WHITE COLLAR-1, a Multifunctional NeurosporaProtein Involved in the Circadian Feedback Loops, Light Sensing, and Transcription Repression of wc-2
2003; Elsevier BV; Volume: 278; Issue: 6 Linguagem: Inglês
10.1074/jbc.m209592200
ISSN1083-351X
AutoresPing Cheng, Yuhong Yang, Lixin Wang, Qiyang He, Yi Liu,
Tópico(s)Photoreceptor and optogenetics research
ResumoWHITE COLLAR-1 (WC-1) and WC-2, the two PAS domain-containing transcription factors, are the positive elements of the circadian feedback loops in Neurospora. In addition, both proteins are essential components for the light input of various blue light responses, including the light entrainment of the circadian clock. Recently, we identified WC-1 as the blue light photoreceptor responsible for these light responses. In this study, we show that the formation of the FRQ-WC complex in vivo, a step critical in closing the circadian negative feedback loop, requires WC-1. In addition, we show that WC-1 negatively regulates the expression ofwc-2 at the level of the transcription, forming another interacting loop. In a wc-1 mutant, we demonstrate that there is alternative protein initiation of WC-1, and the requirements of WC-1 for the light induction of frq and other genes differ significantly, suggesting the existence of different WC complexes in the cell. Consistent with this interpretation, our results show that there are at least two different types of WC-1/WC-2 complexes in vivo, and that the larger WC-1/WC-2 complex contains more than one WC-1 molecule. Using a series ofwc-1 mutants, we show that the WC-1 PASC domain and its C-terminal region are essential for the formation of the WC-1/WC-2 complex. Functional analyses reveal that the DNA-binding domain of WC-1 is required only for the activation of frq in the dark and not for the light function of the protein, confirming that WC-1 is a multifunctional protein with separable protein domains. WHITE COLLAR-1 (WC-1) and WC-2, the two PAS domain-containing transcription factors, are the positive elements of the circadian feedback loops in Neurospora. In addition, both proteins are essential components for the light input of various blue light responses, including the light entrainment of the circadian clock. Recently, we identified WC-1 as the blue light photoreceptor responsible for these light responses. In this study, we show that the formation of the FRQ-WC complex in vivo, a step critical in closing the circadian negative feedback loop, requires WC-1. In addition, we show that WC-1 negatively regulates the expression ofwc-2 at the level of the transcription, forming another interacting loop. In a wc-1 mutant, we demonstrate that there is alternative protein initiation of WC-1, and the requirements of WC-1 for the light induction of frq and other genes differ significantly, suggesting the existence of different WC complexes in the cell. Consistent with this interpretation, our results show that there are at least two different types of WC-1/WC-2 complexes in vivo, and that the larger WC-1/WC-2 complex contains more than one WC-1 molecule. Using a series ofwc-1 mutants, we show that the WC-1 PASC domain and its C-terminal region are essential for the formation of the WC-1/WC-2 complex. Functional analyses reveal that the DNA-binding domain of WC-1 is required only for the activation of frq in the dark and not for the light function of the protein, confirming that WC-1 is a multifunctional protein with separable protein domains. PER-ARNT-SIM WHITE COLLAR-1 FREQUENCY albino-3 vivid amino acid light-regulated element quinic acid nuclear localization signal constant light Endogenous circadian clocks control a wide variety of daily physiological, behavioral, cellular, and biochemical activities in most eukaryotic and certain prokaryotic organisms. The circadian oscillators are networks of positive and negative elements that form the core circadian feedback loops generating the basic circadian rhythmicity (1Dunlap J.C. Cell. 1999; 96: 271-290Abstract Full Text Full Text PDF PubMed Scopus (2325) Google Scholar,2Young M.W. Kay S.A. Nat. Rev. Genet. 2001; 2: 702-715Crossref PubMed Scopus (919) Google Scholar). The positive elements of the loop activate the transcription of the negative elements, whereas the negative elements feedback to block their own activation. In Neurospora,Drosophila, and mammals, the positive elements of the loops are all heterodimeric protein complexes consisting of PER-ARNT-SIM (PAS)1domain-containing transcription factors (1Dunlap J.C. Cell. 1999; 96: 271-290Abstract Full Text Full Text PDF PubMed Scopus (2325) Google Scholar, 2Young M.W. Kay S.A. Nat. Rev. Genet. 2001; 2: 702-715Crossref PubMed Scopus (919) Google Scholar). In each system, the heterodimeric complex activates the transcription of the negative elements, and the protein products of the negative elements close the feedback loop by inhibiting their own transcription through direct physical interaction with the positive elements (3Allada R. White N.E., So, W.V. Hall J.C. Rosbash M. Cell. 1998; 93: 791-804Abstract Full Text Full Text PDF PubMed Scopus (574) Google Scholar, 4Gekakis N. Stankis D. 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In these three systems, the negative elements of the oscillators also activate the expression of one or two of the positive elements, forming positive feedback loops that interlock with the negative ones (10Glossop N.R. Lyons L.C. Hardin P.E. Science. 1999; 286: 766-768Crossref PubMed Scopus (313) Google Scholar, 11Shearman L.P. Sriram S. Weaver D.R. Maywood E.S. Chaves I. Zheng B. Kume K. Lee C.C. van der Horst G.T. Hastings M.H. Reppert S.M. Science. 2000; 288: 1013-1019Crossref PubMed Scopus (1103) Google Scholar, 12Lee K. Loros J.J. Dunlap J.C. Science. 2000; 289: 107-110Crossref PubMed Scopus (283) Google Scholar, 13Cheng P. Yang Y. Liu Y. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 7408-7413Crossref PubMed Scopus (199) Google Scholar).In the Neurospora frq-wc based circadian feedback loops, WHITE COLLAR-1 (WC-1) and WC-2, the two PAS domain-containing transcription factors (containing GATA type zinc-finger DNA binding domains) form heterodimer complexes (14Talora C. Franchi L. Linden H. Ballario P. Macino G. EMBO J. 1999; 18: 4961-4968Crossref PubMed Scopus (197) Google Scholar) and act as the positive components (15Cheng P. Yang Y. Heintzen C. Liu Y. EMBO J. 2001; 20: 101-108Crossref PubMed Scopus (122) Google Scholar, 16Crosthwaite S.K. Dunlap J.C. Loros J.J. Science. 1997; 276: 763-769Crossref PubMed Scopus (436) Google Scholar). On the other hand, two alternatively translated FREQUENCY (FRQ) protein forms are the negative elements (7Aronson B. Johnson K. Loros J.J. Dunlap J.C. Science. 1994; 263: 1578-1584Crossref PubMed Scopus (505) Google Scholar,15Cheng P. Yang Y. Heintzen C. Liu Y. EMBO J. 2001; 20: 101-108Crossref PubMed Scopus (122) Google Scholar, 17Liu Y. Garceau N. Loros J.J. Dunlap J.C. Cell. 1997; 89: 477-486Abstract Full Text Full Text PDF PubMed Scopus (201) Google Scholar, 18Denault D.L. Loros J.J. Dunlap J.C. EMBO J. 2001; 20: 109-117Crossref PubMed Scopus (150) Google Scholar). In constant darkness, the WC-1·WC-2 heterodimeric complex binds to two light-regulated elements (LREs) in the promoter offrq and activates the transcription of frq (13Cheng P. Yang Y. Liu Y. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 7408-7413Crossref PubMed Scopus (199) Google Scholar,16Crosthwaite S.K. Dunlap J.C. Loros J.J. Science. 1997; 276: 763-769Crossref PubMed Scopus (436) Google Scholar, 19Froehlich A.C. Liu Y. Loros J.J. Dunlap J.C. Science. 2002; 297: 815-819Crossref PubMed Scopus (401) Google Scholar). In either wc-1 or wc-2 mutants, the levels of frq RNA and FRQ protein are very low in the dark, and the circadian clock is not functional under normal conditions (13Cheng P. Yang Y. Liu Y. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 7408-7413Crossref PubMed Scopus (199) Google Scholar,16Crosthwaite S.K. Dunlap J.C. Loros J.J. Science. 1997; 276: 763-769Crossref PubMed Scopus (436) Google Scholar, 20Cheng P. Yang Y. Gardner K.H. Liu Y. Mol. Cell. Biol. 2002; 22: 517-524Crossref PubMed Scopus (135) Google Scholar, 21Collett M.A. Garceau N. Dunlap J.C. Loros J.J. Genetics. 2002; 160: 149-158Crossref PubMed Google Scholar). In addition, WC-1 is the limiting factor for the formation of the WC-1/WC-2 complex (13Cheng P. Yang Y. Liu Y. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 7408-7413Crossref PubMed Scopus (199) Google Scholar, 18Denault D.L. Loros J.J. Dunlap J.C. EMBO J. 2001; 20: 109-117Crossref PubMed Scopus (150) Google Scholar).After the transcription of frq, two forms of FRQ protein (large and small FRQ forms) are made (17Liu Y. Garceau N. Loros J.J. Dunlap J.C. Cell. 1997; 89: 477-486Abstract Full Text Full Text PDF PubMed Scopus (201) Google Scholar, 22Garceau N. Liu Y. Loros J.J. Dunlap J.C. Cell. 1997; 89: 469-476Abstract Full Text Full Text PDF PubMed Scopus (293) Google Scholar), and they exist in homodimeric complexes (15Cheng P. Yang Y. Heintzen C. Liu Y. EMBO J. 2001; 20: 101-108Crossref PubMed Scopus (122) Google Scholar). After their amounts reach a certain level, they feedback to repress the transcription of frq by interacting with the WC-1/WC-2 complex (7Aronson B. Johnson K. Loros J.J. Dunlap J.C. Science. 1994; 263: 1578-1584Crossref PubMed Scopus (505) Google Scholar, 15Cheng P. Yang Y. Heintzen C. Liu Y. EMBO J. 2001; 20: 101-108Crossref PubMed Scopus (122) Google Scholar, 18Denault D.L. Loros J.J. Dunlap J.C. EMBO J. 2001; 20: 109-117Crossref PubMed Scopus (150) Google Scholar, 23Merrow M. Franchi L. Dragovic Z. Gorl M. Johnson J. Brunner M. Macino G. Roenneberg T. EMBO J. 2001; 20: 307-315Crossref PubMed Scopus (106) Google Scholar, 24Yang Y. Cheng P. Liu Y. Genes Dev. 2002; 16: 994-1006Crossref PubMed Scopus (119) Google Scholar), thus closing the negative feedback loop. In addition to its role as a negative element of the loop, FRQ positively regulates the expression of both WC-1 and WC-2 through two different mechanisms, forming positive feedback loops interlocked with the negative feedback loop (12Lee K. Loros J.J. Dunlap J.C. Science. 2000; 289: 107-110Crossref PubMed Scopus (283) Google Scholar, 13Cheng P. Yang Y. Liu Y. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 7408-7413Crossref PubMed Scopus (199) Google Scholar, 20Cheng P. Yang Y. Gardner K.H. Liu Y. Mol. Cell. Biol. 2002; 22: 517-524Crossref PubMed Scopus (135) Google Scholar). Our previous data suggest that the positive feedback loops are important for the robustness and stability of the clock (13Cheng P. Yang Y. Liu Y. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 7408-7413Crossref PubMed Scopus (199) Google Scholar).In addition to their essential role in the circadian feedback loop, WC-1 and WC-2 are also essential components in the light input of the clock and other light responses in Neurospora (16Crosthwaite S.K. Dunlap J.C. Loros J.J. Science. 1997; 276: 763-769Crossref PubMed Scopus (436) Google Scholar, 25Ballario P. Vittorioso P. Magrelli A. Talora C. Cabibbo A. Macino G. EMBO J. 1996; 15: 1650-1657Crossref PubMed Scopus (373) Google Scholar, 26Linden H. Macino G. EMBO J. 1997; 16: 98-109Crossref PubMed Scopus (283) Google Scholar, 27Lakin-Thomas P. Coté G. Brody S. Crit. Rev. Microbiol. 1990; 17: 365-416Crossref PubMed Scopus (108) Google Scholar). In true wc-1 or wc-2 null mutants, most if not all light responses are abolished, including the light induction offrq (21Collett M.A. Garceau N. Dunlap J.C. Loros J.J. Genetics. 2002; 160: 149-158Crossref PubMed Google Scholar, 28He Q. Cheng P. Yang Y. Wang L. Gardner K.H. Liu Y. Science. 2002; 297: 840-843Crossref PubMed Scopus (341) Google Scholar). Recently, we (28He Q. Cheng P. Yang Y. Wang L. Gardner K.H. Liu Y. Science. 2002; 297: 840-843Crossref PubMed Scopus (341) Google Scholar) and Froehlich et al. (19Froehlich A.C. Liu Y. Loros J.J. Dunlap J.C. Science. 2002; 297: 815-819Crossref PubMed Scopus (401) Google Scholar) identified WC-1, a flavin dinucleotide (FAD)-containing protein, as the blue light photoreceptor mediating these light responses. The result of the in vitro DNA binding assay suggests that a large WC complex (that is different from the dark complex) binds to the frq LREs in a light-dependent manner to mediate light input of the clock (19Froehlich A.C. Liu Y. Loros J.J. Dunlap J.C. Science. 2002; 297: 815-819Crossref PubMed Scopus (401) Google Scholar). Thus, light irradiation triggers rapid induction offrq transcription, a process that leads to the resetting of the clock (29Crosthwaite S.K. Loros J.J. Dunlap J.C. Cell. 1995; 81: 1003-1012Abstract Full Text PDF PubMed Scopus (300) Google Scholar). Although the level of WC-1 determines the concentrations of the WC complex formed in vivo (13Cheng P. Yang Y. Liu Y. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 7408-7413Crossref PubMed Scopus (199) Google Scholar), the WC-2 PAS domain-mediated WC-1/WC-2 complex formation is important for maintaining the steady state level of WC-1 and its functions in the circadian clock and light responses (20Cheng P. Yang Y. Gardner K.H. Liu Y. Mol. Cell. Biol. 2002; 22: 517-524Crossref PubMed Scopus (135) Google Scholar). Without WC-2 or the formation of a WC-1/WC-2 complex, the level of WC-1 in the cell is low (20Cheng P. Yang Y. Gardner K.H. Liu Y. Mol. Cell. Biol. 2002; 22: 517-524Crossref PubMed Scopus (135) Google Scholar).These previous studies indicate that WC-1 is a protein with at least two roles: it is a circadian positive element in the dark and a photosensing transcriptional activator mediating light responses. Previously, WC-2 was proposed to function as a scaffold protein that mediates the FRQ-WC interaction to close the negative feedback loop (18Denault D.L. Loros J.J. Dunlap J.C. EMBO J. 2001; 20: 109-117Crossref PubMed Scopus (150) Google Scholar); however, the involvement of WC-1 in this interaction is unclear. Although it is clear that WC-2 positively regulates WC-1 by forming the WC complex, it is not known whether WC-1 regulates the expression of WC-2. In addition, based on the data described above, there should be different forms of the WC complexes with distinct functions in the cell, but no in vivo evidence is available about the nature of the different WC complexes. Furthermore, we do not know which domain of WC-1 mediates its interaction with WC-2 and whether different regions of WC-1 have distinct functional roles. In this study, experiments were carried out to address these questions. Together, our results demonstrate that WC-1 is a multifunctional protein with separable protein domains.DISCUSSIONIn constant darkness, WC-1 and WC-2, the two PAS-containing transcription factors, form a complex that binds thecis-acting elements of the frq promoter and activates the transcription of frq, leading to the cycling of frq and the overt rhythmicity (19Froehlich A.C. Liu Y. Loros J.J. Dunlap J.C. Science. 2002; 297: 815-819Crossref PubMed Scopus (401) Google Scholar, 36Loros J.J. Dunlap J.C. Annu. Rev. Physiol. 2001; 63: 757-794Crossref PubMed Scopus (187) Google Scholar). Recently, WC-1 has also been identified as the first fungal blue light photoreceptor that binds FAD as a chromophore and (together with WC-2) binds to the LREs of the frq promoter in a light-dependent manner (19Froehlich A.C. Liu Y. Loros J.J. Dunlap J.C. Science. 2002; 297: 815-819Crossref PubMed Scopus (401) Google Scholar, 28He Q. Cheng P. Yang Y. Wang L. Gardner K.H. Liu Y. Science. 2002; 297: 840-843Crossref PubMed Scopus (341) Google Scholar). Thus, the rapid light-dependent induction of frq transcription mediated by the WC complex is the mechanism that allows the Neurospora clock to be entrained by light (16Crosthwaite S.K. Dunlap J.C. Loros J.J. Science. 1997; 276: 763-769Crossref PubMed Scopus (436) Google Scholar, 29Crosthwaite S.K. Loros J.J. Dunlap J.C. Cell. 1995; 81: 1003-1012Abstract Full Text PDF PubMed Scopus (300) Google Scholar). Therefore, both WC proteins are required for the clock function in the dark and for the light input of the clock. In addition, the WC complex is required for other light responses inNeurospora (27Lakin-Thomas P. Coté G. Brody S. Crit. Rev. Microbiol. 1990; 17: 365-416Crossref PubMed Scopus (108) Google Scholar).The physical interaction between the WC complex and FRQ is a crucial step that closes the Neurospora circadian negative feedback loop (15Cheng P. Yang Y. Heintzen C. Liu Y. EMBO J. 2001; 20: 101-108Crossref PubMed Scopus (122) Google Scholar, 18Denault D.L. Loros J.J. Dunlap J.C. EMBO J. 2001; 20: 109-117Crossref PubMed Scopus (150) Google Scholar). Although WC-2 was previously proposed as a scaffold protein that mediates such an interaction (18Denault D.L. Loros J.J. Dunlap J.C. EMBO J. 2001; 20: 109-117Crossref PubMed Scopus (150) Google Scholar), here we show that the presence of WC-1 is essential for the formation of the FRQ-WC complex (Fig. 1). In mutants without WC-1, despite the normal expression of WC-2, no WC-2-FRQ interaction was detected in vivo. Because WC-1 needs WC-2 to form complexes to maintain its level and because WC-1 is the limiting factor in WC complexes (13Cheng P. Yang Y. Liu Y. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 7408-7413Crossref PubMed Scopus (199) Google Scholar, 18Denault D.L. Loros J.J. Dunlap J.C. EMBO J. 2001; 20: 109-117Crossref PubMed Scopus (150) Google Scholar, 20Cheng P. Yang Y. Gardner K.H. Liu Y. Mol. Cell. Biol. 2002; 22: 517-524Crossref PubMed Scopus (135) Google Scholar), we think that WC proteins can interact only with FRQ in vivo as a WC-1/WC-2 complex. A similar situation may also exist inDrosophila, in which PER and TIM may only interact with the dCLK-CYC heterodimer and not with the free CYC protein (37Bae K. Lee C. Hardin P.E. Edery I. J. Neurosci. 2000; 20: 1746-1753Crossref PubMed Google Scholar).Previously, we showed that FRQ positively regulates the expression of WC-2 and that the PAS domain of WC-2 is required for the formation of the WC complex and the maintenance of the steady state of WC-1 (13Cheng P. Yang Y. Liu Y. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 7408-7413Crossref PubMed Scopus (199) Google Scholar,20Cheng P. Yang Y. Gardner K.H. Liu Y. Mol. Cell. Biol. 2002; 22: 517-524Crossref PubMed Scopus (135) Google Scholar). Here we show that WC-1 negatively regulates the expression ofwc-2 at the level of transcription (Figs. 2 and 3), thus forming another interacting feedback loop. Interestingly, the repression of wc-2 by WC-1 is light independent, suggesting that this function of WC-1 does not require its role in light sensing (Fig. 3 C). Such an interlocked nature of the expression of the two WC proteins may be important for maintaining an appropriate ratio of the two proteins in the cell, allowing them to function properly in the clock and in light responses. The opposite effects of FRQ and WC-1 on wc-2 may help explain the fact that WC-2 is not rhythmically expressed (13Cheng P. Yang Y. Liu Y. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 7408-7413Crossref PubMed Scopus (199) Google Scholar, 18Denault D.L. Loros J.J. Dunlap J.C. EMBO J. 2001; 20: 109-117Crossref PubMed Scopus (150) Google Scholar). Although we could not exclude the possibility that WC-1 regulates wc-2indirectly, these data suggest that in addition to being a transcription activator, WC-1 may function as a transcription repressor as well.Consistent with WC-1 being a multifunctional protein, our data also demonstrate the existence of different forms of the WC complexes and the significantly different requirements of WC-1 for light induction offrq and other genes. The differential requirement of WC-1 for light-induced gene expression is highlighted by our result in thewc-1 4401 mutant (Fig. 4). In this mutant, resulting from alternative protein initiation from a downstream AUG, less than 1% of the normal amount of WC-1 was expressed, and it was still associated with WC-2. However, its light induction of frqwas near normal, whereas the light induction of al-3 andvvd was mostly abolished. A similar situation was also observed for a wc-2 mutant. In thewc-2 ER33 mutant (a point mutation in the zinc finger DNA-binding domain), although its light induction of other genes are abolished, its light induction of frq was close to normal (16Crosthwaite S.K. Dunlap J.C. Loros J.J. Science. 1997; 276: 763-769Crossref PubMed Scopus (436) Google Scholar, 20Cheng P. Yang Y. Gardner K.H. Liu Y. Mol. Cell. Biol. 2002; 22: 517-524Crossref PubMed Scopus (135) Google Scholar, 21Collett M.A. Garceau N. Dunlap J.C. Loros J.J. Genetics. 2002; 160: 149-158Crossref PubMed Google Scholar). Therefore, the light induction of frq, a step critical in the light resetting of the clock, does not require fully functional WC proteins, and needs only a very low amount of a WC complex.Recently, a study by Dragovic et al. (38Dragovic Z. Tan Y. Gorl M. Roenneberg T. Merrow M. EMBO J. 2002; 21: 3643-3651Crossref PubMed Scopus (49) Google Scholar) showed that in some wc mutants, conidiation rhythms could still be driven by light/dark cycles. Although this study suggested the existence of awc-independent photoreceptor in Neurospora that regulates the conidiation process, the interpretation of the results is complicated by the fact that the Δwc-1 strain (generated by RIP) used in that study may not be a real wc-1 null strain. In that wc-1 mutant strain, although the light induction of other genes was eliminated, the light induction offrq could still be observed, a fact that is in conflict with the results obtained in true wc-1 null strains and otherwc-1 mutants (16Crosthwaite S.K. Dunlap J.C. Loros J.J. Science. 1997; 276: 763-769Crossref PubMed Scopus (436) Google Scholar, 19Froehlich A.C. Liu Y. Loros J.J. Dunlap J.C. Science. 2002; 297: 815-819Crossref PubMed Scopus (401) Google Scholar, 28He Q. Cheng P. Yang Y. Wang L. Gardner K.H. Liu Y. Science. 2002; 297: 840-843Crossref PubMed Scopus (341) Google Scholar). Therefore, based on the results we presented here, we think that the wc-1 mutant used by Dragovic et al. (38Dragovic Z. Tan Y. Gorl M. Roenneberg T. Merrow M. EMBO J. 2002; 21: 3643-3651Crossref PubMed Scopus (49) Google Scholar) is most likely not a real null, and it could produce a low level of WC-1 (that could not be detected by regular Western blot analysis) because of alternative protein initiation from a downstream AUG.The differential requirement of WC complex in the light induction of genes and the in vitro DNA binding data (19Froehlich A.C. Liu Y. Loros J.J. Dunlap J.C. Science. 2002; 297: 815-819Crossref PubMed Scopus (401) Google Scholar) suggest the existence of different WC complexes in the cell, a notion that was confirmed by the immunoprecipitation assay in a wt,Myc-WC-1 strain (Fig. 5). Using the extract of this strain, we found that there were at least two types of WC-1-containing protein complexes in the cell: ∼20–30% of WC-1 was found to self-associate to form a large WC-1/WC-2 complex. The larger WC complex may be the large WC-containing complex identified by Froehlich et al. (19Froehlich A.C. Liu Y. Loros J.J. Dunlap J.C. Science. 2002; 297: 815-819Crossref PubMed Scopus (401) Google Scholar) using a in vitro DNA binding assay that binds to the LREs of thefrq promoter in a light-dependent manner and regulates light responses. In contrast to WC-1, no WC-2 was found to self-associate in vivo, although strong self-association was previously observed in vitro (34Ballario P. Talora C. Galli D. Linden H. Macino G. Mol. Microbiol. 1998; 29: 719-729Crossref PubMed Scopus (150) Google Scholar). Because WC-1 needs WC-2 to form a complex to maintain its steady state level, one WC-2 molecule should form complexes with more than one WC-1 molecule in the cell. Our results also confirm the previous results that the majority of WC-1 is in a WC-1/WC-2 heterodimeric complex (18Denault D.L. Loros J.J. Dunlap J.C. EMBO J. 2001; 20: 109-117Crossref PubMed Scopus (150) Google Scholar, 19Froehlich A.C. Liu Y. Loros J.J. Dunlap J.C. Science. 2002; 297: 815-819Crossref PubMed Scopus (401) Google Scholar, 28He Q. Cheng P. Yang Y. Wang L. Gardner K.H. Liu Y. Science. 2002; 297: 840-843Crossref PubMed Scopus (341) Google Scholar). The function of the WC-1/WC-2 heterodimer may be important for the activation offrq and other genes in the dark (19Froehlich A.C. Liu Y. Loros J.J. Dunlap J.C. Science. 2002; 297: 815-819Crossref PubMed Scopus (401) Google Scholar, 39Lewis Z.A. Correa A. Schwerdtfeger C. Link K.L. Xie X. Gomer R.H. Thomas T. Ebbole D.J. Bell-Pedersen D. Mol. Microbiol. 2002; 45: 917-931Crossref PubMed Scopus (83) Google Scholar).To map the WC-1 domain that mediates its interaction with WC-2 and to determine the function of various WC-1 domains, we made a series ofwc-1 mutants. Our results indicate that the PASC domain and its immediate C-terminal region (upstream of the putative NLS) are required for the formation of the WC-1/WC-2 complex, whereas the rest of WC-1 is not essential (Figs. 6 and 7). Although the requirement of the PASC domain for the interaction is consistent with the interaction mediated by the PAS-PAS interaction of the two WC proteins (20Cheng P. Yang Y. Gardner K.H. Liu Y. Mol. Cell. Biol. 2002; 22: 517-524Crossref PubMed Scopus (135) Google Scholar), the involvement of the PASC C-terminal region indicates that PASC alone is not sufficient for such an interaction. Consistent with our previous work of WC-2 (20Cheng P. Yang Y. Gardner K.H. Liu Y. Mol. Cell. Biol. 2002; 22: 517-524Crossref PubMed Scopus (135) Google Scholar), these results show that the formation of the WC complex is essential for their functions in the circadian clock and light responses and for maintaining the steady state level of WC-1. However, the existence of the WC-1/WC-2 complex is not sufficient to be a functional complex, because the PASB domain of WC-1 is required for both of its dark and light functions whereas it is not required for the complex formation (Fig. 8, A and B).One surprising result from the deletion study is that the C-terminal part of the WC-1 protein (starting from the putative NLS and including the zinc finger) is not required for the light induction offrq, al-3, and vvd (Fig. 8). However, the NLS and DNA-binding domain are required for the expression of FRQ in the dark. Thus, by deleting this region of WC-1, we separated the light and dark functions of WC-1. This is in contrast with the mutant lacking the entire LOV domain, in which only the light function but not the dark function of WC-1 is defective (28He Q. Cheng P. Yang Y. Wang L. Gardner K.H. Liu Y. Science. 2002; 297: 840-843Crossref PubMed Scopus (341) Google Scholar). Without the DNA-binding domain of WC-1, how does WC-1 mediate the light-induced gene expression? Because WC-1 forms complexes with WC-2, the zinc finger DNA-binding domain of WC-2 must be responsible for the DNA binding in these mutants. Interestingly, as described above, in awc-2 mutant (ER33) that contains a point mutation in the DNA-binding domain, the light induction of frq was still observed, although the light induction of other genes was defective (16Crosthwaite S.K. Dunlap J.C. Loros J.J. Science. 1997; 276: 763-769Crossref PubMed Scopus (436) Google Scholar, 20Cheng P. Yang Y. Gardner K.H. Liu Y. Mol. Cell. Biol. 2002; 22: 517-524Crossref PubMed Scopus (135) Google Scholar, 21Collett M.A. Garceau N. Dunlap J.C. Loros J.J. Genetics. 2002; 160: 149-158Crossref PubMed Google Scholar). Together, these data suggest that the light induction offrq only requires one DNA-binding domain from either of the WC proteins, but the DNA-binding domain of WC-2 may be essential for binding to the promoters of other light-inducible genes. On the other hand, the DNA-binding domains of both WC proteins may be required for DNA binding and transcription activation of frq in the dark.In conclusion, our results show that WC-1 is required for the formation of the FRQ·WC complex and it negatively regulates the expression ofwc-2. In addition, WC-1 differentially regulates the light induction of frq and other genes, and there are different forms of the WC complexes in the cell, possibly with distinct functions. Together, our data demonstrate that WC-1 is a multifunctional protein involved in the circadian clock, light sensing, and transcription repression in Neurospora. Endogenous circadian clocks control a wide variety of daily physiological, behavioral, cellular, and biochemical activities in most eukaryotic and certain prokaryotic organisms. The circadian oscillators are networks of positive and negative elements that form the core circadian feedback loops generating the basic circadian rhythmicity (1Dunlap J.C. Cell. 1999; 96: 271-290Abstract Full Text Full Text PDF PubMed Scopus (2325) Google Scholar,2Young M.W. Kay S.A. Nat. Rev. Genet. 2001; 2: 702-715Crossref PubMed Scopus (919) Google Scholar). The positive elements of the loop activate the transcription of the negative elements, whereas the negative elements feedback to block their own activation. In Neurospora,Drosophila, and mammals, the positive elements of the loops are all heterodimeric protein complexes consisting of PER-ARNT-SIM (PAS)1domain-containing transcription factors (1Dunlap J.C. Cell. 1999; 96: 271-290Abstract Full Text Full Text PDF PubMed Scopus (2325) Google Scholar, 2Young M.W. Kay S.A. Nat. Rev. Genet. 2001; 2: 702-715Crossref PubMed Scopus (919
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