We discuss the control and maintenance of cellular identity during developmental transitions as they have been studied using direct reprogramming to pluripotency with OCT4, SRY-box 2 (SOX2), Krüppel-like factor 4 (KLF4) and MYC, collectively known as OSKM, with an emphasis on transcriptional induction and epigenetic regulation, as well as on defining molecular features of pluripotent stem cells.
Initial responses to ectopic reprogramming factors in somatic cells are limited to changes in the cell cycle and metabolism, as well as the transcription of epithelial genes, which represent permissive induction of shared genetic modules between pluripotent and differentiated cells.
The pluripotent state can be distinguished from those of differentiated cells by several additional molecular features, including the maintenance of bivalent chromatin at developmental genes, persistence of self-renewal in the absence of epigenetic repressors and dynamic regulation of retrotransposons.
The observed latency and low efficiency of induced pluripotent stem cell generation during direct reprogramming reflect epigenetic barriers that are imposed during differentiation. Once these have been surmounted, direct reprogramming proceeds deterministically to consolidate the pluripotent state.
OCT4, SOX2 and KLF4 cooperatively bind to select cis-regulatory elements of silenced genes embedded in compact chromatin but cannot immediately induce their transcription without additional cofactors, chromatin remodellers and epigenetic modifiers. MYC largely functions independently to enhance transcription at genes that have functions in pluripotent cells as well as in somatic cells.
Differentiating somatic cells are progressively restricted to specialized functions during ontogeny, but they can be experimentally directed to form other cell types, including those with complete embryonic potential. Early nuclear reprogramming methods, such as somatic cell nuclear transfer (SCNT) and cell fusion, posed significant technical hurdles to precise dissection of the regulatory programmes governing cell identity. However, the discovery of reprogramming by ectopic expression of a defined set of transcription factors, known as direct reprogramming, provided a tractable platform to uncover molecular characteristics of cellular specification and differentiation, cell type stability and pluripotency. We discuss the control and maintenance of cellular identity during developmental transitions as they have been studied using direct reprogramming, with an emphasis on transcriptional and epigenetic regulation.
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The authors would like to thank members of the Meissner laboratory, in particular D. Cacchiarelli, J. Charlton, J. Donaghey and A. Arczewska, as well as A. De Los Angeles and T. S. Mikkelsen for thoughtful discussions, and B. E. Bernstein and R. P. Koche for critical reading of the text. A.M. is a New York Stem Cell Foundation Robertson Investigator and is supported by the New York Stem Cell Foundation.
The authors declare no competing financial interests.
Cell fate reprogramming: criteria and concepts (PDF 136 kb)
Epigenetic maintenance in the absence of transcription factor binding (PDF 125 kb)
Resolution of bivalent domains during lineage commitment (PDF 117 kb)
Silencing of retroviral vectors in pluripotent cells (PDF 112 kb)
Defines a cell that can autonomously contribute to all of the tissues of a developing organism, including extra-embryonic and placental tissues, as well as those of the embryo proper. This property is restricted during development to the zygote and the first two cleavage divisions.
The ability of a cell to contribute to all embryonic tissues, including the germ line. Pluripotency is most stringently confirmed by the generation of germline-competent organisms after injection of cells into tetraploidized, embryo-deficient blastocysts.
- Direct reprogramming
Stable, experimentally induced changes in cellular state driven by a defined set of ectopic factors or conditions.
- Chromatin remodellers
ATP-dependent proteins and complexes that change the relative positioning of nucleosomes to support either the activation or repression of a gene.
Co-activator protein with histone acetyltransferase activity, which associates with transcription factor-occupied enhancers that are actively engaged in promoting gene transcription.
- Non-canonical PRC1 complexes
Whereas canonical Polycomb repressive complex 1 (PRC1) contains a chromobox subunit that recognizes PRC2-deposited epigenetic modifications, non-canonical PRC1 complexes are recruited to chromatin by cofactors such as Lys-specific demethylase 2B (KDM2B), which targets unmethylated CpG islands.
- CXXC domain
Cysteine-rich zinc-finger domain found in numerous chromatin-modifying complexes that preferentially binds to unmethylated CpG-rich sequences such as CpG islands.
- Naive or ground state cells
Pluripotent stem cells with properties of the inner cell mass or early epiblast, directed by culturing in media supplemented with leukaemia inhibitory factor and two kinase inhibitors (2i/LIF) that suppress fibroblast growth factor signalling and support WNT signalling.
- Primed state cells
Pluripotent stem cells that require fibroblast growth factor and activin signalling for continuous self-renewal. Associated with non-murine (including human) embryonic stem cells, with phenotypic and molecular features of the post-implantation epiblast.
- Endogenous retroviruses
(ERVs). Genomic retrotransposons originating from exogenous retroviruses, but which propagate intracellularly within germline-competent cell states to enable inheritance to the subsequent generation.
- Krüppel-associated box domain-containing zinc-finger proteins
(KRAB-ZFPs). Zinc-finger proteins that contain an amino-terminal KRAB domain, which interfaces with the tripartite motif-containing protein 28 (TRIM28)–SETDB1 complex to direct repressive histone H3 Lys9 trimethylation, and a variable number of rapidly evolving zinc-finger domains that can confer sequence specificity to emerging repetitive elements.
- Histone H3 variant
Histone H3 variants include H3.1 and H3.2, which are typically incorporated into chromatin during DNA replication, as well as H3.3, which is directed to loci in a replication-independent manner by specific histone chaperones.
The topological coordination of multiple, spatially discrete enhancers to direct the expression of a gene through the Mediator complex. Often, super-enhancers are necessary for the expression of essential cell type-specific genes.
A large, multi-subunit complex that interacts with and spatially juxtaposes transcription factors at promoters and enhancers to coordinate transcription.
- Pioneer factors
Sequence-specific DNA-binding factors that can engage compact chromatin to initiate the formation of nucleosome-free regions.
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Smith, Z., Sindhu, C. & Meissner, A. Molecular features of cellular reprogramming and development. Nat Rev Mol Cell Biol 17, 139–154 (2016). https://doi.org/10.1038/nrm.2016.6
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