Foxd4l1.1 Negatively Regulates Chordin Transcription in Neuroectoderm of Xenopus Gastrula
Abstract
:1. Introduction
2. Materials and Methods
2.1. Ethics Statement
2.2. Cloning of Chrd Genomic DNA
2.3. DNA and RNA Preparation
2.4. Promoter Constructs
2.5. Embryo Injection and Explants Culture
2.6. RT-PCR
2.7. Luciferase Assays
2.8. Smad2- and Smad3-Truncated Protein Construct
2.9. Co-Immunoprecipitation and Western Blotting
2.10. Site-Directed Mutagenesis
2.11. Chromatin Immunoprecipitation
2.12. ChIP-Sequencing Analysis
2.13. eGFP Fluorescence
2.14. Statistical Analysis
3. Results
3.1. Foxd4l1.1 Inhibits Chrd and Dorsal Target Gene Expression
3.2. Foxd4l1.1 Abolishes Mesoderm and Ectoderm Specific Reporter Genes Expression
3.3. Chrd Promoter Contains Foxd4l1.1 Response Element (FRE)
3.4. Foxd4l1.1 Physically Interacts with Smad2/3 via C-Ternimal MH2 Domain
3.5. Foxd4l1.1 Binds to FRE1 and Blocks Smad2/3 Binding to AREs (ARE1 and ARE2) within the Chrd Promoter
4. Discussion
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Acknowledgments
Conflicts of Interest
References
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Constructs | Primer Name | Sequence (5′ to 3′) |
---|---|---|
Upstream primer | Chrd (-2250)_F | GGGGCTAGCGAACGATACTTCAAGGACAAT |
Chrd (-2206)_F | GGGGCTAGCCCACTATCCCCACTAAGATGA | |
Chrd (-2155)_F | GGGGCTAGCAGGCATACTTTGGTTTGTGTGT | |
Chrd (-2075)_F | GGGGCTAGCTGCAAGTCGAGATCATTGTGT | |
Chrd (-1862)_F | GGGGCTAGCAAGAACACAGTGCCAGGCACT | |
Chrd (-1473)_F | GGGGCTAGCCAGTAGGTTAGATGAACTACT | |
Chrd (-790)_F | GGGGCTAGCACACTCTCTACCCCAATTCT | |
Chrd (-386)_F | GGGGCTAGCCTTGACGGCTTTGTTTGCTT | |
Chrd (-198)_F | GGGGCTAGCGTGTGGGTACAGAGCAACAA | |
Downstream primer | Chrd (-2250)_R | GGGCTCGAGTTTTGTGGTTCCAAACGTTCT |
Gene | Primer Name | Sequence (5′ to 3′) | Cycles |
---|---|---|---|
Chrd | Chrd_F | TTAGAGAGGAGAGCAACTCGGGCAAT | 25 |
Chrd_R | GTGCTCCTGTTGCGAAACTCTACAGA | ||
Noggin | Noggin_F | AGTTGCAGATGTGGCTCT | 27 |
Noggin_R | AGTCCAAGAGTCTGAGCA | ||
Gsc | Gsc_F | GCTGATTCCACCAGTGCCTCACCAG | 30 |
Gsc_R | GGTCCTGTGCCTCCTCCTCCTCCTG | ||
Xbra | Xbra_F | GGATCGTTATCACCTCTG | 25 |
Xbra_R | GTGTAGTCTGTAGCAGCA | ||
Ventx1.1 | Ventx1.1_F | CCTTCAGCATGGTTCAACAG | 28 |
Ventx1.1_R | CATCCTTCTTCCTTGGCATCTCCT | ||
Bmp4 | BMP4_F | GCATGTACGGATAAGTCGATC | 25 |
BMP4_R | GATCTCAGACTCAACGGCAC | ||
ODC | ODC_F | GTCAATGATGGAGTGTATGGATC | 25 |
ODC_R | TCCATTCCGCTCTCCTGAGCAC |
Construct | Primer Name | Sequence (5′ to 3′) | Cycles |
---|---|---|---|
Smad2-ΔC | hSmad2_(N-terminal)-F | CCCGAATTCCATGTCGTCCATCTTGCCATT | 25 |
hSmad2_(Linker)-R | GGGCTCGAGTCAAAATGCAGGTTCTGAGT | ||
Smad2-C-ter | hSmad2_(C-terminal)-F | GGTCTCGAGTTATGACATGCTTGAGCAACGC | 25 |
hSmad2_(C-terminal)-R | GGTCTCGAGTTATGACATGCTTGAGCAACGC | ||
Smad3-ΔC | hSmad3_(N-terminal)-F | CCCGAATTCCATGTCGTCCATCCTGCCTTTC | 25 |
hSmad3_(Linker)-R | AAGTCTAGACCGGCTCGCAGTAGGTAAC | ||
Smad3-C-ter | hSmad3_(C-terminal)-F | CTTGAATTCCTACTCAGAACCTGCATTTTGG | 25 |
hSmad3_(C-terminal)-R | CCATCTAGACTAAGACACACTGGAACAGC |
Mutated Site | Primer Name | Sequence (5′ to 3′) | Cycles |
---|---|---|---|
FRE1 | Chrd(−2250)mFRE1_F | TTAAACAGTATAAGGGGATGCTAAAAACACAG | 20 |
Chrd(−2250)mFRE1_R | CTGTGTTTTTAGCATCCCCTTATACTGTTTAA | ||
FRE2 | Chrd(−2250)mFRE2_F | GTCCTGGCATATGAGGGGATTCAGAGCTATCCT | 20 |
Chrd(−2250)mFRE2_R | AGGATAGCTCTGAATCCCCTCATATGCCAGGAC | ||
FRE3 | Chrd(−2250)mFRE3_F | TTCAATCCTTAGCAGGGAATTCCCTCATCTTTC | 20 |
Chrd(−2250)mFRE3_R | GAAAGATGAGGGAATTCCCTGCTAAGGATTGAA |
Primer Name | Sequence (5′ to 3′) | Cycles | |
---|---|---|---|
Chrd (Foxd4l1.1_ChIP) | Chrd(FRE)_F | GTTGCTTCTGTTTTCCACCT | 25 |
Chrd(FRE)_R | GTCTGGCATATCTAGCAGGTC | ||
Chrd (Internal control 1) | Chrd(C1)_F | TGCGCCGACTAAGTTTCCT | 25(qPCR 40) |
Chrd(C1)_R | ATTAGTGACCCATGGCAGG | ||
Chrd (Internal control 2) | Chrd(C2)_F | ACACTCTCTACCCCAATTCT | 25(qPCR 40) |
Chrd(C2)_R | CAGAATGGCATGTGGGAAGA | ||
Chrd (Smad2 ChIP) | Chrd(AREs)_F | CGATACTTCAAGGACAATTG | 24 |
Chrd(AREs)_R | AGGTGGAAAACAGAAGCAAC | ||
Chrd (Smad3 ChIP) | Chrd(AREs)_F | CGATACTTCAAGGACAATTG | 24 |
Chrd(AREs)_R | AGGTGGAAAACAGAAGCAAC | ||
Ventx2.1 (External control) | Ventx2.1_F | CTACAGCACTAGCACTGACT | 28 |
Ventx2.1_R | AGAAAGCTGGAGTTTGGCTGC |
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Kumar, V.; Goutam, R.S.; Umair, Z.; Park, S.; Lee, U.; Kim, J. Foxd4l1.1 Negatively Regulates Chordin Transcription in Neuroectoderm of Xenopus Gastrula. Cells 2021, 10, 2779. https://doi.org/10.3390/cells10102779
Kumar V, Goutam RS, Umair Z, Park S, Lee U, Kim J. Foxd4l1.1 Negatively Regulates Chordin Transcription in Neuroectoderm of Xenopus Gastrula. Cells. 2021; 10(10):2779. https://doi.org/10.3390/cells10102779
Chicago/Turabian StyleKumar, Vijay, Ravi Shankar Goutam, Zobia Umair, Soochul Park, Unjoo Lee, and Jaebong Kim. 2021. "Foxd4l1.1 Negatively Regulates Chordin Transcription in Neuroectoderm of Xenopus Gastrula" Cells 10, no. 10: 2779. https://doi.org/10.3390/cells10102779