Promoter Analysis Of Genes

Published Date: 02 Nov 2017

bachelor thesis

submitted to the faculty of Bioscience of

the Ruprecht-Karls University Heidelberg

Julia Volz

2012

Contents

Introduction

The circadian clock

During one day, the environment subjects organisms to a variation of different conditions, such as darkness and light or different temperatures, which they answer with rhythmic changes in physiology to be able to deal with these changing conditions1. Interestingly, these rhythms persist even when the organisms are kept for instance in total darkness and at a constant temperature level2. Thus, there has to be an internal pacemaker, which maintains rhythms with a period of approximately 24 hours, even when there is no external signal3. This timing machine is called the "circadian clock" (circadian: about one day) and is present as a central pacemaker in the brain and as peripheral clocks in almost every cell of the body4. Since its period is slightly different from 24 h, under normal conditions the clock is synchronised (=entrained) by external cues to the ambient environment2.

The suprachiasmatic nucleus (SCN) in the anterior hypothalamus is the location of the central pacemaker in vertebrates4. Its direct connection with the light-detecting system2 of the eyes through the retinohypothalamic tract highlights the importance of light as a synchronizer for the clock rhythm3. Via its connections to other brain areas and the neuroendocrine system3, the SCN controls systemic functions like the sleep-wake cycle and endocrine activity4, as well as body temperature, immune activity and blood pressure3. In contrast, the peripheral clocks have an influence on rhythmic changes in cell physiology, such as nutrient metabolism, detoxification, membrane trafficking and cell cycle4. Up to 10% of all mammalian genes are clock-controlled and most of them are organ specific3. However, some of them are expressed everywhere and include genes that are part of the clock-mechanism itself3.

The mechanism of the circadian clock consists of an autoregulatory feedback-loop system1. A heterodimer of two transcription factors called circadian locomotor output cycles kaput (CLOCK) and brain and muscle ARNT-like 1 (BMAL1)5 binds to a transcription factor binding side called E-box of in the promoter regions of Period genes (Per1, Per2, Per3) and Cryptochrome genes (Cry1, Cry2) and thereby activates their transcription2. The translation and the posttranslational modification of these proteins take time and cause a delay until they are able to go into the nucleus and to interact with the CLOCK/BMAL1 heterodimer in order to inhibit its activity6. By this, they repress indirectly their own transcription and therefore the amount of PER and CRY decreases and leads to the restart of the cycle by the rising activity of the CLOCK/BMAL1 heterodimer6. This loop is called the "core loop" of the circadian clock and maintains the cyclic activity of CLOCK/BMAL1, which binds in its active phase also to E-boxes of other clock-controlled genes and leads therefore to their oscillating expression2. The "accessory loop" is also an autoregulatory feedback loop and leads to a cyclic expression of BMAL1, and therefore supports, as well as several protein modifications, the core loop in maintaining the correct timing2. The retinoic acid-related orphan receptor response element (RORE) in the promoter of BMAL1 can be bound by RORA, which acts as a transcriptional activator, and by REV-ERB, which acts as a transcriptional repressor. The transcription of these transcription factors in turn is activated by CLOCK/BMAL12.

Glucocorticoid signalling

The SCN is also responsible for the oscillating activity of the hypothalamic-pituitary-adrenal (HPA) axis7, which represents one connection between the hypothalamus and the adrenal gland and causes the production of glucocorticoids in this gland6. Glucocorticoids are steroid hormones, which are released in 1-2 pulses per hour2. However, they also show a circadian rhythm regarding the amplitude of the pulse6. In diurnal (active at day) animals the highest amount of glucocorticoid is released in the early morning, whereas in nocturnal (active at night) animals it occurs in the early night2. They play an important role in stress response and in the immune system function8. Furthermore, they are involved in many organ or tissue functions, for example in the homeostasis of the intermediary metabolism and the cardiovascular system8 as well as in the immune/inflammatory reaction9. About 20% of the mRNA production of the expressed genome is influenced by this group of steroid hormones9.

When the glucocorticoids reach their target tissue, they bind to the glucocorticoid receptors (GRs), which are located in the cytoplasm of the cells6. This receptor belongs to the steroid/ sterol/thyroid/retinoid/orphan nuclear receptor super-family7 and occurs in a complex with heat shock proteins6. In the case of binding, the receptor enters the nucleus, homodimerizes and can act as a transcription factor by binding to glucocorticoid response elements (GREs), enhancer sequences of glucocorticoid target genes7. The two zinc finger domains in the GR are responsible for the dimerization and the DNA binding, whereas the C-terminal ligand- binding domain (LBD) binds glucocorticoids with a high affinity10. AF1 and AF2 are activation domains, which allow the interaction with other transcription factors10. In composite activation or repression, both the GR and the other transcription factor bind to the DNA alter transcription by their interaction10. Another possibility is the binding of the other transcription factor to the DNA and binding of the GR to this factor10. This is also called tethered binding, because the other transcription factor "tethers" the GR to the DNA. Because such a modulation evokes mostly a repression it is called trans-repression10. Furthermore, there are so called non-genomic effects of glucocorticoids, where the influence cannot be attributed to transcriptional regulation6. One example is a glucocorticoid dependent inhibition of the c-Jun N-terminal kinase (JNK), attributed to the binding of JNK to the LBD in the GR10.

Interaction of the peripheral clock and the glucocorticoid pathway

As already mentioned, the central pacemaker regulates the release of the glucocorticoids from the adrenal gland6. But there is evidence to assume that glucocorticoids in turn have an influence on the peripheral clock. For example, treatment with dexamethasone, a synthetic glucocorticoid, leads to a robust oscillating expression of clock genes like Per and Cry and clock-regulates genes in rat-1 fibroblasts, whereas without treatment there is no oscillation observable11. Furthermore, it is possible to use dexamethasone to cause a delay or an advance, dependent on the time of the treatment, in the phase of the peripheral oscillator in liver cells of mice, so it can be used as a substance to entrain the clocks11. With mouse mutants which have no GR in the liver, Balsalobre et al. could show that the GR mediates these phase shifts tissue autonomously11. However, lack of GR in the liver did not perturb clock gene expression in the absence of dexamethasone challenges, indicating that glucocorticoids are not crucially required for establishing the clock synchronisation in the liver11. In contrast, in some brain regions, there is a need for glucocorticoids in order to maintain the oscillating expression of per26. Moreover, Segall et al. demonstrated that only the glucocorticoid corticosterone, procured in a rhythmic manner and not in a constant supply, results in an oscillating per2 expression there12. The study of a mouse mutant, where BMAL1 is knocked down in the adrenal gland, shows an attenuation of the cycling per1 expression but no influence on the per2 expression in the liver, the kidney and the pancreas13. This might be due to the additional regulations of those two clock genes13. The expression of per1 is also directly influenced by the binding of the GR to a promoter element and in this mutant the glucocorticoid cycling is reduced. In contrast per2 is more dependent on the daily changes in body temperature that are present in those mutants than on the hepatic clock13.

Furthermore, there are also protein interactions between members of the glucocorticoid signalling and of the clock mechanism, by which the clock may exert an influence on the glucocorticoid pathway. An overexpression of the CLOCK/BMAL1 heterodimer in two human cell lines, HCT116 and HeLa, for example, leads to a repression of the transcriptional activity mediated by the GR7. The knockdown of the Clock and the Bmal1 mRNA, together and separately, leads to a higher transcription of a glucocorticoid-responsive gene in the presence of dexamethasone7. Considering the fact that the mRNA expression of Clock and Bmal1 occurs in a reverse-phase circadian fashion compared with the activity of the GR, it is likely that the CLOCK/BMAL1 heterodimer causes a circadian repression of the glucocorticoid activated transcriptional activity7. The heterodimer acetylates the GR, which hampers the binding to the GRE7. Another link between both pathways is the observation that also Cry1 and Cry2 interact with the GR5. This association is encouraged by the presence of glucocorticoids and occurs at the C-terminus of the receptor, which is used for the activation or repression of transcription5. In cry1-/-; cry2-/- knockout mice, most genes which are normally repressed by glucocorticoids are not repressed anymore and a considerable number of genes is activated in comparison to the control5. Therefore, it seems that Cry1 and Cry2 inhibit the transcriptional activation activity of the GR5.

The zebrafish as a model organism for clock research and endocrinology

The various techniques established in zebrafish, such as TILLING, morpholinos and in vivo imaging14, as well as its sequenced genome are obvious benefits for zebrafish as a model organism. The robust S-phase rhythms, which occur daily in zebrafish larvae under light-dark cycles and persists even after the transfer into complete darkness, indicates the activity of the clock in the larvae, what enables clock research already in those early stages15. To establish the clock mechanism in the larvae, light-dark cycles are essential15. Moreover, the existence of peripheral clocks in almost every cell type is certain15. About the nature of a potential central peacemaker in the zebrafish not so much is known yet16. The comparison of important members and principles of the endocrine system such as the HPA axis of zebrafish and mammals reveals a high conservation and, therefore, the obtained knowledge might be relevant also for the human endocrine system14.

Moreover, due to mutagenesis screens, an enormous number of mutants with defects in organ systems are available17, which can be used to reveal their contribution to the clock and its influence on the whole animal4. One example is the chokh/rx3 mutant, which is available in two different alleles due to a missense ("weak") or a nonsense ("strong") mutation in the retinal homeobox gene 3 (rx3)18. Both mutant types are affected in the development of the eye and as a result, they are blind18. However, the nonsense mutation leads to a lower number of corticotrope cells in the pituritary4, which normally produce adrenocorticotropic hormone (ACTH)2. ACTH would then stimulate the production of the glucocorticoid cortisol in the adrenal/interrenal gland2, but because it is not present there is a reduced level of cortisol in those mutants4. Using these mutants, Dickmeis et al. have shown that the cell cycle rhythm needs no ocular photoreception4. Furthermore, a rescue experiment with a continuous addition of dexamethasone to the strong rx3 mutants confirmed that deficiency of the glucocorticoid cortisol is the reason for the disruption in the circadian clock controlled cell cycle4, thereby revealing another interaction between the circadian clock and the glucocorticoid pathway. In addition, an increasing number of mutants for human diseases are discovered14.

The work with zebrafish cell culture cells reveals further advantages. In contrast to mammalian cells, zebrafish cells are able to detect light directly and use this information for clock entrainment19. Therefore, no treatments with substances such as dexamethasone or forskolin pulses are needed to make sure that the clocks in all the cells are at the same phase, but they simply have to be exposed to light-dark cycles20. The omission of substances is an advantage in the research about the interaction of the clock with signalling pathways, because then the pathways are not influenced by the substance treatment independently of the clock.

Aim of this project

According to unpublished results from Benjamin and Meltem Weger (Karlsruhe Institute of Technology), both a GRE and an E-box are necessary in a minimal promoter to get a dexamethasone dependent oscillating expression of a luciferase reporter gene. No rhythmic expression is detectable when only one of those two is present in the promoter. However, four E-box elements without a GRE are also able to produce an oscillating expression. This suggests that there is not only protein-protein interaction between members of the clock and the glucocorticoid signalling pathway, as explained in 1.3, but a composite activation or repression (1.2) might play a role as well. This approach was a so-called bottom-up approach, because two regulatory elements from well-known pathways were combined to prove that they interact when they are together in one system21.

To verify those results, a top-down approach shall be made, where you start with a look on the whole system to get information about the mechanism further down21. The first step is to collect experimental data, which will be analysed to reveal correlations which lead to the development of a hypothesis21. This hypothesis can be used as a starting point to set new experiments in order to prove the assumptions21.

In order to do this, Benjamin and Meltem Weger have used the RNA-Seq technique22 to compare the mRNA levels of wildtype zebrafish with those of strong and weak rx3 mutants at different time points. As explained in 1.4, both mutants are blind, but the strong mutant shows a reduced level of the glucocorticoid cortisol. Genes which are expressed in an oscillating fashion in wildtype but not in strong rx3 mutants are candidates to be controlled by glucocorticoids and the clock. To rule out the possibility that the ocular photoreception is the reason for the different expression, the mRNA expression of the weak mutant should look like those of the wildtype in candidate genes.

Since the expression pattern of those genes may refer to the activity of their promoters, these can be tested to see if their circadian expression is dexamethasone responsive. In this case, the promoter can be searched for the presence of E-box and GRE elements and their locations. Furthermore, it can be tested if mutations in those motives lead to a loss of the oscillating expression and thereby if those motives are really responsible for the dexamethasone-inducible oscillatory expression pattern.

In this bachelor thesis, the method used to analyse the activity of the candidate promoters is examined. The promoters are cloned into a luciferase construct which reports their expression pattern via bioluminescence in a zebrafish cell line with entrained clocks and with and without dexamethasone exposure. The protocol should enable to see if there is an oscillating expression only in the case of dexamethasone treatment. When this protocol is established, it is a fast and easy way to examine the circadian behaviour of a large number of promoters in dependence on glucocorticoid signalling. In this way, it is hoped to gain a global overview of how glucocorticoid dependent circadian transcription is regulated, revealing common motifs and motif arrangements driving particular aspects of this regulation. It will also confirm to which extent artificial element combinations (e.g. of E-boxes and GREs) can mimic the behaviour of endogenous promoters.

Materials and Methods

Cloning of the promoters of the genes of interest into the vector pT2:pGL3basicDONR221

vector

The Vector pT2:pGL3basicDONR221 was provided from Benjamin and Meltem Weger and is used to indicate the activity of the promoters of the genes of interest via a luciferase assay. The promoter is cloned in front of a luciferase gene in order to control its expression. The presence of the luciferase can then be detected and conclusions regarding to the activity of the promoter can be drawn. Furthermore, the promoter contains attP1 and attP2 sites and a ccdB fragment for negative selection. The attP1 and attP2 sites enable to insert the promoter into the vector by a clonase reaction, whereby the ccdB fragment is removed. When, due to a wrong clonase reaction, the ccdB fragment is still in the vector, the ccdB protein is build23. This protein causes breaks in the double stranded DNA by inducing the activity of the DNA gyrase and thereby it inhibits the growth of those bacteria23. Due to the ampicillin resistance carried by the vector, only the bacteria with the vector inside can grow on the ampicillin containing agar. Moreover, there are Tol2 sites present, which enable the enzyme transposase to integrate the vector into the genome of the cell line in which the luciferase assay is done.

Polymerase chain reaction (PCR)

To obtain the promoter regions of the genes of interest from genomic zebrafish DNA, a PCR is done. The start of the promoter region in our case is defined as about 900bp upstream of the transcription start and the end about 100 bp downstream. However, to get ideal primer pairs for the desired promoter, this region can vary. The oligonucleotides for the PCR were determined with the program Vector NTI and were ordered from …. They are shown in Table .

Table Oligonucleotides, used for PCR to extract the promoters from genomic zebrafish DNA and the optimal temperature for the annealing step of the PCR.

pdk2

(918 bp)

forward primer

5’-CCTGGAGGGGCTTTTGTAA-3’

55 °C

reverse primer

5’-TTGGTTCAGTCTCGGGTTGT-3’

fdx1b

(906 bp)

forward primer

5’-GCCATCTTTAGCAATTCCACA-3’

55 °C

reverse primer

5’-CATTCCTGATGAGCAAAACG-3’

sc4mol

(823 bp)

forward primer

5’-TGGGAAATCCTTAGCACTACA-3’

56 °C

reverse primer

5’-ACCATTGGATGAGGCAAGAG-3’

ugt5a1

(931 bp)

forward primer

5’-GGCACTATTGCCTGCAGAA-3’

55 °C

reverse primer

5’- ACACACATGCCAGAAGTCCA-3’

MTHFD1L

(1011 bp)

forward primer

5’-TAGCTCCAACCACCTCCAAC-3’

55 °C

reverse primer

5’-GGCGACAGACAGTCTCATCA-3’

apoea

(1016 bp)

forward primer

5’-GCCCAGAATACCCAAGACAA-3’

55 °C

reverse primer

5’-ACACCAGACAGATGGGCTCT-3’

cyp2aa12

(1066 bp)

forward primer

5’-GTACCGCATCTGGTGTGAAC-3’

58 °C

reverse primer

5’-ACGGAGGCCAGGTCTAACTT-3’

NPC1L1

(1033 bp)

forward primer

5’-TGGCTTATTTATCAGGAGTCG-3’

58 °C

reverse primer

5’-GTCCTTACAGGGAACTCTTGG-3’

tceb3

(1023 bp)

forward primer

5’-GCAACTGACGCAGGTAGGTC-3’

55 °C

reverse primer

5’-AAGCTGATGGCAGATTCAGA-3’

pla2g1b

(1029 bp)

forward primer

5’-AGTGAAACACGCACACATGC-3’

55 °C

reverse primer

5’-CAGGTAGGCAAACTGATGCTC-3’

cyp3a65

(1016 bp)

forward primer

5’-AAGCCGCTCTACAGTCAACC-3’

55 °C

reverse primer

5’-CAAGAACGCCAGCAGAGTC-3’

stk35

(804 bp)

forward primer

5’-CCTAGAGAAGGTTGTGCGTGA-3’

58 °C

reverse primer

5’-TCACTTCCTCCATCAATCGTC-3’

STK35

(804 bp)

forward primer

5’-CCTAGAGAAGGTTGTGCGTGA-3’

58 °C

reverse primer

5’-TCACTTCCTCCATCAATCGTC-3’

SLC26A3

(968 bp)

forward primer

5’-GACTTGCTGCATCACAGACA-3’

58 °C

reverse primer

5’-CAGCTGCAAGTGCTCTCAGT-3’

si:ch211-264e16.1

(937 bp)

forward primer

5’-CCTTGTTGGCTCTGGTTGAT-3’

55 °C

reverse primer

5’-TTGGCATCTGTTCATCTTGA-3’

paics

(1026 bp)

forward primer

5’-GATTGCCTCTCGCTTCATTC-3’

-

reverse primer

5’-ACCAGCTGTGTCCCTCTGTC-3’

To be able to insert the promoters into the vector by a clonase reaction, as explained below, the forward primers contain an attB1 overhang and the reverse primers an attB2 overhang.

attB1 : 5’-GGGGACAAGTTTGTACAAAAAAGCAGGCT-forwardprimer sequence-3’

attB2 : 5’-GGGGACCACTTTGTACAAGAAAGCTGGGT-reverse primer sequence-3’

The polymerase chain reaction was carried out with the GoTaq DNA Polymerase and the corresponding 5xGoTaq Green Reaction Buffer (Promega). 25 mM dNTP Mix, 100 µM forward and 100 µM reverse primer, … genomic zebrafish DNA, 0.25 µl polymerase and Buffer were filled up with water to an end volume of 50 µl.

The temperature profile of the program was set as follows:

95°C 2 min

----------------------------------

95°C 30 sec

50-60°C 30 sec repeated 35 times

72°C 1min/kb

----------------------------------

72°C 5 min

The exact temperature in step 3 to receive an optimal yield is primer-dependent and is shown in Table . First, 55°C was used for every promoter and when the result was not sufficient, the temperature was changed. In the case of an unspecific binding of the oligonucleotides the temperature was raised and when no PCR product was amplified, the temperature was reduced.

Gel electrophoresis and gel extraction

40 µl of the PCR product were analysed by gel electrophoresis in a 1% agarose gel with …ethidiumbromid. The run was performed at 110 Volt in the presence of TAE buffer for about 25 minutes and with the 0.1 – 10.0 kb DNA-Ladder from …. The bands with the PCR product were cut out on a UV-light table (75% maximal strength) and purified with the gel extraction kit (from…) as recommended by the manufacturer. Briefly, the gel band was solved in buffer and 5 µl of 3 M potassium acetate were added. The solution was put into a column with a membrane, where the DNA binds. The incubation time of 2 minutes, every time after adding the washing buffer, is a modification of the 0 min proposed in the original protocol to get a purer result. Furthermore, the 30 µl of the elution buffer were pipetted onto the membrane and spun down twice to increase the yield as opposed to the single spin down as suggested in the original protocol.

Clonase reaction

The clonase reaction was done with the MultiSite Gateway® Three-Fragment Vector Construction Kit (Invitrogen). 100 fmol purified PCR product and 150 ng pT2:pGl3basicDONR211 were filled up with TE to a reaction volume of 4.5 µl. After the addition of 0.5 µl BP Clonase II Enzyme Mix (from KIT) and an incubation of 2 hours at room temperature (RT), 0.5 µl Proteinase K (from KIT) was added and the reaction mix was exposed for 10 minutes to 37°C in order to stop the reaction and digest the remaining enzymes in the mix. After this, the reaction mix was kept on ice.

Transformation

3 µl of the clonase reaction mix were added to 25µl of competent bacteria, which were provided from Benjamin and Meltem Weger. After thirty minutes of incubation, the bacteria were put for one minute into a 42°C water bath and 300 µl 1xLB was added. Next, the flask was placed for 1 hour at 37°C in the shaker at … rpm. During this time, the bacteria start to express the ampicillin resistance gene. After incubation, 70 µl were plated on an agar plate with ampicillin. After one day at 37°C, colonies were picked for a mini- or a maxipreparation.

Minipreparation

Single bacterial colonies from the transformation were picked with a 200 µl pipette tip and grown over night in 3ml 1xLB-medium. 2 ml of this bacteria suspension was centrifuged at 13000 rounds per minute (rpm) for 30 seconds at RT. The pellet was resuspended in 250 µl resuspension buffer by vortexing and 250 µl lysis buffer was added. After inverting the tubes 7 times, 300 µl of neutralisation buffer were added and the mix was centrifuged at 13000 rpm for 25 minutes at RT. To the clear supernatant, which contains the plasmid, 800µl isopropanol was added and it was incubated for 30 minutes at -80°C so that the DNA can precipitate. After centrifugation at 13000rpm for 25 minutes at RT, the plasmid was sedimented. It was washed with 500 µl 75% ethanol and resuspended in 50µl H2O.

Digestion

1 µl of each miniprepatation was digested with … ApaI and … EcoRV (from…) for one hour at 37°C to cut out the promoter. This was then analysed by gel electrophorese with a 1% agarose gel at 125V in the presence of TAE buffer for about 25 minutes and with the 0.1 – 10.0 kb DNA-Ladder from ….

Sequencing

Microsynth AG was commissioned to sequence the minipreparations. The results were analysed with Vector NTI and with the ENSEMBL web site.

Maxipreparation

The maxipreparation was done with the Qiagen Plasmid Maxiprep Kit (Qiagen), as recommended in the instructions of the manufacturer.

Cell culture

cell culture

The zebrafish embryonic cell line Pac2 was cultured in 15 ml Pac2-medium in a 75 cm2 bottle in 37°C. At a density of about 100% (once a week) they were washed with PBS and detached with 1 ml of … trypsin for about 3 minutes in 37°C. After adding 7 ml of Pac2-medium, 1ml was returned into the 75 cm2 bottle and filled up to 15 ml with Pac2-medium.

Pac2 Luciferase Assay with Fugene HD

800000 cells per 6well plate were seeded in Pac2-medium and after 24 hours, the medium was changed to L15+FCS. Antibodies were omitted because they disturb the transfection. Furthermore, the transfection complex was prepared by mixing 100 µl L15-medium, 900ng vector DNA, 100 ng transposase and 4 µl FugeneHD (Promega) and after 20 minutes incubation, it was added dropwise to the cells. On the next day, the transfected cells were transferred into 24 wells of a 96well plate, so that in one well, there are about 27000 cells and 250 µl Pac2-medium. At day 4, 200 µl Luciferin-medium and 50 µl treatment solution were added to each well. 8 wells were treated with 60nM dexamethasone, 8 wells were treated with 10nM dexamethasone and the other 8 wells were treated with … DMSO as a control. In these steps of the last day hormone free FCS is used to make sure that there are no other glucocorticoids present.

Pac2 Luciferase Assay with SreenFect

The seeding, the transfer into the 96well plates and the treatment and measurement was done as explained in 2.2.2. However, the transfection complex was prepared differently. The amount of SreenFect (12µl or 15µl) was solved in 300 µl ScreenFect buffer and 1800ng vector DNA was also diluted in 300µl ScreenFect buffer. Then the two solutions were mixed, incubated for 20 minutes and dropwise added to the cells.

Measurement of the transfected cells with a florescence plate reader

The EnVision® XCite Multilabel Plate Reader with stacker automation and enhanced luminescence detection was used to detect the bioluminescence of luciferin and therefore indirectly the presence of luciferase in the cells. The well plates were covered with adhesive film and put into the reader. Between every plate two transparent plates were positioned to guarantee that the light reaches the cells, because they were exposed to light-dark cycles as shown below:

1. 900-2100: light 4. 900-2100: darkness

2100-900: darkness 2100-900: darkness

2. 900-2100: light 5. 900-2100: darkness

2100-900: darkness 2100-900: light

3. 900-2100: light 6. 900-2100: darkness

2100-900: darkness 2100-900: light

The received data was processed with the Biological Rhythms Analysis Software System (BRASS).

Used mediums and buffers

Medium/Buffer

usage

ingredients

TAE buffer

gel electrophorese

TE buffer

clonase reaction

1xLB-Medium

transformation

1x LB-Medium with ampicillin

minipreparation; maxipreparation

1xLB-Medium

ampicillin

resuspension buffer

minipreparation

lysis buffer

minipreparation

neutralisation buffer

minipreparation

L-15-medium without PR

Cell culture

L-15-medium

Cell culture

L15-medium without PR

PR

Pac2-medium

Cell culture

L15-medium

FCS

PenStreb

Gentamycin

L15+FCS

Luciferase Assay

L15-medium

FCS

Luciferin-medium

Luciferase Assay

L-15 medium without PR

hormone free FCS

Luciferin

ScreenFect buffer

Luciferase Assay

50mM NaOAc; pH5.0

Results

Cloning of the promoters of the genes of interest into the vector pT2:pGL3basicDONR221

With the RNA-Seq technique there were genes determined, which are rhythmically expressed in the WT and in the weak rx3 mutant but not in the strong rx3 mutant. In those mutants, as explained above, the amount of cortisol is considerably lower and therefore, their expression seems to be glucocorticoid and clock dependent.

To be able to see the activity pattern of the promoters of those genes related to the clock and the glucocorticoids, they were cloned in front of the luciferase gene of the pT2:pGL3basicDONR221 vector by a recombination reaction. For this purpose the Gateway® technology was used. It refers to the bacteriophage lambda site-specific recombination system, where the vector and the DNA insert need to have specific att sites which were recombined by a clonase enzyme mix.

Therefore, in the PCR to extract the promoters from genomic zebrafish DNA, the forward primers contain the attB1 sites and the reverse primers the attB2 sites, so that the PCR product is flanked with both sites. The primers were designed to amplify about 900bp upstream and about 100bp downstream of the transcription start of the genes. The PCR was done for 16 promoter regions and in 15 cases a PCR product was obtained, so there is high efficiency of 93.75 %. To purify the product, a gel extraction was performed.

Figure recombination reaction in which the attP flanked ccdB region is replaced with the attB flanked promoter from the PCR, catalysed by the BP Clonase enzyme mix (MultiSite Gateway® Three-Fragment Vector Construction Kit Protocol).

With this purified attB containing PCR product and the attP sites in pT2:pGL3basicDONR221 as donor vector the recombination reaction can be done. The clonase enzyme mix leads to a replacement of the attP flanked region with the attB flanked region whereby a mixture of both regions (attL) emerge, as you can see in Figure . The ccdB region which is located between the attP sites in the donor vector can be used for negative selection in the proliferation of the construct in E.coli. If the reaction doesn’t work and therefore, the ccdB region is not replaced, the ccdB protein is built in E.coli and inhibits the growth. The proliferation was done with a transformation of the new cloned vector into E. coli and with a minipreparation from 5 single colonies per promoter. Another selective component is the presence of the ampicillin resistance in the vector, so the bacteria are grown in agar with ampicillin to prevent the existence of bacteria without vector. Due to the digestion of the purified vector with ApaI and EcoRV and a gel electrophorese, the size of the cloned promoter region could be determined because the restriction sites flank the cloning site. This gave the first evidence on constructs with the correct promoter inside.

Figure The percentage of promoters with no, one or two mutations after cloning into the vector and the percentage of unsuccessful cloning with a total amount of 15 promoters

In 14 from 15 cases, 5 minipreparations were enough to find at least 3 positive constructs per promoter, which then could be sequenced to see if there are any mutations occurred. Only for one construct, 15 minipreparations had to be done to have enough positives.

The sequencing results were compared with the known promoter sequence from the ENSEMBL Database. Unfortunately, the genome of different zebrafish species varies and it could happen that the ENSEMBL sequence differs in some cases from the promoter sequence, present in the genome, which was used here. Therefore, when all three sequences show the same bases and only the ENSEMBL sequence is different, no mutation is assumed.

In two from 15 promoters the sequence could not be aligned to the known sequence. The other 13 promoters were successfully cloned into the donor vector pT2:pGL3basicDONR221. Two of them carry at least two mutations and two others contain at least one mutation, whereas in nine cases, there was a construct available, where the promoter shows no difference to the known sequence. In percentage, as you can see in Figure , 86.66% of the recombination reactions were successful and even 60% were without mutations. In Table the amount of mutations in the promoter sequence after the cloning. For every promoter the two constructs were listed which were transfected into the cell line in order to measure the promoter activity. the promoters and the corresponding number of mutations are listed.

Table the amount of mutations in the promoter sequence after the cloning. For every promoter the two constructs were listed which were transfected into the cell line in order to measure the promoter activity.

pdk2

#1

no mutation

#3

1 mutation

fdx1b

#3

1 mutation

#4

no mutation

sc4mol

#2

no mutation

#3

no mutation

ugt5a1

#1

no mutation

#5

no mutation

MTHFD1L

#4

no mutation

#5

no mutation

apoea

#4

1 mutation

#5

no mutation

cyp2aa12

#3

no mutation

#4

no mutation

NPC1L1

#4

1 mutation

#5

no mutation

tceb3

#4

1 mutation

#5

no mutation

pla2g1b

#1

3 mutations

#2

1 mutation

cyp3a65

#1

2 mutations

#4

2 mutations

stk35

#1

1 mutation

#4

1 mutation

STK35

#1

2 mutations

#4

2 mutations

SLC26A3

unsuccessful

si:ch211-264e16.1

un successful

Measurement of the promoter activity under dexamethasone treatment

As explained in 3.1, the promoter was cloned into the pT2:pGL3basicDONR221 vector in front of a Luciferase gene. This vector was then inserted into Pac2 cells by transfection with FugeneHD (from…). The Tol2 sites, existing in the vector, enabled the enzyme transposase to integrate the DNA into the genome of the host cell in order to produce a semi-stable cell line. Without this semi-stability, the signal would go down during the measurement because after a cell division only one daughter cell would still have the construct inside. To produce a stable cell line for each promoter is also possible, but the effort per promoter is higher and it would be impossible to examine such a high number of promoters in this range of time.

The semi-stable cell lines were then treated with two different concentrations of dexamethasone. Dexamethasone is solved in DMSO and to exclude that DMSO induces the effects, the cells were treated with DMSO as a control, too. For the treatment, only hormonefree FCS was used to exclude the possibility of glucocorticoids in the medium. When the promoter was active, luciferase was build. This enzyme is able to catalyse the reaction from luciferin to oxyluciferin, whereby light is emitted. To measure the presence and the amount of produced Luciferase, luciferin was added to the medium and the bioluminescence was measured for six days. Furthermore, the cells were exposed to a normal day-night cycle in the first three days to entrain the clock. The next day they spent in total darkness and the last two days in a twisted rhythm with light in the night and darkness at daytime. However, due to problems with the equipment, not every run could be measured the full time. Every promoter was tested in two different constructs (Table the amount of mutations in the promoter sequence after the cloning. For every promoter the two constructs were listed which were transfected into the cell line in order to measure the promoter activity.). If there was only one or no construct without mutation available, those with mutations were accepted.

Figure The value of the bioluminescence of the control constructs A) Ebox/GRE and B) 4xEbox, measured for 100 hours under light(l)-dark(d) conditions. To see if the transfection was successful, an Ebox/GRE construct in the … vector and a 4xEbox in the … vector was transfected as a control as well. The promoter regions of those constructs contain a minimal promoter with either a clock controlled Ebox element together with a glucocorticoid controlled GRE element or four Ebox elements. Therefore, the oscillating activity of the Ebox/GRE promoter should be dependent on dexamethasone and on the clock. In Figure The value of the bioluminescence of the control constructs A) Ebox/GRE and B) 4xEbox, measured for 100 hours under light(l)-dark(d) conditions. A the run with the Ebox/GRE construct is shown. The difference between the treatments can be seen. Under the DMSO treatment, where no glucocorticoid was available, the curve show no cycling in the dark period between 36 and 84 hours, whereas, under dexamethasone treatment oscillation persists. This means that the clock and the glucocorticoid signalling together created this oscillation pattern. Furthermore, with 60 nM dexamethasone a higher value was achieved than with 10 nM and the DMSO control had even a lower value and the amplitudes of the dexamethasone curves were higher than those of the DMSO curve. This indicated the activating function from the glucocorticoid on the promoter expression. By contrast, the oscillating activity of the 4xEbox promoter should be only dependent on the clock and not on glucocorticoids, because there are only Ebox and no GRE elements present. Therefore the cycling pattern of the DMSO curve in Figure The value of the bioluminescence of the control constructs A) Ebox/GRE and B) 4xEbox, measured for 100 hours under light(l)-dark(d) conditions. B and the similarity of all three curves were estimated. The value of the 4xEbox construct is with around 25000 in a scale, which was expected, whereas the value of the Ebox/GRE construct is substantially lower.

Figure the activity of the NPC1L1#4 promoter under light(l)-dark(d) cycles and three different treatments, measured for 110 hours. IN A) the original data and in B) the detranded data was plotted to subtract the decreases in the baseline level.

In Figure A the curves with the different treatment for the promoter construct NPC1L1 colony 4 are shown. It is clearly visible that the value of each curve decrease drastically during the measurement. As explained before, a decrease of the signal can refer to vector DNA, which is not integrated into the genome. This issue occurred in the measurement of every promoter and a problem-solving approach can be found in 3.3. Nevertheless, to be able to see the circulations better, the decreases in the baseline level were subtracted with the "detrand" function in the BRASS program for all constructs. The resulted curves for the NPC1L1#4 construct are shown in Figure B. The rapid decrease followed by an increase at the beginning is an effect of the detranding due to the rapid decrease in the original curve and has no validity. The further progress of the curve stays approximately on the same level so that a potential oscillation can be seen easier.

As explained for the Ebox/GRE control in Figure The value of the bioluminescence of the control constructs A) Ebox/GRE and B) 4xEbox, measured for 100 hours under light(l)-dark(d) conditions., dexamethasone can have an activating function on the promoter expression. This is also the case for the promoter ugt5a1, which is shown in Figure . Both used constructs (#1, #5) had no mutations and the curve shape is comparable. The treatment with dexamethasone leaded to higher values. In contrast to the Ebox/GRE control, the 60 nM dexamethasone displays a lower value than 10 nM of the glucocorticoid. This means, that the 10nM concentration induced a stronger activation, than the 60 nM concentration of dexamethasone. However, the oscillation which could be seen during the dark-light phase at the beginning of the run did not persist in the total darkness between 60 hours and 108 hours after the start in both cases. Therefore, the dexamethasone induced only an activation of the expression, but had no influence on the oscillation pattern.

Figure the activity of the ugt5a1 promoter #1 (A) and #5 (B) under light(l)-dark(d) cycles, treated with DMSO (red) 10 nM dexamethasone (green) or 60 nM dexamethasone (blue). This promoter shows a dexamethasone induced activation.

Besides an activation of the expression, dexamethasone can also generate a repression what results in a high value DMSO curve and lower value in the dexamethasone curves, because there, the expression is reduced. The promoters displayed in Figure the activity of the fdx1b promoter #3 (A) and #4 (B), of the cyp2aa12 promoter #3 (C) and #4 (D) and of the sc4mol promoter #2 (E) and #3 (F) under light(l)-dark(d) cycles, treated with DMSO (red) 10 nM dexamethasone (green) or 60 nM dexamethasone (blue). All promoters show a dexamethasone induced repression. correspond to this and therefore, they show a dexamethasone dependent repression. Both constructs of the fdx1b promoter (#3: 1 mutation, #4: no mutation) show no oscillation pattern in the long dark phase and are comparable with the DMSO control. This means that dexamethasone cannot lead to a persistence of the oscillation pattern. Whereas, in the mutation free cyp2aa12 constructs the oscillation might persist. However, it persists even in the DMSO control and therefore, cannot be dexamethasone dependent. The sc4mol promoter is the only promoter in Figure the activity of the fdx1b promoter #3 (A) and #4 (B), of the cyp2aa12 promoter #3 (C) and #4 (D) and of the sc4mol promoter #2 (E) and #3 (F) under light(l)-dark(d) cycles, treated with DMSO (red) 10 nM dexamethasone (green) or 60 nM dexamethasone (blue). All promoters show a dexamethasone induced repression., which had a better value height. In both constructs with no mutation an oscillation can be guessed with 10 nM and 60 nM dexamethasone treatment during the long dark period between 36 hours and 84 hours after the start, when you compare it with the non-cycling DMSO control. However, despite the higher value, this is just a weak suggestion and of course not proved with these results. It seemed to be necessary to achieve a higher value to be able to see oscillations in a conclusive extend.

Figure the activity of the fdx1b promoter #3 (A) and #4 (B), of the cyp2aa12 promoter #3 (C) and #4 (D) and of the sc4mol promoter #2 (E) and #3 (F) under light(l)-dark(d) cycles, treated with DMSO (red) 10 nM dexamethasone (green) or 60 nM dexamethasone (blue). All promoters show a dexamethasone induced repression.

In Figure A) the promoter MTHFD1L construct 4 is shown. Due to the higher value of the dexamethasone curves an activation function of the glucocorticoid could be recognized. However, the MTHFD1L construct 5 (Figure B) which carried as well as construct 4 no mutation, displays the lowest value in the 60nM dexamethasone curve. Nevertheless, the highest value in both cases was induced by the 10 nM dexamethasone treatment. So this might be a good concentration for activating the expression of this promoter. There is no oscillation during the long dark phase visible, and therefore the expression seemed not dexamethasone and clock dependent. For the promoter pdk2, the construct 1 without mutation (Figure C) shows a dexamethasone activation of the expression, whereas the construct 3 with one mutation (Figure D) shows a dexamethasone repression. This mutation could be hold responsible for this change, but as seen in the case of the MTHFD1L promoter, even when there is no difference in the sequence the value height in different constructs can change. During the long dark phase, the oscillation might persist, but as in the case of cyp2aa12, there is no difference to the DMSO control visible, and therefore it cannot be dexamethasone dependent.

Figure the activity of the MTHFD1L promoter #4 (A) and #5 (B), of the pdk2 promoter #1 (C) and #3 (D). of the tceb3 promoter #4 (E) and #5 (F) and of the pla2g1b promoter #1 (G) and #2 (H) under light(l)-dark(d) cycles, treated with DMSO (red) 10 nM dexamethasone (green) or 60 nM dexamethasone (blue).

For the tceb3 promoter, it could not be determined if dexamethasone has an activating or a repressive function on the expression, because the graphs of construct 4 with one mutation (Figure E) and construct 5 without mutation (Figure F) differ from each other. During the long dark period no oscillation was detectable and therefore, the glucocorticoid and clock dependent expression seemed to play no role here. The promoter pla2g1b construct 1 (Figure G) with 3 mutations suggested an activation function of dexamethasone regarding to the expression, whereas construct 2 (Figure H) with one mutation inclined to a repression. Furthermore, it seemed not to be a glucocorticoid and clock dependent expression, due to no oscillation in the long dark phase.

The promoters shown in Figure also did not reveal a dexamethasone and clock dependent oscillation, because there is no oscillation during the long dark phase between 60 hours and 108 hours after the start of the measurement detectable.

Figure the activity of the NPC1L promoter #4 (A) and #5 (B), of the cyp3a65 promoter #1 (C) and #4 (D) and of the apoea promoter #4 (E) and #5 (F) under light(l)-dark(d) cycles, treated with DMSO (red) 10 nM dexamethasone (green) or 60 nM dexamethasone (blue).

Construct 4 of the NPC1L promoter (Figure A) carried one mutation and indicated a dexamethasone dependent activation, whereas construct 5 (Figure B) with no mutation shows a repression at the high dexamethasone concentration. In the case of the cyp3a65 promoter construct 1 (Figure C), not transfection occurred, because the value of around 50 is as high as the one of cells without transfection. The run with construct 4 (Figure D) with two mutations displayed a slight repression with 60 nM dexamethasone.Figure the activity of the STK35 promoter #1 (A) and #4 (B) and of the stk35 promoter #1 (C) and #4 (D) under light(l)-dark(d) cycles, treated with DMSO (red) 10 nM dexamethasone (green) or 60 nM dexamethasone (blue).

The DMSO and the 10 nM dexamethasone curves for the promoter apoea are in both constructs at arount the same value, whereas in construct 4 with one mutation, the 10 nM dexamethasone curve is at a higher value and therefore indicates an activation of expression and construct 5 without a mutation shows a slight repression.

In Figure the activity of the STK35 promoter #1 (A) and #4 (B) and of the stk35 promoter #1 (C) and #4 (D) under light(l)-dark(d) cycles, treated with DMSO (red) 10 nM dexamethasone (green) or 60 nM dexamethasone (blue). the results for the measurement with the promoters STK35 and stk35 are shown. The sequencing results for these promoters revealed beside the two mutations in both constructs of STK35 and one mutation in both constructs of the stk35 a high amount of repetetive sequences. All constructs for both promoters showonly slight oscillation as a reaction on the light-dirk cycles at the begining of the measurement and therefore, also no oscillation in the long dark phase. There are also only slight differences acording to the different treatment detectable. The only exeption is STK35 construct one, where a high consentration of dexamethasone seems to induce an activation of the repression.

In conclusion these results are not realy significant due to the low value. The assertions made here are therefore only suggestions which need to be proved with a revised experimental setup. A problem-solving approach can be found in 3.3.

The promoter ugt5a1 shows a dexamethasone dependent activation of expression and the promoters fdx1b, cyp2aa12 and sc4mol a dexamethasone dependent repression. For the other promoters it could not be determined if there is an activation or repressen due to the treatment with the glucocorticoid. The dexamethasone and clock dependent oscillating patter, could only be assumed in the case of sc4mol. However, this is the only promoter with a slightly higher value what suggests that a higher value is nesseary to be able to see an oscillation. Therefore, the possibility that the other promoters could show an oscillation at a higher value is not ruled out.

Improvements to get a higher value in the measurement

To get useful results in the measurement, the efficiency of the integration into the genome of the host cell needs to be increased drastically. There could be many reasons why this was not working properly in the previous experiments and four of them were investigated here.

The first most obvious reason might be a mutation in the Tol2-sites, so that they could not be recognised from the transposase. Therefore, the Tol2 sites from the pT2:pGL3basicDONR221 vector were sequenced and compared with those of the … vector. This was the vector from the control 4xEbox, which was integrated in a rage as it was expected. However, the sequences correlated exactly and therefore a mutation could not be the reason.

Figure the height of the value 24 hours after the treatment, depending on different transposase:DNA ratios. A) value of the promoter pdk2 construct 1 B) value of the Ebox/GRE construct C) value of the 4xEbox control. The DMSO control is shown in black and the 10 nM dexamethasone treatment in grey.The ratio between the amount of utilised DNA and transposase could also be a point of application to improve the experimental structure in order to get better results. Therefore, the normal luciferase assay was done with the promoter pdk2 and the two controls Ebox/GRE and 4xEbox, but with different transposase:DNA ratios. In the previous experiments 100 ng transposase and 900 ng DNA were used what corresponds to a 1:10 ratio. Here, a 1:4 (250ng transposase; 750 ng DNA) and a 1:2 ratio (500 ng transposase; 500 ng DNA) was tested under 10nM dexamethasone treatment and DMSO control. In Figure the height of the value 24 hours after the treatment, depending on different transposase:DNA ratios. A) value of the promoter pdk2 construct 1 B) value of the Ebox/GRE construct C) value of the 4xEbox control. The DMSO control is shown in black and the 10 nM dexamethasone treatment in grey. the value 24 hours after the dexamethasone or DMSO treatment is shown. In the case of the promoter pdk2, colony 1 (Figure the height of the value 24 hours after the treatment, depending on different transposase:DNA ratios. A) value of the promoter pdk2 construct 1 B) value of the Ebox/GRE construct C) value of the 4xEbox control. The DMSO control is shown in black and the 10 nM dexamethasone treatment in grey., A) and 4xEboy (Figure the height of the value 24 hours after the treatment, depending on different transposase:DNA ratios. A) value of the promoter pdk2 construct 1 B) value of the Ebox/GRE construct C) value of the 4xEbox control. The DMSO control is shown in black and the 10 nM dexamethasone treatment in grey., C), there was no significant difference between the height of the value of the dexamethasone treatment and the DMSO control visible. Whereas, the Ebox/GRE value was notably lower under DMSO treatment than under dexamethasone. This means, that at this time point the 4xEbox and the pdk2 promoters were with and without dexamethasone to the same extent active, while the EBox/GRE promoter needed the glucocorticoid for activity. The 1:10 transposase:DNA ratio seemed to be the best choice regarding the pdk2 promoter (Figure the height of the value 24 hours after the treatment, depending on different transposase:DNA ratios. A) value of the promoter pdk2 construct 1 B) value of the Ebox/GRE construct C) value of the 4xEbox control. The DMSO control is shown in black and the 10 nM dexamethasone treatment in grey. A). Its value was about tree time as high as this of the 1:4 or the 1:2 ratios. In the case of Ebox/GRE (Figure the height of the value 24 hours after the treatment, depending on different transposase:DNA ratios. A) value of the promoter pdk2 construct 1 B) value of the Ebox/GRE construct C) value of the 4xEbox control. The DMSO control is shown in black and the 10 nM dexamethasone treatment in grey. B) the 1:10 ratio was at the same level as the 1:4 ratio but the 1:2 ratio was lower. However, the 4xEbox (Figure the height of the value 24 hours after the treatment, depending on different transposase:DNA ratios. A) value of the promoter pdk2 construct 1 B) value of the Ebox/GRE construct C) value of the 4xEbox control. The DMSO control is shown in black and the 10 nM dexamethasone treatment in grey. C) promoter showed the lowest value at the 1:10 transposase:DNA ratio. At the 1:4 and the 1:2 ratios the value was about 25% higher. Regarding to the fact, that this was the only promoter, which was successfully integrated into the DNA of the host cell, even the value at the 1:10 ratio was high enough to get convincing results. In conclusion, the 1:10 transposase/DNA ratio was already a good choice and is therefore also not the reason for the insufficient results.

Figure the value of luciferase assays with the 4xEbox contruct and with different amounts of transposase(TP) at a 1:10 tranposase :DNA ratio measured for 72 hours in light(L)-dark(D)cycles. A) ASSAY with DMSO treatment. B) Assay with dexamethasone(DEX) treatment. C) value of the DMSO and the dexamethasone run 24 hours after the start.

A different amount of transposase but in a maintained 1:10 transposase:DNA ratio could also have an influence on the value. To investigate this, the luciferase assay with the 4xEbox construct was done with three different amounts of transposase (100ng transposase, 50 ng transposase or 25 ng transposase). To maintain the 1:10 transposase:DNA ratio and the total amount of DNA (1µg) a dummy DNA was used, which had nothing to do with the experiment. The probes were measured for 72 hours with light-dark cycles. In Figure A the run with DMSO treated cells and in Figure B the treatment with 10 nM dexamethasone were shown. All curves looked as explained in 3.2 and therefore the assay was successful. Due to the light induced and dexamethasone independent activity, the results of the DMSO and dexamethasone treated cells showed no significant difference. The 25 ng transposase curve had the highest value, followed by the 100 ng curve. The lowest value could be seen on the 50 ng curve. In Figure C, the value 24 hours after the start of the measurement was displayed to clarify that 25 ng transposase with a 1:10 transposase:DNA ratio the higest value was created.

Figure The comparison of the transfection methods FugeneHD and ScreenFect with the 4xEbox and the pdk2#1 construct under DMSO and dexamethasone(dex) treatment measured for 72 hours under light(L)-dark(D) cycles. A) 4XEbox treated with DMSO B) 4xEbox treated with 10nM dexamethasone C)Zoom in B) to see the Screenfect transfection D) pdk2#1 trated with DMSO E) pdk2#1 treated with 10 nM dexamethasone

The constructs were transfected into the cells with FugeneHD. This could also be an interference factor for the integration into the genome. To investigate this assumption, the transfection reagents ScreenFect was tested with the 4xEbox and the pdk2 construct and compared with FugeneHD. Furthermore, two different amounts of ScreenFect were used. The results for both constructs under DMSO and 10nM dexamethasone treatment were shown in Figure The comparison of the transfection methods FugeneHD and ScreenFect with the 4xEbox and the pdk2#1 construct under DMSO and dexamethasone(dex) treatment measured for 72 hours under light(L)-dark(D) cycles. A) 4XEbox treated with DMSO B) 4xEbox treated with 10nM dexamethasone C)Zoom in B) to see the Screenfect transfection D) pdk2#1 trated with DMSO E) pdk2#1 treated with 10 nM dexamethasone. As it is visible in Figure The comparison of the transfection methods FugeneHD and ScreenFect with the 4xEbox and the pdk2#1 construct under DMSO and dexamethasone(dex) treatment measured for 72 hours under light(L)-dark(D) cycles. A) 4XEbox treated with DMSO B) 4xEbox treated with 10nM dexamethasone C)Zoom in B) to see the Screenfect transfection D) pdk2#1 trated with DMSO E) pdk2#1 treated with 10 nM dexamethasone A, B, D and E the transfection with FugeneHD leads in al constructs and independently from the treatment and the amount of SreenFect to the highest value by far. However, as shown in Figure The comparison of the transfection methods FugeneHD and ScreenFect with the 4xEbox and the pdk2#1 construct under DMSO and dexamethasone(dex) treatment measured for 72 hours under light(L)-dark(D) cycles. A) 4XEbox treated with DMSO B) 4xEbox treated with 10nM dexamethasone C)Zoom in B) to see the Screenfect transfection D) pdk2#1 trated with DMSO E) pdk2#1 treated with 10 nM dexamethasone C that represents a zoom in Figure The comparison of the transfection methods FugeneHD and ScreenFect with the 4xEbox and the pdk2#1 construct under DMSO and dexamethasone(dex) treatment measured for 72 hours under light(L)-dark(D) cycles. A) 4XEbox treated with DMSO B) 4xEbox treated with 10nM dexamethasone C)Zoom in B) to see the Screenfect transfection D) pdk2#1 trated with DMSO E) pdk2#1 treated with 10 nM dexamethasone B to have a better look on the ScreenFect transfection, the transfection itself was successful based on the presence of the cycling signal.

Discussion

In order to test the activity of a number of promoters, a technique was tested, where the promoter region was cloned in front of a luciferase gene to control its expression. Therefore, a cloning strategy with the Gateway® technology was used.

The PCR to extract the promoter from genomic zebrafish DNA and to flank this sequence with att sites was with 93.75% efficiency very productive. Only the paics promoter, one of 16 promoter sequences could not be extracted, even though the annealing temperature varied. Maybe the primer pair was not optimal and a new one should be determined. With the remaining promoters the recombination reaction could be done, to insert the promoter into the host vector. The selection method with ccdB to prevent the occurrence of the vector without integrated promoter worked perfectly well, because in all cases, there was something inserted. However, in 13.33% (2 of 15 promoters) of the recombination the reaction was unsuccessful as the inserted sequence did not correspond to the known promoter sequence. Due to the fact, that the inserted sequence was the same in all the sequenced constructs for one promoter, there might have been gone something wrong in the PCR reaction, so that the wrong sequence was flanked by the att sites. The remaining 86.66% were successfully cloned promoters, where in 60% at least one construct with a perfect match to the known promoter sequences, in 13.33% with at least 1 and 13.33% with 2 mutations are available. Mutations can occur due to the UV-light which was used to cut the PCR product out of the gel. Therefore only 75% UV was taken and the work was done as fast as possible. However, mutations could not been totally prevented. In summary it can be said that the cloning strategy was successful and a high amount of promoters could be cloned into the host vector.

To visualise the activity of the promoter, the construct was inserted into Pac2 cells and should be integrated into the genome where the luciferase could be build. Unfortunately, the integration into the genome did not work properly and therefore, no semi-stable cell line could be build. Nevertheless, the build luciferase catalysed the reaction from Luciferin which was added to the medium to oxyluciferin and the resulted bioluminescence could be measured. However, the value of this signal decreased continually during the measurement and was at a very low level. The control with 4xEbox displayed the expected level around 10000 whereas the value of the most promoters was around 300. Due to the fact, that the 4xEbox construct was in another host vector, there could be several reasons why the integration failed. The possibility was tested, if a mutation occurred in the tol2 sites of the host vector, and the transposase could not recognize its point of application. But sequencing showed that there was no difference between the sequence of the tol2 sites in the host vector of the promoters and of the 4xEbox control. Therefore, this could not be the reason. A second possibility to gain a semi-stable cell line seemed to be the change of the DNA:transposase ratio. This was done with tree different ratios for the 4xEbox and the Ebox/GRE constructs and one promoter construct. Thereby, the for the experiments used 10:1 DNA:transposase ratio revealed as the one with the highest value. So the change of this ratio could not evoke better results. The amount of transposase is also a variable factor, which could change possibility of integration. To investigate this, another experiment with different amounts of transposase was done for the 4xEbox construct. To maintain the DNA:transposase ratio, a dummy DNA was used. Here, 25 ng transposase showed a higher value as the 100 ng transposase which was used in the normal experiments. This should be tested with a promoter sequence again and then it could be used as an improvement for the protocol. Another problem could be the transfection reagents FugeneHD, which was used to insert the DNA into the cells. To exclude the possibility of an interference of this substance, two different amounts of Screenfect, another transfection reagent was tested and compared in its efficiency with FugeneHD. However, it showed a drastically lower value and therefore, FugeneHD remained the transfection reagent of choice. In conclusion, the here tested parameters are at its optimal level and not the reason for the low value, except the amount of transposase which can be tied to change from 100 ng to 25 ng in the next experiments. However, the next step would be to try to produce stable cell lines, where the construct is inserted by electroporation for example. This would mean a higher time exposure per promoter but hopefully the results would be more significant.

A look on the bioluminescence measurement results, which showed a low value and a decrease during the measurement as already mentioned above, reveals only the sc4mol promoter as potential candidate to be regulated by the clock and the glucocorticoid pathway. However, this is the only promoter by which a value of about 2200 could be achieved. This is about 5 times higher than the value of the other promoters and might indicate that only with a value in this magnitude and higher the oscillation can be seen. Therefore, it cannot be excluded that the other promoter are also regulated by the clock and the glucocorticoid and this can just not be seen in these results here.

The sc4mol gene encodes for the sterol-C4-methyl oxidase (SMO), which is important in the cholesterol biogenesis24. Cutts et al. recognized early that the synthesis of cholesterol in human lymphoblastic leukemia cells is inhibited by dexamethasone25. Cholesterol is, besides other functions, a precursor for the glucocorticoid cortisol26. Furt