Project description:The interaction between SUMO and ZMYM2 is mediated through specific motifs found in ZMYM2 that are called SIMs (SUMO interacting motifs). We found/characterised three SIMs in ZMYM2 and in vitro experiments showed that mutation in these residues prevent the interaction with SUMO. Microarray transcription profiling was done to study the effects of disrupting the multiSUMO binding activity of ZMYM2, assessing the differences in gene expression in cells expressing wild-type or SIM2 mutant version of ZMYM2.
Project description:It has been suggested that the transcription factor Nanog is essential for the establishment of pluripotency during the derivation of embryonic stem (ES) cells and induced pluripotent stem (iPS) cells. However, successful reprogramming to pluripotency with a growing list of divergent transcription factors, at ever increasing efficiencies, suggests that there may be many distinct routes to a pluripotent state. Here, we have investigated whether Nanog is necessary for reprogramming murine fibroblasts under highly efficient conditions using the canonical reprogramming factors Oct4, Sox2, Klf4 and cMyc. In agreement with prior results, the efficiency of reprogramming Nanog-/- fibroblasts was significantly lower than that of control fibroblasts. However, in contrast to previous findings, we were able to reproducibly generate iPS cells from Nanog-/- fibroblasts that effectively contributed to chimeric mice. Thus while Nanog may be an important mediator of reprogramming it is not required for establishing pluripotency in the mouse, even under standard conditions. In order to further evaluate the equivalency of Nanog null iPSC to nanog null ESCs, we have performed RNAseq on two independent nanog null iPSC lines, as well as Nanog Null ESC, WT ESC and iPSCs as well as MEFs. As a negativve control for reprogramming we have analyzed a partially reprogrammed iPSC line. 2-4 biological replicates each of 7 conditions (WT MEFs, WT ESC, WT iPSC, WT partially reprogrammed iPSC (piPS), Nanog null ESC, Nanog null iPSC clone G2 and Nanog null iPSC clone G5)
Project description:It has been suggested that the transcription factor Nanog is essential for the establishment of pluripotency during the derivation of embryonic stem (ES) cells and induced pluripotent stem (iPS) cells. However, successful reprogramming to pluripotency with a growing list of divergent transcription factors, at ever increasing efficiencies, suggests that there may be many distinct routes to a pluripotent state. Here, we have investigated whether Nanog is necessary for reprogramming murine fibroblasts under highly efficient conditions using the canonical reprogramming factors Oct4, Sox2, Klf4 and cMyc. In agreement with prior results, the efficiency of reprogramming Nanog-/- fibroblasts was significantly lower than that of control fibroblasts. However, in contrast to previous findings, we were able to reproducibly generate iPS cells from Nanog-/- fibroblasts that effectively contributed to chimeric mice. Thus while Nanog may be an important mediator of reprogramming it is not required for establishing pluripotency in the mouse, even under standard conditions. In order to further evaluate the equivalency of Nanog null iPSC to nanog null ESCs, we have performed RNAseq on two independent nanog null iPSC lines, as well as Nanog Null ESC, WT ESC and iPSCs as well as MEFs. As a negativve control for reprogramming we have analyzed a partially reprogrammed iPSC line.
Project description:This experiment was carried to identify potential reprogramming factors that augment Sall1 and Nanog reprogramming efficiency either individually or in combination during co-expression. The intent of the experiment was to understand genes regulated by Sall1 and Nanog during mouse epiblast stem cell reprogramming. Empty: mock transfected cells. Nanog: Nanog gene expressing plasmid. Sall1: Sall1 gene expressing plasmid. Untransfected: Untransfected cells. Sall1Nanog: Sall1 and Nanog expressing plasmids (cotransfection).
Project description:Mouse embryonic stem cells (ESCs) sporadically express preimplantation two-cell-stage (2C) transcripts, including MERVL endogenous retrovirus and Zscan4 cluster genes. Such 2C-like cells (2CLCs) can contribute to both embryonic and extraembryonic tissues when reintroduced into early embryos. We examined global nucleosome occupancy and gene expression in 2CLCs and identified miR-344 as the noncoding molecule that positively controls 2CLC potency. We found that activation of endogenous MERVL or miR-344-2 alone is sufficient to induce 2CLCs with induction of 2C genes and an expanded potency. Mechanistically, miR-344 is activated by the 2C-state driver DUX and post-transcriptionally represses ZMYM2 and LSD1, which recruit the HDAC corepressor to MERVL LTR for transcriptional repression. Consistently, zygotic depletion of Zmym2 compromises the totipotency-to-pluripotency transition during early development. Our studies establish the novel DUX->miR-344--|Zmym2/Lsd1 axis that controls MERVL for expanded stem cell potency.