XB-ART-51022J Biol Chem September 4, 2015; 290 (36): 21925-38.
The Proto-oncogene Transcription Factor Ets1 Regulates Neural Crest Development through Histone Deacetylase 1 to Mediate Output of Bone Morphogenetic Protein Signaling.
The neural crest (NC) is a transient, migratory cell population that differentiates into a large variety of tissues including craniofacial cartilage, melanocytes, and peripheral nervous system. NC is initially induced at the border of neural plate and non-neural ectoderm by balanced regulation of multiple signaling pathways among which an intermediate bone morphogenetic protein (BMP) signaling is essential for NC formation. ets1, a proto-oncogene playing important roles in tumor invasion, has also been implicated in delamination of NC cells. In this study, we investigated Ets1 function in NC formation using Xenopus. Overexpression of ets1 repressed NC formation through down-regulation of BMP signaling. Moreover, ets1 repressed the BMP-responsive gene id3 that is essential for NC formation. Conversely, overexpression of id3 can partially rescue the phenotype of NC inhibition induced by ectopic ets1. Mechanistically, we found that Ets1 binds to id3 promoter as well as histone deacetylase 1, suggesting that Ets1 recruits histone deacetylase 1 to the promoter of id3, thereby inducing histone deacetylation of the id3 promoter. Thus, our studies indicate that Ets1 regulates NC formation through attenuating BMP signaling epigenetically.
PubMed ID: 26198637
PMC ID: PMC4571947
Article link: J Biol Chem
Genes referenced: bmp4 chrd.1 ets1 ets2 etv2 fgf4 foxd3 hdac1 hes4 id3 krt12.4 lrig3 msx1 myc ncam1 odc1 pax3 smad1 snai2 sncg sox2 sox3 szl tbxt twist1 wnt3a zic1
Morpholinos: ets1 MO1 ets1 MO2 ets1 MO3
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|FIGURE 1. Expression of ets1 in NC is regulated by Lrig3 and FGF signaling. A, embryos were injected with chordin (Chd), chordin + wnt3a, or chordin + wnt3a + lrig3MO (L3MO). Animal caps were dissected at stage 9 and cultured to stage 17. Expression of the indicated genes in animal caps was assayed by RT-PCR. Ornithine decarboxylase (odc) was used as an internal standard. WE, uninjected whole embryo; AC, uninjected animal caps; RT−, without reverse transcriptase. B–G, spatial expression pattern of ets1 as detected by in situ hybridization. B, C, and D, dorsal view; D′, anterior view; E, F, and G, lateral view. The white arrow in C indicates the weak ets1 signal starting at stage 13. The ventral blood island is indicated by a white arrow in D′. H, temporal expression of Xenopus ets1a at the indicated stages. I–N, embryos treated with the indicated concentrations of SU5402 from stage 12. ets1 and brachyury (bra) expression was examined at stage 16 by whole-mount in situ hybridization. The expression of the indicated genes was affected in the following percentage of embryos: M, 73% (8 of 11); N, 100% (20 of 20); J, 33% (7 of 21); K, 61% (11 of 18). O, embryos were injected with 10 pg of efgf or 300 pg of wnt3a mRNA, respectively. The caps were dissected from embryos at stage 9 and then cultured to stage 15. Expression of the indicated genes in animal caps was assayed by RT-PCR.|
|FIGURE 2. Overexpression or knockdown of ets1 causes defects in NC derivatives. A and B, ets1 overexpression (500 pg/embryo; 85%; 23 of 27) caused loss of pigment throughout the body and inhibition of head structures. C–F, knockdown of ets1using ets1MO1 (94%; 30 of 32) or ets1MO2 (94%; 33 of 35) showed similar pigment loss and repression of head. G, categories of defects induced by either 30 ng of ets1MO1 separately or by co-injection (inj.) with 250 pg of ets1 mRNA. Numbers at the top indicate total embryos scored at stage 35 from three independent experiments. H and I, either 60 ng of ets1MO1 or ets1MO2 was injected into two-cell stage embryos, the embryos were collected at stage 11, and Ets1 protein was detected by α-Ets1 antibody. Tubulin was used as an internal control. The bands on Western blots (H) were quantified in I. J–L, the expression of cranial nerve marker gene sncg is disrupted in embryos injected with 250 pg of ets1 mRNA (K; 85%, 28 of 33) or 30 ng of ets1MO1 (L; 72%, 18 of 25). con, control. M–P, either ets1-GR or dnets1-GR mRNA (500 pg/embryo) was injected into two-cell stage embryos. The injected embryos were treated with DEX starting at stage 13. Loss of pigment and inhibition of anterior axis were observed in DEX-treated embryos (N, 81%, 26 of 32; P, 74%, 17 of 23) but not in untreated embryos (M, 4%, 1 of 26; O, 0%, 0 of 11). Likewise, the expression of sncg was much reduced in the embryos injected with either ets1-GR or dnets1-GR and sequentially treated with DEX (R, 84%, 26 of 31; T, 84%, 21 of 25) but remained normal in embryos without DEX treatment (Q, 0%, 0 of 29; S, 0%, 0 of 18). U–W, cranial cartilage formation in control (U) and embryos injected with ets1 mRNA (V; 85%, 51 of 60) or ets1MO (W; 83%, 33 of 40) was examined by Alcian blue staining. An asterisk indicates the injected side. con, control.|
|FIGURE 3. Knockdown of ets1 does not obviously affect NC formation but blocks NC migration. A–J, the expression of foxd3 or snail2 was not inhibited by ets1MOs. ets1MO1 or ets1MO2 was injected either separately into both blastomeres (A–D, 60 ng of ets1MO1/embryo; E–H, 60 ng of ets1MO2/embryo) or together with 100 pg of lacZ mRNA into one blastomere (I and J, 30 ng/embryo) at the two-cell stage. The injected embryos were collected at stage 17 and examined for foxd3 (B, 90%, 18 of 20; F, 96%, 24 of 25; I, 95%, 21 of 22) and snail2 (D, 95%, 22 of 23; H, 92%, 23 of 25; J, 90%, 18 of 20) by whole-mount in situ hybridization. The injected side was traced by red X-Gal staining and is marked with an asterisk (I and J). K, overexpression and knockdown of ets1 repressed NC formation in an animal cap assay. Expression of the indicated genes in animal caps injected with either chordin (Chd) + wnt3a, chordin + wnt3a + ets1, or chordin + wnt3a + ets1MO1 was examined by RT-PCR. L–Q, spatial expression pattern of ets2 as detected by in situ hybridization. st, stage. L, P, and Q, lateral view; M and N, vegetal view; O, dorsal view. The black arrow in N indicates weak expression of ets2. R and S, ets1MO1 (30 ng) and lacZ mRNA (100 pg) were co-injected into one dorsal blastomere of four-cell stage embryos, and the embryos were collected at stage 20. Segmentation and extension of cranial NC were blocked at the ets1MO1-injected side (R, 85%, 17 of 20; S, 83%, 20 of 24). T–W, knockdown of ets1 decreased the expression of twist1 in both neurula (T and U) and tail bud embryos (V and W). T, control embryo at stage 20. U, embryos injected with ets1MO1 at one side. V and W, embryos at stage 25 injected with ets1MO1 at one side. At the injected side (U, 85%, 11 of 13; W, 90%, 9 of 10), the twist1 signal stripes were weaker and did not extend laterally as far as those at the uninjected side (T and V). An asterisk indicates the injected side. U′ and U′, transverse sections of embryos shown in U (5 of 5 embryos). ets1MO1 and lacZ mRNA were co-injected at one side of the embryos. In the injected side, NC cells marked by twist1 staining were concentrated laterally to the neural tube and seemed not to detach from the neural plate. In the uninjected side, NC cells extended out of the neural plate, and signal spots were scattered underneath the mesoderm region, suggesting that NC cells migrated into the arches. An asterisk indicates the injected side. Scale bars in U′ and U′ indicate 100 μm. X and Y, Ets1MO2 (30 ng/embryo) was co-injected with lacZ mRNA (100 pg; used as a lineage tracer) into one blastomere of two-cell stage embryos, and embryos were stained for the expression of twist1 (81%, 17 of 21) and snail2 (79%, 11 of 14). The extension of cranial NC was decreased in the ets1MO2-injected side. WE, uninjected whole embryo; AC, uninjected animal caps; RT−, without reverse transcriptase; con, control.|
|FIGURE 4. Overexpression of ets1 represses formation of NC in embryos and in animal caps injected with pax3 and zic1 mRNAs. A–F, the NC markers foxd3 (100%, 10 of 10), snail2 (78%, 7 of 9), and twist1 (80%, 12 of 15) were repressed by overexpression of ets1. G–R, either ets1 or ets1-GR mRNA was co-injected with lacZ mRNA into one dorsal blastomere of four-cell embryos, and embryos were collected at around stage 17. Embryos injected with ets1-GR were treated with dimethyl sulfoxide or DEX from stage 12 to stage 17. Whole-mount in situ hybridization was used to examine the NC marker genes snail2 (J, 71%, 17 of 24; M, 100%, 19 of 19; P, 65%, 13 of 20), foxd3 (K, 63%, 17 of 27; N, 95%, 18 of 19; Q, 62%, 8 of 13), and c-myc (L, 62%, 13 of 21; O, 100%, 20 of 20; R, 60%, 9 of 15). The injected side was traced by LacZ staining. S–V, mesoderm marker genes brachyury (bra; S and T) and chordin (chd; U and V) were examined in embryos injected with ets1. S and U, control embryo; T and V, ets1-injected embryos. W–X′, the mRNA mixtures of pax3 + zic1 or pax3 + zic1 + ets1 were injected into one dorsal blastomere at the four-cell stage, and the expression of foxd3 (W, 90%, 17 of 19; W′, 74%, 29 of 39) and snail2 (X, 94%, 15 of 16; X′, 75%, 24 of 32) was examined by whole-mount in situ hybridization at stage 16. Y, animal cap assays indicated that overexpression of ets1 suppressed the expression of NC maker genes induced by pax3 and zic1 and enhanced neural marker genes. WE, uninjected whole embryo; AC, uninjected animal caps; RT−, without reverse transcriptase; con, control.|
|FIGURE 5. Overexpression of ets1 attenuates BMP signal downstream of Smad1/5/8 phosphorylation and represses id3 through binding to its promoter. A–C, in situ hybridization analysis of the expression of the indicated markers in embryos injected with ets1 mRNA (sox3, 87%, 13 of 15; epi-keratin (epiker), 75%, 12 of 16; zic1, 100%, 22 of 22). An asterisk indicates the injected side. D, neural markers sox3, sox2, and ncam were induced by ets1 overexpression in an animal cap assay. E–G, expression of sizzled was examined in embryos injected with bmp4 mRNA (89%, 32 of 36) alone or together with ets1 mRNA (68%, 27 of 40). H, luciferase assay was performed to assay BMP signaling. chordin (chd) was used as a control. *, p < 0.05 between indicated group and the group only treated with BMP4. I, Western blot showing the level of phosphorylated Smad1/5/8 (p-Smad1/5/8) and total Smad1/5/8 in stage 16 embryos overexpressing ets1. Endogenous tubulin was used as a loading control. J, co-immunoprecipitation was performed using lysates from embryos injected with either ets1-HA, smad1-Myc separately, or both. K and L, expression of id3 in control (K) and ets1-injected embryos (L; 65%, 22 of 34) was examined by in situ hybridization. M–P, expression of foxd3 and snail2 in embryos injected with either the mixture of ets1 and gfp (M, 69%, 24 of 35; O, 61%, 22 of 36) or the mixture of ets1 and id3 (N, 69%, 31 of 45; P, 60%, 29 of 48). Q–R′, cell apoptosis was detected by TUNEL assay (black spots) in embryos with one side injection of ets1 mRNA (100%, 5 of 5). LacZ staining (light blue) was used to indicate the injected side. Q, control embryos; R, ets1-injected embryo; R′, high magnification of framed region in R. S–V, hairy2-GR mRNA (500 pg/embryos) was injected into embryos at the two-cell stage alone or together with ets1 (500 pg/embryos), and DEX was used to treat embryos from stage 13 to stage17. The expression of msx1 was examined using in situ hybridization. Although hairy2 slightly promoted msx1 expression (T, 88%, 23 of 26), co-expression with ets1 enhanced msx1 expression (U, 95%, 19 of 20; V, 96%, 25 of 26). W, the predicated Ets1 binding site in the id3 promoter of X. laevis and the primers used in ChIP. W′, schematic diagram illustrating the predicted SMAD1 and ETS1 binding sites in the human ID3 promoter and the primers used in the ChIP assay. X, ChIP was performed using antibodies against ETS1 in HEK293T cells, and the precipitated DNA fragments were amplified using primer pair 1 (P1) or the GAPDH primer pair, respectively. Endogenous ETS1 can bind to the predicted binding site. The GAPDH primer pair was used as the control. Y and Z, ChIP was done in X. laevis embryos injected with 500 pg of ets1-FLAG. Semiquantitative (Y) and real time PCR (Z) were performed using primer pair 3 (P3) or primer pair control (Pc) (Table 2). WE, uninjected whole embryo; AC, uninjected animal caps; RT−, without reverse transcriptase; con, control; IB, immunoblot. Error bars represent S.D.|
|FIGURE 6. Ets1 physically interacts with HDAC1 through the ETS domain. A, co-IP was carried out with extracts of HEK293T cells transfected with either Ets1-Myc, HDAC1-HA separately, or both using antibodies against Myc or HA. B, co-IP indicates that Ets1-Myc binds to HDAC1-HA in Xenopus embryos. C–E, expression patterns of Xenopus hdac1 (C) and ets1 (D) in Xenopus embryo at stage 17. A schematic diagram of Xenopus hdac1 and ets1 expression (E) indicates overlap between them. F, Ets1 deletion mutants and their binding ability to HDAC1. G, co-IP was carried out to examine the binding of Myc-tagged Ets1 deletion mutants and HDAC1-HA in HEK293T cells. IB, immunoblot; TAD, transactivation domain; SAM, sterile α motif; PNT, pointed.|
|FIGURE 7. The interaction with HDAC1 is required for Ets1 to regulate NC and epidermis formation as well as id3 expression. A–J, embryos were injected with mRNAs of different ets1 deletion mutants (250 pg/embryo) in one side and stained for the expression of foxd3. Injection of ets1F2, ets1F3, and ets1F4 did not apparently affect the expression of foxd3 (A–C) and epi-keratin (epiker; F–H), whereas overexpression of ets1F5 and ets1F6 suppressed these two marker genes at the injected side (foxd3: D, 65%, 13 of 20; E, 94%, 15 of 16; epi-keratin: I, 90%, 9 of 10; J, 93%, 26 of 28). An asterisk indicates the injected side in embryos. K–R, embryos injected with 250 pg of ets1 mRNA were treated with 50 nM TSA from stage 13 to stage 17. foxd3 was repressed by overexpression of ets1 alone (67%, 16 of 24), whereas ectopic foxd3 in ets1-injected embryos was induced after TSA treatment (57%, 8 of 14). The expression of sox3 was expanded by overexpressing ets1 (83%, 15 of 18), whereas TSA treatment reduced the expression of sox3 in the ets1-injected side (93%, 14 of 15). LacZ staining was used to trace the injected side. S, real time PCR analysis of id3 expression in embryos injected with the different mRNAs as indicated or treated with TSA. Results are presented as -fold changes after normalization to ornithine decarboxylase expression. con, control. Error bars represent S.D.|
|FIGURE 8. Ets1 recruits HDAC1 to the id3 promoter and reduces the level of acetylated histone 4. A, ChIP was done using HEK293T cells transfected with either HDAC1-HA alone or Xenopus Ets1. The precipitated DNA fragments were amplified using primer pair 2 (P2) or the GAPDH primer pair. The co-expression of Ets1 promoted the binding of HDAC1 to the ID3 promoter. B–E, ChIP was performed in the embryos injected with the indicated mRNAs. The precipitated DNA fragments were analyzed by semiquantitative PCR (B) or real time PCR (C–E). Error bars represent S.D. Asterisk represents p value <0.05 and double-asterisk represents p value <0.01.|
|FIGURE 9. Proposed model showing Ets1 regulation of BMP signal and NC formation through Hdac1. When ets1 is overexpressed in embryos, it binds to Hdac1, and recruits Hdac1 to the id3 promoter, leading to deacetylation of histone and chromatin condensation, which prevents transcription of id3 and causes inhibition of NC formation. Other BMP targets involved in NC development may also be affected by this mechanism. When ets1F3 or ets1F4 Ets1 mutant is overexpressed, it enhances histone acetylation of id3 promoter and therefore promotes NC formation. eF3, Ets1F3; Ac, acetyl group; P-Smad, phosphorylated Smad.|