Cell Stem Cell

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The Nexus of Tet1 and the Pluripotency Network

Steven A. Jackson1 and Rupa Sridharan1,*1Department of Cell and Regenerative Biology, Wisconsin Institute for Discovery, Epigenetics Theme, University of Wisconsin—Madison,Madison, WI 53706, USA*Correspondence: rsridharan@discovery.wisc.eduhttp://dx.doi.org/10.1016/j.stem.2013.03.007

Resetting DNA methylation and reactivation of pluripotency genes are late events in the formation of iPSCs.Recent work by Costa et al. (2013) and Gao et al. (2013) has examined the role of Tet proteins in the hydrox-ymethylation of pluripotency genes, with the latter replacing Oct4 with Tet1 for reprogramming.

The reprogramming of somatic cells to

pluripotency, although inefficient, has

been demonstrated to occur in a stepwise

manner. Recent studies have clearly

delineated the early cell cycle changes

and transitions to an epithelial state fol-

lowed by hierarchical activation of the

pluripotency network and independence

from exogenous expression of the re-

programming factors (Buganim et al.,

2012; Golipour et al., 2012; Polo et al.,

2012). This profound change in cell iden-

tity is accompanied by several epigenetic

changes, with genome-wide resetting of

DNA methylation status being one of the

last events (Polo et al., 2012). While DNA

methylation was thought to be removed

by passivemeans—dilution upon cell divi-

sion—the recent discovery of the Ten-

Eleven Translocation (Tet1, Tet2, and

Tet3) proteins that oxidize 5-methylcyto-

sine (5mC) to 5-hydroxymethylcytosine

(5hmC) has led to the possibility of active

removal of the methyl mark (Balasubra-

mani and Rao, 2013). Two recent papers

by Costa et al. (2013) and Gao et al.

(2013) (with the latter’s work in this issue

of Cell Stem Cell) now provide a link be-

tween the activation of the pluripotency

network and changes in DNA hydroxyme-

thylation status (Figure 1).

Costa and colleagues performed high-

affinity purification of biotin-tagged

Nanog from ESCs and recovered Tet1

as a novel interactor (Costa et al., 2013).

They next derived partially reprogrammed

clonal lines, starting from either neural

stem cells (NSCs) or MEFs, that can

convert to fully reprogrammed iPSCs

upon overexpression of Nanog and the in-

hibition of the MEK and GSK pathways

(the 2i condition) (Costa et al., 2013).

This Nanog-dependent conversion to an

iPSC state was sensitive to Tet1 levels in

that it was enhanced by overexpression

and inhibited by knockdown of Tet1.

Overexpression of a catalytically mutated

form of Tet1 was also able to cause con-

version of NSC-derived intermediates to

the iPSC state with the same efficiency

as wild-type Tet1 even though global

levels of 5hmC were not elevated equally.

This effect was due to the redundant com-

pensatory function provided by Tet2,

which was upregulated upon expression

of Nanog and Tet1 (WT or mutant) and

was shown to interact with Nanog, albeit

with less affinity. The DNA-binding

domain of Tet1 was dispensable for

enhancing reprogramming from this

partially reprogrammed state, suggesting

that Nanog is the partner that drives Tet1

to the correct genomic loci. This model

was further corroborated by the authors

demonstrating the reduction of Tet1 bind-

ing at shared genomic loci, such as Oct4

and Esrrb, when Nanog was absent.

Taken together, these findings suggest

that rather than a global increase in

5hmC levels, the local effects of Nanog-

mediated Tet1 recruitment may be impor-

tant for the transition to iPSCs. Recent

studies have reported O-linked N-acetyl-

glucosamine transferase (OGT) as amajor

interacting partner of Tet1 and Tet2 in

ESCs (Balasubramani and Rao, 2013).

However, Costa et al. did not report

finding OGT peptides with their Nanog-

Tet1 complex—this suggests that there

are at least two independent complexes

that contain Tet1 in ESCs, which may

perform different functions.

Gao and colleagues focused on the role

of Tet1 in regulating Oct4 expression dur-

ing reprogramming (Gao et al., 2013).

They show that Tet1 overexpression

promoted the formation of completely re-

programmed colonies from fibroblasts,

presumably due to accelerated Oct4 tran-

scriptional activation, when reprogram-

Cell Stem Ce

ming was conducted in the presence

of the Yamanaka factors, Oct4, Sox2,

c-Myc, and Klf4 (OSKM). Remarkably

they found that Tet1 could substitute for

Oct4 in reprogramming experiments and

went on to derive a secondary inducible

system from their Tet1, Sox2, c-Myc,

and Klf4 (TSKM) iPSCs. Primary TSKM-

iPSCs exhibited all the hallmarks of

pluripotency including the ability to

produce animals from tetraploid comple-

mentation. Surprisingly, in the secondary

TSKM system, the additional overexpres-

sion of Tet1 improved reprogramming

efficiency over TSKM alone, suggesting

that the levels of Tet1, while sufficient to

replace Oct4, were still limiting for

increased reprogramming. During TSKM

reprogramming there was a transient in-

crease in global 5mC levels while there

was a linear increase in 5hmC, which

was reflected in an enrichment of both

marks at CpG islands on day 3. It is inter-

esting to note that the timing of increased

levels of 5mC and 5hmC corresponds to a

time when gene expression levels do not

change substantially in OSKM reprog-

ramming (Polo et al., 2012). This may

reflect intermediate states where the

emergence of a new cohort of methylated

and/or demethylated genes, occurring at

low probability and in the right combina-

tion, allows the reprogramming process

to proceed. A future comparison of the

dynamics in OSKM-mediated reprogram-

ming will be useful to determine whether

TSKM reprogramming events are distinct

and what the downstream genomic tar-

gets are in this context.

One interesting difference between the

results obtained by the two studies is

that Gao et al. observed that an increase

in reprogramming efficiency was not

supported by a catalytic mutant of Tet1,

whereas Costa et al. reported the

ll 12, April 4, 2013 ª2013 Elsevier Inc. 387

Figure 1. Tet1 Increases Reprogramming EfficiencyThis schematic representation summarizes recently published work by Costa et al. (2013) and Gao et al.(2013). Tet1 interacts with both Oct4 and Nanog, and a complex including Oct4, Nanog, and Tet1 mayexist. Tet1 (T) can replace Oct4 (O) in the reprogramming cocktail of Sox2 (S), c-Myc (M), and Klf4 (K),and exogenous Tet1 increases and accelerates iPSC colony formation from both OSKM and TSKM re-programming. Reprogramming intermediates derived from neural stem cells can be converted to iPSCswith Nanog and 2i at greater rates in the presence of Tet1 than without it, implying a functional role forthe Nanog-Tet1 interaction.

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opposite result (Gao et al., 2013). While

this difference may be due to a difference

in starting cell types (NSCs versus MEFs)

or stage of reprogramming (intermediate

versus somatic cell), it could also be

because Costa et al. observe an increase

in Tet2 levels (Costa et al., 2013) that was

not reproduced by Gao et al. Strikingly, a

previous report, found that Tet2, but not

Tet1, was activated early in reprogram-

ming and that a loss of Tet2 decreased re-

programming efficiency (Doege et al.,

2012). Whether these results can be ex-

plained merely by the absence of the

redundant Tet protein in the respective

experiments or if each of the Tet proteins

plays an indispensible role remains to be

determined. In support of the idea that

the Tet proteins have unique functions, a

388 Cell Stem Cell 12, April 4, 2013 ª2013 El

recent paper by Piccolo and colleagues

demonstrates that Tet1 and Tet2 have

discrete roles in erasing imprints and

cell-fusion-based reprogramming in B

cell/embryonic germ cell heterokaryons

(Piccolo et al., 2013).

Overall, both Costa et al. and Gao et al.

show that Tet1 is an important compo-

nent of the reprogramming process,

which is particularly intriguing considering

ESCs with a double knockout of Tet1 and

Tet2 maintain pluripotency and can form

viable, fertile offspring (Dawlaty et al.,

2013). Although a subset of double Tet1/

Tet2 knockout pups succumb to perinatal

lethality, it is likely there is redundancy

between Tet1, Tet2, and Tet3 (Dawlaty

et al., 2013). Further, this suggests that

Tet1 is required for the establishment of

sevier Inc.

pluripotency rather than the maintenance

of ESCs. This requirement is in contrast to

the dispensability of the de novo DNA

methyltransferases Dnmt3a and Dnmt3b

for the induction of pluripotency (Pawlak

and Jaenisch, 2011).

While these studies have provided

some links to the function of Tet proteins

in reprogramming, several exciting ques-

tions can be answered by future studies,

including the functional implications of

Nanog/Tet- mediated effects on DNA

demethylation during the transition from

a stochastic to a deterministic phase of

pluripotency gene activation (Buganim

et al., 2012; Golipour et al., 2012) and

the similarity of OSKM and TSKM

reprogramming.

REFERENCES

Balasubramani, A., and Rao, A. (2013). Mol. Cell49, 618–619.

Buganim, Y., Faddah, D.A., Cheng, A.W., Itsko-vich, E., Markoulaki, S., Ganz, K., Klemm, S.L.,van Oudenaarden, A., and Jaenisch, R. (2012).Cell 150, 1209–1222.

Costa, Y., Ding, J., Theunissen, T.W., Faiola, F.,Hore, T.A., Shliaha, P.V., Fidalgo, M., Saunders,A., Lawrence, M., Dietmann, S., et al. (2013).Nature 495, 370–374.

Dawlaty, M.M., Breiling, A., Le, T., Raddatz, G.,Barrasa, M.I., Cheng, A.W., Gao, Q., Powell,B.E., Li, Z., Xu, M., et al. (2013). Dev. Cell 24,310–323.

Doege, C.A., Inoue, K., Yamashita, T., Rhee, D.B.,Travis, S., Fujita, R., Guarnieri, P., Bhagat, G.,Vanti, W.B., Shih, A., et al. (2012). Nature 488,652–655.

Gao, Y., Chen, J., Li, K., Wu, T., Huang, B., Liu, W.,Kou, X., Zhang, Y., Huang, H., Jiang, Y., et al.(2013). Cell Stem Cell 12, this issue, 453–469.

Golipour, A., David, L., Liu, Y., Jayakumaran, G.,Hirsch, C.L., Trcka, D., and Wrana, J.L. (2012).Cell Stem Cell 11, 769–782.

Pawlak, M., and Jaenisch, R. (2011). Genes Dev.25, 1035–1040.

Piccolo, F.M., Bagci, H., Brown, K.E., Landeira, D.,Soza-Ried, J., Feytout, A., Mooijman, D., Hajkova,P., Leitch, H.G., Tada, T., et al. (2013). Mol. Cell, inpress. Published online February 28, 2013. http://dx.doi.org/10.1016/j.molcel.2013.01.032.

Polo, J.M., Anderssen, E., Walsh, R.M., Schwarz,B.A., Nefzger, C.M., Lim, S.M., Borkent, M., Apos-tolou, E., Alaei, S., Cloutier, J., et al. (2012). Cell151, 1617–1632.