Skip to main content
PMC home page
Molecular and Cellular Biology logoLink to Molecular and Cellular Biology
. 1996 Feb;16(2):513–521. doi: 10.1128/mcb.16.2.513

Distinct cis-acting elements mediate clock, light, and developmental regulation of the Neurospora crassa eas (ccg-2) gene.

D Bell-Pedersen 1, J C Dunlap 1, J J Loros 1
PMCID: PMC231029  PMID: 8552078

Abstract

The Neurospora crassa eas (ccg-2) gene, which encodes a fungal hydrophobin, is transcriptionally regulated by the circadian clock. In addition, eas (ccg-2) is positively regulated by light and transcripts accumulate during asexual development. To sort out the basis of this complex regulation, deletion analyses of the eas (ccg-2) promoter were carried out to localize the cis-acting elements mediating clock, light, and developmental control. The primary sequence determinants of a positive activating clock element (ACE) were found to reside in a 45-bp region, just upstream from the TATA box. Using a novel unregulated promoter/reporter system developed for this study, we show that a 68-bp sequence encompassing the ACE is sufficient to confer clock regulation on the eas (ccg-2) gene. Electrophoretic mobility shift assays using the ACE reveal factors present in N. crassa protein extracts that recognize and bind specifically to DNA containing this element. Separate regions of the eas (ccg-2) promoter involved in light induction and developmental control are identified and shown not to be required for clock-regulated expression of eas (ccg-2). The distinct nature of the ACE validates its use as a tool for the identification of upstream regulatory factors involved in clock control of gene expression.

Full Text

The Full Text of this article is available as a PDF (470.3 KB).

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Anderson S. L., Kay S. A. Functional dissection of circadian clock- and phytochrome-regulated transcription of the Arabidopsis CAB2 gene. Proc Natl Acad Sci U S A. 1995 Feb 28;92(5):1500–1504. doi: 10.1073/pnas.92.5.1500. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Anderson S. L., Teakle G. R., Martino-Catt S. J., Kay S. A. Circadian clock- and phytochrome-regulated transcription is conferred by a 78 bp cis-acting domain of the Arabidopsis CAB2 promoter. Plant J. 1994 Oct;6(4):457–470. doi: 10.1046/j.1365-313x.1994.6040457.x. [DOI] [PubMed] [Google Scholar]
  3. Aronin N., Sagar S. M., Sharp F. R., Schwartz W. J. Light regulates expression of a Fos-related protein in rat suprachiasmatic nuclei. Proc Natl Acad Sci U S A. 1990 Aug;87(15):5959–5962. doi: 10.1073/pnas.87.15.5959. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Aronson B. D., Johnson K. A., Dunlap J. C. Circadian clock locus frequency: protein encoded by a single open reading frame defines period length and temperature compensation. Proc Natl Acad Sci U S A. 1994 Aug 2;91(16):7683–7687. doi: 10.1073/pnas.91.16.7683. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Aronson B. D., Johnson K. A., Loros J. J., Dunlap J. C. Negative feedback defining a circadian clock: autoregulation of the clock gene frequency. Science. 1994 Mar 18;263(5153):1578–1584. doi: 10.1126/science.8128244. [DOI] [PubMed] [Google Scholar]
  6. Arpaia G., Loros J. J., Dunlap J. C., Morelli G., Macino G. Light induction of the clock-controlled gene ccg-1 is not transduced through the circadian clock in Neurospora crassa. Mol Gen Genet. 1995 Apr 20;247(2):157–163. doi: 10.1007/BF00705645. [DOI] [PubMed] [Google Scholar]
  7. Arpaia G., Loros J. J., Dunlap J. C., Morelli G., Macino G. The interplay of light and the circadian clock. Independent dual regulation of clock-controlled gene ccg-2(eas). Plant Physiol. 1993 Aug;102(4):1299–1305. doi: 10.1104/pp.102.4.1299. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Beever R. E., Dempsey G. P. Function of rodlets on the surface of fungal spores. Nature. 1978 Apr 13;272(5654):608–610. doi: 10.1038/272608a0. [DOI] [PubMed] [Google Scholar]
  9. Bell-Pedersen D., Dunlap J. C., Loros J. J. The Neurospora circadian clock-controlled gene, ccg-2, is allelic to eas and encodes a fungal hydrophobin required for formation of the conidial rodlet layer. Genes Dev. 1992 Dec;6(12A):2382–2394. doi: 10.1101/gad.6.12a.2382. [DOI] [PubMed] [Google Scholar]
  10. Berlin V., Yanofsky C. Isolation and characterization of genes differentially expressed during conidiation of Neurospora crassa. Mol Cell Biol. 1985 Apr;5(4):849–855. doi: 10.1128/mcb.5.4.849. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Borello U., Ceccarelli E., Giuliano G. Constitutive, light-responsive and circadian clock-responsive factors compete for the different l box elements in plant light-regulated promoters. Plant J. 1993 Oct;4(4):611–619. doi: 10.1046/j.1365-313x.1993.04040611.x. [DOI] [PubMed] [Google Scholar]
  12. Bowler C., Chua N. H. Emerging themes of plant signal transduction. Plant Cell. 1994 Nov;6(11):1529–1541. doi: 10.1105/tpc.6.11.1529. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Brann M. R., Cohen L. V. Diurnal expression of transducin mRNA and translocation of transducin in rods of rat retina. Science. 1987 Jan 30;235(4788):585–587. doi: 10.1126/science.3101175. [DOI] [PubMed] [Google Scholar]
  14. Carattoli A., Cogoni C., Morelli G., Macino G. Molecular characterization of upstream regulatory sequences controlling the photoinduced expression of the albino-3 gene of Neurospora crassa. Mol Microbiol. 1994 Sep;13(5):787–795. doi: 10.1111/j.1365-2958.1994.tb00471.x. [DOI] [PubMed] [Google Scholar]
  15. Corrochano L. M., Lauter F. R., Ebbole D. J., Yanofsky C. Light and developmental regulation of the gene con-10 of Neurospora crassa. Dev Biol. 1995 Jan;167(1):190–200. doi: 10.1006/dbio.1995.1016. [DOI] [PubMed] [Google Scholar]
  16. Dunlap J. C. Closely watched clocks: molecular analysis of circadian rhythms in Neurospora and Drosophila. Trends Genet. 1990 May;6(5):159–165. doi: 10.1016/0168-9525(90)90151-u. [DOI] [PubMed] [Google Scholar]
  17. Dunlap J. C. Genetic analysis of circadian clocks. Annu Rev Physiol. 1993;55:683–728. doi: 10.1146/annurev.ph.55.030193.003343. [DOI] [PubMed] [Google Scholar]
  18. Feinberg A. P., Vogelstein B. A technique for radiolabeling DNA restriction endonuclease fragments to high specific activity. Anal Biochem. 1983 Jul 1;132(1):6–13. doi: 10.1016/0003-2697(83)90418-9. [DOI] [PubMed] [Google Scholar]
  19. Hastings M. Circadian rhythms: peering into the molecular clockwork. J Neuroendocrinol. 1995 May;7(5):331–340. doi: 10.1111/j.1365-2826.1995.tb00766.x. [DOI] [PubMed] [Google Scholar]
  20. Kaldenhoff R., Russo V. E. Promoter analysis of the bli-7/eas gene. Curr Genet. 1993 Nov;24(5):394–399. doi: 10.1007/BF00351847. [DOI] [PubMed] [Google Scholar]
  21. Kornhauser J. M., Nelson D. E., Mayo K. E., Takahashi J. S. Regulation of jun-B messenger RNA and AP-1 activity by light and a circadian clock. Science. 1992 Mar 20;255(5051):1581–1584. doi: 10.1126/science.1549784. [DOI] [PubMed] [Google Scholar]
  22. Lauter F. R., Russo V. E., Yanofsky C. Developmental and light regulation of eas, the structural gene for the rodlet protein of Neurospora. Genes Dev. 1992 Dec;6(12A):2373–2381. doi: 10.1101/gad.6.12a.2373. [DOI] [PubMed] [Google Scholar]
  23. Lauter F. R., Yanofsky C. Day/night and circadian rhythm control of con gene expression in Neurospora. Proc Natl Acad Sci U S A. 1993 Sep 1;90(17):8249–8253. doi: 10.1073/pnas.90.17.8249. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Lavery D. J., Schibler U. Circadian transcription of the cholesterol 7 alpha hydroxylase gene may involve the liver-enriched bZIP protein DBP. Genes Dev. 1993 Oct;7(10):1871–1884. doi: 10.1101/gad.7.10.1871. [DOI] [PubMed] [Google Scholar]
  25. Lehrach H., Diamond D., Wozney J. M., Boedtker H. RNA molecular weight determinations by gel electrophoresis under denaturing conditions, a critical reexamination. Biochemistry. 1977 Oct 18;16(21):4743–4751. doi: 10.1021/bi00640a033. [DOI] [PubMed] [Google Scholar]
  26. Loros J. J., Denome S. A., Dunlap J. C. Molecular cloning of genes under control of the circadian clock in Neurospora. Science. 1989 Jan 20;243(4889):385–388. doi: 10.1126/science.2563175. [DOI] [PubMed] [Google Scholar]
  27. Loros J. J., Dunlap J. C. Neurospora crassa clock-controlled genes are regulated at the level of transcription. Mol Cell Biol. 1991 Jan;11(1):558–563. doi: 10.1128/mcb.11.1.558. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Mittag M., Lee D. H., Hastings J. W. Circadian expression of the luciferin-binding protein correlates with the binding of a protein to the 3' untranslated region of its mRNA. Proc Natl Acad Sci U S A. 1994 Jun 7;91(12):5257–5261. doi: 10.1073/pnas.91.12.5257. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Roberts A. N., Yanofsky C. Genes expressed during conidiation in Neurospora crassa: characterization of con-8. Nucleic Acids Res. 1989 Jan 11;17(1):197–214. doi: 10.1093/nar/17.1.197. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Rusak B., McNaughton L., Robertson H. A., Hunt S. P. Circadian variation in photic regulation of immediate-early gene mRNAs in rat suprachiasmatic nucleus cells. Brain Res Mol Brain Res. 1992 Jun;14(1-2):124–130. doi: 10.1016/0169-328x(92)90019-8. [DOI] [PubMed] [Google Scholar]
  31. Sargent M. L., Briggs W. R. The effects of light on a circadian rhythm of conidiation in neurospora. Plant Physiol. 1967 Nov;42(11):1504–1510. doi: 10.1104/pp.42.11.1504. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Sargent M. L., Briggs W. R., Woodward D. O. Circadian nature of a rhythm expressed by an invertaseless strain of Neurospora crassa. Plant Physiol. 1966 Oct;41(8):1343–1349. doi: 10.1104/pp.41.8.1343. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Sassone-Corsi P. Rhythmic transcription and autoregulatory loops: winding up the biological clock. Cell. 1994 Aug 12;78(3):361–364. doi: 10.1016/0092-8674(94)90415-4. [DOI] [PubMed] [Google Scholar]
  34. Sommer T., Chambers J. A., Eberle J., Lauter F. R., Russo V. E. Fast light-regulated genes of Neurospora crassa. Nucleic Acids Res. 1989 Jul 25;17(14):5713–5723. doi: 10.1093/nar/17.14.5713. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Stehle J. H., Foulkes N. S., Molina C. A., Simonneaux V., Pévet P., Sassone-Corsi P. Adrenergic signals direct rhythmic expression of transcriptional repressor CREM in the pineal gland. Nature. 1993 Sep 23;365(6444):314–320. doi: 10.1038/365314a0. [DOI] [PubMed] [Google Scholar]
  36. Vollmer S. J., Yanofsky C. Efficient cloning of genes of Neurospora crassa. Proc Natl Acad Sci U S A. 1986 Jul;83(13):4869–4873. doi: 10.1073/pnas.83.13.4869. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Wang Z., Deak M., Free S. J. A cis-acting region required for the regulated expression of grg-1, a Neurospora glucose-repressible gene. Two regulatory sites (CRE and NRS) are required to repress grg-1 expression. J Mol Biol. 1994 Mar 18;237(1):65–74. doi: 10.1006/jmbi.1994.1209. [DOI] [PubMed] [Google Scholar]
  38. Wuarin J., Schibler U. Expression of the liver-enriched transcriptional activator protein DBP follows a stringent circadian rhythm. Cell. 1990 Dec 21;63(6):1257–1266. doi: 10.1016/0092-8674(90)90421-a. [DOI] [PubMed] [Google Scholar]
  39. Yarden O., Plamann M., Ebbole D. J., Yanofsky C. cot-1, a gene required for hyphal elongation in Neurospora crassa, encodes a protein kinase. EMBO J. 1992 Jun;11(6):2159–2166. doi: 10.1002/j.1460-2075.1992.tb05275.x. [DOI] [PMC free article] [PubMed] [Google Scholar]

Articles from Molecular and Cellular Biology are provided here courtesy of Taylor & Francis

RESOURCES