Summary
To assess the genetic consequences of induced Pluripotent Stem Cell (iPSC) reprogramming, we sequenced the genomes of ten murine iPSC clones derived from three independent reprogramming experiments, and compared them to their parental cell genomes. We detected hundreds of single nucleotide variants (SNVs) in every clone, with an average of 11 in coding regions. In two experiments, all SNVs were unique for each clone and did not cluster in pathways, but in the third, all four iPSC clones contained 157 shared genetic variants, which could also be detected in rare cells (<1 in 500) within the parental MEF pool. This data suggests that most of the genetic variation in iPSC clones is not caused by reprogramming per se, but is rather a consequence of cloning individual cells, which “captures” their mutational history. These findings have implications for the development and therapeutic use of cells that are reprogrammed by any method.
LCR-Fl is a mammalian bZIP transcription factor containing a basic amino acid domain highly homologous to a domain in the Drosophila Cap 'N' Collar and Caenorbabditis elegans SKN-1 proteins. LCR-Fl binds to API-like sequences in the human p-globin locus control region and activates high-level expression of p-globin genes. To assess the role of LCR-Fl in mammalian development, the mouse Lcrfl gene was deleted in embryonic stem (ES) cells, and mice derived from these cells were mated to produce Lcrfl null animals. Homozygous mutant embryos progressed normally to the late egg cylinder stage at -6.5 days post coitus (dpc), but development was arrested before 7.5 dpc. Lcrfl mutant embryos failed to form a primitive streak and lacked detectable mesoderm. These results demonstrate that LCR-Fl is essential for gastrulation in the mouse and suggest that this transcription factor controls expression of genes critical for the earliest events in mesoderm formation. Interestingly, Lcrfl null ES cells injected into wild-type blastocysts contributed to all mesodermally derived tissues examined, including erythroid cells producing hemoglobin. These results demonstrate that the Lcrfl mutation is not cell autonomous and suggest that LCR-Fl regulates expression of signaling molecules essential for gastrulation. The synthesis of normal hemoglobin levels in erythroid cells of chimeras derived from Lcrfl null cells suggests that LCR-Fl is not essential for globin gene expression. LCR-Fl and the related bZIP transcription factors NF-E2 p45 and NRF2 must compensate for each other in globin gene regulation.
The persistence of fetal hemoglobin in many patients with deletion type beta thalassemias and the expression patterns of human globin genes in transgenic mice suggest that gamma- to beta-globin gene switching results primarily from competition of gamma- and beta-globin genes for interaction with the beta-globin locus control region (LCR). To define regulatory sequences that are essential for the competitive advantage of the gamma gene at early developmental stages, stable transgenic mouse lines were produced with LCR gamma-beta constructs containing deletions of gamma 5'-flanking DNA. All constructs contained the full 22 kb LCR, a 4.1 kb beta-globin gene and a gamma-globin gene with 1348, 383, 202, 130, 72 or 52 bp of 5'-flanking sequence. Primer extension analysis of yolk sac, fetal liver and blood RNA from these lines demonstrated that a region between -202 and -130 of the human gamma-globin gene promoter was required to suppress beta-globin gene expression at early developmental stages. Four transcription factor binding sites within this region [GATA(p), Oct1, GATA(d) and CACCC] were mutated independently in LCR gamma-beta constructs and transgenic mouse lines were produced. Only the gamma CACCC box mutation resulted in high levels of beta-globin gene expression in early embryos. These results demonstrate that the CACCC box of the human gamma-globin gene plays a critical role in human beta-globin gene developmental specificity. The data also suggest that gamma CACCC box binding factors mediate LCR-gamma interactions which normally enhance gamma-globin and suppress beta-globin gene expression in fetal erythroid cells.
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