Nordin K and LaBonne C (2014), Sox5 Is a DNA-binding cofactor for BMP R-Smads ...

XB-ART-50439

Dev Cell 2014 Nov 10;313:374-382. doi: 10.1016/j.devcel.2014.10.003.

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Sox5 Is a DNA-binding cofactor for BMP R-Smads that directs target specificity during patterning of the early ectoderm.

Nordin K , LaBonne C .

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The SoxD factor, Sox5, is expressed in ectodermal cells at times and places where BMP signaling is active, including the cells of the animal hemisphere at blastula stages and the neural plate border and neural crest at neurula stages. Sox5 is required for proper ectoderm development, and deficient embryos display patterning defects characteristic of perturbations of BMP signaling, including loss of neural crest and epidermis and expansion of the neural plate. We show that Sox5 is essential for activation of BMP target genes in embryos and explants, that it physically interacts with BMP R-Smads, and that it is essential for recruitment of Smad1/4 to BMP regulatory elements. Our findings identify Sox5 as the long-sought DNA-binding partner for BMP R-Smads essential to plasticity and pattern in the early ectoderm.

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Species referenced: Xenopus
Genes referenced: babam2 bmp4 chrd foxd3 foxi1 foxi4 h3-3a id3 krt12.4 msx1 myc myod1 npb pax3 six1 smad1 smad10 smad2 smad3 smad4 smad7 snai2 sox10 sox15 sox3 sox5 sox6 sox8 sox9 tfap2a ventx1.2 ventx2.2 zic1
???displayArticle.antibodies??? Myc Ab5 Myc Ab8 Smad1 Ab5 Smad1 Ab7 Smad2 Ab4
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	Figure 1. Sox5 Is Required for NC and NPB Formation (A) In situ hybridization examining FoxD3, Snail2, Sox9, and Sox10 in Xenopus embryos injected with Sox5 MO or Sox5 mRNA. Embryos were collected at midneurula stages (stage 17). Asterisk denotes injected side with β-galactosidase (β-gal) staining (red) as lineage tracer. (B) Explant assay examining Snail2 and Sox9 in Wnt8/Chordin-induced explants that were injected with Sox5 MO. Explants were collected alongside sibling embryos cultured to midneurula stages (stage 17). (C) In situ hybridization examining Six1 and Foxi1c in embryos injected with Sox5 MO or Sox5 mRNA. Embryos were collected at early neurula stages (stage 13). (D) In situ hybridization examining Msx1, Pax3, and Zic1 in embryos injected with Sox5 MO or Sox5 mRNA. Embryos were collected at early neurula stages (stage 13/14). (E) Explant assay examining Msx1, Pax3, and Zic1 in Wnt8/Chordin-induced explants that were injected with Sox5 MO. Explants were collected alongside sibling embryos cultured until early neurula stages (stage 14). Asterisk denotes injected side with β-gal staining (red) as lineage tracer. See also Figure S1.
	Figure 2. Misregulating Sox5 Phenocopies Loss of BMP Signaling (A and B) In situ hybridization examining Sox3 (A) and EPK (B) in embryos injected with Sox5 MO or Sox5 mRNA. Embryos were collected at early neurula stages (stage 13). (C and D) Explant assay examining Sox3 (C) and EPK (D) in explants that were either injected with Sox5 MO or Sox5 mRNA. Explants were collected alongside sibling embryos cultured until early neurula stages (stage 13). (E and F) In situ hybridization examining Sox3 (E) and EPK (F) in embryos injected with Chordin mRNA showing a similar loss of EPK and expanded expression of Sox3. Embryos were collected at early neurula stages (stage 13/14). Asterisk denotes injected side with β-gal (red) as lineage tracer. (G) In situ hybridization examining AP2, Id3, and Vent2 at blastula stages. Embryos were injected with Chordin, Smad7, or Sox5 MO and collected at stage 9. Asterisk denotes injected side with β-gal staining (red) as lineage tracer. See also Figure S2.
	Figure 3. BMP Signaling Requires Sox5 to Activate Transcriptional Targets (A and B) Ectoderm explant assay examining Vent1 (A) and Msx1 (B) in explants injected with BMP4 mRNA or BMP4 mRNA with Sox5 MO. Explants were collected alongside sibling embryos cultured until early neurula stages (stage 14). (C) Western blot using lysates from embryos injected either with BMP4, BMP4/Sox5, BMP4/Sox5MO, or Chordin to examine levels of phosphorylated Smad1 and unphosphorylated Smad1. (D) Western blot using using lysates from embryos injected with Activin, Activin/Sox5, Activin/Sox5MO, Sox5, or Sox5MO to examine levels of phosphorylated Smad2. (E and F) Luciferase assay examining fold activation by the Vent2 promoter, p = 0.0068 (E), or 12XGCCG reporter, p = 0.0227 (F), in the presence of active Smad1/4 complexes or with active Smad1/4 and Sox5 MO. (G) Luciferase assay using the Vent2 reporter and the Vent2 3x Mutant reporter (carrying the same mutations as in Figure S3C). The mutations that reduce Sox5 binding also reduce activation of the reporter by active Smad1/4. Error bars represent SD of three independent experiments. See also Figure S3.
	Figure 4. Sox5 Recruits Smad1/4 to Target Promoters through Physical Association (A) GST pull-down assay using lysates from uninjected embryos or embryos injected with Sox5 or Sox6 mRNA assaying for binding to GST, Smad1-GST, Smad3-GST, or Smad4-GST. Western blots show expressed levels of Sox5 and Sox6. (B) GST pull-down assay using lysates from embryos injected with Sox5 mRNA assaying for binding to GST, Smad1-GST, Smad1-MH1-GST, or Smad1-MH2-GST. (C) Schematic representations of Smad1, Smad1-MH1, and Smad1-MH2 constructs used in pull-down assays. (D) Coomassie staining of expressed GST fusion proteins used for pull-downs in (A) and (B). (E) ChIP-qPCR analysis of Smad1 enrichment on EPK promoter relative to eEF1α promoter. Smad1 enrichment is significantly reduced when Sox5 is depleted. Error bars represent SEM of three independent biological experiments. p = 0.0022. (F) ChIP q-PCR analysis of Smad1 enrichment on Vent2 promoter relative to control eEF1α promoter. Smad1 enrichment is significantly reduced when Sox5 is depleted. Error bars represent SEM of three independent biological experiments. p = 0.0472. (G) ChIP analysis examining Sox5 enrichment on EPK promoter relative to eEF1α promoter. Smad1 enrichment is significantly reduced in the presence of Chordin. Error bars represent SEM of three independent biological experiments. p = 0.0048. (H) ChIP analysis examining Sox5 enrichment on Vent2 promoter relative to eEF1α promoter. Sox5 enrichment is significantly reduced in the presence of Chordin. Error bars represent SEM of three biological experiments. p = 0.0006. See also Figure S4.
	Figure S1. Related to Figure 1. Expression of Sox5 in wildtype embryos and Sox5 is effectively knocked down (A-C) In situ hybridization showing expression of Sox5 with schematics showing location (green) of NPB and premigratory and migratory NC cells at: (A) early neurula (stage 13) (B) mid-neurula (stage 18) (C) tailbud (stage 24) stages. At early neurula stages Sox5 is expressed in the somitic mesoderm and throughout the ectoderm. By stage 18 the pan-ectodermal expression has been lost and robust expression is seen at the NPB. (D-F) 60 μM serial sections through embryos following in situ hybridization for Sox5. Strong expression in mesoderm is seen at stage 13 (D) while expression in the neural crest can be seen at mid-neurula stages (E), and tailbud stages (F). (G) In situ hybridization of wildtype embryos showing Sox5 expression at the two cell (stage 2), morula (stage 6) and blastula (stage 8) stages. Maternal expression was also reported in a proteomics study (Yanai et al., 2011). (H) RT-PCR stage 9 animal pole explants, vegetal bottom explants and whole embryos Sox5 is expressed in both animal and vegetal explants whereas Mix.1 is expressed preferentially in the vegetal pole and Xema in the animal pole. Histone H4 was used to normalize cDNA input. (I) Wild type expression of Sox6 from stage 8 through stage 32. In contrast to Sox5, Sox6 lacks detectable early ectodermal/NC expression. (J) Western blot of lysates from embryos co-injected with Sox5 mRNA and Sox5 morpholino demonstrating that Sox5 translation is blocked by the morpholino. An N-terminally myc tagged Sox5 cannot be targeted by the morpholino. (K) In situ hybridization examining expression of Snail2 in embryos injected with Sox5 mRNA, Sox5 morpholino, or both. Coinjection of Sox5 mRNA rescues the effects of Sox5 morpholino.
	Figure S2. Related to Figure 2. Sox5 misexpression and knockdown causes gene expression defects in gastrula stage embryos. (A) phospho-Histone H3 staining of stage 15 embryos injected with Sox5 mRNA or Sox5 morpholino. No differences in proliferation are seen. (B) TUNEL staining stage 15 embryos injected with Sox5 mRNA or Sox5 morpholino. No differences in apoptosis are seen. DNMT3bmediated apoptosis serves as a positive control. Asterisk denotes side with β-gal staining (red) lineage tracer. (C) In situ hybridization examining expression of MyoD in stage 15 embryos injected with Sox5 mRNA or Sox5 morpholino in micromeres at the 8-cell stage targeting for ectodermal tissue that will give rise to neural crest. No defects in mesoderm are noted. (D) In situ hybridization examining expression of MyoD in stage 15 embryos injected with Sox5 mRNA or Sox5 morpholino in macromeres at the 8 cell stage targeting for mesoderm. Mesoderm formation as assayed by MyoD expression is perturbed, reflecting a role for Sox5 in this tissue. (E) In situ hybridization examining expression of Sox8 in embryos injected with Sox5 mRNA. Embryos were collected at both stage 17 and stage 23. (F) In situ hybridization examining expression of FoxD3 and Snail2 in embryos injected with Sox5* (a shorter Sox5 isoform/splice variant expressed in laevis). Embryos were collected at mid-neurula stages, stage 17. (G) In situ hybridization examining expression of EPK and Sox3 in embryos injected with Sox5. Embryos were collected at early-neurula stages, stage 14. (H) In situ hybridization examining expression of Msx1, Pax3 and Zic1 in embryos injected with Sox5. Embryos were collected at earlyneurula stages, stage 14. Sox5* behaved indistinguishably to Sox5 in these assays. (I-K) In situ hybridization examining expression of Id3 (I), AP2 (J) and Pax3 (K) at gastrula stages (stage 11.5) in embryos injected with Sox5 or Sox5MO. Red arrow denotes blastopore. Asterisk denotes injected side.
	Figure S3. Related to Figure 3. Sox5 is required for BMP target activation (A) Luciferase assay examining fold activation of the Id3 promoter. Reporter activation by active Smad1/4 is significantly diminished when Sox5 is depleted. p=0.0052 (B) Luciferase assay examining fold activation of the 4XCAGA reporter. Reporter activation by active Smad 2/4 is not diminished when Sox5 is depleted. Experiments were repeated three times and error bars represent standard deviation of three replicates. (C) Vent2 BRE sequence within the luciferase reporter. Bolded sequences are within putative Sox5 binding sites that were mutated from AT rich sequence to C rich sequence (red). (D) EMSA using the Vent2 BRE Wildtype sequence or the mutated sequence as a probe. Mutations greatly reduce Sox5 and Sox6 binding to this element. (E,F) Western blots of embryo lysates injected for luciferase assays in Figure 3E,F. Embryos injected for Vent2 reporter (E) and 12XGCCG reporter (F).
	Figure S4. Related to Figure 4. Physical mapping of Sox5 with Smad1 and Sox5 is required to recruit Smad1 to the Msx1 and Id3 promoters. (A) Co-immunoprecipitation examining interaction between Sox5 and Smad1 or Smad4. Lysates from embryos expressing the noted epitope tagged proteins were immunoprecipitated (IP) with anti-flag antibodies and immunoblotted (IB) using anti-myc antibodies. Direct immunoblotting of lysate with either myc, flag, or HA antibodies served as input control (bottom panels). (B) Schematic representations of Sox5 deletion constructs used in GST pulldown assays. (C) GST pulldown assay using Xenopus embryo lysates that were either uninjected, injected with Sox5 wildtype mRNA or Sox5 deletion constructs shown in (B). Lysate was incubated with glutathione beads coupled to GST or Smad1-GST. (D) Western blot showing the expression levels of the Sox5 deletion proteins used in (C). (E) ChIP analysis examining Smad1 enrichment on eEF1α, Id3 and Msx1 promoters. Smad1 enrichment is significantly diminished when Sox5 is depleted. Embryo lysates were immunoprecipitated with antibodies against the epitope tag in Smad1 (myc). Fold Enrichment is obtained by normalizing to uninjected sample. Error bars represent S.E.M. of three independent biological experiments. Id3 p=0.0207, Msx1 p=0.0274. (F) Western blot of embryo lysates injected for Smad1 Chromatin immunoprecipitation (Figure 4E,F). Three biological replicates were used and the expression for factors was analyzed for each experiment. (G) Western blot of embryo lysates injected for Sox5 Chromatin immunoprecipitation (Figure 4G,H). Three biological replicates were used and the expression for factors was analyzed for each experiment.
	sox5 (SRY-box 5) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 2, lateral view, animal up.
	sox5 (SRY-box 5) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 6, lateral view, animal up.
	sox5 (SRY-box 5) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 13, dorsal view, anterior down.
	sox5 (SRY-box 5) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 18, dorsal view, anterior down.
	sox5 (SRY-box 5) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 25, lateral view, anterior right, dorsal up.
	sox6 (SRY-box 6) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 20, dorsal view, anterior down.
	sox6 (SRY-box 6) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 28, lateral view, anterior right, dorsal up.

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