For many years scientists have been attracted to the possibility of changing cell identity. In the last decades seminal discoveries have shown that it is possible to reprogram somatic cells into pluripotent cells and even to transdifferentiate one cell type into another. In view of the potential applications that generating specific cell types in the laboratory can offer for cell-based therapies, the next important questions relate to the quality of the induced cell types. Importantly, epigenetic aberrations in reprogrammed cells have been correlated with defects in differentiation. Therefore, a look at the epigenome and understanding how different regulators can shape it appear fundamental to anticipate potential therapeutic pitfalls. This paper covers these epigenetic aspects in stem cells, differentiation, and reprogramming and discusses their importance for the safety of in vitro engineered cell types. 1. Introduction The genome is organized into particular chromatin structures that have specific roles both in maintaining the overall structure and in gene expression. The fundamental unit of chromatin is the nucleosome, composed of two copies each of four core histones, H2A, H2B, H3, and H4, wrapped by 146？bp of DNA. The recruitment of linker histone H1 and other structural proteins can lead to further condensation and the of higher-order structures, which play additional roles in the organization of chromosomes. Chromatin offers a physical barrier to the efficient recruitment and processivity of the RNA Polymerase II (Poll l) and thus impedes gene transcription [1]. The extent of chromatin condensation is subject to regulation. The N-terminal tails of histones are relatively accessible to enzymatic modifications such as acetylation, methylation, phosphorylation, ubiquitination, and sumoylation. Furthermore, the cytosine residues of DNA can be modified by methylation and hydroxymethylation. These modifications can influence the degree of condensation of chromatin per se or/and facilitate the recruitment of structural or effector proteins, such as remodeling complexes, that directly affect the condensation of chromatin. Certain areas of the genome are organized into heavily condensed chromatin structures, such as centromeric regions, and offer little room for transcriptional regulation. These areas are enriched in H3K9 methylation and marked by the presence of structural proteins such as HP1 (heterochromatin protein 1), which contribute to maintain high levels of condensation that play mainly structural roles in the organization of chromosomes.
References
[1]
C. Hodges, L. Bintu, L. Lubkowska, M. Kashlev, and C. Bustamante, “Nucleosomal fluctuations govern the transcription dynamics of RNA polymerase II,” Science, vol. 325, no. 5940, pp. 626–628, 2009.
[2]
T. Jenuwein and C. D. Allis, “Translating the histone code,” Science, vol. 293, no. 5532, pp. 1074–1080, 2001.
[3]
J. K. Stock, S. Giadrossi, M. Casanova et al., “Ring1-mediated ubiquitination of H2A restrains poised RNA polymerase II at bivalent genes in mouse ES cells,” Nature Cell Biology, vol. 9, no. 12, pp. 1428–1435, 2007.
[4]
A. O'Loghlen, A. M. Mu？oz-Cabello, A. Gaspar-Maia, et al., “MicroRNA regulation of Cbx7 mediates a switch of Polycomb orthologs during ESC differentiation,” Cell Stem Cell, vol. 10, no. 1, pp. 33–46, 2012.
[5]
X. Shen, Y. Liu, Y. J. Hsu et al., “EZH1 mediates methylation on histone H3 lysine 27 and complements EZH2 in maintaining stem cell identity and executing pluripotency,” Molecular Cell, vol. 32, no. 4, pp. 491–502, 2008.
[6]
L. A. Boyer, K. Plath, J. Zeitlinger et al., “Polycomb complexes repress developmental regulators in murine embryonic stem cells,” Nature, vol. 441, no. 7091, pp. 349–353, 2006.
[7]
V. Azuara, P. Perry, S. Sauer et al., “Chromatin signatures of pluripotent cell lines,” Nature cell biology., vol. 8, no. 5, pp. 532–538, 2006.
[8]
T. I. Lee, R. G. Jenner, L. A. Boyer, et al., “Control of developmental regulators by Polycomb in human embryonic stem cells,” Cell, vol. 125, no. 2, pp. 301–313, 2006.
[9]
D. Pasini, A. P. Bracken, J. B. Hansen, M. Capillo, and K. Helin, “The Polycomb group protein Suz12 is required for embryonic stem cell differentiation,” Molecular and Cellular Biology, vol. 27, no. 10, pp. 3769–3779, 2007.
[10]
D. Landeira, S. Sauer, R. Poot et al., “Jarid2 is a PRC2 component in embryonic stem cells required for multi-lineage differentiation and recruitment of PRC1 and RNA Polymerase II to developmental regulators,” Nature Cell Biology, vol. 12, no. 6, pp. 618–624, 2010.
[11]
D. Pasini, K. H. Hansen, J. Christensen, K. Agger, P. A. C. Cloos, and K. Helin, “Coordinated regulation of transcriptional repression by the RBP2 H3K4 demethylase and Polycomb-Repressive Complex 2,” Genes and Development, vol. 22, no. 10, pp. 1345–1355, 2008.
[12]
L. Ho, J. L. Ronan, J. Wu et al., “An embryonic stem cell chromatin remodeling complex, esBAF, is essential for embryonic stem cell self-renewal and pluripotency,” Proceedings of the National Academy of Sciences of the United States of America, vol. 106, no. 13, pp. 5181–5186, 2009.
[13]
O. Yildirim, R. Li, J. H. Hung, et al., “Mbd3/NURD complex regulates expression of 5-hydroxymethylcytosine marked genes in embryonic stem cells,” Cell, vol. 147, no. 7, pp. 1498–1510, 2011.
[14]
N. Y. Chia, Y. S. Chan, B. Feng et al., “A genome-wide RNAi screen reveals determinants of human embryonic stem cell identity,” Nature, vol. 468, no. 7321, pp. 316–320, 2010.
[15]
S. Efroni, R. Duttagupta, J. Cheng et al., “Global transcription in pluripotent embryonic stem cells,” Cell Stem Cell, vol. 2, no. 5, pp. 437–447, 2008.
[16]
A. Gaspar-Maia, A. Alajem, F. Polesso et al., “Chd1 regulates open chromatin and pluripotency of embryonic stem cells,” Nature, vol. 460, no. 7257, pp. 863–868, 2009.
[17]
K. Agger, P. A. C. Cloos, J. Christensen et al., “UTX and JMJD3 are histone H3K27 demethylases involved in HOX gene regulation and development,” Nature, vol. 449, no. 7163, pp. 731–734, 2007.
[18]
H. Jiang, A. Shukla, X. Wang, W. Y. Chen, B. E. Bernstein, and R. G. Roeder, “Role for Dpy-30 in ES cell-fate specification by regulation of H3K4 methylation within bivalent domains,” Cell, vol. 144, no. 4, pp. 513–525, 2011.
[19]
Y. S. Ang, S. Y. Tsai, D. F. Lee et al., “Wdr5 mediates self-renewal and reprogramming via the embryonic stem cell core transcriptional network,” Cell, vol. 145, no. 2, pp. 183–197, 2011.
[20]
A. Adamo, B. Sesé, S. Boue et al., “LSD1 regulates the balance between self-renewal and differentiation in human embryonic stem cells,” Nature Cell Biology, vol. 13, no. 6, pp. 652–659, 2011.
[21]
M. H. Kagey, J. J. Newman, S. Bilodeau et al., “Mediator and cohesin connect gene expression and chromatin architecture,” Nature, vol. 467, no. 7314, pp. 430–435, 2010.
[22]
R. L. Davis, H. Weintraub, and A. B. Lassar, “Expression of a single transfected cDNA converts fibroblasts to myoblasts,” Cell, vol. 51, no. 6, pp. 987–1000, 1987.
[23]
I. Wilmut, A. E. Schnieke, J. McWhir, A. J. Kind, and K. H. S. Campbell, “Viable offspring derived from fetal and adult mammalian cells,” Nature, vol. 385, no. 6619, pp. 810–813, 1997.
[24]
K. Takahashi and S. Yamanaka, “Induction of pluripotent stem cells from mouse embryonic and adult fibroblast cultures by defined factors,” Cell, vol. 126, no. 4, pp. 663–676, 2006.
[25]
K. Takahashi, K. Tanabe, M. Ohnuki et al., “Induction of pluripotent stem cells from adult human fibroblasts by defined factors,” Cell, vol. 131, no. 5, pp. 861–872, 2007.
[26]
M. Nakagawa, M. Koyanagi, K. Tanabe et al., “Generation of induced pluripotent stem cells without Myc from mouse and human fibroblasts,” Nature Biotechnology, vol. 26, no. 1, pp. 101–106, 2008.
[27]
J. B. Kim, H. Zaehres, G. Wu et al., “Pluripotent stem cells induced from adult neural stem cells by reprogramming with two factors,” Nature, vol. 454, no. 7204, pp. 646–650, 2008.
[28]
D. Huangfu, K. Osafune, R. Maehr et al., “Induction of pluripotent stem cells from primary human fibroblasts with only Oct4 and Sox2,” Nature Biotechnology, vol. 26, no. 11, pp. 1269–1275, 2008.
[29]
M. Stadtfeld, M. Nagaya, J. Utikal, G. Weir, and K. Hochedlinger, “Induced pluripotent stem cells generated without viral integration,” Science, vol. 322, no. 5903, pp. 945–949, 2008.
[30]
I. H. Park, N. Arora, H. Huo et al., “Disease-specific induced pluripotent stem cells,” Cell, vol. 134, no. 5, pp. 877–886, 2008.
[31]
H. Zhou, S. Wu, J. Y. Joo et al., “Generation of induced pluripotent stem cells using recombinant proteins,” Cell Stem Cell, vol. 4, no. 5, pp. 381–384, 2009.
[32]
L. Warren, P. D. Manos, T. Ahfeldt et al., “Highly efficient reprogramming to pluripotency and directed differentiation of human cells with synthetic modified mRNA,” Cell Stem Cell, vol. 7, no. 5, pp. 618–630, 2010.
[33]
M. Ieda, J. D. Fu, P. Delgado-Olguin et al., “Direct reprogramming of fibroblasts into functional cardiomyocytes by defined factors,” Cell, vol. 142, no. 3, pp. 375–386, 2010.
[34]
T. Vierbuchen, A. Ostermeier, Z. P. Pang, Y. Kokubu, T. C. Südhof, and M. Wernig, “Direct conversion of fibroblasts to functional neurons by defined factors,” Nature, vol. 463, no. 7284, pp. 1035–1041, 2010.
[35]
E. Szabo, S. Rampalli, R. M. Risue？o et al., “Direct conversion of human fibroblasts to multilineage blood progenitors,” Nature, vol. 468, no. 7323, pp. 521–526, 2010.
[36]
F. Anokye-Danso, C. M. Trivedi, D. Juhr et al., “Highly efficient miRNA-mediated reprogramming of mouse and human somatic cells to pluripotency,” Cell Stem Cell, vol. 8, no. 4, pp. 376–388, 2011.
[37]
R. Lister, M. Pelizzola, Y. S. Kida et al., “Hotspots of aberrant epigenomic reprogramming in human induced pluripotent stem cells,” Nature, vol. 471, no. 7336, pp. 68–73, 2011.
[38]
A. Gore, Z. Li, H. L. Fung et al., “Somatic coding mutations in human induced pluripotent stem cells,” Nature, vol. 471, no. 7336, pp. 63–67, 2011.
[39]
L. C. Laurent, I. Ulitsky, I. Slavin et al., “Dynamic changes in the copy number of pluripotency and cell proliferation genes in human ESCs and iPSCs during reprogramming and time in culture,” Cell Stem Cell, vol. 8, no. 1, pp. 106–118, 2011.
[40]
L. A. Boyer, I. L. Tong, M. F. Cole et al., “Core transcriptional regulatory circuitry in human embryonic stem cells,” Cell, vol. 122, no. 6, pp. 947–956, 2005.
[41]
M. Pardo, B. Lang, L. Yu et al., “An expanded Oct4 interaction network: implications for stem cell biology, development, and disease,” Cell Stem Cell, vol. 6, no. 4, pp. 382–395, 2010.
[42]
D. L. C. van den Berg, T. Snoek, N. P. Mullin et al., “An Oct4-centered protein interaction network in embryonic stem cells,” Cell Stem Cell, vol. 6, no. 4, pp. 369–381, 2010.
[43]
E. Meshorer, D. Yellajoshula, E. George, P. J. Scambler, D. T. Brown, and T. Misteli, “Hyperdynamic plasticity of chromatin proteins in pluripotent embryonic stem cells,” Developmental Cell, vol. 10, no. 1, pp. 105–116, 2006.
[44]
B. E. Bernstein, T. S. Mikkelsen, X. Xie et al., “A bivalent chromatin structure marks key developmental genes in embryonic stem cells,” Cell, vol. 125, no. 2, pp. 315–326, 2006.
[45]
K. P. Koh, A. Yabuuchi, S. Rao et al., “Tet1 and Tet2 regulate 5-hydroxymethylcytosine production and cell lineage specification in mouse embryonic stem cells,” Cell Stem Cell, vol. 8, no. 2, pp. 200–213, 2011.
[46]
K. Williams, J. Christensen, M. T. Pedersen et al., “TET1 and hydroxymethylcytosine in transcription and DNA methylation fidelity,” Nature, vol. 473, no. 7347, pp. 343–348, 2011.
[47]
M. M. Dawlaty, K. Ganz, B. E. Powell et al., “Tet1 is dispensable for maintaining pluripotency and its loss is compatible with embryonic and postnatal development,” Cell Stem Cell, vol. 9, no. 2, pp. 166–175, 2011.
[48]
I. M. Min, J. J. Waterfall, L. J. Core, R. J. Munroe, J. Schimenti, and J. T. Lis, “Regulating RNA polymerase pausing and transcription elongation in embryonic stem cells,” Genes and Development, vol. 25, no. 7, pp. 742–754, 2011.
[49]
M. Vermeulen, K. W. Mulder, S. Denissov et al., “Selective anchoring of TFIID to nucleosomes by trimethylation of histone H3 lysine 4,” Cell, vol. 131, no. 1, pp. 58–69, 2007.
[50]
J. Simon, A. Chiang, W. Bender, M. J. Shimell, and M. O'Connor, “Elements of the Drosophila bithorax complex that mediate repression by Polycomb group products,” Developmental Biology, vol. 158, no. 1, pp. 131–144, 1993.
[51]
M. Ku, R. P. Koche, E. Rheinbay et al., “Genomewide analysis of PRC1 and PRC2 occupancy identifies two classes of bivalent domains,” PLoS Genetics, vol. 4, no. 10, Article ID e1000242, 2008.
[52]
A. Tanay, A. H. O'Donnell, M. Damelin, and T. H. Bestor, “Hyperconserved CpG domains underlie Polycomb-binding sites,” Proceedings of the National Academy of Sciences of the United States of America, vol. 104, no. 13, pp. 5521–5526, 2007.
[53]
H. Wu, A. C. D'Alessio, S. Ito et al., “Dual functions of Tet1 in transcriptional regulation in mouse embryonic stem cells,” Nature, vol. 473, no. 7347, pp. 389–393, 2011.
[54]
J. Wang, S. Rao, J. Chu et al., “A protein interaction network for pluripotency of embryonic stem cells,” Nature, vol. 444, no. 7117, pp. 364–368, 2006.
[55]
G. Gargiulo, S. Levy, G. Bucci et al., “NA-Seq: a discovery tool for the analysis of chromatin structure and dynamics during differentiation,” Developmental Cell, vol. 16, no. 3, pp. 466–481, 2009.
[56]
K. Cui, C. Zang, T. Y. Roh et al., “Chromatin signatures in multipotent human hematopoietic stem cells indicate the fate of bivalent genes during differentiation,” Cell Stem Cell, vol. 4, no. 1, pp. 80–93, 2009.
[57]
T. S. Mikkelsen, M. Ku, D. B. Jaffe et al., “Genome-wide maps of chromatin state in pluripotent and lineage-committed cells,” Nature, vol. 448, no. 7153, pp. 553–560, 2007.
[58]
R. D. Hawkins, G. C. Hon, L. K. Lee et al., “Distinct epigenomic landscapes of pluripotent and lineage-committed human cells,” Cell Stem Cell, vol. 6, no. 5, pp. 479–491, 2010.
[59]
T. Burgold, F. Spreafico, F. de Santa et al., “The histone H3 lysine 27-specific demethylase Jmjd3 is required for neural commitment,” PLoS ONE, vol. 3, no. 8, Article ID e3034, 2008.
[60]
I. Issaeva, Y. Zonis, T. Rozovskaia et al., “Knockdown of ALR (MLL2) reveals ALR target genes and leads to alterations in cell adhesion and growth,” Molecular and Cellular Biology, vol. 27, no. 5, pp. 1889–1903, 2007.
[61]
F. de Santa, M. G. Totaro, E. Prosperini, S. Notarbartolo, G. Testa, and G. Natoli, “The histone H3 lysine-27 demethylase Jmjd3 links inflammation to inhibition of Polycomb-mediated gene silencing,” Cell, vol. 130, no. 6, pp. 1083–1094, 2007.
[62]
J. M. Terme, B. Sesé, L. Millán-Ari？o, et al., “Histone H1 variants are differentially expressed and incorporated into chromatin during differentiation and reprogramming to pluripotency,” The Journal of Biological Chemistry, vol. 286, no. 41, pp. 35347–35357, 2011.
[63]
S. Epsztejn-Litman, N. Feldman, M. Abu-Remaileh et al., “De novo DNA methylation promoted by G9a prevents reprogramming of embryonically silenced genes,” Nature Structural and Molecular Biology, vol. 15, no. 11, pp. 1176–1183, 2008.
[64]
N. Feldman, A. Gerson, J. Fang et al., “G9a-mediated irreversible epigenetic inactivation of Oct-3/4 during early embryogenesis,” Nature Cell Biology, vol. 8, no. 2, pp. 188–194, 2006.
[65]
B. Feng, J. H. Ng, J. C. D. Heng, and H. H. Ng, “Molecules that promote or enhance reprogramming of somatic cells to induced pluripotent stem cells,” Cell Stem Cell, vol. 4, no. 4, pp. 301–312, 2009.
[66]
D. Huangfu, R. Maehr, W. Guo et al., “Induction of pluripotent stem cells by defined factors is greatly improved by small-molecule compounds,” Nature Biotechnology, vol. 26, no. 7, pp. 795–797, 2008.
[67]
T. S. Mikkelsen, J. Hanna, X. Zhang et al., “Dissecting direct reprogramming through integrative genomic analysis,” Nature, vol. 454, no. 7200, pp. 49–55, 2008.
[68]
R. Sridharan, J. Tchieu, M. J. Mason et al., “Role of the murine reprogramming factors in the induction of pluripotency,” Cell, vol. 136, no. 2, pp. 364–377, 2009.
[69]
J. M. Polo, S. Liu, M. E. Figueroa et al., “Cell type of origin influences the molecular and functional properties of mouse induced pluripotent stem cells,” Nature Biotechnology, vol. 28, no. 8, pp. 848–855, 2010.
[70]
K. Kim, A. Doi,, B. Wen, et al., “Epigenetic memory in induced pluripotent stem cells,” Nature, vol. 467, pp. 285–290, 2010.
[71]
S. Boue, I. Paramonov, M. J. Barrero, and J. C. I. Belmonte, “Analysis of human and mouse reprogramming of somatic cells to induced pluripotent stem cells. What is in the plate?” PLoS ONE, vol. 5, no. 9, Article ID e12664, 2010.
[72]
R. P. Koche, Z. D. Smith, M. Adli et al., “Reprogramming factor expression initiates widespread targeted chromatin remodeling,” Cell Stem Cell, vol. 8, no. 1, pp. 96–105, 2011.
[73]
P.C. Taberlay, T. K. Kelly, C. C. Liu, et al., “Polycomb-repressed genes have permissive enhancers that initiate reprogramming,” Cell, vol. 147, no. 6, pp. 1283–1294, 2011.
[74]
C. Bock, E. Kiskinis, G. Verstappen et al., “Reference maps of human es and ips cell variation enable high-throughput characterization of pluripotent cell lines,” Cell, vol. 144, no. 3, pp. 439–452, 2011.
[75]
R. Lister, M. Pelizzola, R. H. Dowen et al., “Human DNA methylomes at base resolution show widespread epigenomic differences,” Nature, vol. 462, no. 7271, pp. 315–322, 2009.
[76]
Y. Ohi, H. Qin, C. Hong et al., “Incomplete DNA methylation underlies a transcriptional memory of somatic cells in human iPS cells,” Nature Cell Biology, vol. 13, no. 5, pp. 541–549, 2011.
[77]
O. Bar-Nur, H. A. Russ, S. Efrat, and N. Benvenisty, “Epigenetic memory and preferential lineage-specific differentiation in induced pluripotent stem cells derived from human pancreatic islet beta cells,” Cell Stem Cell, vol. 9, no. 1, pp. 17–23, 2011.
[78]
M. H. Chin, M. J. Mason, W. Xie et al., “Induced pluripotent stem cells and embryonic stem cells are distinguished by gene expression signatures,” Cell Stem Cell, vol. 5, no. 1, pp. 111–123, 2009.
[79]
A. Doi, I. H. Park, B. Wen et al., “Differential methylation of tissue-and cancer-specific CpG island shores distinguishes human induced pluripotent stem cells, embryonic stem cells and fibroblasts,” Nature Genetics, vol. 41, no. 12, pp. 1350–1353, 2009.
[80]
F. Gonzalez, S. Boue, and J. C. I. Belmonte, “Methods for making induced pluripotent stem cells: reprogramming a la carte,” Nature Reviews Genetics, vol. 12, no. 4, pp. 231–242, 2011.
[81]
E. Szabo, S. Rampalli, R. M. Risue？o et al., “Direct conversion of human fibroblasts to multilineage blood progenitors,” Nature, vol. 468, no. 7323, pp. 521–526, 2010.
[82]
T. Vierbuchen, A. Ostermeier, Z. P. Pang, Y. Kokubu, T. C. Südhof, and M. Wernig, “Direct conversion of fibroblasts to functional neurons by defined factors,” Nature, vol. 463, no. 7284, pp. 1035–1041, 2010.
[83]
M. Widschwendter, H. Fiegl, D. Egle et al., “Epigenetic stem cell signature in cancer,” Nature Genetics, vol. 39, no. 2, pp. 157–158, 2007.
[84]
V. K. Rakyan, T. A. Down, S. Maslau et al., “Human aging-associated DNA hypermethylation occurs preferentially at bivalent chromatin domains,” Genome Research, vol. 20, no. 4, pp. 434–439, 2010.
