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. 2012 Aug 30;488(7413):652-5.
doi: 10.1038/nature11333.

Early-stage epigenetic modification during somatic cell reprogramming by Parp1 and Tet2

Claudia A Doege  1 , Keiichi Inoue, Toru Yamashita, David B Rhee, Skylar Travis, Ryousuke Fujita, Paolo Guarnieri, Govind Bhagat, William B Vanti, Alan Shih, Ross L Levine, Sara Nik, Emily I Chen, Asa Abeliovich
Affiliations

Early-stage epigenetic modification during somatic cell reprogramming by Parp1 and Tet2

Claudia A Doege et al. Nature. .

Abstract

Somatic cells can be reprogrammed into induced pluripotent stem cells (iPSCs) by using the pluripotency factors Oct4, Sox2, Klf4 and c-Myc (together referred to as OSKM). iPSC reprogramming erases somatic epigenetic signatures—as typified by DNA methylation or histone modification at silent pluripotency loci—and establishes alternative epigenetic marks of embryonic stem cells (ESCs). Here we describe an early and essential stage of somatic cell reprogramming, preceding the induction of transcription at endogenous pluripotency loci such as Nanog and Esrrb. By day 4 after transduction with OSKM, two epigenetic modification factors necessary for iPSC generation, namely poly(ADP-ribose) polymerase-1 (Parp1) and ten-eleven translocation-2 (Tet2), are recruited to the Nanog and Esrrb loci. These epigenetic modification factors seem to have complementary roles in the establishment of early epigenetic marks during somatic cell reprogramming: Parp1 functions in the regulation of 5-methylcytosine (5mC) modification, whereas Tet2 is essential for the early generation of 5-hydroxymethylcytosine (5hmC) by the oxidation of 5mC (refs 3,4). Although 5hmC has been proposed to serve primarily as an intermediate in 5mC demethylation to cytosine in certain contexts, our data, and also studies of Tet2-mutant human tumour cells, argue in favour of a role for 5hmC as an epigenetic mark distinct from 5mC. Consistent with this, Parp1 and Tet2 are each needed for the early establishment of histone modifications that typify an activated chromatin state at pluripotency loci, whereas Parp1 induction further promotes accessibility to the Oct4 reprogramming factor. These findings suggest that Parp1 and Tet2 contribute to an epigenetic program that directs subsequent transcriptional induction at pluripotency loci during somatic cell reprogramming.

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Conflict of interest statement

The authors declare no competing financial interests.

Figures

Figure 1
Figure 1. Parp1 promotes OSKM-mediated iPSC generation
a, Diagram of proteomic strategy to identify candidate epigenetic modification (EM) factors. b, Unsupervised hierarchical clustering analysis (Spearman rank correlation) of mass spectrometry data from nuclear extracts of MEFs (n = 3), iPSCs (n = 3) and ESCs (n = 1). The scale bar represents the correlation height (= 1 − Abs[correlation]). c, Dual-colour heat map for expression levels of 29 proteins highly enriched in both the iPSC and ESC samples (relative to MEFs). The colour scale bar represents the spectral count. Candidate EM factors were divided into six groups for further functional testing. d, Functional screen of candidate EM factors for promotion of somatic cell reprogramming in OSKM-MEFs. EM candidates were transduced together as a single pool of 29 genes, as 6 subpools, or as individual factors from group 6 (as in c). Alkaline phosphatase-positive (AP+) iPSC colonies were counted at day 14 after transduction with OSKM. e, Diagram of time-course analyses of iPSC reprogramming. f, Gene expression time course of endogenous Parp1, Nanog and Oct4. g, Immunocytochemistry analysis of WT or Parp1−/− d4-OSKM-MEFs and d4-CONT-MEFs with an antibody against Parp1 (upper panels; red), and counterstained with Sytox nuclear marker (lower panels; green). Increased Parp1 expression in d4-OSKM-MEFs is quantified on the right (Parp1HI; defined as mean plus 2 s.d. or greater than the expression level in d4-CONT-MEFs); modified nuclear morphology apparent in d4-OSKM-MEFs is as described previously. Results in d, f and g are shown as means and s.d. for three independent experiments. Asterisk, P < 0.05; three asterisks, P < 0.001.
Figure 2
Figure 2. Parp1 activities during iPSC reprogramming
a, Functional analysis of Parp1 mutants for rescue of iPSC colony formation in Parp1−/− OSKM-MEFs. Cultures were transduced with green fluorescent protein (GFP), WT Parp1, or Parp1 mutants encoding a catalytic domain missense mutation (CAT mutant; E988K), deletion of the catalytic domain (ΔCAT), deletion of the DNA-binding and automodification domains (CAT-only), or triple missense mutation of the DNA-binding domain (DBD mutant; C21G/C125G/L139P). Zn, zinc fingers; BRCT, BRCA1 carboxy terminus. b, Schematic representation of the Nanog locus transcription start site (TSS) region. Indicated are HpaII/MspI sites (green bars) and amplicons for ‘exon 1/intron 1’ and ‘intron 1’ regions(thick greylines). bp,base pairs. c, Parp1 ChIP analyses of the cultures as indicated, presented as the relative enrichment to glyceraldehyde-3-phosphate dehydrogenase (GAPDH). d, Content of 5hmC assessed by GlucMS-qPCR (as a percentage of total cytosine). e, Content of 5mC, quantified by subtraction of 5hmC content (as in d) from the total methylated cytosine (5mC + 5hmC, as determined by HpaII digestion insensitivity; see Supplementary Fig. 3f). Results in a and c–e are shown as means and s.d. for three independent experiments. Asterisk, P < 0.05; two asterisks, P < 0.01; three asterisks, P < 0.001.
Figure 3
Figure 3. Tet2 is required for 5hmC formation at the Nanog locus
a, Immunocytochemistry of d4-OSKM-MEFs and d4-CONT-MEFs with an antibody against 5hmC (ref. 5) (upper panels; red) and counterstained with Sytox nuclear marker (lower panels; green). Representative images show increased 5hmC in d4-OSKM-MEFs, as quantified on the right (5hmCHI; defined as mean plus 2 s.d. or greater above the level in d4-CONT-MEFs). b, Time course of Tet1 and Tet2 gene expression assessed by qPCR (relative to ESC level). c, OSKM-mediated iPSC colony formation assay (AP+) in shRNA-mediated Tet2 knockdown (Tet2 KD; blue) and non-silencing control shRNA (mock KD; black)-treated MEFs. d, Diagram of the Nanog locus; regions are the same as in Fig. 2b. e, Tet2 ChIP-qPCR at the exon 1/intron 1 amplicon. f, g, Content of 5hmC in the cultures indicated, assessed by hMeDIP of the exon 1/intron 1 region (f, relative to GAPDH) or GlucMS-qPCR intron 1 amplicon (g, as a percentage of total cytosine). h, Content of 5mC at the intron 1 amplicon, quantified by subtraction of 5hmC (as in g) from the total methylated cytosine levels (as in Supplementary Fig. 4k; determined by HpaII sensitivity assay; as a percentage of total cytosine). Results in a–c and e–h are shown as means and s.d. for three independent experiments. Asterisk, P < 0.05; two asterisks, P < 0.01; three asterisks, P < 0.001.
Figure 4
Figure 4. Impact of Parp1 and Tet2 on chromatin state and Oct4 accessibility at the Nanog and Esrrb loci
a–d, H3K4me2 (a, c) and H3K27me3 (b, d) ChIP-qPCR at Nanog or Esrrb amplicons in cultures as indicated. e–h, Oct4 ChIP-qPCR. Results are shown as means and s.d. for three independent experiments. Asterisk, P < 0.05; two asterisks, P < 0.01; three asterisks, P < 0.001.
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