A Unique Oct4 Interface Is Crucial for Reprogramming to Pluripotency

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A unique Oct4 interface is crucial for reprogramming to pluripotency

Daniel Esch1,5, Juha Vahokoski2,5, Matthew R. Groves2, Vivian Pogenberg2, Vlad Cojocaru1, Hermann vom Bruch1, Dong Han1, Hannes C. A. Drexler1, Marcos J. Araúzo-Bravo1, Calista K. L. Ng3,4, Ralf Jauch3, Matthias Wilmanns2,6 and Hans R. Schöler1,6

Terminally differentiated cells can be reprogrammed to Oct6 complex10, although this linker is shorter than the Oct1 linker pluripotency by the forced expression of Oct4, Sox2, Klf4 and and similar to the Oct4 linker (17 amino acids), suggesting that Oct6 1,2 c-Myc . However, it remains unknown how this leads to the contains an unstructured linker, just like Oct1. Although POUS and multitude of epigenetic changes observed during the POUHD have been vigorously studied, the linker has received little reprogramming process. Interestingly, Oct4 is the only factor attention, but it has been shown to be important in the maintenance that cannot be replaced by other members of the same family of pluripotency11. POU proteins bind as monomers to the so-called to induce pluripotency3–5. To understand the unique role of octamer motif of DNA or form distinct homodimers based on the Oct4 in reprogramming, we determined the structure of its pseudo-palindromic PORE and the palindromic MORE sequence mo- POU domain bound to DNA. We show that the linker between tifs. They also heterodimerize with other POU members, as well as with the two DNA-binding domains is structured as an α-helix and members of the Sox family through the HMG (DNA-binding) domain exposed to the protein’s surface, in contrast to the unstructured (SOX2HMG). Oct4 is a POU family member specific to the mammalian linker of Oct1. Point mutations in this α-helix alter or abolish germline, encoding a transcription factor that is found in oocytes and the reprogramming activity of Oct4, but do not affect its other is zygotically expressed in pluripotential cells of the pregastrulation fundamental properties. On the basis of mass spectrometry embryo12. The protein is primarily expressed in pluripotential cells and studies of the interactome of wild-type and mutant Oct4, we in the germ cell lineage, and its function has been proved to be critical propose that the linker functions as a protein–protein for both13. Most interestingly, Oct4 cannot be replaced by any other interaction interface and plays a crucial role during POU factor, as such a transcription factor cocktail does not lead to the reprogramming by recruiting key epigenetic players to Oct4 successful derivation of induced pluripotent stem (iPS) cells. target genes. Thus, we provide molecular insights to explain Here we investigated the features that make Oct4 unique within how Oct4 contributes to the reprogramming process. the POU family members and their role in the reprogramming process. We have crystallized the POU domain of Oct4 (Oct4POU) Proteins of the POU (Pit1, Oct1/Oct2, UNC-86) family contain a on the PORE DNA-binding motif complex and determined the bipartite DNA-binding domain, comprising a POU-specific domain structure of the complex at 2.8 Å resolution (Supplementary Table S1), POU 7 (POUS) with four α-helices and a POU homeodomain (POUHD) with similarly to the already known Oct1 :PORE structure . In contrast 6,7,10,14 three α-helices. These subdomains are tethered by a linker region that to previous structures , most of the POUS–POUHD connecting is not only hypervariable in both sequence and length but also exhibits linker, except for residues 87–89, is visible in our structure (Fig. 1b,c no homology to the different POU factors (Fig. 1a). Although the and Supplementary Fig. S1a–c). Although the protein–DNA contacts atomic structures of both POU subdomains of Oct1 complexed with are similar to those found in Oct1POU:DNA complexes (Supplementary DNA have been elucidated, the properties of the linker connecting the Fig. S1d)6,7, the amino-terminal part of the linker is folded as an α-helix.

POUS and POUHD remain elusive, as the linker structure has not been This additional α-helix 5 (α5) interacts with helices α2 and α4 of POUS visible in previously solved structures6–9. In addition, the linker was (Fig. 1b,c) mostly by Van der Waals interactions, except for one specific also not revealed in the recently determined crystal structure of the hydrogen bond between Tyr 25 of the POUS and Gln 81 of the linker.

1Department for Cell and Developmental Biology, Max Planck Institute for Molecular Biomedicine, Röntgenstrasse 20, Münster D-48149, Germany. 2EMBL-Hamburg, Notkestrasse 85, Hamburg D-22603, Germany. 3Laboratory for Structural Biochemistry, Genome Institute of Singapore, 60 Biopolis Street, Singapore 138672, Singapore. 4School of Biological Sciences, Nanyang Technological University, Singapore 639798, Singapore. 5These authors contributed equally to this work. 6Correspondence should be addressed to M.W. or H.R.S. (e-mail: [email protected] or ofﬁ[email protected])

Received 22 October 2012; accepted 17 December 2012; published online 3 February 2013; DOI: 10.1038/ncb2680

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a Pit1 MD SP E I RE L EQ F ANE FK VRR I K L G Y TQ TN VGE A L AA VHGS E F SQT T I CR F E N LQ LS FK NA CK L K A I L S KWL E E A E Oct1 E E PSD L E E L EQ F A KT FK QRR I K L G F TQGD VG L AMGK L YGND F SQT T I SR F E A L N LS FK NMCK L K P L L E KWLND A E Oct2 E E PSD L E E L EQ F A RT FK QRR I K L G F TQGD VG L AMGK L YGND F SQT T I SR F E A L N LS FK NMCK L K P L L E KWLND A E Oct6 E D AP S SDD L EQ F A KQ FK QRR I K L G F TQ AD VG L A L GT L YGNV F SQT T I CR F E A LQ LS FK NMCK L K P L LNKWL E E T D Oct4 DMKA L QK E L EQ F A K L LK QKR I T L G Y TQ AD VG L T L GV L FGK V F SQT T I CR F E A LQ LS L K NMCK LR P L L E KWVE E A D 1 20 30 40 50 60 70 75 α1 α2 α3 α4

Pit1 Q V G A L Y N - - E K V G A N E ------15 Oct1 N L S S D S T A S S P S A L N S P G L G A E G L N - 26 Oct2 T M S V D S S L P S P N Q L S S P S L G F D G L P G 27 Oct6 S S S G S P T N L D K I A A Q G ------17 Oct4 N N E N L Q E I C K S E T L V Q ------17 76 77 78 79 80 81 82 α5

Pit1 R K R K R R T T I S V A A K D A L E R H F G E H S K P S S Q E I MRMA E E L N L E K E V V R VWF CN R R QR E K R V K Oct1 R R R K K R T S I E T N I R V A L E K S FME NQ K P T S E D I T L I A E Q L NME K E V I R VWF CN R R QK E K R I N Oct2 R R R K K R T S I E T NV R F A L E K S F L A NQ K P T S E E I L L I A E Q L HME K E V I R VWF CN R R QK E K R I N Oct6 R K R K K R T S I E V GV KG A L E S H F L K C P K P S A H E I T G L AD S L Q L E K E V V R VWF CN R R QK E K RMT Oct4 A R K R K R T S I E N R V RWS L E TM F L K C P K P S L QQ I T H I ANQ L G L E K D V V R VWF CN R R QK GK R S S 93 100 110 120 130 152 α1 α2 α3

bc α2 α1 V71 N76 D75 L23 α1 α1 D72 E78 K69 D73 α4 α3 α3 N77 α5 α4 N79 W70 α2 Q81 Y25 L37 L80 T33 E82 α5 I83 α2 V36 α2 D29 α5 3 S86 α L32 C84 α3 α4 K85 α1 α1 V90 Q91 α2 A92

Figure 1 The crystallographic structure of the Oct4POU:PORE complex. corresponding number. (c) Magniﬁed view of the linker and its neighbouring (a) Sequence alignment between the DNA-binding domains of different protein residues. The residues with at least one atom within 7 Å of any POU proteins. Strictly conserved residues are highlighted in yellow. The atom in the linker are shown in a ball-and-stick representation coloured α-helices deﬁning the protein tertiary structure are indicated underneath according to the type of residue (lysine in dark blue; aspartic acid and the alignment coloured in green for POUS, in red for the linker and in glutamic acid in red; asparagine in dark orange; glutamine in light orange; POU blue for POUHD.(b) Schematic representation of the Oct4 homodimer cysteine, serine and threonine in pink; valine, leucine and isoleucine in POU bound to the PORE DNA. Oct4 is coloured by domain (POUS in green light blue; tyrosine and tryptophan in purple). The hydrogen bonds are and POUHD in blue). The linker is highlighted in red. Residues that were shown as black dotted lines. The residues that were not resolved in the not visible in the electron density map are inferred with a dotted line. electron density map are shown as a dotted red line. This colouring is The DNA is shown in yellow. The α-helices are labelled according to the maintained throughout this manuscript.

POU Val 36 of the POUS plays a prominent role by interfacing with the built comparative models of Oct4 bound as a homodimer or HMG carboxy-terminal part of the linker α5 (Supplementary Fig. S1b). The heterodimer with Sox2 to four distinct DNA elements and found two residues Asn 79 and Leu 80 are completely exposed to the surface in that the residues of the linker, although situated in the proximity of the linker structure and are invariant among all known Oct4 sequences the Oct4POU:Sox2HMG interaction interface, do not contribute to this but are not conserved in other members of the Oct family (Fig. 1a), interaction with the exception of Glu 82 and Lys 85, which may form suggesting a distinct functional role in Oct4. The amino acid residues transient salt bridges with complementary residues in Sox2 (Supple- of the helix point away from POUS, with the exception of Gln 81, which mentary Fig. S2). To investigate whether α5 plays an important function is located in a microenvironment comprising residues in helix α2 of in reprogramming, we designed a set of three Oct4 variants (Fig. 2b). POUS (Supplementary Fig. S1b). Interestingly, the amino acids of helix First, as Oct6 has already been shown to have no reprogramming 4 α5 are also highly conserved in other Oct4 orthologues (Fig. 2a). potential , we replaced the linker in Oct4 with the linker from Oct6. The Oct4POU:PORE structure lacks electron density between residues The other two mutants, V36K and Q81R, were designed to interfere

Cys 84 and Leu 89 (Fig. 1b,c and Supplementary Fig. S1c). To complete specifically with the POUS-linker interface in α5, and their biological our model and to visualize the linker on other DNA motifs, we function was assessed in a reprogramming assay (Fig. 2c–e).

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a Human N N E N L Q E I C K A E T L – V Q A d PhaseGFP SSEA-1 AP Mouse N N E N L Q E I C K S E T L – V Q A Xenopus N N E N L Q E I I S R G Q I I P Q V Zebrafish N S E N P Q D M Y K I E R V F V D T WT Medaka T S E N P Q D M Y K I E R V F A D T

Oct6 b 176 92 151 N-terminal C-terminal linker

TADPOUS Linker POUHD TAD V36K

WT ...LGVLF... NNENLQEICKSETLVQA Q81R Oct6 SSSGSPTNLDKIAAQGR linker ...LGVLF...... LGKLF... NNENLQEICKSETLVQA e V36K 7 days 14 days 20 days WT 34 163 200 Oct6 linker 0 0 0 Q81R ...LGVLF... NNENLREICKSETLVQA V36K 6 94 145 Q81R 0 0 0 Xenopus ...LGVLF... NNENLQEIISRGQIIPQV 400 Zebrafish ...LGVLF... NSENPQDMYKIERVFVDT 350 300 Medaka ...LGVLF... TSENPQDMYKIERVFADT 250 20 days 14 days colonies 200 + 150 7 days

c GFP 7 days 14 days 20 days 100 Mouse 34 191 218 50 Xenopus 30 179 211 Zebrafish 000 0 Medaka 000 WT Oct6 linker V36K Q81R

f 1,600 450 400 1,400 350 1,200 300 20 days 1,000 250 14 days 800 colonies

+ 200 7 days 600 150 GFP 400

100 Oct4 expression 50 200 0 0 Mouse Xenopus Zebrafish Medaka WT Oct6 linker V36K Q81R

Figure 2 The effect of mutations in the Oct4 linker on reprogramming are shown under phase contrast and GFP ﬂuorescence. In the right activity. (a) Sequence alignment of the Oct4 linker region from various columns, immunohistochemical staining of the iPS cell colonies for species. The conserved residues from the mammalian Oct4 that are SSEA-1 and AP is shown. Scale bars, 75 µm. (e) The number of also conserved in at least one other species analysed are shown in GFP-positive iPS cell colonies formed at different time points during yellow. (b) Representation of mouse Oct4 protein (WT) and different reprogramming (7, 14 and 20 days) with proteins containing different genetic modiﬁcations of the linker region. Amino acids that are linker mutations in comparison with mouse WT Oct4. The data from replaced are coloured in red. (c) The number of GFP-positive iPS one of two independent reprogramming experiments are shown (for the cell colonies formed by replacing the linker with the corresponding complete data, see Supplementary Table S2). (f) The relative transcript sequence from other species. The data from one of three independent levels of the mutations observed with the qRT–PCR experiment. The reprogramming experiments are shown (for the complete data see mean values of three replicates started from the same reprogramming Supplementary Table S2). (d) In the left columns, iPS cell colonies experiment are shown with the error bars representing the s.d.

The reprogramming efficiency of the Oct4 mutants was compared viral Oct4 variants using quantitative PCR with reverse transcription with that of the wild-type (WT) Oct4 protein in parallel fashion (see (qRT–PCR; Fig. 2f). Viral transcript levels were found to be comparable, Supplementary Methods). Using OG2 mouse embryonic fibroblast and thus they could not account for the loss-of-function phenotype. (MEF) cells that drive green fluorescent protein (GFP) expression We characterized the mutants in terms of protein turnover, protein under control of the Pou5f1 (Oct4) distal enhancer, reprogramming localization, DNA binding and transactivation potential compared efficiency was scored by counting the number of GFP-positive with WT Oct4 (Supplementary Fig. S3). The Oct4 with the linker colonies. Indeed, the Oct4 with the linker from Oct6 and the from Oct6 showed reduced DNA-binding activity to the W and PORE Q81R mutation led to a complete loss-of-function phenotype, as motifs, which is reflected by reduced transactivation activity, possibly not a single GFP-positive colony had formed within 20 days of providing an explanation for the observed loss-of-function phenotype. reprogramming (Fig. 2d,e). As viral titres play a vital role in the Interestingly, the Q81R mutation did not affect essential transcription reprogramming process, we analysed the expression levels of the factor properties of the protein.

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a 450 b 1,000 400 350 300 100 20 days 250 14 days

colonies 200 7 days + 150 10 GFP

100 Oct4 expression 50 0 1 WT 76A 77A 78A 79A 80A 81A 82A WT 76A 77A 78A 79A 80A 81A 82A c Phase Oct4 DAPI Overlay d 0 8 16 24 30 96 (h) Oct4 WT WT Tubulin

Oct4 76A 76A Tubulin

Oct4 77A 77A Tubulin

Oct4 78A 78A Tubulin

Oct4 79A 79A Tubulin

Oct4 80A 80A Tubulin

e W sequence PORE sequence

Oct1 f 7

Oct6 6 Oct4 Oct6 5 Oct4 4 3 2 1 Relative luciferase activity 0 WT 76A 77A 78A 79A 80A Neg Oct4 mutant

Probe Probe

WT 76A 77A 78A 79A 80A Neg Neg 80A 79A 78A 77A 76A WT

Figure 3 Characterization of alanine mutations on the linker segment. (a) The (DAPI). Scale bars, 50 µm. (d) The in vivo stability of the Oct4 mutants number of GFP-positive iPS cell colonies formed for the corresponding is compared with that of WT Oct4 by using cycloheximide inhibition of alanine mutations on the linker segment was calculated. The data from translation over 96 h. (e) Oct4 alanine mutations were assessed for the one of three independent reprogramming experiments are shown (for the ability to bind to the W and PORE sequences. (f) The relative transcriptional complete data, see Supplementary Table S2). (b) The relative transcript activity of WT Oct4 and Oct4 with alanine mutations was measured using levels for WT Oct4 and Oct4 with alanine mutations on the linker segment an oligomerized octamer-containing oligonucleotide as an enhancer (6W; were assessed using qRT–PCR. The mean values from three replicates ref. 25). The mean values of three independent biological replicates started started from the same reprogramming experiment are shown with the s.d. as from the same reprogramming experiment are shown with the error bars error bars. (c) Localization of the alanine mutations on the linker segments representing the s.d. Uncropped versions of the electrophoretic mobility as shown by phase contrast and Oct4 immunostaining with counterstaining images are shown in Supplementary Fig. S5.

To confirm our findings and further map the amino acids important Xenopus linker was found to be functional, proteins with either the to the reprogramming process, we replaced the mouse Oct4 linker medaka and zebrafish linker sequences did not give rise to any iPS with its Xenopus, zebrafish and medaka orthologues, and tested for cell colonies (Fig. 2c and Supplementary Fig. S4a and Table S2). This reprogramming activity. Although the chimaeric protein with the data set confirmed the importance of the linker region in the biological

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acChd4 Mta3 120 200 110 180 100 160 140 90 120 80 100 70 80 Peptide counts (%) Peptide counts (%) 60 80A WT 80A WT

Msh6 Msh2 160 140 120 140 100 120

b 80 100 60

Peptide counts (%) Peptide counts (%) 80 80A WT 80A WT

Smarca4 Smarcc1

300 140 Q81 250 L80 N77 120 N76 200 N79 150 100 E82 E78 100 80

Peptide counts (%) 50 Peptide counts (%)

80A WT 80A WT

d Acin1 Cul4b Ddb1 Hnrnpu Kpna2 Actr3 Ahnak Ankrd17 Kpna3 Msh6 Nudc Pp2r1a Psmb6 Ap2m1 Atp1a1 Atxn10 Rpa1 Ssrp1 Supt16h Xrcc6

Bzw2 Calu Cct2 Pardo

Cct3 Cct4 Cct5

Cct7 Cnn3 Cnot1 Common

Ctnnb1 Dbt Dnaja2 14 Chd4 Ctbp2 Eef2 Elavl1 Etf1 Hcfc1 Flnb Fubp3 Gcn1l1 10 Hdac1 Hdgf Hk2 Hsp1 8 Hells 46 Mta2 Hspg2 Ilf3 Lamb1 Mta3 Mcm5 Nono Numa1 Ogt Paf1 Parp1 Ppp1cb Smarca4

Ppp1cc Rfc4 Rpn1 Smarcc1

Rpn2 Serpinh1 Snd1 Berg Tcof1 Tcp1 Tpr1 Ewsr1 Hdac1 Msh2 Prmt1 Upf1 Rbm14 Ruvbl2 Set Smc1a Ding

Figure 4 Surface view of the Oct4POU:PORE complex. (a) The The whiskers correspond the minimum and maximum observed values of the solvent-accessible surfaces of the DNA-binding domains of Oct4 are percentage of peptide counts normalized to the average peptide counts in shown in green for POUS, in blue for POUHD, and in red for the linker. The the case of the WT Oct4; the box spans from the ﬁrst to the third quartile; DNA is shown in yellow ribbons. (b) A magniﬁed view of the linker surface the bold line in the box corresponds to the median. (d) Comparison of rotated by 90◦ relative to that in a. The residues mutated to alanine are our Oct4 interactome with the three published Oct4 network studies15–17. highlighted. The colour scheme is the same as in a, except for the linker Overlapping proteins in all three published data sets are listed. Candidates residues that are coloured according to the type of residue as in Fig. 1. that were under-represented in the L80A mutant and detected by all three (c) Boxplots of candidate proteins Chd4, Msh6 and Smarca4, which are studies are highlighted in red, and candidates from only one published data under-represented in the L80A pulldowns in comparison with the WT Oct4. set are highlighted in orange.

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Table 1 The intensity of the proteins that are commonly detected in all three replicates and the published interactome studies on Oct4. Mt-154 Mt-406 Mt-432 Wt-154 Wt-406 Wt-432

Chd4 18,981,917 14,797,190 1,991,505 30,508,831 19,737,231 2,628,261 Ctbp2 15,134,948 24,162,382 8,881,419 21,109,575 29,267,851 4,120,841 Hcfc1 2,872,290 7,161,581 2,483,793 4,919,465 6,742,908 1,882,718 Hdac1 23,159,511 35,204,275 33,317,271 21,012,796 26,978,942 15,619,726 Hells 6,516,943 3,330,736 1,576,757 5,214,580 2,790,398 1,006,093 Mta2 16,089,147 11,496,410 885,722 16,500,153 11,496,410 344,610 Mta3 4,249,351 8,467,608 1,143,120 2,146,365 7,731,482 1,564,548 Ogt 4,258,756 1,143,120 2,842,064 3.104,843 1,064,624 3,983,403 Smarca4 6,033,749 11,952,742 48,944 6.631,674 11,084,689 183,747 Smarcc1 20,729,606 42,283,859 2,508,807 29,998,777 33,317,271 1,740,677

function of Oct4 during reprogramming and provided a basis for a pulled down most of the interacting proteins with comparable detailed mutagenesis screen. The sequence alignment of the linker affinity (Supplementary Table S3). Within the intersection of the regions in the mouse, Xenopus, zebrafish and medaka revealed that three published data sets, only two proteins exhibited a significantly mouse and Xenopus linkers share only a few amino acids that are not reduced intensity in the mutant interactome (Fig. 4c,d, Table 1 and conserved in zebrafish and medaka (Fig. 2a). Supplementary Table S4). The first was Smarca4, a helicase of the BAF Next, we investigated whether the linker residues exposed to the remodelling complex previously shown to improve reprogramming surface of the protein may be required for the biological activity of efficiency using the iPS cell methodology18. Furthermore, Smarca4 is Oct4. We performed an alanine scan on the most exposed and highly known to interact with key epigenetic repressors and activators19, and conserved linker segment, including residues of α5 (Asn 76–Glu 82, we propose that it is recruited by Oct4 to downstream target genes Figs 1a and 2a). The Oct4 mutations Q81A and E82A showed a to enforce euchromatin/heterochromatin transitions of Oct4 target reprogramming efficiency comparable to that of the WT Oct4 (Fig. 3a). genes during reprogramming. It is worth noting that we detected In marked contrast, the point mutations N76A, N77A and N79A exclusively esBAF components and not pBAF components in our led to the formation of significantly fewer iPS cell colonies (Fig. 3a interactome study, highlighting the selectivity of this interaction and and Supplementary Fig. S4a and Table S2). The strongest effect was its importance for the induction of pluripotency. The second protein obtained with L80A, which abolished any iPS cell colony formation was Chd4, a helicase of the NuRD complex, which also is known to in the investigated time period. qRT–PCR analysis demonstrated safeguard pluripotency by maintaining the bivalent mark (H3K273m) that viral overexpression of the mutant proteins was comparable to in embryonic stem cells20. In the mutant pulldown, the amount of that of the WT protein (Fig. 3b) and thus could not account for the Chd4 was found to be significantly reduced (Fig. 4c,d). When only loss-of-function phenotype. As expression levels of the mutants and one published data set was used for filtering, Msh6 was found to be the WT protein were comparable, we examined the localization of another potential interaction candidate. Msh6 recognizes T/G DNA the mutants by immunohistochemistry (Fig. 3c), protein turnover by mismatches and could be involved in the DNA nucleotide excision cycloheximide-mediated inhibition of translation (Fig. 3d), sequence- process after deamination of methylcytosines by AID and APOBEC specific DNA-binding activity (Fig. 3e), heterodimer formation with (refs 21,22). However, Msh6 −/− embryonic stem cells have been Sox2 by electrophoretic mobility shift assay (EMSA) and modelling shown to give rise to viable offspring. In contrast to the quadruple (Supplementary Fig. S2), and transactivation potential by the luciferase mutant (76A, 77A, 79A, 80A), the L80A mutant exhibited only a assay (Fig. 3f). We could not find any significant differences in partial loss-of-function phenotype in the rescue experiments with comparison to the WT protein. Examination of our models revealed TC4 cells23 (Supplementary Fig. S4b). This observation may explain that these amino acids are entirely exposed to the surface of the protein why only a mild reduction in intensity was observed. The rescue (Fig. 4a,b). Thus, we deduced that mutating the L80A residue could experiment also confirms that the integrity of the linker and the potentially disturb an interaction surface with yet unknown additional identified amino acids are important not only for reprogramming, factors. As the Oct4 L80A mutant led to complete loss-of-function in but also to maintain the pluripotent state. reprogramming, we used this variant for further investigations. Here we show that a unique property of the linker distinguishes Oct4 To elucidate the molecular mechanism underlying the biological from other members of the POU family and that the integrity of the function of this linker residue, we designed strep-tagged WT Oct4 and linker is essential for successful reprogramming. Our data also strongly L80A Oct4 constructs. Nuclear extracts were prepared in triplicates, indicate that the linker plays an important role in reprogramming by and protein complexes were analysed by mass spectrometry to enable recruiting key epigenetic players to sites occupied by Oct4, suggesting label-free quantification of the interacting proteins. that Oct4 may serve as a recruiting platform during the epigenetic The role of the Oct4 interactome in embryonic stem cells has transition from a differentiated to a pluripotent cell state. A more been assessed by three independent studies15–17, which have described detailed analysis of the orchestration of the series of events mediating numerous transcription factors and major remodelling complexes this transition and the interaction of the epigenetic players with to be direct interaction partners of Oct4. Here we compared the the linker interface will provide further insights into the molecular interactome of the L80A mutant with that of the WT protein by processes underlying reprogramming and may prompt future label-free mass spectrometry in a quantitative manner. The mass studies to direct induced transdifferentiation towards different cell spectrometry analysis revealed that the WT and L80A constructs lineages24.

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METHODS 6. Klemm, J. D., Rould, M. A., Aurora, R., Herr, W. & Pabo, C. O. Crystal structure of the Oct-1 POU domain bound to an octamer site: DNA recognition with tethered Methods and any associated references are available in the online DNA-binding modules. Cell 77, 21–32 (1994). version of the paper. 7. Reményi, A. et al. Differential dimer activities of the transcription factor Oct-1 by DNA-induced interface swapping. Mol. Cell 8, 569–580 (2001). Note: Supplementary Information is available in the online version of the paper 8. Reményi, A. et al. Crystal structure of a POU/HMG/DNA ternary complex suggests differential assembly of Oct4 and Sox2 on two enhancers. Genes Dev. 17, 2048–2059 (2003). ACKNOWLEDGEMENTS 9. Williams, D. C. Jr, Cai, M. & Clore, G. M. Molecular basis for synergistic X-ray diffraction experiments were performed on the ID23-1 beamline at the transcriptional activation by Oct1 and Sox2 revealed from the solution structure European Synchrotron Radiation Facility (ESRF), Grenoble, and X12 and X13 of the 42-kDa Oct1.Sox2.Hoxb1–DNA ternary transcription factor complex. J. Biol. at EMBL Hamburg, DESY. We are grateful to E. Fioravanti of the ESRF for Chem. 279, 1449–1457 (2004). providing assistance in using beamline ID23-1. We thank G. Bourenkov for 10. Jauch, R., Choo, S. H., Ng, C. K. & Kolatkar, P. R. Crystal structure of the dimeric invaluable assistance in crystallographic problem resolution and data collection. Oct6 (POU3f1) POU domain bound to palindromic MORE DNA. Proteins 79, We thank A. Nolte for help with mass spectrometry measurements. We also 674–677 (2011). thank R. Grindberg for critically reading, and A. Malapetsas and J. Bruder for 11. Nishimoto, M. et al. Oct-3/4 maintains the proliferative embryonic stem cell state editing the manuscript. We acknowledge the financial support from DFG (SPP1356 via specific binding to a variant octamer sequence in the regulatory region of the Pluripotency and Cellular Reprogramming priority program) AJ DFG SI 1695/1-2 UTF1 locus. Mol. Cell Biol. 25, 5084–5094 (2005). and AJ DFG CO 975/1. 12. Pesce, M., Gross, M. K. & Schöler, H. R. In line with our ancestors: Oct-4 and the mammalian germ. Bioessays 20, 722–732 (1998). 13. Kehler, J. et al. Oct4 is required for primordial germ cell survival. EMBO Rep. 5, AUTHOR CONTRIBUTIONS 1078–1083 (2004). D.E. and J.V. performed the experiments and wrote the manuscript; J.V., M.R.G. 14. Klemm, J. D. & Pabo, C. O. Oct-1 POU domain-DNA interactions: cooperative and V.P. collected and analysed the crystallographic data; V.C. performed the binding of isolated subdomains and effects of covalent linkage. Genes Dev. 10, comparative modelling experiments and wrote the manuscript; H.v.B. performed 27–36 (1996). the cycloheximide experiments; H.C.A.D. performed the MS experiments; M.J.A-B. 15. Van den Berg, D. L. et al. An Oct4-centered protein interaction network in embryonic analysed the proteomic data sets; D.H. performed the rescue experiments; and stem cells. Cell Stem. Cell 6, 369–381 (2010). C.N. assessed the heterodimer formation ability of Oct4 and Sox2. All authors 16. Pardo, M. et al. An expanded Oct4 interaction network: implications for stem cell commented on the manuscript; and R.J., M.W. and H.R.S. as co-senior authors biology, development, and disease. Cell Stem Cell 6, 382–395 (2010). supervised the project and assisted in writing the manuscript. 17. Ding, J., Xu, H., Faiola, F., Ma’ayan, A. & Wang, J. Oct4 links multiple epigenetic pathways to the pluripotency network. Cell Res. 22, 155–167 (2012). COMPETING FINANCIAL INTERESTS 18. Singhal, N. et al. Chromatin-remodeling components of the BAF complex facilitate reprogramming. Cell 141, 943–955 (2010). The authors declare no competing financial interests. 19. Trotter, K. W. & Archer, T. K. The BRG1 transcriptional coregulator. Nucl. Recept. Signal. 6, e004 (2008). Published online at www.nature.com/doifinder/10.1038/ncb2680 20. Hu, G. & Wade, P. A. NuRD and pluripotency: a complex balancing act. Cell Stem Reprints and permissions information is available online at www.nature.com/reprints Cell 10, 497–503 (2012). 21. Rai, K. et al. DNA demethylation in zebrafish involves the coupling of a deaminase, 1. Takahashi, K. & Yamanaka, S. Induction of pluripotent stem cells from a glycosylase, and gadd45. Cell 135, 1201–1212 (2008). mouse embryonic and adult fibroblast cultures by defined factors. Cell 126, 22. Bhutani, N. et al. Reprogramming towards pluripotency requires AID-dependent DNA 663–676 (2006). demethylation. Nature 463, 1042–1047 (2010). 2. Yu, J. et al. Induced pluripotent stem cell lines derived from human somatic cells. 23. Niwa, H., Miyazaki, J. & Smith, A. G. Quantitative expression of Oct-3/4 defines Science 318, 1917–1920 (2007). differentiation, dedifferentiation or self-renewal of ES cells. Nat. Genet. 24, 3. Shi, Y. et al. Induction of pluripotent stem cells from mouse embryonic fibroblasts by 372–376 (2000). Oct4 and Klf4 with small-molecule compounds. Cell Stem Cell 3, 568–574 (2008). 24. Adachi, K. & Schöler, H. R. Directing reprogramming to pluripotency by transcription 4. Nakagawa, M. et al. Generation of induced pluripotent stem cells without Myc from factors. Curr. Opin. Genet. Dev. 22, 416–422 (2012). mouse and human fibroblasts. Nat. Biotechnol. 26, 101–106 (2008). 25. Schöler, H. R., Balling, R., Hatzopoulos, A. K., Suzuki, N. & Gruss, P. Octamer 5. Feng, B. et al. Reprogramming of fibroblasts into induced pluripotent stem cells with binding proteins confer transcriptional activity in early mouse embryogenesis. EMBO orphan nuclear receptor Esrrb. Nat. Cell Biol. 11, 197–203 (2009). J. 8, 2551–2557 (1989).

METHODS DOI: 10.1038/ncb2680

METHODS amino acids (NEAA; PAA) and 1×β-mercaptoethanol (Invitrogen). OG2 MEFs Purification and assembly of the Oct4POU:PORE complex. The POU domain were isolated as described previously1. Mouse embryonic stem cell and iPS cell of mouse Oct4 (Oct4POU) was cloned previously7 (mouse Pou5f1 residues cultures were maintained in DMEM containing 15% knockout serum replacement 131–282) and overexpressed in the bacterial strain BL21 (DE3) RIL (Novagen) (Invitrogen), 5% FBS, 1× PSG, 1× NEAA, and 1 × β-mercaptoethanol, with in ZYM-5052 at 20 ◦C. The pellet was resuspended (50 mM HEPES at pH 7.5, 1000 units of leukaemia inhibitory factor (LIF) on feeder layers of gamma- 250 mM NaCl and 0.01% thioglycerol), lysed by sonication and centrifuged irradiated MEFs or 2,000 units of LIF under feeder-free conditions, as previously (18,000g). The remaining pellet was resuspended (50 mM HEPES at pH 7.5, described35. 6 M Gu–HCl and 20 mM imidazole at pH 8.0), and the denatured fraction was centrifuged (18,000g). The Oct4POU protein was purified using immobilized Virus production. HEK293T cells were seeded (density of 2.2 × 106 cells per metal affinity chromatogram (Ni–NTA, QIAGEN) and eluted (50 mM HEPES 100-mm dish). The following day, cells were transfected with pMX-based retroviral at pH 7.5, 6 M Gu–HCl and 300 mM imidazole at pH 8.0). The Oct4POU:PORE vectors using Fugene 6 (Roche). Cells were incubated overnight at 37 ◦C with 5% 0 0 (5 -TCACATTTGAAAGGCAAATGGA-3 ; ref. 7) complex was formed and refolded CO2. The medium was replaced 24 h after transfection with 6 ml fresh medium. in a one-step process, dialysing all components (15 mg Oct4POU, 1 mg TEV protease The virus-containing supernatant was collected and filtered (0.45 µm, Millex-HV, and 200 nmol DNA in 5–8 ml total volume) in buffer (50 mM HEPES at pH 7.5, Millipore) 48 h after infection. The supernatant was supplemented with 6 µg ml−1 150 mM NaCl and 5 mM dithiothreitol) at 4 ◦C overnight and purifying by size protamine sulphate (Sigma-Aldrich) before infection. Viral stocks were generated exclusion chromatography (16/60 Superdex 75 column, GE Healthcare; 50 mM simultaneously to ensure equivalent virus production among different experiments. HEPES at pH 7.5, 150 mM NaCl and 1 mM dithiothreitol). Fractions containing the complex (verified by EMSA and SDS–PAGE) were pooled and concentrated Reprogramming and rescue assay. MEFs were seeded in a gelatinized 6-well plate by ultrafiltration after incubation for 15–30 min at room temperature (relative (density 5 ×104 cells per well). Simultaneously, viral supernatant was added on the molecular mass cutoff 10,000; Millipore). OG2 MEF cells. Viral expression was analysed by qRT–PCR. The supernatant was removed and cells were washed twice with PBS 24 h after infection. The medium Crystallization and structure determination. The Oct4POU:PORE complex was was replaced with embryonic stem cell medium (high-glucose DMEM with LIF) crystallized by vapour diffusion in the presence of 320 mM Na/K phosphate (pH 72 h after infection. Cells were maintained in culture until GFP-positive colonies 5.7), using the EMBL Hamburg HTP screening facility26. The crystal was collected appeared. The number of colonies obtained with the WT and with each mutant one day after setting up the drops and transferred into a solution with glycerol, Oct4 were compared after 7, 14 and 20 days. For the rescue assay, the proteins were which was added stepwise to final concentrations of 8, 20 and 38%. An X-ray overexpressed in TC4 cells. The morphology of the colonies was assessed after 96 h data set covering 90◦ was collected at beamline ID23-1 at the ESRF Grenoble. The of doxycycline treatment23. data were integrated and reduced with XDS and scaled using XSCALE (ref. 27). The P41 space group with a (h,−k,−l) twinning operator (twinning fraction 0.29) Strep-tagged purification of proteins. SF lentivirus encoding strep-tagged WT was determined after analysing the reflections using PHENIX.XTRIAGE (ref. 28). Oct4 and the mutants was produced in HEK293T cells by co-transfection with The structure was solved by molecular replacement using PHASER (ref. 29) with pCL-Eco. SF lentivirus tomato was used as a negative control. The virus-containing the Oct1POU:PORE complex (1HF0) as the template7. One dimeric Oct4POU:DNA supernatant was filtered. OG2 embryonic stem cells were infected. Cells (50 × 106) complex per asymmetric unit was found. The refinement was performed stepwise were collected, and extracts were applied to Strep-Tag/Strep-Tactin affinity columns. in REFMAC 5.6 (ref. 30) using restrained non-crystallography symmetry operators Pulldown quality was assessed by western blot analysis for Oct4 using SC-9081 and manual model building in COOT (ref. 31). Ramachandran plots were calculated antibody (Santa Cruz). using MOLPROBITY (ref. 32; 93.7% of residues were in the most favoured regions, and 5.2% in the allowed regions). Structure determination details are given in Mass spectrometry. Three independent experiments were analysed by mass Supplementary Table S1. spectrometry (MS) for label-free quantification. Proteins from the strep-tagged pulldown were processed for liquid chromatography (LC)–MS/MS analysis with the EMSA. Proteins were overexpressed in BL21(DE3) bacteria and purified using filter-aided sample preparation method as described previously36. Proteins eluted immobilized metal affinity chromatography followed by ion-exchange chromatog- from the Strep-Tactin beads with desthiobiotin were reduced by dithiothreitol raphy and gel filtration. Whole-cell extracts were prepared as described previously33. (0.1 M; 45 min at 56 ◦C) followed by alkylation for 20 min in the dark at room Cells (2.2×106) were scraped in lysis buffer (20 mM HEPES, 150 mM NaCl, 0.2 mM temperature using 55 mM iodoacetamide. After exchanging the urea buffer to 32 EDTA at pH 8.0 and 25% glycerol). DNA fragments were labelled with [γ - P] ATP 50 mM NH4HCO3, proteins were digested with trypsin (2.5 µg per sample) by using polynucleotide kinase. The protein sequences are listed in Supplementary incubation overnight at 37 ◦C. Peptides were collected by centrifugation, desalted Table S5, DNA sequences in Supplementary Table S6 and primers in Supplementary using STAGE-Tips37 and analysed by LC-MS/MS with an Easy-nLC nanoflow system Table S7. (Proxeon Biosystems) that was online-coupled to an LTQ Orbitrap Velos mass spectrometer (Thermo Scientific) through a nanoelectrospray source (Proxeon Luciferase assay. The transactivation potential was tested with the Dual-Glo Biosystems) and a fused-silica capillary emitter column filled with C18 reversed- Luciferase Assay System (Promega) by measuring luciferase activity after 48 h. phase material (Dr.Maisch, ReproSil-Pur 120, C18-AQ, 3 µm). Peptides were loaded HEK293T cells (4 × 105) were transfected with 100 ng of effector DNA, 100 ng at the maximum flow rate at 200 bar and separated at 250 nl min−1 running a linear of pTK-RL (Renilla luciferase) and 800 ng of reporter constructs (6W 37tk-Luc; gradient from 7 to 35% Buffer B in 90 min, 35–60% Buffer B in 20 min and 60–98% refs 25,34). Buffer B in 6 min (Buffer A: 0.5% acetic acid; Buffer B: 80% acetonitrile and 0.5% acetic acid). qRT–PCR analysis. Total RNA was extracted using the RNeasy Mini Kit The mass spectrometer was operated in the positive-ion mode (spray voltage (QIAGEN). Complementary DNAs were synthesized with the High Capacity cDNA 2.2 kV, heated capillary maintained at 225 ◦C) using Xcalibur software, automati- Archive Kit (Applied Biosystems). Transcript levels were determined using the ABI cally switching to a data-dependent mode between survey scans in the mass range PRISM Sequence Detection System 7900. Gene expression was normalized to the of m/z 300–1650 and MS/MS acquisition. Collision-induced MS/MS spectra from housekeeping gene Hprt1. the 15 most intense ion peaks in the MS were collected (target value of the Orbitrap survey scan was 1,000,000, resolution 60,000, and the lock mass 445.12). Precursor Localization of Oct4. NIH3T3 cells were infected with pMX virus and collected ion charge state screening was enabled, excluding unassigned charge states as well after 24 h. Cells were fixed in 4% paraformaldehyde (PFA) for 10 min and quenched as singly charged species. Dynamic exclusion was activated, allowing maximum 500 with 50 mM glycine in PBS. The cell membrane was permeated with Triton X-100. entries and a retention period of 180 s. Nuclear localization was probed with Oct4 antibody (Santa Cruz, sc-8628, 1:200). Raw data were processed with MaxQuant software (v. 1.0.13.13) and the Mascot Nuclei were counterstained with Hoechst (Sigma-Aldrich H2261). database. Data were searched against the International Protein Index sequence database (mouse IPI, v. 3.60) concatenated with reversed-sequence versions of all SSEA-1 and AP staining. Cells were fixed with 4% PFA for stage-specific entries. The parameters were: minimum length of 6 amino acids, maximum of 2 embryonic antigen 1 (SSEA-1) staining with SSEA-1 antibody (Developmental missed cleavages, fixed carbamidomethylation of cysteines, variable oxidation of Studies Hybridoma Bank, MC-480, 1:50). Naphtol and fast red were used for alkaline methionines and acetylation at the N termini. Maximum allowed mass deviation phosphatase (AP) staining (15 min in dark). was 7 ppm for MS and 0.5 Da for MS/MS scans. Proteins were considered identified if there were at least two matching peptides, with one unique to the protein. Cell culture. OG2 MEF, HEK293T, HEK293 and NIH3T3 cells were maintained The false discovery rate (FDR) was 1% for both the peptide and the protein in low-glucose DMEM containing 10% FBS (Biowest), 1× PSG, 1× non-essential identifications.

DOI: 10.1038/ncb2680 METHODS

Modelling Oct4–DNA complexes. We built 100 comparative models in MOD- 28. Adams, P. D. et al. PHENIX: a comprehensive Python-based system for ELLER (ref. 38) for each of the four Oct4POU:Oct4POU:DNA or Oct4POU: macromolecular structure solution. Acta Crystallogr. D 66, 213–221 (2010). Sox2HMG:DNA complexes formed on the following DNA elements: PORE 29. McCoy, A. J. et al. Phaser crystallographic software. J. Appl. Crystallogr. 40, (templates: Oct4POU:PORE (3L1P), Oct1POU:PORE (1HFO; ref. 7), Oct1POU: 658–674 (2007). Sox2HMG:FGF4 (1GT0; ref. 8)); MORE (templates: Oct1POU:MORE (1E3O; ref. 7), 30. Murshudov, G. N., Vagin, A. A. & Dodson, E. J. Refinement of macromolecular Oct6POU:MORE (2XSD; ref. 10), 3L1P); FGF4 (templates: 3L1P, 1GT0 and Oct1POU: structures by the maximum-likelihood method. Acta Crystallogr. D 53, 240–255 (1997). Sox2HMG:HOXB1 (1O4X; ref. 9)); and HOXB1 (templates: 3L1P, 1O4X, 1GT0). 31. Emsley, P., Lohkamp, B., Scott, W. G. & Cowtan, K. Features and development of We refined the loop between residues Cys 84 and Leu 89 and generated 25 loop Coot. Acta Crystallogr. D 66, 486–501 (2010). 38,39 models for each model. The refinement procedure included energy minimiza- 32. Chen, V. B. et al. MolProbity: all-atom structure validation for macromolecular tion and molecular dynamics simulations. We selected the 25 best-scoring models crystallography. Acta Crystallogr. D 66, 12–21 (2010). (using the normalized DOPE score) and clustered them according to the root mean 33. Sauter, P. & Matthias, P. Coactivator OBF-1 makes selective contacts with both the square deviation of the linker. POU-specific domain and the POU homeodomain and acts as a molecular clamp on DNA. Mol. Cell Biol. 18, 7397–7409 (1998). POU Primary accession codes. Protein Data Bank (the Oct4 :PORE structure): 34. Tomilin, A. et al. Synergism with the coactivator OBF-1 (OCA-B, BOB-1) is mediated 3L1P. by a specific POU dimer configuration. Cell 103, 853–864 (2000). Proteome Commons Database (the WT and mutant Oct4 interactome data): 35. Kim, J. B. et al. Pluripotent stem cells induced from adult neural stem cells by T7mU/n8vauq1d4M3P8ZEnECJx7AV3CqyDygo7RXFfAmRAC/KV6Xf3JZYU04F reprogramming with two factors. Nature 454, 646–650 (2008). n2NlBngbErGvevwXkY6eNvv8tnVmEJkAAAAAAAAglw== 36. Wisniewski, J. R., Zougman, A., Nagaraj, N. & Mann, M. Universal sample preparation method for proteome analysis. Nat. Methods 6, 359–362 (2009). Reference accession codes. Uniprot (Swissprot): P20263 (mouse Pou5f1 se- 37. Rappsilber, J., Ishihama, Y. & Mann, M. Stop and go extraction tips for quence). matrix-assisted laser desorption/ionization, nanoelectrospray, and LC/MS sample pretreatment in proteomics. Anal. Chem. 75, 663–670 (2003). 26. Mueller-Dieckmann, J. The open-access high-throughput crystallization facility at 38. Sali, A. & Blundell, T. L. Comparative protein modelling by satisfaction of spatial EMBL Hamburg. Acta Crystallogr. D 62, 1446–1452 (2006). restraints. J. Mol. Biol. 234, 779–815 (1993). 27. Kabsch, W. Evaluation of single-crystal X-ray diffraction data from a position- 39. Fiser, A., Do, R. K. & Sali, A. Modeling of loops in protein structures. Protein Sci. 9, sensitive detector. J. Appl. Crystallogr. 21, 916–924 (1988). 1753–1773 (2000).

SUPPLEMENTARY INFORMATION Esch et al. - A unique Oct4 interface is crucial for reprogramming to pluripotency

DOI: 10.1038/ncb2680Supplementary Figure 1

Figure S1 Details of the Oct4POU:PORE complex structure (see Oct4POU protomers in two different orientations. The type of map is 2Fo-Fc complementary information in Figure 1). (a) Superposition of the and the contour level has been set to 1.0 . (d) Interactions between Oct4POU Oct4POU:PORE and Oct1POU:PORE complex structures. Oct4POU and 4and the PORE motif. In the figure, a NUCPLOT diagram of the crystal the DNA are coloured as in main Figure 1. Oct1POU is shown in grey. structure of Oct4POU:PORE is presented. After the interacting residue, the The missing residues in the linkers of Oct1POU and Oct4POU are inferred corresponding chain identifier is indicated (A or B). The amino and hydroxy with dotted lines (grey for Oct1POU; red for Oct4POU). (b) Surface view groups forming a hydrogen bond are shown in blue and red upper case highlighting the cavity of the POUS domain in which Q81 is docked. (c) letters, respectively. Residues in the POUS domain surrounding this cavity Stereo images of the the electron density map of residues 70 to 86 in both are labelled.

Esch et al. - A unique Oct4 interface is crucial for reprogrammingSUPPLEMENTARY to pluripotency INFORMATION

Supplementary Figure 2

5 Figure S2 Models of Oct4POU bound to four DNA elements. Each panel Oct4POU:Sox2HMG:FGF4 ternary complex. (e) Model in which K85 and E87 shows a view of a complete model and a zoomed-in surface view of the of the Oct4 linker establish additional interaction with residues from the linker region. The colouring is as in Figure 1. The Sox2HMG is shown in C-terminal tail of Sox2HMG. (f) Model in which K85 forms a hydrogen bond pink. (a), (b) Oct4POU:PORE complex (homodimer). (a) Model in which with D29 of the POUS domain, while E87 is exposed to the surface. (g), the residues E87, T88, and L89, missing in the crystal structure, are (h) Oct4POU:Sox2HMG:HOXB1 (UTF1-like) ternary complex. (g) Model with exposed to the surface. (b) Model in which K85 is oriented towards the highlighted residues oriented as in (e). (h) Model with the highlighted POU POUS, establishing a salt bridge with D29. (c), (d) Oct4 :MORE residues oriented as in (f). (i) Electrophoretic mobility shift assay (EMSA) complex (homodimer). (c) Model in which E87, T88, V90 are exposed using the purified Oct4POU and Sox2HMG on the Nanog probe. The L80A to the surface. (d) Model in which E87 is oriented toward the interface mutant is able to form a heterodimer with Sox2, comparable to the WT between the POUS and the N-terminal tail of the POUHD domain. (e), (f) Oct4:Sox2 heterodimer.

SUPPLEMENTARYEsch et al. - A unique INFORMATION Oct4 interface is crucial for reprogramming to pluripotency

Supplementary Figure 3

Figure S3 Characterization of the initial mutations of the linker segment proximal octamer enhancer (6W) sequence, by using bacterially expressed (see complementary information in Figure 2). (a) The cellular distribution purified protein and crude cellular extracts from 293T cells. (d) The of the mutant proteins is analyzed by phase contrast (Phase) and Oct4 relative transcriptional activity of WT Oct4 was compared with that of immunostainings (Oct4) with DAPI counterstaining (DAPI). Scale bar is the mutants by the luciferase assay using 6W. The mean values of three 50 μm (b) The in vivo stability of Oct4 mutants is compared with that of replicates started from different cell extracts from the same reprogramming WT Oct4 by using cycloheximide inhibition of translation over 96 hours. experiment are shown with the error bars representing the standard (c) Oct4 alanine mutations were tested for the ability to bind to the deviation.

SUPPLEMENTARY INFORMATION Esch et al. - A unique Oct4 interface is crucial for reprogramming to pluripotency

Supplementary Figure 4

7 Figure S4 Characterization of the Oct4 mutants from the alanine scan and cells were performed as described23. In contrast to the reprogramming the Oct4 orthologues (see complementary information in Figures 2 and 3) experiments, here the L80A mutant had a rescue index of 0.2, while (a) Fluorescence stereolupe images of (green fluorescent protein-positive the quadrupole mutant showed a complete loss-of-function phenotype. [GFP+] and stagespecific embryonic antigen 1–positive [SSEA-1+]) colonies Scale bar is 100 μm. The mean values of three independent experiments during reprogramming for proteins with orthologue linker exchanges and (biological replicates) are shown with the error bars representing the from the alanine scan. Scale bar is 5 mm (b) Rescue experiments with TC4 standard deviation.

Esch et al. - A unique Oct4 interface is crucial for reprogramming to pluripotency

SUPPLEMENTARYSupplementary INFORMATION Figure 5

Figure S5 Uncropped electrophoretic mobility images showed in Figures 2 and 3. 8