XB-ART-50982J Biol Chem. August 14, 2015; 290 (33): 20273-83.
JmjC Domain-containing Protein 6 (Jmjd6) Derepresses the Transcriptional Repressor Transcription Factor 7-like 1 (Tcf7l1) and Is Required for Body Axis Patterning during Xenopus Embryogenesis.
Tcf7l1 (also known as Tcf3) is a bimodal transcription factor that plays essential roles in embryogenesis and (embryonic and adult) stem cells. On the one hand, Tcf7l1 works as transcriptional repressor, via the recruitment of Groucho-related transcriptional co-repressors, to repress the transcription of Wnt target genes, and on the other, it activates Wnt target genes when Wnt-activated β-catenin interacts with it. However, how its activity is modulated is not well understood. Here, we demonstrate that a JmjC-domain containing protein, Jmjd6, interacts with Tcf7l and derepresses Tcf7l. We show that Jmjd6 binds to a region of Tcf7l1 that is also responsible for Groucho interaction, thus making it possible that Jmjd6 binding displaces the Groucho transcriptional co-repressor from Tcf7l1. Moreover, we manifest that Jmjd6 antagonizes the repression effect of Tcf7l1 on target gene transcription and is able to enhance the β-catenin-induced gene activation, and vice versa, inhibition of Jmjd6 activity compromises the gene activation in both cells and Xenopus early embryos. We also show that jmjd6 is both maternally and zygotically transcribed during Xenopus embryogenesis. Loss of Jmjd6 function causes defects in anterio-posterior (A-P) body axis formation and downregulation of genes that are involved in A-P axis patterning. The results elucidate a novel mechanism underlying the regulation of Tcf7l1 activity and the regulation of embryonic body axis formation.
PubMed ID: 26157142
PMC ID: PMC4536435
Article link: J Biol Chem.
Genes referenced: ag1 cer1 chrd.1 dkk1 foxa4 gsc jarid2 jmjd6 nodal1 nodal3.1 odc1 otx2 sia1 tbl1x tcf7l1 xpo1
Morpholinos referenced: jmjd6 MO1 tcf7l1 MO2
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|FIGURE 1. Conservation of Jmjd6 in multicellular organisms. A, sequence similarity analysis for Jmjd6 proteins in human (JMJD6. Accession number: NP_055982.2), Xenopus laevis (XlJmjd6a. Accession number: NP_001085948.1), zebrafish (DrJmjd6. Accession number: NM_170761.2), fruitfly (DmJmjd6. Accession number: NP_651026.1), and in Trichoplax adhaerens (TaJmjd6. Accession number: XM_002107775.1). B, phylogenetic tree shows the evolutionary distances between the Jmjd6 proteins in different species. Sequence comparison was made using the online program (http://www.ebi.ac.uk/Tools/msa/clustalo/) with default settings.|
|FIGURE 2. Jmjd6 interacts with Tcf7l1. A, Co-IP detection of the interaction between overexpressed Jmjd6 and Tcf7l1 in HEK293T cells. B, overexpressed Tcf7l1 precipitated endogenous JMJD6 in HEK293T cells. C, nuclear co-localization of Jmjd6 and Tcf7l1 in HEK293T cells, as detected by immunofluorescence staining. DAPI staining reveals nuclei. D and E, test of the knockdown efficiency of miJMJD6-1. Transfection of the plasmid for miJMJD6-1 did not cause a significant reduction in both the transcript (D) and protein (E) levels of JMJD6 in HEK293T cells. F and G, test of the knockdown efficiency of miJMJD6-2 and the effect of JMJD6 knockdown on the transcription of TCF7l1 and β-Catenin. Transfection of the plasmid for miJMJD6-2 resulted in a significant decrease in both the transcript (F) and protein (G) levels of JMJD6. Meanwhile, the transcription of TCF7L1 and β-Catenin was not affected in response to the efficient JMJD6 knockdown. GAPDH in (D) and (F) was used as a loading control for RT-PCR detection of the JMJD6 transcript, and β-Actin in (E) and (G) was used as a loading control for the detection of JMJD6 protein with immunoblotting. RT-: transcription without reverse transcriptase. H, detection of the effect of endogenous JMJD6 knockdown on the interaction between exogenous Jmjd6 and Tcf7l1.|
|FIGURE 3. Mapping of the region in Tcf7l1 protein for Jmjd6 interaction. A, domain structure, the construction of the deletion mutants of Tcf7l1 used in the study, and their binding affinities to Jmjd6 after Co-IP assays. +++: Very strong binding; ++: strong binding; +: weak binding; +/-: trace or no binding. B and C, Tcf7l1 deletion mutants exhibited different binding affinities to Jmjd6 as shown by Co-IP assays. D, immunofluorescence showed that the aa 1-323 region of Tcf7l1 primarily distributed in the cytosol, whereas addition of an NLS to the region re-localized it into nucleus. E, addition of a nuclear localization Downloaded from http://www.jbc.org/ at Childrens Hospital Medical Center on July 28, 2015 13 signal (NLS) to the 1-323 region of Tcf7l1 did not increase its binding affinity to Jmjd6.|
|FIGURE 4. Jmjd6 enhances Wnt signaling. A, domain structure and the construction of the deletion mutants of Xenopus laevis Jmjd6a used in the study. B, subcellular localizations of the Jmjd6 mutants revealed by immunofluorescence. Jmjd6 without the C-terminal region lost the nuclear localization and distributed ubiquitously throughout the whole cell. C, Jmjd6 enhanced strongly β-catenin-stimulated reporter activity, whereas the deletion mutants of Jmjd6 did not show such an enhancing effect. Error bars represent the standard error of the mean (SEM) of four replicates. **p<0.01. NS: not significant. D, Tcf7l1 overexpression showed strong repressive effect on the β-catenin-stimulated reporter activity, whereas co-transfection of the plasmid for Jmjd6 led to a significant alleviation of the repressive effect. Error bars represent the SEM of four replicates. *p<0.05, **p<0.01. E, Jmjd6 is required for the β-catenin-stimulated transcription. In HEK293T cells, β-catenin stimulated strongly the reporter activity. Simultaneous knockdown of endogenous JMJD6 compromised the stimulation of the reporter activity, which was then rescued by the co-transfection of the plasmid for Xenopus laevis Jmjd6a. Error bars represent the SEM of four replicates. *p<0.05, **p<0.01.|
|FIGURE 5. The spatio-temporal expression patterns of jmjd6a and jmjd6b during the embryogenesis of Xenopus laevis. A, temporal expression of jmjd6a (Accession number: NM_001092479) and jmjd6b (Accession number: NM_001087045) in different stages of embryos detected with RT-PCR. Expression of odc was used as a loading control. RT-: transcription without reverse transcriptase. B and C, spatial expression patterns of jmjd6a (B) and jmjd6b (C) detected with whole mount in situ hybridization. Embryo stages are indicated at the top of each panel. a: anterior view, with the dorsal at the top of the panel; ba: branchial arch; bl: blastopore lip; br: brain; ey: eye; fb: forebrain; hb: hindbrain; l: lateral view, with the anterior to the left; la: lateral view, with animal pole at the top; ld: lateral-dorsal view, with the animal pole at the top; mb: midbrain; np: neural plate; nt: neural tube; ov: otic vesicle.|
|FIGURE 6. Jmjd6 is required for Xenopus embryonic development. A, Co-IP detection of the interaction between Jmjd6 and Tcf7l1 in Xenopus embryos. Each 300 pg of mRNA for tagged Jmjd6 or/and Tcf7l1 was injected into 2-cell stage embryos. At gastrula stage, embryos were collected and subjected to Co-IP assays. B, design of an antisense morpholino oligonucleotide, Jmjd6MO, against both the transcripts of jmjd6a and jmjd6b. Translational start site of each transcript is underlined. C, immunoblotting detection of the knockdown efficiency of the Jmjd6MO, as compared with the standard control morpholino (ctrlMO). One nanogram of mRNA for HA-tagged Jmjd6a that contains the Jmjd6MO binding site was injected alone or co-injected together with 20 ng of either ctrlMO or Jmjd6MO into Xenopus embryos. Embryos were collected at gastrula stage and subjected to immunoblotting. D, typical Xenopus embryos after injection of ctrlMO or different doses of Jmjd6MO, showing severe developmental defect in response to Jmjd6 knockdown. The defect became stronger in response to a higher dose of injected Jmjd6MO. Embryos are shown in lateral view, with the anterior being placed to the right of each panel. E, numbers and percentages of total and defect embryos in the experiments in (D). F, different changes in the expression of genes that are involved in anterio-posterior body axis patterning in gastrula (for cer1, dkk1, gsc and chrd) and neurula (for xag2) embryos after injection of 20 ng of ctrlMO or Jmjd6MO. v: vegetal view, with the dorsal being orientated to the top of each panel; a: anterior view, with the dorsal being placed to the top. G, numbers and percentages of embryos showing different changes in gene expression after Jmjd6 knockdown.|
|FIGURE 7. Jmjd6 mediates Tcf7l1-regulated gene transcription. A, microarray result shows that Jmjd6 knockdown in Xenopus laevis embryos enhanced the repressive effect of Tcf7l1 on the transcription of Wnt/β-catenin target genes involved in germ layer induction and patterning, but not enhance the effect on other genes. B, in Xenopus late blastula embryos, injection of the mRNA (200 pg) for dominant-negative Tcf7l1 that lacks the β-Catenin binding site led to the repression of Wnt target genes. Co-injection of Jmjd6a mRNA (1 ng) reversed the repression effect. All embryos are shown in dorsal view, with the animal pole being orientated to the top. C, numbers and percentages of embryos showing different changes in gene expression in response to injection of dnTcf7l1 and Jmjd6 mRNA, as shown in the experiments in (B). D, knockdown of endogenous Tcf7l1 in Xenopus late blastula embryos via injection of Tcf7l1MO (40 ng) upregulated the Wnt target gene, whereas simultaneous injection of Jmjd6a mRNA (1 ng) enhanced the upregulation of gene expression and even caused ectopic transcription. In the left and middle panels, embryos are shown as dorsal view, with the animal pole being placed to the top. In the right panel, animal pole is shown to view ectopic gene expression. E, numbers and percentages of embryos showing different changes in gene expression in response to injection of Tcf7l1MO and Jmjd6 mRNA, as shown in the experiments in (D). F, injection of Tcf7l1MO (40 ng) caused an increase in the expression of the Wnt target gene in Xenopus blastula embryos. However, the increase was severely weakened when Jmjd6MO (20 ng) was injected at the same time. G, numbers and percentages of embryos showing different changes in gene expression in response to injection of Tcf7l1MO and Jmjd6MO, as shown in the experiments in (F).|
|FIGURE 8. A model demonstrating the function of Jmjd6 in regulating the activity of Tcf7l1. In the absence of Wnt activation, Tcf7l1 recruits Groucho-related proteins, functions as a transcriptional repressor to inhibit the transcription of Wnt target genes. Otherwise, Wnt activation leads to β-catenin displacement of Groucho from Tcf7l1 and turns Tcf7l1 into an activator. Jmjd6 interacts with Tcf7l1 in the Groucho-binding domain, leading to alleviation of the repression activity of Tcf7l1 even in the absence of Wnt activation. This interaction enhances the transcriptional activation that is stimulated by Wnt signaling. See text for details.|
|jmjd6 (jumonji domain containing 6) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 3, lateral view, animal up.|
|jmjd6 (jumonji domain containing 6) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 20, lateral view, anterior left, dorsal up.|
|jmjd6 (jumonji domain containing 6) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 28, lateral view, anterior left, dorsal up.|