Bokelmann K, Brockmöller J, Tzvetkov MV.

Impact of Promoter Polymorphisms on the Transcriptional Regulation of

the Organic Cation Transporter OCT1 (SLC22A1). J Pers Med. 2018;8(4):42.

This journal club builds on material covered in the molecular techniques collaborative learning session in week 9 and
lectures from weeks 9 and 10.

1. What was the hypothesis the authors were testing in this study? Hint: read the abstract and the introduction
to find this answer
Common OCT1 promoter SNPs contribute to high variability of OCT1 expression.

 (i) What methods were used to address the aims of this study Hint: read the introduction and methods
(headings) to find this answer (ii) What information do these assays provide? 
Cell culture and transfection - to measure luciferase activity
EMSA - common affinity electropheresis technique used to study protein-DNA or protein-
RNA interactions

2. In figure 1 the promoter region of OCT1 gene which codes for the organic cation transporter is shown, along
with the binding sites of USF1/2 and HNF4. Single nucleotide polymorphisms (SNPs) identified in the OCT1
promoter are also shown. (i) OCT1 belongs to the SLC transporter superfamily, very briefly describe the role these
proteins play in cells. (ii) HNF4 is an orphan type II nuclear receptor, describe what the term orphan means in this
context and how this receptor signals. (iii) Define the term SNP. 
i) The SLC transporter superfamily regulate organic cationic fluxes at the plasma
membrane, or in order words, the solute transporter into and out of cellular organelles,
from the blood and into the human liver.
ii) Orphan receptors are receptors that have no known endogenous ligand that binds to
it.
iii) SNP is abbrevaited for Short Nucleotide Polymorphisms, which is a type of genetic
variation or polymorphism that involves the variation of a single base pair.

3. Answer the following questions about Figure 2

• What the aim is of this experiment?

To determine nuclear protein binding of USF1/2 to an E-box in the OCT1 promoter

region of the A-allele using EMSA.

• What probes were used in this EMSA? What do the letters represent?
Probes carried OCT1 promoter SNPs
Letter represent single nucleotide bases
• What were the probes incubated with before being run on the gel? Hint: The figure legend will help you answer
this.
The probes were incubated with nuclear extracts from HepG2 and Hep3B cells

• Describe the results that were observed. Note: the bands at the very top of the gel is sample still present in the
wells (this is often cut off before imaging, but hasn’t been in this case) and it appears the authors have cropped
the picture so we can’t see the bottom of the gel.

• What conclusions can you draw from this data? Hint: have they addressed their aims? How do these results link
to the hypothesis being tested?
There was clear nuclear protein binding for the SNPs, rs58812592 and rs6935207,
whereby rs6935207 was highly allele-specific.

4. Answer the following questions about Figure 3

• What is being measured on the Y-axis of each graph?
Relative luciferase activity

• What is being varied on the X-axis of each graph?

Different SNPs

• What was the aim of this experiment? Hint: think about what was cloned into the vector (i.e. was it the whole
OCT1 promotor or just part of it or was another promotor used?). The figure legend will help you work this out.

To determine the effect of SNPs on the constitutive SV40 promoter activity.

• Describe the results that were observed.

All effects showed similar results in all three cells, although -1795G>A showed
significance in Hep3B cells with the greatest luciferase activity. Meanwhile, -1795G>A
and -1620T>C exhibited similar luciferase activity in HepG2 cells. The WT and Variant
strains of -1795G>A in all three cell lines were significantly different (WT < Var luciferase
activity).

• What conclusions can you draw from this data? Hint: have they addressed their aims? How do the results compare
between the three cell lines? What is the effect of the SNPs in the OCT1 promotor?

The -1795A-allele showed a 2.5 fold increase in promoter activity in Hep3B, 1.6 fold in
HepG2 and 1.4 fold increase in Huf7 cells. There was strong functional binding only to
the A-allele and they detected allele-specific activity for the region around -1795G>A
only in the artificially shortened SV40 promoter construct.
5. Answer the following questions about Figure 4C and 4D
• What question are these experiments investigating?
What are the in vitro effects of the -1795G>A SNP on the binding of transcription factor
NF-Y and OCT1 promoter activity.

• What probes were used in the EMSA in figure 4C?

Two labelled probes containing -1795G and -1795A.

• What were the probes incubated with before being run on the gel?
The probes were incubated with nuclear extracts from HepG2 and Hep3B cells.

• Describe the results that were observed in the EMSA. Note: the bands at the very top of the gel is sample still
present in the wells (this is often cut off before imaging but hasn’t been here) and it appears the authors have
cropped the picture so we can’t see the bottom of the gel.
Strong evidence of NF-Y binding, leading to an allele-specific increase of activity in the
pGL3 promoter constructs.

• What experimental conditions are being examined in figure 4D?

Cloning of the OCT1 promoter region spanning from -1853 to -61 bp from the
translational start codon in the pGL3-basic vector.

• What is being measured in figure 4D?

Luciferase activity of different SNPs in all three cell lines (HepG2, Hep3B, Huf7).

• Describe the results that were observed in figure 4D.

Significant increase in luciferase activity in WT OCT1 promoter compared to the empty
vector in all cell lines that were investigated.

• What conclusions can you draw about the -1795G>A SNP from data presented in figures 4 C &D?
There are no effects of -1795G>A SNP on the OCT1 promoter region to contribute to
OCT1 expression.

6. Answer the following questions about Figure 5 C, D & E.

• What was the aim of this part of the study?
To determine if -1795G>A SNP shows pharmacokinetic effect on fenoterol, sumatriptan and proguanil.
• What is being measured on the Y-axis of 5C, D & E?
Area under the time-concentration curves of fenoterol, sumatriptan and proguanil.

• What is being compared on the X-axis of figures 5C, D & E?

Amino acid mutations

• Describe the results that were observed.

• What conclusions can you draw from this data?

No effects on the pharmacokinetics of fenoterol, sumatriptan and proguanil in healthy
volunteers.

7. Answer the following questions about Figure 6

• What question are these experiments investigating?
Does -201C>G SNP affect the binding of the transcription factors (USF1/2) and affect
OCT1 promoter activity.

• What probe(s) was used in figure 6A?

A labelled probe containing the -201C probe

• What was the probe(s) incubated with before being run on the gel?
The probe was incubated with nuclear extracts from HepG2 and Hep3B cells,
respectively in the absence or presence of unlabelledprobes(coldcompetition/antibodies)

• Describe the results that were observed in the EMSA. Note: the bands at the very top of the gel is sample still
present in the wells (this is often cut off before imaging, but hasn’t been here)

• What experimental conditions are being investigated in figure 6B?

• What is being measured in figure 6B?

Luciferase activity
• Describe the results that were observed in figure 6B.

Significant allele-specific changes were detected in the native OCT1 promoter activity in
dependence of the -201C>G allele.

• What conclusions can you draw from data presented in figures 6 A & B?
Binding of USF1/2 to an E-box in the OCT1 promoter at -200 to -195 bp was confirmed.

8. Answer the following questions about Figure 7

• What question is this experiment investigating?

Does -1620 T>C SNP affect OCT1 promoter activity?

• What experimental conditions are being investigated in figure 7?

Luciferase reporter gene assay

• What is being measured in figure 7?

Relative uciferase activity

• Describe the results that were observed in figure 7.

There were no significant changes in luciferase activity between -1620 T and C SNPs in
all cell lines investigated.

• What conclusions can you draw from data presented in figure 7?

The -1620T>C SNPS does not affect native OCT1 promoter activity in all cell lines
investigated.

9. Having completed the questions above answer the following questions.

• Summarise the overall findings of this study based on your critical analysis of the paper. Do the authors’
conclusions agree with your conclusions based on their data?
2 out of 10 SNPs that were analysed in the OCT1 promoter region (-1795G>A and
-201C>G) showed functional in vitro effects for OCT1 expression.
• Suggest the next step in this research. What hypothesis would you test? What methods would you use to
test your hypothesis?