MCB

2050 Tutorial 1: Seminar Application Assignment

Gene Regulation

Before attending your seminar use the tutorial readings and/or the relevant lecture notes to
complete the following questions.

Fill in each blank with one of the following terms, not all terms are used, some more than
once: antibody, transcription factor, promoter, luciferase, open reading frame, TBP, upstream,
downstream, primers, reporter plasmid, DNA, RNA, TATA box, transcription factor, repressors,
enhancer.
1. is a subunit of TFIID that binds the element of the core .
Other types of regulatory elements include which function to repress gene
expression and which function to increase expression when bound to their
respective _.

2. In order to use a reporter plasmid to study the regulatory elements of a gene of interest
the of the gene of interest is inserted of the open reading frame
of a reporter gene like .

3. When performing a deletion mapping experiment many versions of a are

constructed with particular regions of the deleted. Analyzing the effects of
these deletions provides insight into the function of the promoter element.

4. When using Chromatin immunoprecipitation (ChIP) to determine if a specific

transcription factor binds a particular promoter element specific to the
transcription factor of interest is used to precipitate the transcription factor with its
bound _____________. The promoter element isolated can be amplified with PCR. In
this case ______________are designed to flank the element in question in order to
amplify this region only.

INTRODUCTION:

SOX4 is a member of the SOX gene family which consists of transcription factors involved in
the development and differentiation of cells and organs, as well as the initiation and
development of cancers. Previous studies have reported that SOX4 is highly expressed in over
20 malignant tumors and promotes malignant phenotypes. However, many of the SOX4-
regulated genes are still unknown.

The gene CXCR7 encodes for a chemokine (polypeptide that serve as key regulators of
metastasis). The activation of CXCR7 may promote cell migration, tumorigenesis and anti-
apoptosis. CXCR7 is highly expressed in various cancer cells.
In this tutorial we want to investigate if SOX4 binds to the regulatory sequence of CXCR7 and if
it effects its expression.

In this seminar we will examine the use of a reporter plasmid assay to study the DNA elements
within the CXCR7 promoter that regulate the expression of the CXR7 gene. These elements
MUST bind a transcription factor in vivo in order to function.

Usually reporter plasmid assays are followed by confirming the transcription factor (protein)
that associates with these promoter elements (DNA). Here Chromatin immunoprecipitation
(ChIP) is conducted to isolate the transcription factor bound to the promoter element in vivo.

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MCB 2050 Tutorial 3: Seminar Application Assignment
Gene Regulation

Question 1: Reporter Plasmids

In order to study the transcriptional regulation of
CXCR7 you must first understand the elements
of its promoter. You construct a luciferase
reporter plasmid which contains the promoter
region of the CXCR7 gene (sequences -150 to
+1, relative to the transcription start site) inserted
upstream of the luciferase open reading frame
(ORF). The luciferase protein is an enzyme
whose activity can be easily measured. The
measured enzyme activity directly reflects the
activity of the promoter of the CXCR7 gene
because expression of the luciferase gene (luc+)
is regulated exclusively by the CXCR7 promoter.

You then generate different versions of the plasmid with regions of the CXCR7 promoter
mutated (A-D). Next you transfect each plasmid into two different cancer cell lines, Y and Z,
which have high and low expression, respectively of CXCR7, and measure reporter activity. The
results are shown below. Note: - no activity, +/++/++++ high activity

i) Can you predict what kind of regulatory element is A? Explain briefly its function, why
does its mutation affect activity so dramatically?

ii) What kind of regulatory element is D and what type of transcription factor does it bind?
Why does its mutation reduce transcription in the Y line? Is this element operating in the
Z cell line?

iii) What kind of regulatory element is C? Why is its mutation increasing transcription in the
Z line? Is this element operating in the Y cell line?

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MCB 2050 Tutorial 1: Seminar Application Assignment
Gene Regulation

Question 2: From your deletion mapping experiment (Question 1) you have identified a
potential enhancer sequence (element D) within the CXCR7 promoter that stimulates the high
levels of expression observed in cell line Y. Examination of this region reveals a DNA
sequence, AACAAAG (motif 1) which is consistent with the consensus SOX4 binding motif. You
wish to confirm that SOX4 does in fact bind this motif of the CXRC7 promoter in vivo using the
chromatin immunoprecipitation assay (ChIP).

i) Briefly explain the steps of chromatin immunoprecipitation as relevant to the DNA and protein
in question.

ii) Indicate in the figure the location of the primers you will use if the DNA product is ~ 100 bp.

iii) What control will you use to ensure the antibody-protein binding was specific to SOX4?

iv) The schematic below depicts the agarose gel following ChIP for normal cells (N) with normal (+)
CXCR7 expression, and a cancer cell line Y with high (++++) CXCR7 expression. Fill out the PCR
products (when using the above primers) if SOX4 does in fact bind these motifs in the CXCR7 promotor
in vivo. The first two lanes are from ChIP experiments using a control antibody (C), the second two
lanes are from ChIP experiments using the anti-SOX4 antibody.

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MCB 2050 Tutorial 3: Seminar Application Assignment
Gene Regulation

Question 3: GRADED QUESTION Work with your break-out groups to complete this question.
Submit this page to the CourseLink Dropbox within 15 min. after your seminar is over. You can
submit a word document, pdf or image file. Your lowest application assignment grade will be
dropped.

Analysis of the cell line Z promoter confirms that the SOX4 binding motif is NOT present.
The chart below summarizes the SOX4 and CXCR7 expression levels for the three cell lines,
normal (N), Y and Z.

SOX4 Expression CXCR7 Expression

N —normal expression (+) N — normal expression (++)

Y — high expression (+++) Y — high expression (++++)

Z — very high expression (++++) Z — low expression (++)

ChIP experiments in each cell line N, Y, Z was performed using an antibody against SOX4.
Parallel ChIP experiments with control (C) antibody against a protein that does not bind DNA
was also conducted. The precipitated DNA from all samples was amplified using the primers
specific for the promoter region of CRCX7. The SOX4 binding site in the promoter is shown.
The products are run on an agarose gel and visualized with DNA dye.

i)The schematic on the right depicts the agarose gel

analyzing SOX4 binding to the promoter region of
CXCR7. Fill out the PCR products of the ChIP from
all 3 cell lines. (0.25)

ii) Despite having a high expression of SOX4 in

cell line Z, there is low expression of the CXCR7
gene, so you perform a ChIP experiment to
analyze TBP (TATA binding protein) binding to the
CXCR7 promoter in all 3 cell lines.

• In the figure above indicate the location of your

primers if your PCR product is ~100 bp (0.25).

• Fill out the schematic to the right (0.25) and

indicate which antibody you will use (0.25).