Jessica Dausman

Direct Reprogramming of Terminally Differentiated Mature B Lymphocytes To Pluripotency

Pluripotent cells can be derived from fibroblasts by ectopic expression of defined transcription factors. A fundamental unresolved question is whether terminally differentiated cells can be reprogrammed to pluripotency. We utilized transgenic and inducible expression of four transcription factors (Oct4, Sox2, Klf4, and c-Myc) to reprogram mouse B lymphocytes. These factors were sufficient to convert nonterminally differentiated B cells to a pluripotent state. However, reprogramming of mature B cells required additional interruption with the transcriptional state maintaining B cell identity by either ectopic expression of the myeloid transcription factor CCAAT/enhancer-binding-protein-alpha (C/EBPalpha) or specific knockdown of the B cell transcription factor Pax5. Multiple iPS lines were clonally derived from both nonfully and fully differentiated B lymphocytes, which gave rise to adult chimeras with germline contribution, and to late-term embryos when injected into tetraploid blastocysts. Our study provides definite proof for the direct nuclear reprogramming of terminally differentiated adult cells to pluripotency.Pluripotent cells can be derived from fibroblasts by ectopic expression of defined transcription factors. A fundamental unresolved question is whether terminally differentiated cells can be reprogrammed to pluripotency. We utilized transgenic and inducible expression of four transcription factors (Oct4, Sox2, Klf4, and c-Myc) to reprogram mouse B lymphocytes. These factors were sufficient to convert nonterminally differentiated B cells to a pluripotent state. However, reprogramming of mature B cells required additional interruption with the transcriptional state maintaining B cell identity by either ectopic expression of the myeloid transcription factor CCAAT/enhancer-binding-protein-α (C/EBPα) or specific knockdown of the B cell transcription factor Pax5. Multiple iPS lines were clonally derived from both nonfully and fully differentiated B lymphocytes, which gave rise to adult chimeras with germline contribution, and to late-term embryos when injected into tetraploid blastocysts. Our study provides definite proof for the direct nuclear reprogramming of terminally differentiated adult cells to pluripotency.

Loss of genomic methylation causes p53-dependent apoptosis and epigenetic deregulation

Cytosine methylation of mammalian DNA is essential for the proper epigenetic regulation of gene expression and maintenance of genomic integrity. To define the mechanism through which demethylated cells die, and to establish a paradigm for identifying genes regulated by DNA methylation, we have generated mice with a conditional allele for the maintenance DNA methyltransferase gene Dnmt1. Cre-mediated deletion of Dnmt1 causes demethylation of cultured fibroblasts and a uniform p53-dependent cell death. Mutational inactivation of Trp53 partially rescues the demethylated fibroblasts for up to five population doublings in culture. Oligonucleotide microarray analysis showed that up to 10% of genes are aberrantly expressed in demethylated fibroblasts. Our results demonstrate that loss of Dnmt1 causes cell-type–specific changes in gene expression that impinge on several pathways, including expression of imprinted genes, cell-cycle control, growth factor/receptor signal transduction and mobilization of retroelements.

Generation of mice from wild-type and targeted ES cells by nuclear cloning

Acknowledgements We thank T. Perry for critical and useful comments on the manuscript. R.Y. acknowledges financial support from ProBio America. Teruhiko Wakayama1,3, Hiroyuki Tateno1, Peter Mombaerts2 & Ryuzo Yanagimachi1 1University of Hawaii Medical School, Department of Anatomy and Reproductive Biology, Honolulu, Hawaii, USA. 2The Rockefeller University, New York, New York, USA. 3Present address: The Rockefeller University, New York, NY, USA. Correspondence should be addressed to T.W. (e-mail: wakayat@rockvax.rockefeller.edu).

A 450 kb Transgene Displays Properties of the Mammalian X-Inactivation Center

X inactivation results in inactivation of one X chromosome to compensate for gene dosage differences between mammalian females and males. It requires the X-inactivation center (Xic) and Xist in cis. We report that introducing 450 kb of murine Xic/Xist sequences onto autosomes activates female dosage compensation in male ES cells. Xist is induced upon differentiation and can be expressed from both endogenous and ectopic loci, suggesting that elements for counting and choosing Xs are present in the transgene. Differentiating transgenic ES cells undergo excessive cell death. Postnatally, Xist is expressed only from the transgene. Ectopic Xist RNA structurally associates with the autosome and may inactivate a marker gene in cis. These results argue that the Xic is contained within 450 kb and that these sequences are sufficient for chromosome counting, choosing, and initiation of X inactivation.

X Chromosome Inactivation Is Mediated by Xist RNA Stabilization

Low level Xist expression can be detected from both active X chromosomes (Xa) in female embryonic stem cells prior to X inactivation. After differentiation, Xist is expressed at high levels only from the inactive X chromosome (Xi). Differentiating female cells increase Xist expression from the Xi prior to silencing low level Xist expression from the Xa. The transition from low level to high level expression is regulated by the stabilization of Xist transcripts at the Xi. We suggest that these developmentally modulated changes in Xist expression are regulated by several different mechanisms: factors that stabilize Xist transcripts at the Xi, an activity that blocks this stabilization at the Xa, and a mechanism that silences low level Xist expression from the Xa.