2.8.1 Plasmid generation for functional analysis Figure 1:
Figure 1: pCpGl-basic: Klug and Rheli239 designed a CpG-free vector for promoter analysis experiments. This vector contained a multiple cloning site in front of a firefly Luciferase gene.
Various constructs were cloned into the multiple cloning site. The figure was adopted from:
Klug and Rehli. Functional analysis of promoter CpG methylation using a CpGfree luciferase reporter vector. Epigenetics. 2006; 1(3): 127-130
PCpGl-basic, a CpG-free Luciferase containing vector (Klug and Rheli,239see figure 1), was used to analyze CpG-methylation dependent functional aspects of the AREG promoter and the exon 2.
Human genomic DNA was used as input for a standard PCR containing primers which harbor locusspecific parts and 5’- overhang sequences. In the overhang sequence
the forward primers contained a BamHI restriction site whereas the reverse primers con-tained a HindIII restriction site. The primer sequences are shown in the supplementary material 8.1. They were used to amplify the AREG promoter sequence, the AREG exon 2 and the exon 2 sequence divided into two parts. The temperature program of the PCR was as follows:
After PCR the PCR products as well as pCpGl-basic were digested with BamHI and HindIII as described in 2.2.7. Isolated linearized pCpGl-basic was then dephosphory-lated (see 2.2.10), ligated with the isodephosphory-lated PCR product (2.2.11), transformed into PIR1, identified and isolated (2.4.2 and 2.4.3). Due to theBamHI andHindIII restriction sites, plasmids were generated with forward oriented inserts.
For the generation of the promoter-containing plasmids, pCpGl-basic was digested with HindIII and NcoI. The CpG-free EF1-promoter was amplified by PCR from the control plasmid pCpGl-CMV-EF1 using the same program as shown above. Primers were used having 5’ overhangs which contain either a HindIII restriction site (forward primer) or an NcoI restriction site (reverse primer). To create a CpG-free Tata-binding motif, two oligonucleotides were used, which anneal to a DNA double strand Tata motif containing sequence with 3’-overhangs on both strands. The 3’ overhangs could be ligated into digested HindIII and NcoI restriction sites. The EF1 -PCR product as well as the Tata-binding motif forming oligonucleotides were ligated with the linearized plasmid to form pCpGl-Tata and pCpGl-EF1. Plasmids were transformed and isolated (see 2.4.2 and 2.4.3).
PCpGl-basic, pCpGl-Tata, pCpGl-EF1 and pCpGl-AREG-promoter were used to create plasmids harboring the exon 2 insert sequence in both orientations. First, the plasmids were linearized withBamHI. The exon 2 insert as well as the exon 2 parts were created as described above and digested withHindIII andBamHI. For the exon 2 reverse plasmid (pCpGl-AREG-exon2-R), the insert was created directly using primers without 5’ overhang. The single-stranded overhangs of both the linearized plasmids as well as the digested PCR products were filled to doublestranded DNA using the Klenow fragment (see 2.2.9). The plasmids were then dephosphorylated and ligated with the PCR products (see 2.2.10 and 2.2.11). Plasmids were transformed and isolated as described in section 2.4.2 and 2.4.3.
Finally, plasmids, harboring specific mutations were necessary to analyze the influ-ence of the CTCF-binding site on the function of the exon 2 region.
pCpGl-AREG-exon2-AREG-promoter, AREG-promoter and pCpGl-AREG-exon2-R-Tata were mutated using the kit ”Phusion site-directed mutagenesis kit” and the 5’
phosphorylated primers CTCFtoHINDIII 1/2 (see: 2.2.5). These primers bind down-stream the CTCF-binding site on both strands of the plasmid and have a 5’- overhang that form aHindIII restriction site (see figure 2). The formation of PCR products was performed as described by the manufacturer. The PCR-temperature program was as follows.
30 seconds - 98 ℃ 40x
{10 seconds - 98 ℃, 30 seconds - 71 ℃, 2 minutes - 72℃} 10 minutes - 72℃
HOLD 4 ℃
Circular plasmids were created by ligation. Afterwards, the plasmids were transformed into PIR1 bacteria, isolated and characterized as described in 2.4.2 and 2.4.3.
All generated plasmids are listed in supplementary section 8.2. The sequences of all plasmids are shown in supplementary section 8.3.
For methylation dependent promoter function analysis, several plasmids needed to be in vitro methylated. Methylation was performed as described in section 2.2.8.
Methylated plasmids were analyzed for their methylation by restriction digest using the methylation sensitive restriction enzyme AvaI. One example, on how methylated plasmids were identified, is shown in supplementary section 8.5.
Figure 2:
Figure 2: Principle of plasmid mutagenesis: Phosphorylated primers which bind downstream a CTCF-binding site on both strands of a plasmid and which harbor sequence unspecific parts, forming together a HindIII restriction site (HindIIIa, HindIIIb) were used in the ”Phusion site-directed mutagenesis kit” to create linear products to form new plasmids after ligation.
2.8.2 Cell transfection and sample preparation
Plasmids were transfected either in two replicates in 12 well plates or in three replicates in a 96 well Costar Cluster-Plate as shown in section 2.3.6. All pCpGl-basic derived plasmids, which contain a Firefly Luciferase gene were transfected simultaneously with a Renilla Luciferase gene containing control plasmid. After replacing the transfection medium with fresh medium, cells were incubated in the cell incubator over night. On the next day the medium was completely removed from the plate and each well was washed once with 1x PBS.
12 well plate assay 200 µl 1x Passive Lysis Buffer (PLB: Dual-Luciferaser Reporter (DLRT M Assay) were pipetted into each well. Cells were scraped from the plate and transferred into a new reaction tube. The tubes were stored at -80 ℃.
96 well plate assay 20 µl 1x PLB (Dual-Luciferaser Reporter Assay) were pipetted into each well. The plate was shaken for 30 minutes at 600 rpm on the plate shaker IKA MTS 2/4, sealed and afterwards stored at -80 ℃.
2.8.3 Detection of Firefly and Renilla Luciferase activity
Luminescence measurement using Lumat LB 9507 20 µl of PLB -lysed cells were transferred into a 5 ml polypropylene tube (Sarstedt) and mixed with 90 µl of Lu-ciferase Assay Reagent II (LARII: Dual-LuLu-ciferaser Reporter (DLRT M) Assay). Firefly luminescence was measured for 10 seconds. Afterwards, 90µl of 1x Stop & Glow reagent was tranferred into the glas vial and mixed carefully. Renilla luminescence was then measured for 10 seconds.
Luminescence measurement on Luminoscan RS The Costar Cluster-Plate, used for transfection, was thawed to room temperature and shaken for 10 minutes at 600 rpm.
Alternatively, 20µl of PLB -lysed cells were transferred into the wells of a 96-well Costar Cluster-Plate. Firefly and Renilla luminescence were measured as follows:
1. Start a clock. Pipet 70 µl of Luciferase Assay Reagent II into the first well of the plate
2. Wait 10 seconds.
3. Pipet 70 µl of Luciferase Assay Reagent II into the second well of the plate 4. Wait 10 seconds.
5. Repeat that procedure until a maximum of 8 wells were pipetted.
6. Load the plate into the plate reader.
7. When 2 minutes are passed from the first pipet step, start measuring luminescence.
8. Measure each well for 10 seconds in high gain and collect the data.
9. When 4 minutes are passed from the first pipet step, start measuring again.
10. Measure each well for 10 seconds in high gain and collect the data.
11. When 6 minutes are passed from the first pipet step, start measuring again.
12. Measure each well for 10 seconds in high gain and collect the data.
13. Remove plate from the reader, stop the clock and repeat the whole procedure for the same wells but pipetting Stop & Glow reagent instead of LARII.
14. Repeat all steps, to measure all wells on the plate.
The described procedure ensured that every well was measured at the same time after pipetting the substrate solution and that each well was measured 3 times independently.