Retinoic acid-mediated down-regulation of Oct3/4 coincides with the loss of promoter occupancy in vivo.

Oct3/4, a hallmark of the earliest stages of embryogenesis, is expressed in undifferentiated embryonal carcinoma (EC) and embryonic stem (ES) cells. Oct3/4 gene expression is dependent on the promoter region, the proximal enhancer and the newly identified distal enhancer. We have analysed in vivo occupancy of these elements. In undifferentiated EC and ES cells, strong footprints were detected at specific sites of all three regulatory elements. These were promptly lost upon RA treatment in ES cells and in P19 EC cells, in parallel with sharply reduced Oct3/4 mRNA levels. Thus, the occupancy of regulatory elements is coupled with Oct3/4 expression, and RA treatment causes coordinated factor displacement, leading to extinction of gene activity. In F9 EC cells, footprint was first abolished at the proximal enhancer. However, this loss of binding site occupancy did not result in a decrease in Oct3/4 mRNA levels. The partial factor displacement seen in F9 EC cells, combined with the observation that EC and ES cells utilize the proximal and distal enhancers in differential manner, indicate the complex pattern of Oct3/4 gene regulation, which could reflect a cell type- and lineage-specific expression of the gene in vivo.     

Extrinsic factors derived from mouse embryonal carcinoma cell lines maintain pluripotency of mouse embryonic stem cells through a novel signal pathway

Embryonic carcinoma (EC) cells, which are malignant stem cells of teratocarcinoma, have numerous morphological and biochemical properties in common with pluripotent stem cells such as embryonic stem (ES) cells. However, three EC cell lines (F9, P19 and PCC3) show different developmental potential and self‐renewal capacity from those of ES cells. All three EC cell lines maintain self‐renewal capacity in serum containing medium without Leukemia Inhibitory factor (LIF) or feeder layer, and show limited differentiation capacity into restricted lineage and cell types. To reveal the underlying mechanism of these characteristics, we took the approach of characterizing extrinsic factors derived from EC cells on the self‐renewal capacity and pluripotency of mouse ES cells. Here we demonstrate that EC cell lines F9 and P19 produce factor(s) maintaining the undifferentiated state of mouse ES cells via an unidentified signal pathway, while P19 and PCC3 cells produce self‐renewal factors of ES cells other than LIF that were able to activate the STAT3 signal; however, inhibition of STAT3 activation with Janus kinase inhibitor shows only partial impairment on the maintenance of the undifferentiated state of ES cells. Thus, these factors present in EC cells‐derived conditioned medium may be responsible for the self‐renewal capacity of EC and ES cells independently of LIF signaling.     

Rex1/Zfp42 is dispensable for pluripotency in mouse ES cells

Background  

Rex1/Zfp42 has been extensively used as a marker for the undifferentiated state of pluripotent stem cells. However, its function in pluripotent stem cells including embryonic stem (ES) cells remained unclear although its involvement in visceral endoderm differentiation in F9 embryonal carcinoma (EC) cells was reported.     

The Activin A-Dependent Proliferation of PCC3/A/1 Embryonal Carcinoma Cells in Serum-Free Medium

Examination of the growth requirements of murine embryonal carcinoma cells (EC cells) or embryonic stem cells (ES cells) in serum-free medium revealed that PCC3 EC cells required activin A to grow and/or survive in such medium. In the absence of activin A, PCC3 cells began to disintegrate within 3 days under any serum-free conditions examined. P19 and AT805 EC cells grew even in serum-free medium without activin A but their growth rates were slightly facilitated by its addition. F9 EC cells also grew in the medium without activin A and its addition somewhat inhibited their growth rate. Three independently isolated ES cell lines and feeder-dependent PSA-1 EC cells also grew in serum-free medium without activin A if leukemia inhibitory factor (LIF) was supplemented. The addition of activin A had little effect on their growth rates. These findings suggest that PCC3 EC cells are a sort of nutritional mutant requiring activin A, thus making them useful in stidies on the growth regulatory mechanisms of EC/ES cells and/or the action of activin on EC/ES cells.     

Cooperativity between DNA methyltransferases in the maintenance methylation of repetitive elements.

We used mouse embryonic stem (ES) cells with systematic gene knockouts for DNA methyltransferases to delineate the roles of DNA methyltransferase 1 (Dnmt1) and Dnmt3a and -3b in maintaining methylation patterns in the mouse genome. Dnmt1 alone was able to maintain methylation of most CpG-poor regions analyzed. In contrast, both Dnmt1 and Dnmt3a and/or Dnmt3b were required for methylation of a select class of sequences which included abundant murine LINE-1 promoters. We used a novel hemimethylation assay to show that even in wild-type cells these sequences contain high levels of hemimethylated DNA, suggestive of poor maintenance methylation. We showed that Dnmt3a and/or -3b could restore methylation of these sequences to pretreatment levels following transient exposure of cells to 5-aza-CdR, whereas Dnmt1 by itself could not. We conclude that ongoing de novo methylation by Dnmt3a and/or Dnmt3b compensates for inefficient maintenance methylation by Dnmt1 of these endogenous repetitive sequences. Our results reveal a previously unrecognized degree of cooperativity among mammalian DNA methyltransferases in ES cells.