Nuclear Reprogramming of Human Somatic Cells by Xenopus Egg Extract Requires BRG1

Abstract

Animal cloning by nuclear transplantation in amphibia was demonstrated almost half a century ago and raised the question of the mechanisms and genes involved in nuclear reprogramming. Here, we demonstrate nuclear reprogramming of permeabilized human cells using extracts from Xenopus laevis eggs and early embryos. We show upregulation of pluripotency markers Oct-4 and germ cell alkaline phosphatase (GCAP) in 293T cells and human primary leukocytes. Reprogrammed leukocytes had a limited life span and did not express surface antigens characteristic of pluripotent cells, indicating that reprogramming was incomplete. Reprogramming activity was detected in egg and early embryo extracts until early blastula stage. Late blastula-stage extracts were not only inactive but also inhibitory to reprogramming. Screening for factors required for reprogramming identified the chromatin remodeling ATPase BRG1. Antibody depletion of BRG1 protein or expression of dominant-negative BRG1 abolished the reprogramming ability of amphibian extracts. Conversely, overexpression of BRG1 in Xenopus animal caps extended their competence from blastula to gastrula stage to respond to basic fibroblast growth factor (bFGF) treatment with induction of the mesodermal marker Xbra. Dissection of the molecular machinery using a simplified assay system may aid in achieving complete nuclear reprogramming of somatic cells for regenerative medicine.

Epigenetic Reprogramming ofOCT4andNANOGRegulatory Regions by Embryonal Carcinoma Cell Extract

“…Transcriptional activation of Oct4 in mouse fibroblasts treated with ES cell extract requires ATP hydrolysis, most likely for nuclear import of transcription factors Landsverk et al, 2002) and for the activity of chromatin remodeling complexes (Kingston and Narlikar, 1999;Aalfs et al, 2001). Oct4 activation by ES cell extract or Xenopus egg extract also requires the Brg1 subunit of the SWI/SNF complex (Hansis et al, 2004;Taranger et al, 2005). Promoter-specific targeting of SWI/SNF may involve H3K4 (hyper)methylation on OCT4 and NANOG and prime the loci for further transcription-permissive remodeling (H3K4 methylation per se is not sufficient to allow transcription as H3K4 is methylated on both OCT4 and NANOG in 293T cells).…”

Stretching the limits: Stem cells in regeneration science

“…Hakelien et al (2002) reported the reprogramming of 293T fibroblasts in vitro by extract of Jurkat cells into cells that partially resembled T-lymphocytes and to cells with some characteristics of neurons using a neuronal cell precursor extract. Hansis et al (2004) exposed human 293T kidney cells and primary leukocytes to Xenopus oocyte extract and detected up-regulation of the pluripotency markers alkaline phosphatase and Oct4. Two candidate reprogramming molecules in Xenopus oocyte extract have been identified, the chromatin remodeling ATPase, BRG1 (Hansis et al, 2004), and a tumor-associated protein, Tpt1 (Koziol et al, 2007).…”

“…Hansis et al (2004) exposed human 293T kidney cells and primary leukocytes to Xenopus oocyte extract and detected up-regulation of the pluripotency markers alkaline phosphatase and Oct4. Two candidate reprogramming molecules in Xenopus oocyte extract have been identified, the chromatin remodeling ATPase, BRG1 (Hansis et al, 2004), and a tumor-associated protein, Tpt1 (Koziol et al, 2007). Antibody depletion of BRG1 from the extract abolished its reprogramming activity.…”

Reprogramming of gene expression following nuclear transfer to theXenopusoocyte

“…As mentioned before, one other advantage of the Xenopus system is the large amount of material available, opening the way for biochemical analysis of oocyte/egg components. By combining oocyte extract analysis and nuclear transfer, it was shown that Oct4 reactivation required the activity of several components of the oocyte such as the chromatin remodelling factor Brg1 (Hansis et al, 2004) and the transcription factor Tpt1 (Koziol et al, 2007).…”