NCX) inhibitor KBR7943 induced a rapid increase in [Ca2+]i. Taken together, increased [Ca2+]i is mainly mediated by Ca2+ release from intracellular stores via IP3Rs. In addition, RyRs function in a portion of hESCs, thus indicating heterogeneity of the Ca2+-signaling machinery in hESCs; maintenance of low [Ca2+]i is mediated by uptake of cytosolic Ca2+ into the ER via SERCA and extrusion of Ca2+ out of cells via NCX and PMCA in hESCs.

Plant Homeo Domain Finger Protein 8 Regulates Mesodermal and Cardiac Differentiation of Embryonic Stem Cells Through Mediating the Histone Demethylation of pmaip1.

Histone demethylases have emerged as key regulators of biological processes. The H3K9me2 demethylase plant homeo domain finger protein 8(PHF8), for example, is involved in neuronal differentiation, but its potential function in the differentiation of embryonic stem cells (ESCs) to cardiomyocytes is poorly understood. Here, we explored the role of PHF8 during mesodermal and cardiac lineage commitment of mouse ESCs (mESCs). Using a phf8 knockout (ph8(-/Y) ) model, we found that deletion of phf8 in ESCs did not affect self-renewal, proliferation or early ectodermal/endodermal differentiation, but it did promote the mesodermal lineage commitment with the enhanced cardiomyocyte differentiation. The effects were accompanied by a reduction in apoptosis through a caspase 3-independent pathway during early ESC differentiation, without significant differences between differentiating wide-type (ph8(+/Y) ) and ph8(-/Y) ESCs in cell cycle progression or proliferation. Functionally, PHF8 promoted the loss of a repressive mark H3K9me2 from the transcription start site of a proapoptotic gene pmaip1 and activated its transcription. Furthermore, knockdown of pmaip1 mimicked the phenotype of ph8(-/Y) by showing the decreased apoptosis during early differentiation of ESCs and promoted mesodermal and cardiac commitment, while overexpression of pmaip1 or phf8 rescued the phenotype of ph8(-/Y) ESCs by increasing the apoptosis and weakening the mesodermal and cardiac differentiation. These results reveal that the histone demethylase PHF8 regulates mesodermal lineage and cell fate decisions in differentiating mESCs through epigenetic control of the gene critical to programmed cell death pathways. Stem Cells 2016;34:1527-1540.