Suppression of the imprinted gene NNAT and X-Chromosome gene activation in isogenic human iPS cells

Genetic comparison between human embryonic stem cells and induced pluripotent stem cells has been hampered by genetic variation. To solve this problem, we have developed an isogenic system that allows direct comparison of induced pluripotent stem cells (hiPSCs) to their genetically matched human embryonic stem cells (hESCs). We show that hiPSCs have a highly similar transcriptome to hESCs. Global transcriptional profiling identified 102-154 genes (\u3e2 fold) that showed a difference between isogenic hiPSCs and hESCs. A stringent analysis identified NNAT as a key imprinted gene that was dysregulated in hiPSCs. Furthermore, a disproportionate number of X-chromosome localized genes were over-expressed in female hiPSCs. Our results indicate that despite a remarkably close transcriptome to hESCs, isogenic hiPSCs have alterations in imprinting and regulation of X-chromosome genes. © 2011 Teichroeb et al

Telomeres, DNA damage signaling molecules and cellular aging

grantor: University of TorontoNormal human cells have a finite life span and undergo senescence after a fixed number of divisions. This process appears to involve some form of genetic memory by which normal cells count the number of divisions they undergo before senescence is reached. The telomere hypothesis proposed that loss of telomeric DNA at the end of human chromosomes acts as a mitotic clock, counting each cell division. Once a critical length of telomeric DNA is reached, senescence is initiated. Questions addressed in this thesis are: (1) How does telomere shortening cause cell cycle exit? (2) Is telomere shortening one of the factors which causes senescence? To address the first question, a model is proposed in chapter two in which telomere shortening is perceived by the cell as DNA damage and that this signal activates a DNA damage signaling pathway leading to senescence. In chapter three, we show that post-translational activation of p53 protein is one factor responsible for upregulation of p21WAF1 in aging cells and PARP (poly (ADP-ribose) polymerase) is involved in the regulation of p53 protein. We found that, either inhibition of PARP or loss of p53 led to extension of life span in normal human fibroblasts. Loss of three genes in our model (PARP, p53 and p21) led to extension of cellular life span. In contrast, loss of ATM gene led to accelerated telomere shortening and premature senescence. We conclude that these DNA damage signaling molecules are involved in regulation of cellular senescence. Answering the second question required reconstitution of telomerase activity in normal human cells. We show in chapter four of this thesis that telomerase activity can be reconstituted in normal cells by forced expression of hTERT, the catalytic subunit of human telomerase. This activity is sufficient to elongate telomeric DNA and extend the replicative life span of cells. These findings provide evidence consistent with the telomere hypothesis and indicate that telomere shortening is one factor which initiates cellular senescence by activation of a DNA damage signaling cascade. Furthermore they indicate that telomere elongation may be sufficient to prevent senescence and render normal human cells immortal.Ph.D

Suppression of the Imprinted Gene NNAT and X- Chromosome Gene Activation in Isogenic Human iPS

Genetic comparison between human embryonic stem cells and induced pluripotent stem cells has been hampered by genetic variation. To solve this problem, we have developed an isogenic system that allows direct comparison of induced pluripotent stem cells (hiPSCs) to their genetically matched human embryonic stem cells (hESCs). We show that hiPSCs have a highly similar transcriptome to hESCs. Global transcriptional profiling identified 102–154 genes (.2 fold) that showed a difference between isogenic hiPSCs and hESCs. A stringent analysis identified NNAT as a key imprinted gene that was dysregulated in hiPSCs. Furthermore, a disproportionate number of X-chromosome localized genes were over-expressed in female hiPSCs. Our results indicate that despite a remarkably close transcriptome to hESCs, isogenic hiPSCs have alterations in imprinting and regulation of X-chromosome genes

Genetic Hypervariability in Two Distinct Deuterostome Telomerase Reverse Transcriptase Genes and their Early Embryonic Functions

Functional proteins of complex eukaryotes within the same species are rather invariant. A single catalytic component of telomerase TERT is essential for an active telomerase complex that maintains telomeres. Surprisingly, we have identified two paralogous SpTERT-L and SpTERT-S genes with novel domains in Strongylocentrotus purpuratus (purple sea urchin). The SpTERT-S and SpTERT-L genes were differentially expressed throughout embryogenesis. An unusual germline nucleotide substitution and amino acid variation was evident in these TERTs. The hypervariability of SpTERT-S haplotypes among different individuals reached unprecedented levels of π > 0.2 in exon 11 region. The majority of nucleotide changes observed led to nonsynonymous substitutions creating novel amino acids and motifs, suggesting unusual positive selection and rapid evolution. The majority of these variations were in domains involved in binding of SpTERT to its RNA component. Despite hypervariability at protein level, SpTERT-S conferred telomerase activity, and its suppression during early embryogenesis led to arrest at late mesenchymal blastula. Domain exchange and embryo rescue experiments suggested that SpTERT may have evolved functions unrelated to classic telomerase activity. We suggest that telomerase has a specific and direct function that is essential for integration of early polarity signals that lead to gastrulation. Identification of these unique hypervariable telomerases also suggests presence of a diversity generation mechanism that inculcates hypervariable telomerases and telomere lengths in germline