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Transcriptional Regulation of Genes Encoding Axonemal Central Apparatus Proteins. Ruth Jaimez Melgoza

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Dra. Ruth Jaimez Melgoza
Dra. Ruth Jaimez Melgoza

This document describes a study examining the transcriptional regulation of genes encoding axonemal central apparatus proteins. The researchers analyzed the promoter region of the PF20 gene, which encodes proteins important for sperm motility. They tested luciferase reporter activity driven by varying lengths of the PF20 promoter region in different cell types, and in the presence of potential transcription factors like PF20s, hFH-4, and SPZ1. They found the 0.2-0.3 kb region of the PF20 promoter responded most strongly to hFH-4, suggesting this region contains a response element for this transcription factor. Further mutational analysis of this region could help map the specific response element.

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"Transcriptional Regulation of Genes Encoding Axonemal Central Apparatus Proteins" Center for Research on Reproduction and Women’s Health January 11, 2006. Ruth Jaimez Melgoza Ph.D.
• Spermatogenesis is a complex process that leads to the formation of male gametes. • Sperm motility is essential for fertilization in vivo. • Flagellum is composed of a number of cytoskeletal elements whose proper assembly is critical for subsequent motility.
Eddy, 1988
Studies of central apparatus ultraestructure • Chlamydomonas. Goodenough and Heuser, 1985. • Sperm of squid. Linck et al., 1981. • Sperm of rat. Olson and Linck, 1977. • Cilia of Tetrahymena. Warner, 1981. • Cilia of mussel. Warner and Satir, 1974. reinhardtii
Brokaw, 1972; Smith and Lefebvre, 1997
‘9 + 2’
• Many genes and their expression in proteins have been characterized. • PF20 gene encodes two mayor transcripts (2.5 and 1.4 kb). This transcripts are expressed in different patterns during spermatogenesis yielding proteins of 71 and 35 kDa • Knockout mice in PF6, SPAG6, PF20 are infertile, with respiratory abnormalities and hydrocephaly. Zhang, 2002, 2004, 2005; Horowitz, 2005
Horowitz, 2005 RelativeLuciferaseActivity
Abnormal flagella • Relative common in fertile humans (12-25 %). • Infertile men (18-30 %). • Immotile cilia syndrome or Kartagener’s syndrome. Male sterility, chronic respiratory problems. – Lack of dynein arms. – Lack of outer dynein arms. – Disorganized axoneme. – Abnormal short spokes. – Lack of the central sheath. – Genes mutated. (Hunter, 1986; Pelfrey, 1982; Afzelius, 1976, 1979, 1981).
Methods • Cell culture (CHO, MDCK, BEAS-2B, GC-4) • Making Constructs • PCR • Cloning • Transformation • Transfection • DNA extraction and purification • LUC assay • Western Blotting
PF20L serie. Promotor. (2.0-0.1 Kb) pGL3 Luciferase reporter vector PF20S. Transcription factor Mammalian expression system Renilla reporter vector. SPZ1 and hHF-4
1 tgcatttgaa ttttcaaaac attaatgatc aggtacatat agaacatatg ttcagttgta 61 attgggtaat acatcttggg tgagtttaaa atttgaaatc tactacgtgg aagtaaatag 121 attagcaact cagtttctat cttggtcatt tggaagcttg acataatgga catacataca 181 tatatatata tatatatatt ttgatgcatg tgtgtaaaat atctcttttc catttgcaca 241 cctttgaata aacatctctg acttctctac cacacatagt taatactgac tgaagtttgt 301 atttcaggac tctgtcaaac catttcttat gtcaagcgct ggccatgtct aaaagaatta 361 tgccttacta aataacaatt cctatatatt aagctcttta aaatattaaa tttgggcttg 421 tcacagtcac aatattttaa gcatatttta tttacattac ttattgaaaa tatctggtgt 481 actggaaatg ataagaatct ttacatattt tactttccat tttttttcca ttattttata 541 tatagtaagg cggctcttat aagtctttat aagggaaccg aaagccgtag ttcaaggagc 601 ctaactttaa gtaagacaat tagtaagcac tagaatataa acctgtttgt agaagatgat 661 acattacatt caaagacaac aggctttgta gccgacagaa taaacattag aagctccaag 721 ggatggtcct ccatgtgtct cttgtttctg cctgtccttt gaggcatcag cagtgcctga 781 ccattccgca cccaggccac ttttcagggt tcagtttaca gtcaaccatt tgaaagatga 841 agccagatgt aagccaatgg atcccccatt ttcagaattc ctcttctgta tttttgcttc 901 tttgcattga atgggcactc agaacgtttg ctgtgagcaa actcttgaac tcttcagata 961 ccattgtgac ctttattcta agacgcaaaa ccaagtgtaa tggaagcagc tcatattcct 1021 tatctctgta tgctctgaat aggtggctaa tgctctggtg atttatagct aataacaaca 1081 cttgaggcag gtgacattat cctcgggcct aggtgtggct gtagacagga agtaatgcaa 1141 acggattcca accttcaggt tcttagtcac ccctctagaa gttaccagta tttttttttt 1201 aattaaataa acgtgagctt ttatttaatt atcaaccaga gttcattaag gaatgtagaa 1261 ttaaaaaaga aaaagaatct gaaagatggt cttaatgatg cctatgtata tacactatat 1321 aaaccagaag tttctatcac agcaatgaga cacatgattg gacaaattag tcagttctct 1381 cctgccccct ccttcttgtc ctcctcgcta ttccttctct ttcccctttc catctcctcc 1441 ctgcccttct tccctgctcc cttccctctg aggtagctca ttagttctta actacatgcc 1501 ctatcccaaa tatcacctat tagactttat gttagggtca taggtagctc ataatttttt 1561 atggatgtga gtttacctca gcaaaaatca atggtataat ttgatacgac tgggactatt 1621 ccaagtccgt gtgtaggaag ccgcggtcca aacagatgtc cacataaaag aattaaaaga 1681 ttgctagaaa ggatgtaata aaggtgagca cccctacttt tcattgctag gggtttaagg 1741 agctcctcaa aaatggttcc tagaactttc cccacttctt ctagaaatac agagacaagt 1801 atcccacctt ttcttaagtc ttcccttccc cagaaatatt tgaggttctc tacacatcta 1861 aaaattgagg accgactttg gcatgtgata aacaaagagt aaaggaaagc aatgaagaaa 1921 aaggaagccg aaaagaatgg cgttaaagca ggcaagcaag caagcaagca agcaagcaag 1981 caagcaagca agcaagcaag caagcaagca aacaaaatga aaacaaaaac acccaaacaa 2041 gtagcttgtg taaatgtaat atgagtccct ggttctgggc ttcaggtctg cagtccaagc 2101 ttcctggggc gtctggggta actaggcaac gcgttgccgt agtaactgcg gacgcgcaga 2161 gggcttgacc ggggcctttt ggtgccccgc tggtcggggt gaggggtccc gggttct*cgc 2221 gagagt*cgcg acgccagtcg gag*ggg*ca*cc *gaggggccgc agttg*caagc atggctgctc 2281 cgtctggggg tcccg Sequence of mouse pf20 promoter Putative transcription factor Binding sites. HNF-3beta, SOX17, CREB, SPZ1, TBP, Hunchback Asterisks indicate potencial trans cripcion start sites (5’ RACE) Horowitz, 2005
HFh-4 forkhead family member • Hepatocyte nuclear factor-3/forkhead homologue 4. • Play important roles in development, cell-specific gene expression and oncogenesis. • Has a unique spatial and temporal pattern of expression during embryogenesis restricted to the lung (bronchial and bronchiolar epithelia), choriod plexus, oviduct and testes. (Brody, 1997; Weigel, 1989; Hackett, 1995; Lim, 1997)
SPZ1 a bHLH-Zip • Basic helix-loop-helix family of transcription factors • Regulatory networks of many developmental pathways. • SPZ1 is expressed specifically in testis and epididymis. • Crucial role in spermatogenesis by regulating cell proliferation or differentiation through binding to specific DNA sequences. (Hsu, 2001; Srivastava, 1997; Rawls, 1997; Murre, 1994; Feder, 1993).
Experimental Design LUC activity Pf20L promoter (Kb) 2.0 1.5 0.5 0.3 0.2 0.1 Cell types CHO MDCK GC-4 BEAS-2B Transcription factors PF20s hFH-4 SPZ1 Reporter Expression
0 10 20 30 40 50 60 70 80 90 1000 1 2 3 4 5 6 7 8 9 pf20s 0 10 20 30 40 50 0 50 100 150 200 pgl3 2.0 1.5 0.5 0.3 0.2 0.1 CHO GC-4 MDCK BEAS pgl3 2.0 1.5 0.5 0.3 0.2 0.1 pgl3 2.0 1.5 0.5 0.3 0.2 0.1 pgl3 2.0 1.5 0.5 0.3 0.2 0.1 P target empty PF20S Luciferase activity (fold compared to control) * * * * * * * * * * * * *
0 5 10 15 20 25 30 35 hFH4 pgl3 2.0 1.5 0.5 0.3 0.2 0.1 CHO GC-4 MDCK BEAS pgl3 2.0 1.5 0.5 0.3 0.2 0.1 pgl3 2.0 1.5 0.5 0.3 0.2 0.1 pgl3 2.0 1.5 0.5 0.3 0.2 0.1 PcDNA3 empty hFH4 Luciferase activity (fold compared to control) 0 1 2 3 4 5 0 2 4 6 8 10 12 14 0 50 100 150 200 250 300 350 * * *
0 5 10 15 20 25 30 35 SPZ1 pgl3 2.0 1.5 0.5 0.3 0.2 0.1 CHO GC-4 MDCK BEAS pgl3 2.0 1.5 0.5 0.3 0.2 0.1 pgl3 2.0 1.5 0.5 0.3 0.2 0.1 pgl3 2.0 1.5 0.5 0.3 0.2 0.1 PcDNA3 empty SPZ1 Luciferase activity (fold compared to control) 0 2 4 6 8 10 12 14 16 0 20 40 60 80 100 120 140 0 1 2 3 4 5 6 7
Conclusions • LUC activity depends of transcription factor, cell environment and promoter region. • Beas-2B cells are the best host. • There is a PF20s response element between 0.1 and 0.2 kb. • The 0.2 and 0.3 kb region looks like the most promising target for identifying a hFH-4 response element. • “SPZ1 looks unimpressive” (Dr. Strauss). • hFH-4 is a known transcription factor and doesn’t have a known target gene.
Next? • Generate random mutants in that area to pin point the response element and map it.
1 tgcatttgaa ttttcaaaac attaatgatc aggtacatat agaacatatg ttcagttgta 61 attgggtaat acatcttggg tgagtttaaa atttgaaatc tactacgtgg aagtaaatag 121 attagcaact cagtttctat cttggtcatt tggaagcttg acataatgga catacataca 181 tatatatata tatatatatt ttgatgcatg tgtgtaaaat atctcttttc catttgcaca 241 cctttgaata aacatctctg acttctctac cacacatagt taatactgac tgaagtttgt 301 atttcaggac tctgtcaaac catttcttat gtcaagcgct ggccatgtct aaaagaatta 361 tgccttacta aataacaatt cctatatatt aagctcttta aaatattaaa tttgggcttg 421 tcacagtcac aatattttaa gcatatttta tttacattac ttattgaaaa tatctggtgt 481 actggaaatg ataagaatct ttacatattt tactttccat tttttttcca ttattttata 541 tatagtaagg cggctcttat aagtctttat aagggaaccg aaagccgtag ttcaaggagc 601 ctaactttaa gtaagacaat tagtaagcac tagaatataa acctgtttgt agaagatgat 661 acattacatt caaagacaac aggctttgta gccgacagaa taaacattag aagctccaag 721 ggatggtcct ccatgtgtct cttgtttctg cctgtccttt gaggcatcag cagtgcctga 781 ccattccgca cccaggccac ttttcagggt tcagtttaca gtcaaccatt tgaaagatga 841 agccagatgt aagccaatgg atcccccatt ttcagaattc ctcttctgta tttttgcttc 901 tttgcattga atgggcactc agaacgtttg ctgtgagcaa actcttgaac tcttcagata 961 ccattgtgac ctttattcta agacgcaaaa ccaagtgtaa tggaagcagc tcatattcct 1021 tatctctgta tgctctgaat aggtggctaa tgctctggtg atttatagct aataacaaca 1081 cttgaggcag gtgacattat cctcgggcct aggtgtggct gtagacagga agtaatgcaa 1141 acggattcca accttcaggt tcttagtcac ccctctagaa gttaccagta tttttttttt 1201 aattaaataa acgtgagctt ttatttaatt atcaaccaga gttcattaag gaatgtagaa 1261 ttaaaaaaga aaaagaatct gaaagatggt cttaatgatg cctatgtata tacactatat 1321 aaaccagaag tttctatcac agcaatgaga cacatgattg gacaaattag tcagttctct 1381 cctgccccct ccttcttgtc ctcctcgcta ttccttctct ttcccctttc catctcctcc 1441 ctgcccttct tccctgctcc cttccctctg aggtagctca ttagttctta actacatgcc 1501 ctatcccaaa tatcacctat tagactttat gttagggtca taggtagctc ataatttttt 1561 atggatgtga gtttacctca gcaaaaatca atggtataat ttgatacgac tgggactatt 1621 ccaagtccgt gtgtaggaag ccgcggtcca aacagatgtc cacataaaag aattaaaaga 1681 ttgctagaaa ggatgtaata aaggtgagca cccctacttt tcattgctag gggtttaagg 1741 agctcctcaa aaatggttcc tagaactttc cccacttctt ctagaaatac agagacaagt 1801 atcccacctt ttcttaagtc ttcccttccc cagaaatatt tgaggttctc tacacatcta 1861 aaaattgagg accgactttg gcatgtgata aacaaagagt aaaggaaagc aatgaagaaa 1921 aaggaagccg aaaagaatgg cgttaaagca ggcaagcaag caagcaagca agcaagcaag 1981 caagcaagca agcaagcaag caagcaagca aacaaaatga aaacaaaaac acccaaacaa 2041 gtagcttgtg taaatgtaat atgagtccct ggttctgggc ttcaggtctg cagtccaagc 2101 ttcctggggc gtctggggta actaggcaac gcgttgccgt agtaactgcg gacgcgcaga 2161 gggcttgacc ggggcctttt ggtgccccgc tggtcggggt gaggggtccc gggttct*cgc 2221 gagagt*cgcg acgccagtcg gag*ggg*ca*cc *gaggggccgc agttg*caagc atggctgctc 2281 cgtctggggg tcccg Sequence of mouse pf20 promoter Putative transcription factor Binding sites. HNF-3beta, SOX17, CREB, SPZ1, TBP, Hunchback Asterisks indicate potencial trans cripcion start sites (5’ RACE) Horowitz, 2005
Jerome F. Strauss III, M.D; Ph.D. § Rita Leite (cell culture, transfections, transformations, luciferase assay, interpreter) § Zhibing Zhang (constructs, PCR, cloning, western blot) § Jennifer Wood (DNA purification, mutagenesis) § Hongyan Wang (mutagenesis) § Amy Brown
Transcriptional Regulation of Genes Encoding Axonemal Central Apparatus Proteins. Ruth Jaimez Melgoza
Making constructs Design primer and request to CORE Topo TA cloning PCR Check products in agarose gel 1.5 %, gel extraction 10 µl Topo cloning reaction Transforming TOP10F’, pick up white colonies DNA extraction miniprep (100 µl elution) ECORI digestion Sequence CORE correct orientation Double digestion XhoI, KpnI Put products in Agarose gel 1% DNA gel extraction (10 µl elution) LigationTransforming DH5α DNA extraction (100 µl elution) Double digestion XhoI, KpnI to confirm ligation Put products in Agarose gel 1% picture. Sequence CORE correct orientation If the sequence is correct do transforming In DH5α and maxiprep
Transcriptional Regulation of Genes Encoding Axonemal Central Apparatus Proteins. Ruth Jaimez Melgoza
SPZ1 and hHF-4
reinhardtii

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Transcriptional Regulation of Genes Encoding Axonemal Central Apparatus Proteins. Ruth Jaimez Melgoza

  • 1. "Transcriptional Regulation of Genes Encoding Axonemal Central Apparatus Proteins" Center for Research on Reproduction and Women’s Health January 11, 2006. Ruth Jaimez Melgoza Ph.D.
  • 2. • Spermatogenesis is a complex process that leads to the formation of male gametes. • Sperm motility is essential for fertilization in vivo. • Flagellum is composed of a number of cytoskeletal elements whose proper assembly is critical for subsequent motility.
  • 4. Studies of central apparatus ultraestructure • Chlamydomonas. Goodenough and Heuser, 1985. • Sperm of squid. Linck et al., 1981. • Sperm of rat. Olson and Linck, 1977. • Cilia of Tetrahymena. Warner, 1981. • Cilia of mussel. Warner and Satir, 1974. reinhardtii
  • 5. Brokaw, 1972; Smith and Lefebvre, 1997
  • 7. • Many genes and their expression in proteins have been characterized. • PF20 gene encodes two mayor transcripts (2.5 and 1.4 kb). This transcripts are expressed in different patterns during spermatogenesis yielding proteins of 71 and 35 kDa • Knockout mice in PF6, SPAG6, PF20 are infertile, with respiratory abnormalities and hydrocephaly. Zhang, 2002, 2004, 2005; Horowitz, 2005
  • 9. Abnormal flagella • Relative common in fertile humans (12-25 %). • Infertile men (18-30 %). • Immotile cilia syndrome or Kartagener’s syndrome. Male sterility, chronic respiratory problems. – Lack of dynein arms. – Lack of outer dynein arms. – Disorganized axoneme. – Abnormal short spokes. – Lack of the central sheath. – Genes mutated. (Hunter, 1986; Pelfrey, 1982; Afzelius, 1976, 1979, 1981).
  • 10. Methods • Cell culture (CHO, MDCK, BEAS-2B, GC-4) • Making Constructs • PCR • Cloning • Transformation • Transfection • DNA extraction and purification • LUC assay • Western Blotting
  • 11. PF20L serie. Promotor. (2.0-0.1 Kb) pGL3 Luciferase reporter vector PF20S. Transcription factor Mammalian expression system Renilla reporter vector. SPZ1 and hHF-4
  • 12. 1 tgcatttgaa ttttcaaaac attaatgatc aggtacatat agaacatatg ttcagttgta 61 attgggtaat acatcttggg tgagtttaaa atttgaaatc tactacgtgg aagtaaatag 121 attagcaact cagtttctat cttggtcatt tggaagcttg acataatgga catacataca 181 tatatatata tatatatatt ttgatgcatg tgtgtaaaat atctcttttc catttgcaca 241 cctttgaata aacatctctg acttctctac cacacatagt taatactgac tgaagtttgt 301 atttcaggac tctgtcaaac catttcttat gtcaagcgct ggccatgtct aaaagaatta 361 tgccttacta aataacaatt cctatatatt aagctcttta aaatattaaa tttgggcttg 421 tcacagtcac aatattttaa gcatatttta tttacattac ttattgaaaa tatctggtgt 481 actggaaatg ataagaatct ttacatattt tactttccat tttttttcca ttattttata 541 tatagtaagg cggctcttat aagtctttat aagggaaccg aaagccgtag ttcaaggagc 601 ctaactttaa gtaagacaat tagtaagcac tagaatataa acctgtttgt agaagatgat 661 acattacatt caaagacaac aggctttgta gccgacagaa taaacattag aagctccaag 721 ggatggtcct ccatgtgtct cttgtttctg cctgtccttt gaggcatcag cagtgcctga 781 ccattccgca cccaggccac ttttcagggt tcagtttaca gtcaaccatt tgaaagatga 841 agccagatgt aagccaatgg atcccccatt ttcagaattc ctcttctgta tttttgcttc 901 tttgcattga atgggcactc agaacgtttg ctgtgagcaa actcttgaac tcttcagata 961 ccattgtgac ctttattcta agacgcaaaa ccaagtgtaa tggaagcagc tcatattcct 1021 tatctctgta tgctctgaat aggtggctaa tgctctggtg atttatagct aataacaaca 1081 cttgaggcag gtgacattat cctcgggcct aggtgtggct gtagacagga agtaatgcaa 1141 acggattcca accttcaggt tcttagtcac ccctctagaa gttaccagta tttttttttt 1201 aattaaataa acgtgagctt ttatttaatt atcaaccaga gttcattaag gaatgtagaa 1261 ttaaaaaaga aaaagaatct gaaagatggt cttaatgatg cctatgtata tacactatat 1321 aaaccagaag tttctatcac agcaatgaga cacatgattg gacaaattag tcagttctct 1381 cctgccccct ccttcttgtc ctcctcgcta ttccttctct ttcccctttc catctcctcc 1441 ctgcccttct tccctgctcc cttccctctg aggtagctca ttagttctta actacatgcc 1501 ctatcccaaa tatcacctat tagactttat gttagggtca taggtagctc ataatttttt 1561 atggatgtga gtttacctca gcaaaaatca atggtataat ttgatacgac tgggactatt 1621 ccaagtccgt gtgtaggaag ccgcggtcca aacagatgtc cacataaaag aattaaaaga 1681 ttgctagaaa ggatgtaata aaggtgagca cccctacttt tcattgctag gggtttaagg 1741 agctcctcaa aaatggttcc tagaactttc cccacttctt ctagaaatac agagacaagt 1801 atcccacctt ttcttaagtc ttcccttccc cagaaatatt tgaggttctc tacacatcta 1861 aaaattgagg accgactttg gcatgtgata aacaaagagt aaaggaaagc aatgaagaaa 1921 aaggaagccg aaaagaatgg cgttaaagca ggcaagcaag caagcaagca agcaagcaag 1981 caagcaagca agcaagcaag caagcaagca aacaaaatga aaacaaaaac acccaaacaa 2041 gtagcttgtg taaatgtaat atgagtccct ggttctgggc ttcaggtctg cagtccaagc 2101 ttcctggggc gtctggggta actaggcaac gcgttgccgt agtaactgcg gacgcgcaga 2161 gggcttgacc ggggcctttt ggtgccccgc tggtcggggt gaggggtccc gggttct*cgc 2221 gagagt*cgcg acgccagtcg gag*ggg*ca*cc *gaggggccgc agttg*caagc atggctgctc 2281 cgtctggggg tcccg Sequence of mouse pf20 promoter Putative transcription factor Binding sites. HNF-3beta, SOX17, CREB, SPZ1, TBP, Hunchback Asterisks indicate potencial trans cripcion start sites (5’ RACE) Horowitz, 2005
  • 13. HFh-4 forkhead family member • Hepatocyte nuclear factor-3/forkhead homologue 4. • Play important roles in development, cell-specific gene expression and oncogenesis. • Has a unique spatial and temporal pattern of expression during embryogenesis restricted to the lung (bronchial and bronchiolar epithelia), choriod plexus, oviduct and testes. (Brody, 1997; Weigel, 1989; Hackett, 1995; Lim, 1997)
  • 14. SPZ1 a bHLH-Zip • Basic helix-loop-helix family of transcription factors • Regulatory networks of many developmental pathways. • SPZ1 is expressed specifically in testis and epididymis. • Crucial role in spermatogenesis by regulating cell proliferation or differentiation through binding to specific DNA sequences. (Hsu, 2001; Srivastava, 1997; Rawls, 1997; Murre, 1994; Feder, 1993).
  • 15. Experimental Design LUC activity Pf20L promoter (Kb) 2.0 1.5 0.5 0.3 0.2 0.1 Cell types CHO MDCK GC-4 BEAS-2B Transcription factors PF20s hFH-4 SPZ1 Reporter Expression
  • 16. 0 10 20 30 40 50 60 70 80 90 1000 1 2 3 4 5 6 7 8 9 pf20s 0 10 20 30 40 50 0 50 100 150 200 pgl3 2.0 1.5 0.5 0.3 0.2 0.1 CHO GC-4 MDCK BEAS pgl3 2.0 1.5 0.5 0.3 0.2 0.1 pgl3 2.0 1.5 0.5 0.3 0.2 0.1 pgl3 2.0 1.5 0.5 0.3 0.2 0.1 P target empty PF20S Luciferase activity (fold compared to control) * * * * * * * * * * * * *
  • 17. 0 5 10 15 20 25 30 35 hFH4 pgl3 2.0 1.5 0.5 0.3 0.2 0.1 CHO GC-4 MDCK BEAS pgl3 2.0 1.5 0.5 0.3 0.2 0.1 pgl3 2.0 1.5 0.5 0.3 0.2 0.1 pgl3 2.0 1.5 0.5 0.3 0.2 0.1 PcDNA3 empty hFH4 Luciferase activity (fold compared to control) 0 1 2 3 4 5 0 2 4 6 8 10 12 14 0 50 100 150 200 250 300 350 * * *
  • 18. 0 5 10 15 20 25 30 35 SPZ1 pgl3 2.0 1.5 0.5 0.3 0.2 0.1 CHO GC-4 MDCK BEAS pgl3 2.0 1.5 0.5 0.3 0.2 0.1 pgl3 2.0 1.5 0.5 0.3 0.2 0.1 pgl3 2.0 1.5 0.5 0.3 0.2 0.1 PcDNA3 empty SPZ1 Luciferase activity (fold compared to control) 0 2 4 6 8 10 12 14 16 0 20 40 60 80 100 120 140 0 1 2 3 4 5 6 7
  • 19. Conclusions • LUC activity depends of transcription factor, cell environment and promoter region. • Beas-2B cells are the best host. • There is a PF20s response element between 0.1 and 0.2 kb. • The 0.2 and 0.3 kb region looks like the most promising target for identifying a hFH-4 response element. • “SPZ1 looks unimpressive” (Dr. Strauss). • hFH-4 is a known transcription factor and doesn’t have a known target gene.
  • 20. Next? • Generate random mutants in that area to pin point the response element and map it.
  • 21. 1 tgcatttgaa ttttcaaaac attaatgatc aggtacatat agaacatatg ttcagttgta 61 attgggtaat acatcttggg tgagtttaaa atttgaaatc tactacgtgg aagtaaatag 121 attagcaact cagtttctat cttggtcatt tggaagcttg acataatgga catacataca 181 tatatatata tatatatatt ttgatgcatg tgtgtaaaat atctcttttc catttgcaca 241 cctttgaata aacatctctg acttctctac cacacatagt taatactgac tgaagtttgt 301 atttcaggac tctgtcaaac catttcttat gtcaagcgct ggccatgtct aaaagaatta 361 tgccttacta aataacaatt cctatatatt aagctcttta aaatattaaa tttgggcttg 421 tcacagtcac aatattttaa gcatatttta tttacattac ttattgaaaa tatctggtgt 481 actggaaatg ataagaatct ttacatattt tactttccat tttttttcca ttattttata 541 tatagtaagg cggctcttat aagtctttat aagggaaccg aaagccgtag ttcaaggagc 601 ctaactttaa gtaagacaat tagtaagcac tagaatataa acctgtttgt agaagatgat 661 acattacatt caaagacaac aggctttgta gccgacagaa taaacattag aagctccaag 721 ggatggtcct ccatgtgtct cttgtttctg cctgtccttt gaggcatcag cagtgcctga 781 ccattccgca cccaggccac ttttcagggt tcagtttaca gtcaaccatt tgaaagatga 841 agccagatgt aagccaatgg atcccccatt ttcagaattc ctcttctgta tttttgcttc 901 tttgcattga atgggcactc agaacgtttg ctgtgagcaa actcttgaac tcttcagata 961 ccattgtgac ctttattcta agacgcaaaa ccaagtgtaa tggaagcagc tcatattcct 1021 tatctctgta tgctctgaat aggtggctaa tgctctggtg atttatagct aataacaaca 1081 cttgaggcag gtgacattat cctcgggcct aggtgtggct gtagacagga agtaatgcaa 1141 acggattcca accttcaggt tcttagtcac ccctctagaa gttaccagta tttttttttt 1201 aattaaataa acgtgagctt ttatttaatt atcaaccaga gttcattaag gaatgtagaa 1261 ttaaaaaaga aaaagaatct gaaagatggt cttaatgatg cctatgtata tacactatat 1321 aaaccagaag tttctatcac agcaatgaga cacatgattg gacaaattag tcagttctct 1381 cctgccccct ccttcttgtc ctcctcgcta ttccttctct ttcccctttc catctcctcc 1441 ctgcccttct tccctgctcc cttccctctg aggtagctca ttagttctta actacatgcc 1501 ctatcccaaa tatcacctat tagactttat gttagggtca taggtagctc ataatttttt 1561 atggatgtga gtttacctca gcaaaaatca atggtataat ttgatacgac tgggactatt 1621 ccaagtccgt gtgtaggaag ccgcggtcca aacagatgtc cacataaaag aattaaaaga 1681 ttgctagaaa ggatgtaata aaggtgagca cccctacttt tcattgctag gggtttaagg 1741 agctcctcaa aaatggttcc tagaactttc cccacttctt ctagaaatac agagacaagt 1801 atcccacctt ttcttaagtc ttcccttccc cagaaatatt tgaggttctc tacacatcta 1861 aaaattgagg accgactttg gcatgtgata aacaaagagt aaaggaaagc aatgaagaaa 1921 aaggaagccg aaaagaatgg cgttaaagca ggcaagcaag caagcaagca agcaagcaag 1981 caagcaagca agcaagcaag caagcaagca aacaaaatga aaacaaaaac acccaaacaa 2041 gtagcttgtg taaatgtaat atgagtccct ggttctgggc ttcaggtctg cagtccaagc 2101 ttcctggggc gtctggggta actaggcaac gcgttgccgt agtaactgcg gacgcgcaga 2161 gggcttgacc ggggcctttt ggtgccccgc tggtcggggt gaggggtccc gggttct*cgc 2221 gagagt*cgcg acgccagtcg gag*ggg*ca*cc *gaggggccgc agttg*caagc atggctgctc 2281 cgtctggggg tcccg Sequence of mouse pf20 promoter Putative transcription factor Binding sites. HNF-3beta, SOX17, CREB, SPZ1, TBP, Hunchback Asterisks indicate potencial trans cripcion start sites (5’ RACE) Horowitz, 2005
  • 22. Jerome F. Strauss III, M.D; Ph.D. § Rita Leite (cell culture, transfections, transformations, luciferase assay, interpreter) § Zhibing Zhang (constructs, PCR, cloning, western blot) § Jennifer Wood (DNA purification, mutagenesis) § Hongyan Wang (mutagenesis) § Amy Brown
  • 24. Making constructs Design primer and request to CORE Topo TA cloning PCR Check products in agarose gel 1.5 %, gel extraction 10 µl Topo cloning reaction Transforming TOP10F’, pick up white colonies DNA extraction miniprep (100 µl elution) ECORI digestion Sequence CORE correct orientation Double digestion XhoI, KpnI Put products in Agarose gel 1% DNA gel extraction (10 µl elution) LigationTransforming DH5α DNA extraction (100 µl elution) Double digestion XhoI, KpnI to confirm ligation Put products in Agarose gel 1% picture. Sequence CORE correct orientation If the sequence is correct do transforming In DH5α and maxiprep

Editor's Notes

  • #14: Spz1, a novel bHLH-Zip protein, is specifically expressed in testis. Hsu SH, Shyu HW, Hsieh-Li HM, Li H. Institute of Molecular Biology, Academia Sinica, 11529, Taipei, Taiwan. We isolated a novel bHLH-Zip gene designated Spz1 from a mouse testis cDNA library. Spz1 is expressed specifically in the testis and epididymis. Immunofluorescence staining detected Spz1 protein in the nuclei of LFG6 Leydig cells. The ability of Spz1 protein to bind to the bHLH consensus-binding site, the E-box, was confirmed by EMSA, and a 9-bp asymmetric target site was identified by random selection and PCR amplification. Hormonal regulation of Spz1 was investigated and downregulation of Spz1 expression by testosterone and retinoic acid was found. This nuclear transcription factor may play a crucial role in spermatogenesis by regulating cell proliferation or differentiation through binding to specific DNA sequences like other bHLH-Zip molecules.
  • #15: Spz1, a novel bHLH-Zip protein, is specifically expressed in testis. Hsu SH, Shyu HW, Hsieh-Li HM, Li H. Institute of Molecular Biology, Academia Sinica, 11529, Taipei, Taiwan. We isolated a novel bHLH-Zip gene designated Spz1 from a mouse testis cDNA library. Spz1 is expressed specifically in the testis and epididymis. Immunofluorescence staining detected Spz1 protein in the nuclei of LFG6 Leydig cells. The ability of Spz1 protein to bind to the bHLH consensus-binding site, the E-box, was confirmed by EMSA, and a 9-bp asymmetric target site was identified by random selection and PCR amplification. Hormonal regulation of Spz1 was investigated and downregulation of Spz1 expression by testosterone and retinoic acid was found. This nuclear transcription factor may play a crucial role in spermatogenesis by regulating cell proliferation or differentiation through binding to specific DNA sequences like other bHLH-Zip molecules.