Histone variant macroH2A confers resistance to nuclear reprogramming

Gurdon and collaborators report reversible X chromosome inactivation in epiblast stem cells (EpiSCs) that seems to be determined by macroH2A1 deposition. These findings are of rather general interest as they highlight the epigenetic state of repressed loci as determinant for reprogramming efficiency

Expression of the myodystrophic R453W mutation of lamin A in C2C12 myoblasts causes promoter-specific and global epigenetic defects.

International audienceAutosomal dominant Emery-Dreifuss muscular dystrophy (EDMD) is characterized by muscle wasting and is caused by mutations in the LMNA gene encoding A-type lamins. Overexpression of the EDMD lamin A R453W mutation in C2C12 myoblasts impairs myogenic differentiation. We show here the influence of stable expression of the R453W and of the Dunnigan-type partial lipodystrophy R482W mutation of lamin A in C2C12 cells on transcription and epigenetic regulation of the myogenin (Myog) gene and on global chromatin organization. Expression of R453W-, but not R482W-lamin A, impairs activation of Myog and maintains a repressive chromatin state on the Myog promoter upon induction of differentiation, marked by H3 lysine (K) 9 dimethylation and failure to hypertrimethylate H3K4. Cells expressing WT-LaA also fail to hypertrimethylate H3K4. No defect occurs at the level of Myog promoter DNA methylation in any of the clones. Expression of R453W-lamin A and to a lesser extent R482W-lamin A in undifferentiated C2C12 cells redistributes H3K9me3 from pericentric heterochromatin. R453W-lamin A also elicits a redistribution of H3K27me3 from inactive X (Xi) and partial decondensation of Xi, but maintains Xist expression and coating of Xi, indicating that Xi remains inactivated. Our results argue that gene-specific and genome-wide chromatin rearrangements may constitute a molecular basis for laminopathies

Reprogrammed gene expression in a somatic cell-free extract

We have developed a somatic cell-free system that remodels chromatin and activates gene expression in heterologous differentiated nuclei. Extracts of stimulated human T cells elicit chromatin binding of transcriptional activators of the interleukin-2 (IL-2) gene, anchoring and activity of a chromatin-remodeling complex and hyperacetylation of the IL-2 promoter in purified exogenous resting T-cell nuclei. The normally repressed IL-2 gene is transcribed in nuclei from quiescent human T cells and from various non-T-cell lines. This demonstrates that somatic cell extracts can be used to reprogram gene expression in differentiated nuclei. In vitro reprogramming may be useful for investigating regulation of gene expression and for producing replacement cells for the treatment of a wide variety of diseases

Induction of Dedifferentiation, Genomewide Transcriptional Programming, and Epigenetic Reprogramming by Extracts of Carcinoma and Embryonic Stem Cells

Functional reprogramming of a differentiated cell toward pluripotency may have long-term applications in regenerative medicine. We report the induction of dedifferentiation, associated with genomewide programming of gene expression and epigenetic reprogramming of an embryonic gene, in epithelial 293T cells treated with an extract of undifferentiated human NCCIT carcinoma cells. 293T cells exposed for 1 h to extract of NCCIT cells, but not of 293T or Jurkat T-cells, form defined colonies that are maintained for at least 23 passages in culture. Microarray and quantitative analyses of gene expression reveal that the transition from a 293T to a pluripotent cell phenotype involves a dynamic up-regulation of hundreds of NCCIT genes, concomitant with down-regulation of 293T genes and of indicators of differentiation such as A-type lamins. Up-regulated genes encompass embryonic and stem cell markers, including OCT4, SOX2, NANOG, and Oct4-responsive genes. OCT4 activation is associated with DNA demethylation in the OCT4 promoter and nuclear targeting of Oct4 protein. In fibroblasts exposed to extract of mouse embryonic stem cells, Oct4 activation is biphasic and RNA-PolII dependent, with the first transient rise of Oct4 up-regulation being necessary for the second, long-term activation of Oct4. Genes characteristic of multilineage differentiation potential are also up-regulated in NCCIT extract-treated cells, suggesting the establishment of “multilineage priming.” Retinoic acid triggers Oct4 down-regulation, de novo activation of A-type lamins, and nestin. Furthermore, the cells can be induced to differentiate toward neurogenic, adipogenic, osteogenic, and endothelial lineages. The data provide a proof-of-concept that an extract of undifferentiated carcinoma cells can elicit differentiation plasticity in an otherwise more developmentally restricted cell type