Letters pubs.acs.org/acschemicalbiology
Blue Light-Mediated Manipulation of Transcription Factor Activity In Vivo Shinji Masuda,*,‡,|| Yuki Nakatani,† Shukun Ren,‡ and Mikiko Tanaka† ‡
Center for Biological Resources & Informatics, Tokyo Institute of Technology, Yokohama 226-8501, Japan Earth-Life Science Institute, Tokyo Institute of Technology, Tokyo 152-8550, Japan † Graduate School of Bioscience & Biotechnology, Tokyo Institute of Technology, Yokohama 226-8501, Japan ||
S Supporting Information *
ABSTRACT: We developed a novel technique for manipulating the activity of transcription factors with blue light (termed “PICCORO”) using the bacterial BLUF-type photoreceptor protein PixD. The chimeric dominant-negative T-box transcription factor No Tail formed heterologous complexes with a PixD decamer in a light-dependent manner, and these complexes affected transcription repressor activity. When applied to zebrafish embryos, PICCORO permitted regulation of the activity of the mutant No Tail in response to 472-nm light provided by a light-emitting diode.
T
dimer.12 Gel-shift analysis confirmed that the Ntl-EnR octamer binds to the target DNA of Ntl, and complex species, formed with Ntl-EnR, showed higher molecular weights than did those formed with Ntl (SI Figure S1b). These results indicate that the Engrailed repressor domain induces aggregation of Ntl, which is required for transcription repression (SI Figure S1c). Thus, transcription of the target genes may be regulated by the extent of aggregation. We next established a method to control the aggregation of dominant-negative transcription factors with blue light. We utilized a blue-light photoreceptor called PixD, which is responsible for phototaxis in the cyanobacterium Synechocystis sp. PCC6803.13−15 PixD senses 320- to 500-nm light, as shown in its absorption spectrum (SI Figure S2). PixD forms a large oligomeric complex with the response regulator-like protein PixE (forming PixD10−PixE4 or PixD10−PixE5 complexes) in the dark. Light excitation of PixD alters the conformation of the proteins and breaks the PixD decamer into dimers.16−18 We hypothesized that the light-dependent changes to PixD−PixE oligomers could be used for the artificial control of transcription factor activity by blue light. Specifically, if the PixE portion that is required for PixD binding was fused with a transcription factor, the chimeric transcription factor might form heterologous complexes with PixD that would inhibit its repressor activity (Figure 1a). We first tested complex formation between PixD and the PixE-fused chimeric transcription factor in vitro. Yeast twohybrid analysis indicated that the PixE N terminus (amino acids 1−256; full-length PixE has 380 amino acids) interacted with
he ability to control gene expression experimentally is essential for deciphering many cellular, developmental, and physiological processes. Light is an attractive signal for the artificial manipulation of gene expression because it has high spatial and temporal precision. Several technologies involving light-inducible gene activation or silencing have been developed.1−6 Although these systems allow spatiotemporal analysis of target genes, the target gene must be placed downstream of specific promoters, a process that is timeconsuming and applicable to a limited number of genes. In contrast, our approach is aimed at controlling the transcription factors themselves through light-switchable manipulation of their repressor ability at the protein level. This system has advantages over previous techniques, especially for physiological characterization of transcription factors involved in developmental processes. Dominant-negative transcription factors have been generally used to investigate signaling pathways involved in complex cellular processes.7 Potent dominant-negative transcription factors can be created by fusing the Engrailed repressor domain to a DNA-binding domain of a transcription factor.7−9 Because the exact mechanisms of how the Engrailed repressor domain functions to modulate repression are unknown,7 we first compared the biochemical properties of a zebrafish T-box transcription factor Ntl 10 and its dominant-negative mutant, Ntl-EnR.11 The mutant protein was constructed by fusing the Engrailed repressor domain to the DNA-binding domain of Ntl,11 which suppresses posterior notochord and adaxial tissue development when functional. Blue native PAGE analysis indicated that Ntl and Ntl-EnR exist as a dimer and an octamer, respectively (Supporting Information (SI) Figure S1a). Dimer formation of Ntl is consistent with a previous observation in which T-box transcription factors bind to their target DNA as a © 2013 American Chemical Society
Received: March 12, 2013 Accepted: September 17, 2013 Published: September 24, 2013 2649
dx.doi.org/10.1021/cb400174d | ACS Chem. Biol. 2013, 8, 2649−2653
ACS Chemical Biology
Letters
may block the repressor activity of NtlPixE8. Thus, PixD may function as a light-dependent antirepressor of NtlPixE in vitro. We next tried to block the repression of NtlPixE function in vivo. For this purpose, we constructed a transgenic zebrafish expressing pixD, called Tg(EF1α:PixD). The EF1α promoter was used to constitutively express pixD, and the DNA construct was introduced into the chromosome using the Tol2 system (see Methods). All tested F3 recombinants harbored pixD on the chromosome (SI Figure S5a,b). Expression of pixD in Tg(EF1α:PixD) was confirmed with RT-PCR (SI Figure S5c). We first checked the concentration dependency of the NtlPixE−dependent ntl phenotype. The ntl phenotype was categorized into four classes according to severity.19 In this study, we used the term ‘ntl phenotype’ to describe individuals showing class I and II ntl phenotypes (i.e., a nearly complete loss of somites in the tail and loss of axial tissues in the trunk).19 Embryos that were injected with ntlPixE mRNA showed reduced notochord development (Figure 2a), as was
Figure 1. PixD complex−dependent transcription control (PICCORO). (a) In this study, the PixE N terminus (PixE_N) was fused to the dominant-negative mutant Ntl-EnR (referred to as NtlPixE in the text). Ntl-EnR binds to the target DNA as an octamer for repressing transcription. In the dark, PixD forms complexes with NtlPixE and inhibits the repressor activity of NtlPixE. Upon light illumination, NtlPixE dissociates from the PixD complex and inhibits transcription of target genes. (b) The soluble fraction of lysates from E. coli that expressed NtlPixE was used as the source of NtlPixE. Lane 1, DNA probe only; lane 2−10, increasing concentrations of NtlPixE incubated with a DNA probe in the absence or presence of 2 μg purified PixD; lanes 2 and 6, 20 μg NtlPixE lysate; lanes 3 and 7, 40 μg NtlPixE lysate; lanes 4 and 8, 60 μg NtlPixE lysate; lanes 5 and 9, 80 μg NtlPixE lysate. The mixture for lane 10 was set up as for lane 9 but was incubated under lightilluminated conditions before electrophoresis. Asterisks (*) indicate a contaminating nonspecific DNA band.
Figure 2. PICCORO controls tail formation in zebrafish. (a) Injection of ntlPixE mRNA into WT zebrafish results in suppression of notochord development. Bar = 0.5 mm. (b) Tg(EF1α:PixD) embryos were injected with ntlPixE mRNA and then were incubated under dark or light-illuminated conditions provided by a light-emitting diode (λmax = 472 nm, 250 μmol m−2 s−1). Bar = 0.2 mm. (c) WT and Tg(EF1α:PixD) embryos were injected with ntlPixE mRNA and incubated under dark or light-illuminated conditions as in b. In situ hybridization for tbx6 was performed in four-somite embryos, and staining was compared with uninjected embryos (Control). Arrows indicate unusual distribution in the tbx6 expression.
PixD (SI Figure S3). To test our strategy, we used the dominant-negative transcription factor Ntl-EnR. The Nterminal amino acids (1−256) of PixE were attached to the N terminus of Ntl-EnR, and the chimeric transcription factor was designated NtlPixE. The C terminus of PixE was deleted from the construct because it was not necessary for binding to PixD (SI Figure S3). The complex formed between purified PixD and NtlPixE was examined with blue native PAGE analysis. NtlPixE itself formed a large ∼600-kDa complex that most likely is NtlPixE8 (SI Figure S4a). Formation of heterologous complexes (PixD10−NtlPixE1, PixD10−NtlPixE2, and/or PixD10− NtlPixE4) was observed in the dark (Supporting Figure S4b). Light illumination resulted in dissociation of the complex (SI Figure S4c). Gel-shift analysis indicated that NtlPixE8 specifically binds to the target DNA of Ntl (Figure 1b). Formation of the binary NtlPixE8−DNA complex was reduced by addition of PixD, although we observed additional faint bands that indicated the formation of ternary PixD10−NtlPixE2−DNA and/or PixD10− NtlPixE4−DNA complexes. Formation of the ternary complex
observed with ntl-EnR mRNA injection.11 The frequency of the ntl phenotype was dose dependent, and the nearly saturating concentration of ntlPixE mRNA was ∼0.3 μg/μL (SI Figure S6). We next analyzed PixD-dependent regulation of NtlPixE activity in the Tg(EF1α:PixD) zebrafish. We injected 0.2 μg/ μL ntlPixE mRNA, a concentration that should lead to subtle effects of PixD on NtlPixE function. When embryos were incubated in the dark after injection, 71.9% and 26.2% of ntl phenotypes were observed in WT and Tg(EF1α:PixD) zebrafish, respectively (Table 1; P < 0.0001), indicating that PixD represses NtlPixE activity in the dark. 2650
dx.doi.org/10.1021/cb400174d | ACS Chem. Biol. 2013, 8, 2649−2653
ACS Chemical Biology
Letters
Table 1. Frequency of the ntl Phenotype Caused by NtlPixE a genotype WT Tg(EF1α:PixD)
conditionb
ntl phenotype (%)f
n
χ2
P value
dark high lightb dark high lightb low lightc
71.9 73.5 26.2 47.4 44.9
110 59 56 56 49
0.03d 21.29d 3.87e 3.42e
0.8625d
