Methods for regulating gene expression using light

Details Methods for regulating gene expression using light Methods for regulating gene expression using light

2002-08-26

2004-05-11

McKelvey, Terry (Department: 1636)

Chemistry: molecular biology and microbiology

Micro-organism, tissue cell culture or enzyme using process...

Recombinant dna technique included in method of making a...

C435S375000, C530S350000, C536S024100

Reexamination Certificate

active

06733996

ABSTRACT:

BACKGROUND OF THE INVENTION
Light provides essential phase information for all circadian systems, and it has been asserted that rhythms evolved from PAS/LOV domain-mediated light responses (Crosthwaite, et al. (1997)
Science
276:763). A bacteriophytochrome mediates light input in cyanobacteia (Thresher, et al. (1998)
Science
282:1490), while phytochromes and cryptochromes play this role in plants (Harmer, et al. (2001)
Annu. Rev. Cell Dev. Biol
. 17:215). Insect clocks use cryptochrome with additional input from opsin-based pigments in the compound eye (Harmer, et al. (2001)
Annu. Rev. Cell Dev. Biol
. 17:215); in mammals, cryptochromes may sense light (Thresher, et al. (1998)
Science
282:1490) but recent work has focused on melanopsin as the mammalian circadian photoreceptor (Provencio, et al. (2000)
J. Neurosci
. 20:600; Hattar, et al. (2002)
Science
295:1065; Berson, et al. (2002)
Science
295:1070).
In Neurospora, a wide range of processes is light sensitive, including suppression and phase shifting of circadian rhythms, phototropism of perithecial beaks (Linden, et al. (1997)
Fungal Genet. Biol
. 22:141), and carotenoid biosynthesis (Payen (1843)
Ann. Chim. Phys
. 9:5). Until recently, the photoreceptor(s) involved in these blue light-influenced processes had not been identified, but screens in Neurospora for mutants involved in light perception and signaling repeatedly turned up two indispensable loci, wc-1 and wc-2 (Linden, et al. (1997)
Mol. Gen. Genet
. 254:111). WC-1 and WC-2 are nuclear transcription factors containing trans-activation and zinc-finger (Zn-finger) DNA binding domains (Ballario, et al. (1996)
EMBO J
. 15:1650; Linden and Macino (1997)
EMBO J
. 16:98). They form a White Collar Complex (WCC) by heterodimerizing via PAS (PER ARNT SIM) domains (Ballario, et al. (1998)
Mol. Microbiol
. 29:719; Denault, et al. (2001)
EMBO J
. 20:109) and act as positive elements in light signaling through direct binding of DNA (Ballario, et al. (1996)
EMBO J
. 15:1650; Linden and Macino (1997)
EMBO J
. 16:98). In a true wc-2
K0
strain, all examined light responses are lost (Collett, et al. (2002)
Genetics
160:149). This requirement provided evidence that either WC-1 or WC-2 is the photoreceptor, or that they both are required to mediate the response of an unidentified, or duplicated, receptor (Linden, et al. (1997)
Fungal Genet. Biol
. 22:141; Briggs (1999)
Annu. Rev. Cell Dev. Biol
. 15:33; Loros and Dunlap (2001)
Annu. Rev. Physiol
. 63:757).
Parallel investigations by Froehlich, et al. ((2002)
Science
297(5582):815-819) and He, et al. ((2002)
Science
297(5582):840-843) reveal that WC-1 is indeed the photoreceptor. By deleting the LOV (light, oxygen, voltage) domain of WC-1, He, et al. ((2002)
Science
297(5582):840-843) demonstrate that despite near normal induction of the circadian clock gene frequency (frq) in the dark and the maintenance of WC-2 and FRQ interactions, light responses are abolished. They further demonstrate that the flavin, FAD, copurifies with the WCC and may mediate the phototransduction mechanism. Similarly, Froehlich et al. ((2002)
Science
297(5582):815-819) performed reconstitution experiments and demonstrate that WC-1 interacts with WC-2 to bind to light responsive DNA elements. Furthermore, in the presence of FAD and light, WC-1 initiates the photoresponse event.
In Neurospora, generation of circadian rhythms is dependent on WCC-mediated rhythmic production of frq transcript and protein, both of which are central clock components (Aronson, et al. (1994)
Science
263:1578; Garceau, et al. (1997)
Cell
89:469). Light causes a rapid induction of frq message, the central means by which light influences the clock (Crosthwaite, et al. (1995)
Cell
81:1003). In the absence of WC-1 or WC-2, light induction of frq is completely abolished, highlighting the central role of WC-1 and WC-2 in light input to the clock (Collett, et al. (2002)
Genetics
160:149; Crosthwaite, et al. (1997)
Science
276:763). Other loci also regulated by WCC include al-1 and con-10 (Li and Schmidhauser (1995)
Dev. Biol
. 169:90-95; Linden, et al. (1997)
Mol. Gen. Genet
. 254:111-118; Linden, et al. (1997)
EMBO J
. 16:98-109).
Current methods of controlling gene expression include inducible promoters, such as those responsive to heavy metal ions (Mayo, et al. (1982)
Cell
29:99-108; Brinster, et al. (1982)
Nature
296:39-42; Searle, et al. (1985)
Mol. Cell. Biol
. 5:1480-1489), heat shock (Nouer, et al. (1991) in Heat Shock Response, ed. Nouer, CRC, Boca Raton, Fla., pp. 167-220), hormones (Lee, et al. (1981)
Nature
294:228-232; Hynes, et al. (1981)
Proc. Natl. Acad. Sci. USA
78:2038-2042; Klock, et al. (1987)
Nature
329:734-736; Israel and Kaufman (1989)
Nucl. Acids Res
. 17:2589-2604), or drugs such as tetracycline (Gatz, et al. (1992)
Plant J
. 2:397-404; U.S. Pat. No. 5,814,618) or IPTG (Labow, et al. (1990)
Mol. Cell. Biol
. 10:3343-3356; Baim, et al. (1991)
Proc. Natl. Acad. Sci. USA
88:5072-5076). However, these systems have generally suffered from exogenous inducer molecules (e.g., heavy metal ions, heat shock or steroid hormones) evoking pleiotropic effects, which can complicate analyses. The present invention provides a system of regulating gene expression using the readily available, inexpensive, non-toxic inducer, light.
SUMMARY OF THE INVENTION
This invention relates to methods of regulating gene expression using components of the Neurospora light-responsive White Collar Complex (WCC). In particular, this invention provides a WC-1/WC-2 transactivator which when bound to the flavin cofactor, FAD, is useful for regulating expression, in the presence of light, of a gene operatively-linked to one or more light-responsive regulatory sequences. The invention further provides light-responsive regulatory sequences which bind the WC-1/WC-2 transactivator.
These and other aspects of the present invention are set forth in more detail in the following description of the invention.
DETAILED DESCRIPTION OF THE INVENTION
The present invention provides methods, nucleic acid molecules and proteins for the regulation of gene expression in cells or organisms in a highly controlled manner. Regulation of gene expression by the system of the invention involves at least two components: A gene which is operatively-linked to a regulatory sequence and a transcriptional activator which binds to the regulatory sequence and activates transcription of the gene.
The system of the invention utilizes components of the White Collar Complex of Neurospora to stimulate gene expression in cells using light as an inducer. The White Collar complex is comprised of two proteins, WC-1 and WC-2. When in the presence of the cofactor FAD, the WCC is able to absorb light and subsequently activate transcription of a gene operatively-linked to a light-responsive regulatory sequence which interacts with WCC. Thus, in a host cell, transcription of a gene, operatively-linked to one or more light-responsive regulatory sequences, is stimulated by WC-1/WC-2 by altering the fluence or wavelength of light exposed to the host cell.
Kits which contain the components of the regulatory system described herein, are also encompassed by the invention.
The frq promoter was fused to a reporter gene, hph, and the resulting construct, pYL40B, was transformed into a frq+ strain. Light treatment of transformants resulted in a marked increase in hph transcript level, similar to that of frq. Because only the frq promoter sequence was fused to hph, it was determined that light induction of the hph transcript, and consequently of endogenous frq message, is controlled at the transcriptional level.
Cis-acting elements mediating light induction of frq were identified by transforming frq promoter deletion constructs into a frq
K0
strain and testing for light induction of frq message. Deletion of two light-responsive regulatory sequences, also referred to herein as light-responsive elements (LRES), in the frq promoter decreased light induction of frq message. A ~50% reduction was measu

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