"Transcriptional Regulation of Genes Encoding Axonemal Central Apparatus Proteins"

Center for Research on Reproductionand 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

Re

lati

ve

Lu

cif

era

se

Ac

tiv

ity

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 factorMammalian 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 factorBinding sites. HNF-3beta,SOX17, CREB, SPZ1, TBP,Hunchback

Asterisks indicate potencial transcripcion 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 DesignLUC activity

Pf20L promoter(Kb)

2.01.50.50.30.20.1

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 40 500 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

hFH4pgl3 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

SPZ1pgl3 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 factorBinding sites. HNF-3beta,SOX17, CREB, SPZ1, TBP,Hunchback

Asterisks indicate potencial transcripcion 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

Making constructs

Design primer and request to CORE Topo TA cloning PCR

Check products in agarosegel 1.5 %, gel extraction10 l

Topo cloningreaction

Transforming TOP10F’,pick up white colonies

DNA extraction miniprep(100 l elution)

ECORI digestion

Sequence COREcorrect orientation

Double digestionXhoI, KpnI

Put products in Agarose gel 1%

DNA gel extraction(10 l elution)

LigationTransformingDH5

DNA extraction(100 l elution)

Double digestionXhoI, KpnI to confirmligation

Put products in Agarose gel 1%picture.

Sequence COREcorrect orientation

If the sequence is correct do transformingIn DH5 and maxiprep

SPZ1 and hHF-4

reinhardtii