Team:Freiburg/Project/induction

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Figure 3: Position of the target loci on the SEAP plasmid pKM602. The tested target loci are located in front of the SEAP promoter. Several distances and combinations of the related crRNAs were tested.

Introduction

The aim of this project was to control Cas9 (CRISPR associated protein 9) activity by an external 4-OHT (4-hydroxytamoxifen) stimulus: Only when 4- OHT is applied, Cas9 should enter the nucleus. Thus, Cas9 was fused to the 4-OHT inducible receptor domain ERT2 (mutated hormone binding domain of the human estrogenic receptor). To facilitate the detection of the fusion protein a HA tag and the fluorescent protein mCherry were added.

The native estrogenic receptor (ER) acts as an enhancer: Upon hormone binding this protein stimulates the transcription of specific genes. It consists mainly of a DNA and a hormone binding domain (HBD) [1].
Steroid receptors as ER require chaperones (mostly Hsp90) to be functional: They open the hydrophobic hormone binding cleft and carry them after binding of the appropriate ligand with the help of immunophilins along the cytosceleton to the nucleus [2]. For the glucocorticoid receptor (GR) this interaction is crucial for nuclear import [3] . Even within the nucleus most steroid receptors are in complex with Hsp90 [4]. Without a ligand steroid receptors move continuously into and out of the nucleus. When the adequate hormone is bound GR localization becomes nuclear within a half life of about 4,5 min [2].
Tamoxifen is a competitive inhibitor of ER. For that reason it is widely used for treatment of breast cancer [5]. In the body of higher vertebrates Tamoxifen becomes metabolized to 4-OHT [6]. This form is more potent in inhibiting the ER which leads to a stronger repression of estrogen-dependent cell growth [7] .
In 1993 Danielian et al. [8] mutated the murine ER, so that it is no more sensitive to estrogen, but becomes active after binding of tamoxifen. Littlewood et al. [9] took the HBD of this mutated ER and fused it to the c-Myc protein. This way they were able to control Myc-induced proliferation and apoptosis. Later on the DNA recombinase Cre was fused with a more tamoxifen sensitive human HBD mutant named ERT2 [10]. For the regulation 4-OHT is preferred to tamoxifen because it yields a higher effect with lower concentrations [11].
Besides the inhibition of the ER, tamoxifen can decrease the cellular ATP level via uncoupling of the mitochondrial membrane potential as well as the adenine nucleotide transferase concentration within the mitochondrial membrane. But the derivate 4-OHT is much less harmfull [7].

According to Leone et al. [12] the mechanism of the regulation of the ERT2-Cas9 fusion protein should be the following: Without 4-OHT the protein remains in the cytoplasm, due to the shielding of the NLS by Hsp90s. When 4-OHT is applied to the cell it diffuses through the cell membrane and bind to ERT2 (Fig. 1 (1.) ). Because of this the heat shock proteins should separate from ERT2, which allows the fusion protein to enter the nucleus, where Cas9 (when in complex with crRNA and tracrRNA) can target - and regulate - the desired genes (Fig. 1 (2.) ).

Fig. 1: Proposed mechanism Without (Hydroxy)tamoxifen in the cell the fusion protein (Cas9, NLS & ERT2) is in complex with Hsp90 chaperones, which should cover the NLS. After tamoxifen binding (1) to the HBD, the chaperones separate and the fusion protein will be imported into the nucleus (2).

Assembled plasmids

Due to unknown sterical side effects, 5 constructs with different NLS and ERT2 positions were assembled (see also Fig. 2):
First of all a NLS and an ERT2 were fused to both ends of the fusion protein, as a Cas9-GFP fusion protein with a NLS on both sides was shown by Cong et al. [13] to be localized exclusively in the nucleus within HEK cells. The from ERT2 in the inactive state acquired Hsp90s were thought to cover these NLSs and therefore inhibit the nuclear import of the fusion protein. (pIG3005)
Secondly ERT2 was only fused to the N- or the C-terminal NLSs, because Cong et al. [13] showed a difference in nuclear import efficiency due to the position of the NLS. According to these studies the C-terminal NLS should have a higher impact on nuclear localization, but fusing ERT2 N-terminal leads to a tighter hormone mediated control [14]. (pIG3006: ERT2 only C-terminal & pIG3007: ERT2 only N-terminal)
Thirdly constructs contain only one NLS (with ERT2). This should reduce the amount of uninduced nuclear import, while the size of the protein is lower than pIG3005. (pIG3008: NLS and ERT2 only C-terminal & pIG3009: NLS and ERT2 only N-terminal)
At last for every NLS variation a control was assembled: Cas9-mCherry without a NLS (pIG3001), NLSs fused to both ends (pIG3002) and one NLS fused to the C- (pIG3003) and to the N-terminus (pIG3004).

All planned plasmids could be successfully cloned by Gibson assembly.

Fig. 2: Assembled fusion proteins All fusion proteins used for the hormone induction are pictured with all their essential parts. pIG3001 - 4: controls; pIG3005 - 9: test constructs; HA: HA tag; ERT2: hormone receptor domain; NLS: nuclear localization sequence.

Results

Influence of the NLS on nuclear import

Fluorescent pictures taken with the confocal microscope clearly show that pIG3001 is mostly localized in the cytoplasm, whereas with pIG3002 - 4 transfected cells display a much stronger red fluorescence in the nucleus. It seems that this nuclear localization is even stronger for pIG3003 & 4 (Fig. 3).

Fig. 3: Fluorescence pictures of HeLa cells expressing the control constructs Pictures were taken with a confocal fluorescence microscope. mCherry: fluorophor (part of the Cas9-ERT2 fusion protein); DRAQ5: DNA staining; pIG3001: Cas9-ERT2 fusion protein without a NLS; pIG3002: Cas9-ERT2 fusion protein with a NLS on both ends; pIG3003: Cas9-ERT2 fusion protein with a C-terminal NLS; pIG3004: Cas9-ERT2 fusion protein with a N-terminal NLS.

Effects of 4-OHT induction on nuclear import

The red fluorescence of the Cas9-ERT2 fusion proteins is mostly localized in the nucleus both with 4-OHT and EtOH (negative control, as 4-OHT is dissolved in EtOH) treatment, a difference is not detectable, which is visible for pIG3007 & 9 in Fig. 4.

Fig. 4: Fluorescence pictures of HeLa cells expressing pIG3006 & 8 (ERT2 at the C-terminus) Pictures taken with a confocal fluorescence microscope were chosen as an example for the look of the cell expression the Cas9-ERT2 fusion proteins. mCherry: fluorophor (part of the Cas9-ERT2 fusion protein); DRAQ5: DNA staining; pIG3006: Cas9-ERT2 fusion protein with a NLS on both ends and a ERT2 at the C-terminus; pIG3008: Cas9-ERT2 fusion protein with a C-terminal NLS and ERT2. The very bright, round red shapes are mostly dead cells.

Quantitative analysis reveals a slight increase of the ratio of the mean nuclear red fluorescence intensity to the cytoplasmic upon 4-OHT treatment for pIG3005 (1.5-fold), pIG3007 (2-fold) and pIG3009 (1.3-fold), whereas this ratio drops upon 4-OHT treatment for pIG3006 (2-fold) and pIG3008 (1.6-fold), but for all within the standard deviation. All values range between the positive and the negative controls (Fig. 5).

Fig. 5: Ratio of the mean intensity of nuclear and cytoplasmic fluorescence For each bar the mean intensities of nuclear and cytoplasmic red fluorescence of at least 7 cells on one picture taken by the confocal microscope were determined using ImageJ. This was done by selecting a region in the nucleus and in the cytoplasm of each cell. The figure shows the ration of nuclear to cytoplasmic fluorescence (background subtracted) with the standard deviation. The grey bars represent the controls (no treatment): pIG3001 = negative control (no NLS); pIG3002 - 4 = positive controls (one or two NLSs, respectively). The blue bars represent the test constructs with either 0.1 % EtOH or 1 µM 4-OHT treatment over night.

Effects of 4-OHT on Cas9 functionality

As another aproach to regulate Cas9 activity, HEK cells were transfected with a constitutiv on SEAP plasmid, a RNA plasmid containing the tracrRNA and a crRNA, that is complementary to a sequence at the beginning of the SEAP coding sequence, and the Cas9-ERT2 fusion constructs, which contain two NLS. We hoped that the Hsp90s binding to ERT2 when there is no 4-OHT would sterically hinder Cas9 from binding to the target DNA.
Fig. 6 shows only very little differences in SEAP expression between hormone and ethanol treatment (in both directions) but 1.5- till 3-fold repression when Cas9-ERT2 is transfected with a crRNA.

Fig. 6: Repression of SEAP expression The SEAP activities of technical triplicates were measured after the addition of a certain amount of pNPP (substrate of SEAP). The bars represent the mean with standard deviation. red: controls without Cas9-ERT fusion proteins; blue: tests with Cas9-ERT fusion proteins; 1/5: cotransfection of a RNA plasmid containing a crRNA targeting the SEAP coding region at the first 1/5; 1/10: cotransfection of a RNA plasmid containing a crRNA targeting the SEAP coding region at the first 1/10; -/-: no RNA plasmid transfected; 4-OHT: treatment with 4-OHT; EtOH: treatment with EtOH.

Toxicity of 4-OHT

In order to test if 4-OHT has some lethal side effects on HeLa cells, untransfected cells were treated with different 4-OHT concentrations (10 µM, 1 µM and 0.1 µM), EtOH (1 % and 0.1 %) or none of them in two independent experiments for 18 respectively 24 h.
At the first experiment cells were observed by microscopy: All cells looked healthy, only the cells treated with 10 µM 4-OHT had a quite compact shape, whereas all other had rather stretched shapes.
With the second experiment the number of dead cells in the supernatant were determined. The well with untreated cells contained the lowest number of dead cells, the well with cells treated with 0.1 % EtOH the highest. Treatment with 4-OHT caused a little lower number of dead cells (Fig. 7).

Fig. 7: Number of dead cells in supernatant of HeLa cell culture after different treatment Cells were treated with 4-OHT or EtOH as shown in the figure for 18 h. Cells within the removed supernatant and 0.5 ml PBS of a washing step were counted and dead cells were calculated. 1 µM = 1 µM 4-OHT; 0.1 µM = 0.1 µM 4-OHT.

Discussion

Influence of the NLS on nuclear import

The lack of a NLS leads to an almost exclusive cytoplasmic localization of the Cas9-mCherry fusion protein. The presence of a NLS increases the nuclear import about 14- till 17-fold. Surprisingly one NLS seems to affect this stronger than two, but this difference is not significant due to the overlapping standard deviations. The pictures may be misleading to this conclusion because of a different cell shape between pIG3002 and pIG3003/4 which is caused by a different cell density.
The observations that one NLS is at least as strong as two and that the N-terminal NLS is as strong as the C-terminal one do not agree with the results of Cong et al. [13]. They showed that two NLSs lead to the strongest nuclear import, whereas a C-terminal NLS has a lower effect and a N-terminal NLS causes almost no nuclear import. This divergence to our findings may be because Cong et al. used HEK cells (instead of HeLa cells) and a Cas9-GFP fusion protein for their experiments. As GFP is derived from the fluorophor of Aequorea victoria [15] and mCherry is a monomeric derivative of the fluorophor of Discosoma sp. [16], it should have a different tertiary structure, which may cause another exposure of at least the C-terminal NLS.

Effects of 4-OHT induction

None of the 5 test constructs showed a big difference in subcellular localization after treatment with 4-OHT or EtOH; all values range within the standard deviations (Fig. 5). So no fusion protein could be termed hormone inducible. But because of the high standard deviations, because there were not so many cells on the pictures and because the regions where the mean fluorescence was measured had to be chosen personally, the analysis that was performed is not really trustworthy. This would then be a explanation for a stronger nuclear localization with EtOH treatment.
The pictures of pIG3006 show the tendency of more nuclear localization after 4-OHT treatment (Fig. 5), whereas the quantitative analysis demonstrate the opposite. The problem may be that with EtOH treatment there is a wide variety of cells with different fusion protein distributions, as indicated with the high standard deviation.

Effects of 4-OHT on Cas9 functionality

Also the functionality of Cas9 could not be regulated with hormone induction: The observed repression between Cas9 with and without crRNA due to Cas9 binding to the coding sequencing of SEAP and therefore hindering the transcription is independent of hormone addition. With and without 4-OHT there is only a very slight difference; in most samples the repression is even stronger with ethanol addition than with tamoxifen (Fig. 7).

Toxicity of 4-OHT

4-OHT in concentrations below 1 µM does not seem to be toxic to HeLa cells. If it can be assumed that most dead cells will detach from the well, then the toxicity assay we performed shows that the number of dead cells does not increase with 4-OHT treatment in compare to EtOH. It is more likely that the solvent EtOH has a negative effect on HeLa cells. But to be sure about this, this assey would have to be repeated more times.
The change in shape cells show after treatment with 10 µM 4-OHT may also occur because of a high cell density.
As HeLa cells do not have an ER [1], only the weak effects of 4-OHT on mitochondria [7] could harm the cells. But for this the tested concentrations are most probably too low.

References