Dorsal specification by β-catenin is temporally uncoupled from the onset of target gene expression
The maternal Wnt/β-catenin pathway in Xenopus
(and zebrafish) specifies dorsal cell fates before the MBT under conditions of global transcriptional repression. Two classes of dorsal genes are expressed in response to maternal β-catenin (Yang et al., 2002b
): genes such as siamois
are expressed at the MBT (), whereas genes exemplified by xnr5
are transcribed as early as the 256-cell stage, bypassing preMBT global transcriptional repression ().
β-catenin target genes are poised for expression before the MBT
Maternal β-catenin is required to activate the dorsal gene expression program (Heasman et al., 2000
), but it is not yet clear which β-catenin targets are required for dorsal development. Siamois
induces complete secondary axes and rescues dorsal development in ventralized embryos (Lemaire et al., 1995
), whereas combined loss of siamois
and the closely related gene twin
blocks dorsal development (Ishibashi et al., 2008
; Laurent et al., 1997
), indicating that siamois
play essential, instructive roles in dorsal induction. However, these experiments leave open the possibility that dorsal development depends on additional targets of maternal Wnt/β-catenin signaling. To test whether siamois
expression is sufficient for dorsal development in β-catenin-deficient embryos, β-catenin was depleted by injection of a morpholino oligonucleotide (βMO), which blocked dorsal development (, panel ii
), as described (Heasman et al., 2000
); expression of siamois
in these embryos rescued dorsal development to the same extent as β-catenin itself (, panels iii
). Therefore, establishment of zygotic siamois
expression represents an essential patterning event driven by the maternal Wnt/β-catenin pathway.
It is paradoxical, then, to consider how a transcription factor such as β-catenin functions during a period of global transcriptional repression. One possibility is that, while β-catenin is present throughout cleavage stages, it only functions at the MBT to activate target genes such as siamois
. This seems unlikely, as β-catenin is required for preMBT transcription of xnr5
and dorsal specification by β-catenin is complete by the 32-cell stage (Yang et al., 2002b
). However, those experiments did not directly address the requirement for early β-catenin function in the induction of siamois
at the MBT. We therefore inhibited Wnt pathway activity at discrete times before the MBT using a dexamethasone-inducible form of Tcf3 that is unable to bind β-catenin (ΔNTcf3-GR) and thereby inhibits endogenous β-catenin/Tcf3 transactivation. While inhibition of β-catenin/Tcf3 transactivation at the 4-cell stage significantly reduces siamois
expression after the MBT, inhibition at the 32-cell stage has little or no effect (). This indicates that, as early as the 32-cell stage, dorsal cell fates have been specified and “locked-in”, as the embryo is no longer sensitive to inhibition of β-catenin/Tcf3 transactivation. Notably, six cell divisions separate the 32-cell stage from the MBT, indicating that the information imparted to promoters must be inherited through multiple cell divisions following dorsal specification. We thus conclude that dorsal specification by β-catenin is temporally uncoupled from the onset of dorsal target gene expression.
β-catenin establishes poised chromatin architecture at target promoters before the MBT
We next hypothesized that β-catenin binds to target promoters and poises them for activation before the MBT so that they may be expressed following the large-scale activation of the zygotic genome. In general, transcriptionally poised loci are bound by initiating (CTD pSer5) RNA Pol II and marked by H3K9/14ac and H3K4me3 prior to the onset of transcription (Guenther et al., 2007
). To test whether these promoters bear marks of poised chromatin architecture before the onset of mRNA expression, we performed chromatin immunoprecipitation (ChIP) assays (Blythe et al., 2009
) for these marks of poised loci and for β-catenin in 1000-cell stage embryos, the earliest preMBT time-point (prior to the onset of siamois
expression) where it is feasible, in our hands, to perform ChIP ().
Although transcription is constrained before the MBT, phosphorylated forms of RNA Pol II corresponding to both initiating (CTD pSer5) and elongating (CTD pSer2) are detected before the MBT in whole embryo lysates (Figure S1
), albeit at lower levels than after the MBT. To assess whether initiating or elongating RNA Pol II associates with β-catenin target genes, we performed ChIP against CTD pSer5- and CTD pSer2-RNA Pol II (), comparing samples collected before the onset of siamois
expression (1000-cell) with those collected after the onset of expression (Stage 9, blastula, see ). As a positive control, we compared RNA Pol II occupancy at the xnr6
locus, which is transcribed during both the 1000-cell stage and at Stage 9 (see ). As expected, similar levels of both forms of RNA Pol II are found associated with the xnr6
locus before and after the MBT (). Consistent with our hypothesis, the siamois
loci are bound by initiating RNA Pol II before the onset of their expression, at levels similar to those found after the MBT (, left panel); however the amount of elongating RNA Pol II associated with siamois
increases after the MBT (, right panel). These observations indicate that the siamois
loci are bound by initiating RNA Pol II prior to the onset of their expression, as expected for poised loci.
In addition to binding the promoters of preMBT expressed genes, xnr5
, β-catenin also binds the siamois
promoters before the MBT (). We also tested whether β-catenin could bind later (zygotic Wnt) targets during preMBT stages. Before the MBT, β-catenin does not bind a cluster of Tcf/Lef binding sites flanking the zygotic Wnt target gene myf5
(Yang et al., 2002a
), although it binds these sites after the MBT ( and data not shown), indicating that β-catenin binding is restricted to maternal Wnt target genes before the MBT. The siamois
promoters are also marked by H3K9/14ac before the onset of their expression (). However, while β-catenin binding is restricted to maternal Wnt target genes, H3K9/14ac binding is more widepread: the promoters of all genes tested—maternal and zygotic β-catenin target genes and a negative control locus, myosin light chain 2
)—contain H3K9/14ac before the MBT. Finally, maternal Wnt target promoters are marked before the MBT with another marker of poised loci, H3K4me3 (). Taken together, these data demonstrate that the β-catenin target genes siamois
arrive at the MBT poised for expression.
These observations raise the possibility that β-catenin plays an instructive role in establishing poised chromatin architecture at the siamois and xnr3 loci. In support of this hypothesis, depletion of β-catenin reduces H3K4me3 at the siamois and xnr3 promoters (). In addition, inhibition of β-catenin/Tcf3 transactivation by ΔNTcf3 reduces β-catenin binding to the siamois and xnr3 promoters and blocks H3K4me3 (). In contrast, ΔNTcf3 has less of an effect on H3K9/14ac at the poised siamois and xnr3 promoters, suggesting that preMBT acetylation at siamois and xnr3 is established, at least in part, independently of β-catenin.
β-catenin and RNA Pol II establish H3K4me3 at poised promoters
Dorsal specification by β-catenin prior to the 32-cell stage is sensitive to inhibition of RNA Pol II (Yang et al., 2002b
). Deposition of H3K4me3 at promoters also correlates with occupancy of initiating polymerase (Ng et al., 2003
), and RNA Pol II is also required for β-catenin-mediated H3K4me3 in Drosophila
(Parker et al., 2008
). We therefore tested whether preMBT RNA Pol II function is necessary for the establishment of other marks of poised loci. Knocking down β-catenin has no effect on the occupancy of initiating RNA Pol II at the poised siamois
promoters (), indicating that initiating RNA Pol II is established at poised loci independently of β-catenin. In addition, RNA Pol II function is not required for binding of β-catenin; however, inhibition of Pol II greatly reduces H3K4me3 at the siamois
promoters, but does not affect H3K9/14ac ().
In summary, we find that the maternal Wnt target genes siamois and xnr3 arrive at the MBT poised for activation. β-catenin functions, in collaboration with RNA Pol II, to establish the H3K4me3 mark in particular, whereas these promoters are bound by initiating RNA Pol II and H3K9/14ac even when Wnt/β-catenin signaling is inhibited. We conclude that β-catenin is required for the establishment of poised chromatin architecture at these loci, thereby priming them for activation at the MBT.
β-catenin associates with a Histone H3 (R8) methyltransferase activity in early Xenopus embryos
We next hypothesized that, during dorsal specification (between the 4- and 32-cell stage), β-catenin interacts with a chromatin-modifying activity that functions to establish poised chromatin architecture at target promoters. To identify such a factor, we immunoprecipitated β-catenin from 8- to 32-cell stage embryos and performed in vitro
histone acetyl- or methyl-transferase (HAT or HMT) assays. During dorsal specification, β-catenin interacts with a HMT activity that specifically methylates Histone H3 but not H4 (). Under these assay conditions, we were unable to detect an associated HAT activity (Figure S2A
). β-catenin is predicted to interact with several functionally different macromolecular complexes (Gottardi and Gumbiner, 2001
). We determined that β-catenin interacts with this HMT activity in a high molecular weight complex that is unique to a subset of cellular β-catenin (Figure S2B-D
β-catenin associates with a Histone H3(R8) methyltransferase before the MBT
To identify the residue on Histone H3 targeted by the β-cat/HMT complex, we performed β-catenin IP/HMT assays using as the substrate recombinant H3.3 (rH3.3) with alanine point mutations at candidate target residues () on the H3 N-terminal tail previously shown to be methylated: arginines (R) 2, 8, 17, 26 and lysines (K) 4 and 9 (Bedford and Clarke, 2009
; Kouzarides, 2007
). As with H3, the β-cat/HMT significantly methylates rH3.3(WT) over background. Importantly, mutation of K4 has no effect on H3 methylation, suggesting that the H3K4me3 observed at the poised siamois
promoters is indirectly established by β-catenin (, , see Discussion). These observations also rule out R2, 17, and 26 as the major methyl acceptor sites for the β-cat/HMT. In contrast, mutation of either R8 or K9 prevents H3 methylation by the β-cat/HMT. Similarly, while the β-cat/HMT methylates an unmodified H3 (1-15) peptide (, lane 2) to a similar level as full-length H3 (data not shown), modification of H3 peptides pre-modified at either R8 (asymmetric dimethyl) or K9 (acetyl and trimethyl) prevents methylation by the β-cat/HMT (, lanes 3, 6, and 7). Thus, in addition to targeting either position R8 or K9, the β-cat/HMT is also sensitive to the modification status of these residues.
H3K9 methylation is generally associated with heterochromatin and transcriptional repression (Kouzarides, 2007
), and is therefore an unlikely target residue for the β-cat/HMT. Several ChIP experiments failed to detect H3K9me2 and –me3 at the siamois
promoters between the 1000-cell stage and MBT, and H3K9me1 levels were not sensitive to stabilization of β-catenin by LiCl treatment ( and data not shown). On the other hand, the effect of H3R8 methylation on transcriptional control is poorly understood. To test whether β-catenin activity regulates H3R8 methylation at target promoters, we generated an antibody that specifically detects asymmetrically dimethylated H3R8 (H3R8me2a) (Figure S2E and F
), and confirmed that the H3R8me2a modification occurs in vivo
. Subsequently, we measured H3R8 methylation at the siamois
promoter by ChIP. H3R8me2a associates with the siamois
promoter at the MBT and symmetric H3R8 dimethylation (H3R8me2s) is not detected (). This result was confirmed with an independent source of anti-H3R8me2a antibody ( and Figure S2G
). To test whether association of H3R8me2a correlates with β-catenin activity, we exposed preMBT embryos to a pulse of LiCl, which stabilizes β-catenin throughout the embryo. One hour after the LiCl pulse, H3R8me2a increased dramatically at the siamois
promoters ( and data not shown), indicating that asymmetric H3R8 methylation correlates with preMBT β-catenin activity. Furthermore, knockdown of β-catenin reduced H3R8me2a at the siamois
promoters before the MBT (). Thus, the β-cat/HMT asymmetrically dimethylates H3R8 and is sensitive to the modification state of H3K9. Our results also indicate that β-catenin interacts with a type I (asymmetric) arginine HMT in early Xenopus
β-catenin recruits the H3R8 methyltransferase Prmt2 to target loci
We undertook a candidate-based approach to identify the β-catenin-associated arginine HMT. Of the ten members of the protein arginine methyltransferase (Prmt) family, three have been shown to methylate Histone H3 in vitro
: Carm1, Prmt5, and Prmt6 (Guccione et al., 2007
; Hyllus et al., 2007
; Pal et al., 2004
; Schurter et al., 2001
). Of these, only the type II, symmetric HMT Prmt5 specifically targets R8 (Pal et al., 2004
). By expressing myc-tagged Prmts in Xenopus
embryos and immunoprecipitating β-catenin, we found that none of the Prmts known to methylate H3 (Carm1, Prmt5, and Prmt6) co-purified with β-catenin (data not shown). However, Prmt2, which is most closely related to Carm1 and Prmt6, co-immunoprecipitates with β-catenin (). Although recombinant β-catenin and Prmt2 do not interact directly in vitro (data not shown), GST-tagged Prmt2 interacts with β-catenin in Xenopus
embryo lysates in a temperature and ATP-dependent manner (). Coupled with our observation that the β-cat/HMT complex is large (Figure S2B-D
), we propose that an unknown catalytic activity is required for Prmt2 and β-catenin to interact within a large macromolecular complex.
β-catenin interacts with the Histone H3(R8) methyltransferase Prmt2
By sequence, Prmt2 is most closely related to the type I HMTs Carm1 and Prmt6, but its substrate preference has not been determined because recombinant Prmt2 has little to no activity in vitro
(Lakowski and Frankel, 2009
; Scott et al., 1998
). Also, to our knowledge, HMT activity associated with endogenous Prmt2 has not been reported. Interestingly, endogenous Prmt2 immunoprecipitated from mouse embryonic stem cells methylates Histone H3 (). Likewise, myc-Prmt2 expressed in Xenopus
embryos methylates Histone H3, and this activity requires H3R8 (). In these experiments, purified Prmt2 has HMT activity towards Histone H3, reflecting either the activity of Prmt2 itself or the activity of another HMT that co-immunoprecipitates with Prmt2. Further investigation will be required to determine the factors that regulate endogenous Prmt2 catalysis.
Importantly, Prmt2 binds the siamois promoter in preMBT embryos () as determined by ChIP. Furthermore, β-catenin knockdown reduced Prmt2 occupancy at the siamois promoter, suggesting that β-catenin recruits Prmt2 to target promoters. We therefore conclude that Prmt2 represents the HMT activity that associates with β-catenin in early Xenopus embryos based on the observations that β-catenin interacts with Prmt2, Prmt2 has a HMT activity directed at H3 that is sensitive to the R8A mutation, and β-catenin recruits Prmt2 to target genes during the preMBT period.
Maternal Prmt2 is necessary for dorsal specification
Based on the above observations, we predicted that recruitment of Prmt2 by β-catenin to dorsal target genes would be essential for specifying the dorsal developmental program. Therefore, we tested whether loss of Prmt2 function in early embryos would affect dorsal specification. Xenopus
Prmt2 is expressed maternally, and knockdown of Prmt2 in fertilized embryos only weakly affects expression of siamois
(data not shown). Therefore, we knocked down Prmt2 in oocytes and generated maternally depleted Prmt2 (prmt2-
) embryos by the host transfer method (Mir and Heasman, 2008
embryos generated by antisense DNA are impaired in dorsal development (). These embryos develop with a range of dorso-ventral morphologies, displaying completely ventralized, anterior-truncated (partially ventralized), and normal phenotypes within single clutches (, N=66 embryos from six independent experiments). Importantly, depletion of Prmt2 reduces the expression of both siamois
following the MBT ( and Figure S3A and B
), indicating that Prmt2 function is necessary for the activation of maternal Wnt/β-catenin dependent transcription. These ventralized phenotypes are specific to loss of Prmt2 function, as expression of mouse Prmt2 mRNA in prmt2-
embryos rescues siamois
expression and partially rescues the morphological phenotype (, N=26 from three independent experiments). We also replicated the knockdown of siamois
with a translational blocking morpholino oligonucleotide against Prmt2
). However, while the morpholino generated a more severe knockdown of siamois
, it was more difficult to generate viable MBT-stage embryos, possibly due to a general requirement for Prmt2 in the regulation of chromatin structure beyond its proposed role in the Wnt pathway. Alternatively, the antisense DNA yielded a partial knockdown of maternal prmt2
message that allowed for recovery of viable embryos at the expense of the severity of the phenotype ( and Figure S3
). Nonetheless, these observations strongly support the conclusion that recruitment of Prmt2 to dorsal gene promoters is a necessary step in establishing the dorsal gene expression program.
Maternal Prmt2 is necessary for dorsal specification
Directing Prmt2 to β-catenin target promoters is sufficient to drive dorsal specification in the absence of β-catenin
β-catenin interacts with chromatin via the Tcf/Lef family of DNA-binding factors (Behrens et al., 1996
; Molenaar et al., 1996
), and previous investigations have exploited this interaction to target factors of interest to Tcf/Lef binding sites and test their effects on target gene expression (Vleminckx et al., 1999
). Therefore, we generated chimeric proteins () between Prmt2 and the DNA binding domain of Lef-1 (ΔNLef1) to direct Prmt2 to target genes and evaluate its effect on dorsal specification. Chimeras between ΔNLef1 and a SAM-binding mutant of Prmt2 (Prmt2GG
)(Qi et al., 2002
), or wild type Carm1, Prmt5, and Prmt6 were also generated as controls. All chimeric proteins were expressed to similar levels in blastula stage embryos ( and data not shown).
Directing Prmt2 to β-catenin target gene promoters is sufficient to drive dorsal specification
Recruitment of Prmt2 to β-catenin target genes is sufficient to specify the dorsal developmental program, as expression of the Prmt2:ΔNLef1 chimera in ventral blastomeres converts them to dorsal progenitors (N=172, ), similar to activation of Wnt/β-catenin signaling. Ventral expression of the ΔNLef1 DNA binding domain alone did not induce secondary axes (N=31, data not shown). To determine whether Prmt2-induced dorsal cell fates are dependent on its catalytic activity, we tested the ability of the Prmt2GG mutant to induce secondary axes. At similar levels of expression, Prmt2GG:ΔNLef1 induced significantly fewer secondary axes (N=81, , histogram), and these axes were typically truncated (, photo) compared to those observed with Prmt2:ΔNLef1, indicating that dorsal specification by Prmt2 is dependent on its HMT activity. However, Prmt2GG nonetheless conferred some dorsal axis inducing activity; therefore we cannot rule out that additional factors that interact with Prmt2 contribute to Prmt2-dependent dorsal specification.
In a complementary approach, we tested whether Prmt2:ΔNLef1 could rescue β-catenin loss of function. Embryos depleted for β-catenin develop with a ventralized phenotype (; also ), whereas expression of Prmt2:ΔNLef1 in β-catenin-depleted embryos restores the full range of dorsal and anterior structures (), remarkably similar to control embryos (), albeit typically with a single eye (86% rescue, N=172). The anterior defects in Prmt2:ΔNLef1-rescued β-MO embryos could result from activation of the posteriorizing zygotic Wnt target genes, as expression of Prmt2:ΔNLef1 alone in dorsal blastomeres did not affect dorsal specification, but did induce a weak posteriorized phenotype (data not shown, N=29). Importantly, expression of Prmt2:ΔNLef1 in β-MO embryos rescues organizer gene expression, whereas the ΔNLef1 DNA binding domain alone has minimal activity ().
To assess the specificity of Prmt2:ΔNLef1, we also tested whether other Prmts could rescue dorsal specification in β-catenin depleted embryos. Prmt5 shares target residue specificity with Prmt2, but symmetrically dimethylates H3R8 (Pal et al., 2004
), and Prmt5:ΔNLef1 is unable to rescue dorsal specification in β-catenin depleted embryos (—0% Rescue, N=31)). On the other hand, the robust transcriptional activator Carm1 asymmetrically methylates H3 R2, R17, and R26, with only a weak activity towards R8 (Chen et al., 2000
; Schurter et al., 2001
). Expression of Carm1:ΔNLef1 is toxic to embryos shortly after gastrulation, so phenotypic rescue could not be scored. However, at gastrula stages, Carm1:ΔNLef1 does not rescue siamois
expression (). Finally, Prmt6, which targets H3R2 (Guccione et al., 2007
; Hyllus et al., 2007
), also did not rescue dorsal specification (not shown, 0% rescue, N=30). Thus, of the Prmts tested, only the recruitment of Prmt2 to β-catenin target promoters is sufficient to rescue dorsal specification, demonstrating the unique role of Prmt2 in the regulatory events that establish the transcriptional network driving dorsal development in Xenopus