Kdm6b confers Tfdp1 with the competence to activate p53 signalling in regulating palatogenesis

Epigenetic regulation plays extensive roles in diseases and development. Disruption of epigenetic regulation not only increases the risk of cancer, but can also cause various developmental defects. However, it is still unclear how epigenetic regulators coordinate with tissue-specific regulatory factors during morphogenesis of specific organs. Using palatogenesis as a model, we reveal the functional significance of Kdm6b, a H3K27me3 demethylase, in regulating embryonic development. Our study shows that Kdm6b plays an essential role in neural crest development, and loss of Kdm6b disturbs p53 pathway-mediated activity, leading to complete cleft palate along with cell proliferation and differentiation defects. Furthermore, activity of H3K27me3 on the promoter of p53 is precisely controlled by Kdm6b, and Ezh2 in regulating p53 expression in cranial neural crest cells. More importantly, Kdm6b renders chromatin accessible to the transcription factor Tfdp1, which binds to the promoter of p53 along with Kdm6b to specifically activate p53 expression during palatogenesis. Collectively our results highlight the important role of the epigenetic regulator Kdm6b and how it cooperates with Tfdp1 to achieve its functional specificity in regulating p53 expression, and further provide mechanistic insights into the epigenetic regulatory network during organogenesis.

Loss of Kdm6b in CNC-derived cells disturbs p53 pathway-mediated activity 212 In order to identify the downstream targets of Kdm6b in the palatal mesenchyme, we performed RNA-seq 213 analysis of palatal tissue at E12.5. The results showed that more genes were downregulated than 214 upregulated in the palatal mesenchyme in Wnt1-Cre;Kdm6b fl/fl mice ( Figure 3A), which is consistent with 215 the function of Kdm6b in removing the repressive mark H3K27me3. We further used Ingenuity Pathway Analysis (IPA) and Gene Ontology (GO) analysis to analyze the pathways that were most disturbed in the 217 palatal mesenchyme in Kdm6b mutant mice. Surprisingly, both analyses indicated that pathways involving 218 p53 might be disturbed in the palatal mesenchyme in Wnt1-Cre;Kdm6b fl/fl mice ( Figure 3B-C).

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The tumor suppressor p53 plays prominent roles in regulating DNA damage response, including arresting 220 cell growth for DNA repair, directing cellular senescence, and activating apoptosis (Mijit et al. 2020).

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Mutation of p53 is a major cause of cancer development (Williams and Schumacher 2016   Collectively, these results suggest that p53's function in DNA damage response is impaired in Wnt1-        Figure 7N). This data further indicated that p53 is a direct downstream target 420 of Tfdp1.    Mouse heads were dissected and fixed in 95% EtOH overnight at room temperature. Staining was 553 performed as previously described (Rigueur and Lyons 2014). Briefly, 95% EtOH was replaced with 100% acetone for 2 days and then samples were incubated in Alcian blue solution (80% EtOH, 20% glacial 555 acetic acid, and 0.03% (w/v) Alcian blue 8GX (Sigma, A3157) for 1-3 days. Samples were then destained 556 with 70% EtOH and incubated in 95% EtOH overnight. After incubation, samples were pre-cleared with 557 1% KOH and then incubated in Alizarin red solution (0.005% (w/v) Alizarin red (Sigma, A5533) in 1% 558 (w/v) KOH) for 2-5 days. After clearing samples with 1% KOH, they were stored in 100% glycerol until 559 analysis.  Immunofluorescence assay 573 Cryosections and paraffin sections prepared as described above were used for immunofluorescence assays.

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Sections were dried for 2 hours at 55℃. Paraffin sections were deparaffinized and rehydrated before 575 antigen retrieval. Heat mediated antigen retrieval was used to process sections (Vector, H-3300) and then samples were blocked for 1 hour in blocking buffer at room temperature (PerkinElmer, FP1020). Primary 577 antibodies diluted in blocking buffer were incubated with samples overnight at 4℃. After washing with 578 PBST (0.1% Tween20 in 1xPBS), samples were then incubated with secondary antibodies at room 579 temperature for 2 hours. DAPI (Sigma, D9542) was used for nuclear staining. All images were acquired 580 using Leica DMI 3000B and Keyence BZ-X710/810 microscopes. Detailed information about primary 581 and secondary antibodies is listed in Table S1. Cryosections and paraffin sections were prepared as described above, and cell death was analyzed using 590 TUNEL staining according to the manufacturer's protocol (Invitrogen, C10245). Images were acquired 591 using Keyence BZ-X710/810 microscope.  Table S2. 599 Palate samples from control and Wnt1-Cre;Kdm6b fl/fl mice were collected at E12.5 for RNA isolation with  Table S3.  Then the sample was washed twice with PBS, resuspended in Hypotonic Buffer and incubated at 4℃ for 619 10 minutes to obtain nuclei, which were then resuspended in Digestion Buffer. After chromatin was sheared to 100-500 bp fragments using Shearing Cocktail, 10 µg chromatin with H3K27me3 antibody 621 (CST 9733s, 1:50), DP1 antibody (Abcam ab124678, 1:10) or Immunoglobulin G negative control (2 µg) 622 was added to Column Conditioning Buffer to make up the final volume of 1000 µl. Immunoprecipitation 623 (IP) slurry was mixed thoroughly and incubated on an end-to-end rotor for 1 hour at 4 ℃. An equivalent 624 amount of chromatin was set as an input. After 1 hour incubation, IP slurry was purified using 625 Chromatrap® spin column at room temperature and chromatin was eluted using ChIP-seq elution buffer.

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Chromatin sample and input were further incubated at 65 ℃ overnight to reverse cross-linking. DNA was 627 purified with Chromatrap® DNA purification column after proteinase K treatment. ChIP eluates, negative 628 control and input were assayed using real-time qPCR. Primers were designed using the promoter region 629 of p53. Detailed information is available in Table S3.

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For western blot, palate tissue was dissected from control and Wnt1-Cre;Kdm6b fl/fl mice at E13.5. The 632 tissue sample was lysed using RIPA buffer (Cell Signaling, 9806) with protease inhibitor (Thermo Fisher 633 Scientific, A32929) for 20 minutes on ice followed by centrifugation at 4℃ to remove tissue debris.

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After washing with TBST, membrane was incubated with secondary antibody for 2 hours at room 639 temperature and signals were detected using SuperSignal West Femto (Thermo Fisher Scientific, 34094) 640 and Azure 300 (Azure Biosystems).

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For CoIP, palate tissue was dissected from control mice at E13.5 and 60-80 mg tissue was combined as 642 one sample for each replicate. After lysing using RIPA buffer, 60 µl of the protein extract was mixed with sample buffer and boiled at 98 ℃ to serve as input. The remaining protein extract was incubated with 644 primary antibody at 4 ℃ overnight. Protein G beads from GE Healthcare (GE Healthcare, 10280243) were 645 used to purify the target protein and then the protein sample was analyzed using western blot. Detailed 646 information about primary and secondary antibodies is listed in Table S4.  Table S3. siRNA sequence and plasmid information are listed in Table S5 656 and Table S6. Known transcription factor biding motifs were analyzed by HOMER (Zhang et al. 2008;Heinz et al. 2010). 665 Quality files for sequencing is listed in Table S7 666 Cell differentiation assay 667 Palatal tissue was dissected from control and Wnt1-Cre;Kdm6b fl/fl mice at E13.5 and cultured as previously