Activation of Wnt signaling increases expression of pluripotent factors, nanog
. It can also activate expression of mesodermal differentiation factors, including brachyury
17, 20, 25
. Although Wnt signaling in ESCs directs two opposite outputs, enhancement of self-renewal and differentiation, Wnt-responsive differentiation factors should be transcriptionally repressed to maintain pluripotency. Here, we report Smek as a specific transcriptional repressor for ESC differentiation. We show that Smek is required for repression of brachyury
gene expression, a DNA-binding transcription factor that is a direct target of Wnt signaling and is required for mesoderm formation. This repression occurs when the Smek/PP4c complex binds HDAC1. Deficiency of either Smek or PP4c displaces HDAC1 from the brachyury
promoter and increases its transcription in ESCs. Furthermore, loss of pluripotency in either Smek- or PP4c-deficient ESCs suggests that the PP4 complex has an important role in maintaining pluripotent ESCs.
Nuclear β-catenin converts the Tcf/Lef-repressive complex into a transcriptional active form by releasing HDAC1 from this complex 6, 26
. However, transcription of the brachyury
gene is silent despite the presence of nuclear β-catenin in ESCs 27, 28, 29
. We show that overexpression of Smek inhibited Tcf/Lef-mediated transcription induced by β-catenin. Smek and HDAC1 simultaneously occupied the Tcf/Lef binding motif. The increase of β-catenin in ESCs does not affect the occupancy of Smek and HDAC1 at the brachyury
promoter. These data indicate that by recruiting HDAC1, Smek inhibits the transcription activity of Tcf/Lef. This represents a new mechanism by which ESCs restrain the potential of Wnt target genes to promote differentiation despite the presence of nuclear β-catenin.
Tcf/Lef proteins interact with HDAC1 through the co-repressor Groucho/TLE to repress transcription 5
. However, the interaction between Groucho/TLE and Tcf/Lef family members is restricted. For example, human Tcf1 and X. laevis
Tcf3 bind to Groucho/TLE, but not human Lef1, mouse Tcf3, or mouse Tcf4 30
. This indicates that Tcf/Lef proteins may interact directly with HDAC1, or require interaction with other conserved co-repressors. Smek proteins may mediate the interaction of HDAC1 to Tcf/Lef family proteins. Although we were not able to examine binding of Smek and Tcf/Lef proteins at the endogenous protein level due to lack of antibodies of immunoprecipitation quality, we were able to demonstrate that both Smek1 and Smek2 bind to Lef1, Tcf1, Tcf3, and Tcf4 when overexpressed in ESCs (data not shown).
We show that depletion of Smek leads to loss of ESC pluripotency, which occurred due to induction of differentiation rather than by regulation 31
of pluripotency factors. The pluripotency factors Oct4, Sox2, and Nanog, which promote self-renewal, are well-characterized in ESCs. Although they can occupy the promoters of a large set of development-related genes to repress their transcription in the ESC state, they play a dominant role in activating transcription of pluripotency maintenance genes 32
. In addition, depletion of Smek
did not cause increase of Oct4, Sox2, and Nanog expression. This indicates that the Smek repression complex does not directly regulate expression of pluripotency factors, but rather selectively directs expression of genes important for differentiation. Given that knockdown of Smek
induced expression of early mesodermal markers, such as brachyury
, but not endoderm and ectoderm markers, role of Smek in ESCs is to maintain pluripotency by assisting the inhibition of mesoderm commitment.
Although treatment of ESCs with Wnt proteins can disrupt Smek-mediated transcriptional repressive complex and induce brachyury
expression, it is not sufficient to stimulate mesoderm differentiation. Activation of Wnt signaling increases expression of nanog
as well as brachyury 23, 24
. The increase of pluripotency factors, thus, may favor ESC self-renewal rather than differentiation into mesoderm. On the other hand, sustained Wnt-signaling activation can induce differentiation of ESC. A recent study showed that long-term treatment of ESCs with Wnt3a protein in the presence of LIF induces differentiation into bipotent mesendodermal cells that produce mesoderm 33
. Similarly to this, we show that depletion of Smek proteins induces mesoderm differentiation. Therfore, it is likely that stable expression of shRNA for Smek1
leads to sustained activation of Wnt/β-catenin signaling.
In conclusion, our study has revealed a function of PP4 in transcriptional repression that contributes to silencing of key development-related genes in pluripotent ESCs. Such a function represents a transcriptional regulatory mechanism for key Wnt-responsive development-related genes in ESCs that, to our knowledge, has not been described before. We demonstrated here that expression of the Wnt target gene brachyury
in the ESC state can be completely suppressed by the Smek/PP4c complex recruiting HDAC1 at the promoter of this gene, and that this complex is critical for the inhibition of early mesoderm differentiation gene expression. Furthermore, we suggest a specific function for the Smek/PP4c phosphatase complex in chromatin remodeling. Depletion of HDAC1 simultaneously increases histone H3 phosphorylation and deacetylation, stimulating the change of chromatin structure 22
. Consistent with this, loss of the Smek/PP4c phosphatase complex induces histone H3 phosphorylation as well as deacetylation 12
. Thus, Wnt signaling could displace HDAC1 from inactive chromatin through disruption of the Smek complex during differentiation, opening up the chromatin structure at the brachyury
gene locus for initiation of transcription 34
, and subsequently β-catenin translocated into nucleus could enhance or maintain the transcription.