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In prokaryotes and yeasts, DNA polymerase proofreading (PPR) and DNA mismatch repair (MMR) cooperatively counteracts replication errors leading to repeat sequence destabilization (i.e. insertions/deletions of repeat units). However, PPR has not thus far been regarded as a mechanism stabilizing repeat sequences in higher eukaryotic cells. In a human cancer cell line, DLD-1, which carries mutations in both MSH6 and the Exo domain of POLD1, we previously observed that mononucleotide microsatellites were markedly destabilized whereas being stable in the simple MMR-defective backgrounds. In this study, we introduced the Exo domain mutation found in DLD-1 cells into MSH2-null HeLa cell clones, using CRISPR/Cas9 system. In the established Exo-/MMR-mutated HeLa clones, mononucleotide repeat sequences were remarkably destabilized as in DLD-1 cells. In contrast, dinucleotide microsatellites were readily destabilized in the parental MMR-deficient backgrounds, and the instability was not notably increased in the genome-edited HeLa clones. Here, we show an involvement of the Exo domain functions of DNA polymerase delta in mononucleotide repeat stabilization in human cells, which also suggests a possible role division between DNA polymerase and MMR in repeat maintenance in the human genome.

Cardiogenesis can be induced in vitro in ES cells, though it is difficult to distinguish cardiac-specific genes, since embryoid bodies simultaneously differentiate into multiple lineages. In the present study, transient serum removal during culture greatly enhanced cardiogenesis, and reduced generation of endothelial and hematopoietic cells. Using DNA microarray analysis of 24 differentiated sample cultures including cardiogenesis-enhanced cells, we successfully selected genes up-regulated in embryoid bodies that had undergone cardiogenic differentiation. Besides contractile protein genes, cardiac transcriptional regulatory genes, such as Nkx2-5, Gata4/5, Mef2c, and Myocd, were primary constituents of the first 100 genes chosen as cardiogenesis-associated genes. Further, whole mount in situ hybridization analysis of 13 genes containing non-characterized ones confirmed that most of them were specifically expressed in the heart region of mouse embryos from E9.5-10.5. Based on our results, we consider that the present profiling method may be useful to identify novel genes important for cardiac development.

We previously reported that c-Jun NH2-terminal kinase (JNK)/stress-activated protein kinase-associated protein 1 (JSAP1), a scaffold protein for JNK signaling, is important in embryonic stem (ES) cells during neurogenesis. In that study, we also observed the altered expression of mesodermal marker genes, which indicated that JSAP1 is involved in the differentiation of mesodermal lineages. Here, we investigated the function of JSAP1 in cardiomyocyte development using JSAP1-null ES cells, and found that cardiomyogenesis was impaired in the JSAP1-null mutant. The JSAP1 deficiency resulted in lower gene expression of the cardiac transcription factor Nkx2.5 and contractile proteins. In contrast, the mutant showed a significantly higher expression of mesoderm-related markers other than those of the cardiomyocyte lineage. Together, these results suggest that JSAP1 may be important for the differentiation of the mesodermal lineages, functioning as a positive factor for cardiomyocyte differentiation, and as an inhibitory factor for differentiation into other lineages.

Differentiation of embryonic stem (ES) cells into cardiac myocytes requires activation of a cardiac-specific gene program. Histone acetyltransferases (HATs) and histone deacetylases (HDACs) govern gene expression patterns by being recruited to target genes through association with specific transcription factors. One of the HATs, p300, serves as a coactivator of cardiac-specific transcription factors such as GATA-4. The HAT activity of p300 is required for acetylation and DNA binding of GATA-4 and its full transcriptional activity as well as for promotion of a transcriptionally active chromatin configuration. However, the roles of HATs and HDACs in post-translational modification of GATA-4 during the differentiation of ES cells into cardiac myocytes remain unknown. In an ES cell model of developing embryoid bodies, an acetylated form of GATA-4 and its DNA binding increased concomitantly with the expression of p300 during the differentiation of ES cells into cardiac myocytes. Treatment of ES cells with trichostatin A (TSA), a specific HDAC inhibitor, induced acetylation of histone-3/4 near GATA sites within the atrial natriuretic factor promoter. In addition, TSA augmented the increase in an acetylated form of GATA-4 and its DNA binding during the ES cell differentiation. Finally, TSA facilitated the expression of green fluorescence protein under the control of the cardiac-specific Nkx-2.5 promoter and of endogenous cardiac beta-myosin heavy chain during the differentiation. These findings demonstrate that acetylation of GATA-4 as well as of histones is involved in the differentiation of ES cells into cardiac myocytes.

Wnt signaling plays a crucial role in the control of morphogenesis in several tissues. Herein, we describe the role of Wnt11 during cardiac differentiation of embryonic stem cells. First, we examined the expression profile of Wnt11 during the course of differentiation in embryoid bodies, and then compared its expression in retinoic acid-treated embryoid bodies with that in untreated. In differentiating embryoid bodies, Wnt11 expression rose along with that of Nkx2.5 expression and continued to increase. When the embryoid bodies were treated with retinoic acid, Wnt11 expression decreased in parallel with the decreased expression of cardiac genes. Further, treatment of embryoid bodies with medium containing Wnt11 increased the expression of cardiac marker genes. Based on these results, we propose that Wnt11 plays an important role for cardiac development by embryoid bodies, and may be a key regulator of cardiac muscle cell proliferation and differentiation during heart development.

To explore the role of ErbB-mediated signaling in cardiogenesis of ES cells, we examined the expression of ErbB receptors as well as effects of a ligand and inhibitors using Nkx2.5GFP ES cells, in which the GFP gene was knocked-in to the Nkx2.5 locus to monitor cardiac differentiation. Although all ErbB receptors were expressed in developing embryoid bodies, expression of ErbB4 was almost exclusively found in differentiated cardiomyocytes. Heregulin beta1, a ligand of ErbB receptors, enhanced the generation of Nkx2.5/GFP(+) cardiomyocytes in embryoid bodies, while AG1478 and PD153035, inhibitors of ErbBs, drastically blocked the generation of Nkx2.5/GFP(+) cardiomyocytes. These results suggest that the signaling pathway mediated by ErbBs is important in the induction and differentiation of cardiomyocytes from ES cells.

The MEF2 gene family encodes a MADS-box transcription factor which regulates expression of many muscle-specific genes. We examined the expression of the MEF2 genes in mouse embryonal carcinoma P19 cells before and after in vitro muscle differentiation induced by dimethyl sulfoxide (DMSO). At least three different MADS/MEF2 domains (MEF2A, 2B and 2D) were isolated from P19 cells with the MOPAC technique (mixed oligonucleotides primed amplification of cDNA). Although two of the MADS/MEF2 domain sequences were identical to those of human MEF2A and MEF2D, another domain sequence was similar but not identical to that of human MEF2B. While the transcription of MEF2A and MEF2D was up-regulated during differentiation of P19 cells, the MEF2B homologue was abundantly transcribed in undifferentiated P19, F9 and ES cells and down-regulated in adult heart, skeletal muscle or brain, suggesting that the murine MEF2B homologue might have a function distinct from other members of the MEF2 gene family in embryogenesis and development.