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.

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.

3. Eddy, 1988

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

6. ‘9 + 2’

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

8. Horowitz, 2005
RelativeLuciferaseActivity

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

26. SPZ1 and hHF-4

27. reinhardtii