Tutorial- week 3

Question 1

Learning Outcomes

Answer 1

Identify what aspect of the transcription process would be the best to monitor within a specific question and be able to match this with an appropriate method that could be used to measure this.

Apply the principles governing the control of transcription and explain why some methods are better suited to answer experimental questions than others.

Predict the outcome of a suggested experimental method and what potential problems you may encounter, including how you might get around these issues.

1. Transcription Initiation:
   Key Aspect to Monitor: The binding of transcription factors, RNA polymerase recruitment to promoters, and transcription initiation rate.

Method:

Chromatin Immunoprecipitation (ChIP):
Application: ChIP is used to monitor the binding of specific transcription factors and RNA polymerase to DNA at promoters.
Why This Method: It allows you to investigate protein-DNA interactions and identify which regions of the genome are being targeted by transcription factors or RNA polymerase at the moment of transcription initiation.
Potential Problems:
Low-resolution binding data.
ChIP requires antibodies specific to your transcription factor or polymerase, which can be hard to obtain.
Solution: Use ChIP-seq for higher resolution, combining ChIP with sequencing technology to gain a comprehensive view of binding sites.

2. Transcription Elongation:
   Key Aspect to Monitor: The movement of RNA polymerase along the DNA, transcription elongation speed, and changes in RNA synthesis rates.

Method:

Nascent RNA Sequencing (NET-seq):
Application: NET-seq captures RNA polymerase activity and produces a high-resolution map of where RNA polymerases are transcribing.
Why This Method: This method is well-suited for studying real-time transcription elongation and identifying sites where RNA polymerase pauses.
Potential Problems:
It provides only a snapshot and may not show the full dynamics of transcription.
The technique can be technically challenging and labor-intensive.
Solution: Combine NET-seq with time-course experiments to track transcription dynamics over time.

3. Transcription Termination:
   Key Aspect to Monitor: The point where transcription terminates and RNA polymerase disengages from DNA.

Method:

RNA-seq:
Application: RNA-seq is used to analyze the abundance and structure of transcripts, including the 3’ end where transcription terminates.
Why This Method: It provides a broad and detailed view of the full range of transcripts, including where transcription starts and stops.
Potential Problems:
It may not specifically highlight termination events without careful mapping.
Solution: Use 3’-end focused RNA-seq to specifically enrich for RNA sequences near transcription termination sites.

4. Transcriptional Output (Gene Expression Levels):
   Key Aspect to Monitor: The overall transcriptional output, including mRNA abundance.

Method:

Quantitative PCR (qPCR):
Application: qPCR is widely used to measure the amount of specific mRNAs and quantify transcriptional activity.
Why This Method: It’s a fast, sensitive, and quantitative method to assess transcription levels of specific genes.
Potential Problems:
It is gene-specific and may not provide a full transcriptome-wide view.
Solution: Use RNA-seq for transcriptome-wide analysis, or perform qPCR in conjunction with a broader method like RNA-seq to validate specific findings.

5. Epigenetic Regulation of Transcription:
   Key Aspect to Monitor: The influence of chromatin structure and histone modifications on transcription regulation.

Method:

ATAC-seq (Assay for Transposase-Accessible Chromatin with sequencing):
Application: This method maps chromatin accessibility, giving insights into how open or closed regions of chromatin correlate with active or repressed transcription.
Why This Method: ATAC-seq provides a global view of chromatin accessibility and can show how chromatin changes influence transcription.
Potential Problems:
It only shows accessibility and doesn’t directly measure transcription.
Solution: Combine ATAC-seq with RNA-seq to link chromatin structure changes with gene expression.

Matching Methods to Questions:

Question: How does the presence of a transcription factor affect transcription of a specific gene?

Method: Use ChIP-seq to identify where the transcription factor binds, and RNA-seq to measure changes in gene expression in response to the factor.
Why: ChIP-seq identifies binding, while RNA-seq tracks the output of gene expression.

Question: How fast does RNA polymerase move across a gene?

Method: NET-seq to monitor real-time RNA polymerase positions along the gene.
Why: NET-seq gives high-resolution data on RNA polymerase activity and its pausing events during elongation.

Question: What are the transcription termination sites for a gene?

Method: 3’-end RNA-seq to enrich and identify the exact termination points.
Why: This allows focused analysis of RNA ends to find termination regions accurately.

Predicting Experimental Outcomes & Troubleshooting:
Outcome of NET-seq: You can observe transcription elongation and pausing. One potential issue might be difficulty capturing rare or weak transcriptional pauses, which can be addressed by deeper sequencing.
ChIP-seq: It should show transcription factor binding sites, but non-specific antibody binding might lead to false positives. Use highly specific antibodies and controls to reduce background noise.

In summary, selecting the right method for transcription analysis depends on the specific aspect of transcription you’re studying, such as initiation, elongation, or regulation by epigenetic factors. Methods like ChIP-seq, RNA-seq, and NET-seq each have strengths in addressing particular experimental questions, but they also come with challenges that can be mitigated by combining approaches or refining techniques.

Question 2

What is a detectable output of transcription?

Answer 2

A detectable output of transcription refers to measurable products or signals that indicate transcription is occurring. The primary outputs of transcription can be RNA molecules and various related biochemical markers that indicate different stages of the transcription process.

Key Detectable Outputs of Transcription:

formation of proteins can be a a detectable output of transcription
promotors binding to certain proteins

1. RNA Transcripts (mRNA, rRNA, tRNA, etc.):

The most direct output of transcription is the synthesis of RNA molecules, such as:
Messenger RNA (mRNA): Carries genetic information from DNA to ribosomes for protein synthesis.
Ribosomal RNA (rRNA): A component of ribosomes, essential for translation.
Transfer RNA (tRNA): Helps translate mRNA into amino acid sequences.
Non-coding RNAs: Such as miRNAs or lncRNAs, involved in gene regulation.

Detection Methods:

qPCR (Quantitative PCR): Measures the amount of specific mRNAs.
RNA-seq (RNA Sequencing): Provides a genome-wide view of all RNA molecules being transcribed.
Northern Blotting: Detects specific RNA sequences.
In Situ Hybridization: Detects the location of RNA within cells or tissues.

2. Nascent RNA:

This refers to newly synthesized RNA that is still being produced by RNA polymerase. Detecting nascent RNA can help track real-time transcription activity.

Detection Methods:

NET-seq (Nascent Elongating Transcript Sequencing): Captures nascent RNA being transcribed by RNA polymerase.
Global Run-On Sequencing (GRO-seq): Measures transcriptionally active regions by detecting nascent RNA.

3. Chromatin State and Epigenetic Marks:

Changes in chromatin accessibility or epigenetic modifications (such as histone modifications) are indirect outputs of transcription and can indicate transcriptional activity.

Detection Methods:

ChIP-seq (Chromatin Immunoprecipitation Sequencing): Detects histone modifications or transcription factor binding.
ATAC-seq (Assay for Transposase-Accessible Chromatin with Sequencing): Measures chromatin accessibility, indicating regions where transcription may be active.

4. Protein-RNA Complexes:

Ribonucleoprotein (RNP) complexes: Formed during transcription as the RNA transcript is processed (splicing, capping, etc.). These complexes can indicate transcriptional progress.

Detection Methods:

CLIP-seq (Crosslinking and Immunoprecipitation Sequencing): Identifies RNA-protein interactions.

5. RNA Polymerase Activity:

The binding of RNA polymerase to DNA and its movement along the gene during transcription is a direct indicator of transcription activity.

Detection Methods:

ChIP-seq for RNA Polymerase II: Tracks RNA polymerase binding to gene promoters and coding regions.
NET-seq: Captures RNA polymerase positions on actively transcribed genes.

6. Transcription-Associated Signals:

Some detectable outputs are indirect, such as changes in gene expression levels, which reflect transcription rates.

Detection Methods:

Reporter Assays: Fuse a gene promoter to a reporter gene (e.g., GFP or luciferase) to monitor transcription activity based on fluorescence or luminescence.
Western Blotting for Transcription Factors: Can measure the activity of transcription factors that modulate transcription rates.

Summary:
The detectable output of transcription includes newly synthesized RNA molecules, chromatin changes, and RNA polymerase activity. The most common methods to measure these outputs include qPCR, RNA-seq, and ChIP-seq, each providing valuable insights into different stages of the transcription process.

Question 3

What methods could you use to measure the outputs?

Answer 3

There are several methods to measure the outputs of transcription, depending on which aspect of transcription you want to assess. These methods can be used to detect RNA levels, RNA polymerase activity, transcription factor binding, or even chromatin changes that are associated with transcriptional regulation.

1. Quantitative PCR (qPCR)
   What it measures: The quantity of specific RNA transcripts.
   How it works: RNA is reverse-transcribed into complementary DNA (cDNA), which is then amplified. The amount of cDNA is quantified using fluorescent dyes or probes.
   Application: Measuring the expression levels of specific genes.
   Advantages: Highly sensitive and quantitative for specific gene targets.
   Limitations: Limited to measuring the abundance of known transcripts; not genome-wide.
2. RNA Sequencing (RNA-seq)
   What it measures: Genome-wide RNA expression.
   How it works: RNA is converted to cDNA and sequenced. The number of sequences corresponding to each gene is counted to determine gene expression levels.
   Application: Identifying and quantifying RNA transcripts across the entire genome.
   Advantages: High-throughput, captures the entire transcriptome, and can identify novel transcripts.
   Limitations: Expensive and requires bioinformatics analysis.
3. Northern Blotting
   What it measures: Specific RNA transcripts and their size.
   How it works: RNA is separated by gel electrophoresis and transferred to a membrane. A labeled probe is used to detect specific RNA sequences.
   Application: Measuring specific mRNA levels and transcript sizes.
   Advantages: Provides information on RNA size and abundance.
   Limitations: Low sensitivity compared to qPCR and RNA-seq; labor-intensive.
4. Chromatin Immunoprecipitation (ChIP)
   What it measures: DNA regions bound by transcription factors, RNA polymerase, or histone modifications.
   How it works: Chromatin is cross-linked, and antibodies specific to transcription factors or histone marks are used to pull down the associated DNA. The DNA is then sequenced (ChIP-seq) or analyzed by qPCR (ChIP-qPCR).
   Application: Identifying where transcription factors or RNA polymerase bind, or where histone modifications occur.
   Advantages: High specificity for transcription factor or chromatin modifications.
   Limitations: Requires high-quality antibodies; results can vary depending on the quality of the immunoprecipitation.
5. Nascent RNA Sequencing (NET-seq)
   What it measures: Nascent (newly transcribed) RNA still attached to RNA polymerase.
   How it works: RNA polymerase is immunoprecipitated, and the RNA it is synthesizing is sequenced to map transcriptional activity.
   Application: Studying real-time transcription elongation and RNA polymerase activity.
   Advantages: Provides high-resolution information on RNA polymerase positioning and activity.
   Limitations: Requires specialized protocols and can be technically challenging.
6. Global Run-On Sequencing (GRO-seq)
   What it measures: Nascent RNA produced during transcription.
   How it works: Cells are permeabilized, and nascent RNA synthesis is allowed to continue in the presence of labeled nucleotides. The labeled RNA is sequenced.
   Application: Measures transcriptional activity and polymerase density across the genome.
   Advantages: Direct measurement of transcriptional activity.
   Limitations: Technically demanding and expensive.
7. Reporter Gene Assays
   What it measures: Transcriptional activity from a specific promoter.
   How it works: A gene of interest’s promoter is cloned upstream of a reporter gene (e.g., luciferase or GFP). The activity of the promoter is assessed by measuring the reporter’s signal.
   Application: Investigating promoter activity in response to specific stimuli.
   Advantages: Simple, real-time monitoring of transcriptional activity.
   Limitations: Limited to studying one gene at a time.
8. RT-PCR (Reverse Transcription PCR)
   What it measures: Specific RNA transcripts.
   How it works: RNA is reverse transcribed to cDNA, and then the cDNA is amplified by PCR to detect the presence of specific RNA sequences.
   Application: Detecting and quantifying specific mRNA levels.
   Advantages: Simple and sensitive for detecting specific transcripts.
   Limitations: Not genome-wide and semi-quantitative compared to qPCR.
9. Single-Molecule RNA Fluorescence In Situ Hybridization (smFISH)
   What it measures: Localization and abundance of RNA at the single-molecule level.
   How it works: Fluorescently labeled probes hybridize to RNA molecules within cells. The probes are detected using microscopy.
   Application: Visualizing the spatial distribution of transcripts within a cell.
   Advantages: High sensitivity and spatial resolution.
   Limitations: Labor-intensive and limited to specific targets.
10. ATAC-seq (Assay for Transposase-Accessible Chromatin Sequencing)
    What it measures: Chromatin accessibility, indirectly indicating active transcription regions.
    How it works: A transposase enzyme inserts sequencing adapters into accessible regions of chromatin. The accessible DNA is then sequenced.
    Application: Identifying regions of open chromatin that may be actively transcribed.
    Advantages: High-throughput and reveals regulatory regions.
    Limitations: Provides indirect evidence of transcription; it measures accessibility, not transcription itself.

Summary:
For measuring specific RNA transcripts: Use qPCR, RNA-seq, or Northern Blotting.

For studying protein-DNA interactions: Use ChIP-seq.

For tracking RNA polymerase activity: Use NET-seq or GRO-seq.

For monitoring promoter activity: Use Reporter Gene Assays.

For studying chromatin accessibility: Use ATAC-seq.

Each method has specific advantages and limitations depending on what aspect of transcription you’re investigating, whether it’s the transcription rate, RNA abundance, or protein-DNA interactions.

Question 4

Basics of PCR

Answer 4

Polymerase Chain Reaction (PCR) is a widely used technique in molecular biology that allows for the amplification of a specific segment of DNA. PCR can make millions of copies of a specific DNA sequence from a small starting amount of DNA. Here’s a breakdown of the basic steps and key components of PCR:

Key Components of PCR:

1. DNA Template: The DNA containing the target sequence you want to amplify.
2. Primers: Short sequences of single-stranded DNA that are complementary to the regions flanking the target DNA. Two primers are used: a forward primer and a reverse primer.
3. DNA Polymerase: An enzyme that synthesizes new DNA strands. A heat-stable DNA polymerase, like Taq polymerase, is commonly used.
4. Nucleotides (dNTPs): The building blocks of DNA (adenine, thymine, cytosine, guanine) that DNA polymerase uses to synthesize new strands.
5. Buffer Solution: Maintains the optimal environment (pH, salt concentration) for the DNA polymerase to function.
6. Magnesium Ions (Mg²⁺): Required for the activity of DNA polymerase.

Steps of PCR:
1. Denaturation (94-98°C):

The reaction mixture is heated to separate the double-stranded DNA into single strands. This step breaks the hydrogen bonds between the two strands of DNA.
Purpose: To make the DNA accessible for the primers to bind.

2. Annealing (50-65°C):

The reaction mixture is cooled to allow the primers to bind (anneal) to their complementary sequences on the single-stranded template DNA.
The temperature for this step depends on the melting temperature of the primers.
Purpose: Primers bind to the target regions flanking the DNA sequence that will be amplified.

3. Extension/Elongation (72°C):

The temperature is raised to 72°C, the optimal temperature for Taq polymerase. The DNA polymerase adds nucleotides to the 3’ ends of the primers, extending the DNA and synthesizing the new strands.
Purpose: To create new strands of DNA by copying the template strand.

4. Repeat (Cycle):

The cycle of denaturation, annealing, and extension is repeated 20-40 times. Each cycle doubles the amount of DNA, leading to exponential amplification of the target sequence.

Final Step:
Final Extension (72°C for 5-10 minutes): After the last cycle, a final extension step is typically added to ensure that any remaining single-stranded DNA is fully extended.

Applications of PCR:
- DNA Cloning: Amplifying DNA for insertion into vectors.
- Genetic Testing: Detecting mutations or identifying genetic sequences.
- Forensics: Amplifying DNA from crime scenes (e.g., STR analysis).
- Medical Diagnostics: Detecting pathogens like viruses or bacteria (e.g., COVID-19 tests).
- Gene Expression: Quantifying the amount of a specific RNA transcript after reverse transcription to cDNA (RT-PCR).

Advantages of PCR:
- Specificity: PCR allows for the amplification of a specific DNA segment.
- Sensitivity: PCR can detect even very small amounts of DNA.
- Speed: PCR can be completed in a few hours.

Limitations:
- Contamination Risk: Even tiny amounts of contaminating DNA can lead to false-positive results.
- Primer Design: Requires knowledge of the target DNA sequence to design specific primers.
- Errors in Amplification: Errors can be introduced during the process, especially if the polymerase has low fidelity.

PCR is a fundamental technique that has revolutionized molecular biology by enabling the precise and rapid amplification of DNA for a variety of research and diagnostic purposes.

1. mRNA binds to primers called random hexamers
2. reverse transcriptase then comes along and fills in the gaps to form cDNA
3. the PCR is heated up during denaturing at around 95 degrees
4. Denaturing seperates the strands and allows us to use specific primers to bind to the forward and reverse strand
5. taq polymerase then will complete the new seqences dring the elongation phase at 70 degrees
6. then cycle 2 occurs and we generate 2 more copies

Question 5

Factors affecting PCR:

Answer 5

Primer Design:
-Melting temperature (Tm)
-GC content - they have 3 hydrogen bonds so require higher temps to break
-Salt content (sodium acetate)
-Other buffer components (like denaturant

Tm= (number of A+T) * 2 + (number of G+C) * 4Annealing temperature should be 2-4oC below this

Question 6

How do we know we have the right product?

Answer 6

Sometimes your PCR may lead to the production of several bands on your gel, of different sizes (see below), how can we know that we have the correct product- can use blastx programe

Question 7

What method is best? From papers that you have read you think that p53 increases transcription from the PUMA promoter.

Answer 7

To investigate whether p53 increases transcription from the PUMA promoter, you would need methods that can measure the transcriptional activity of the PUMA promoter in response to p53, and evaluate the direct interaction of p53 with the promoter region.

Here’s how you would apply principles of transcription control to select the most suitable methods:

1. Reporter Gene Assays (Best Method for Functional Analysis):
   Principle: Reporter gene assays allow you to directly measure promoter activity by using a reporter gene, such as luciferase, GFP, or β-galactosidase, which produces a detectable signal.

How it works: You can clone the PUMA promoter upstream of a reporter gene in a plasmid vector. Then, transfect this construct into cells along with p53 (or induce endogenous p53). If p53 activates the PUMA promoter, the reporter gene will be transcribed, producing a measurable signal (e.g., luminescence or fluorescence).

Why it’s suited: This method directly tests whether p53 can activate transcription from the PUMA promoter and quantifies the level of activation. It’s a functional assay that directly measures transcriptional output in live cells.

Advantages:

Simple, quantitative, and can measure real-time promoter activity.
Can test promoter mutations or deletions to map p53 binding sites.
Provides direct evidence of promoter activation by p53.
Limitations:

Only measures activity of the cloned promoter fragment, not endogenous transcription.
Reporter activity might not fully reflect the complexity of chromatin structure or endogenous regulatory elements.

2. Chromatin Immunoprecipitation (ChIP) with qPCR or ChIP-seq (Best for Direct Binding Evidence):
   Principle: ChIP assays allow you to determine if p53 directly binds to the PUMA promoter region on the chromatin and recruits transcriptional machinery.

How it works: Use antibodies against p53 to immunoprecipitate DNA-bound p53 from chromatin. After pulling down p53-bound DNA, perform qPCR or ChIP-seq to identify whether p53 binds directly to the PUMA promoter region.

Why it’s suited: This method directly tests the physical interaction between p53 and the PUMA promoter, providing evidence of whether p53 is binding to the promoter in its native chromatin context.

Advantages:

Confirms direct binding of p53 to the promoter.
Can analyze binding in the context of endogenous gene regulation.
Provides high-resolution binding information (ChIP-seq).
Limitations:

It doesn’t measure transcriptional output directly, just p53 binding.
Technically demanding and requires high-quality antibodies.

3. Quantitative RT-PCR (qRT-PCR) (Good for Measuring Endogenous Transcription):
   Principle: Measures the mRNA levels of the PUMA gene to see if p53 increases transcription from the endogenous promoter.

How it works: After inducing p53 expression (or activating it through stress or DNA damage), extract total RNA and reverse transcribe it into cDNA. Perform qPCR using primers specific to the PUMA transcript. Compare mRNA levels of PUMA in p53-expressing cells versus controls.

Why it’s suited: qRT-PCR provides direct evidence of increased endogenous transcription of PUMA, which reflects transcriptional activation by p53 at the natural genomic locus.

Advantages:

Measures endogenous transcription levels of the PUMA gene.
Quantitative and sensitive.
Can be performed in various conditions (e.g., p53 overexpression or stress-induced p53 activation).
Limitations:

Doesn’t directly show that p53 is binding to the PUMA promoter.
Only measures mRNA levels, not promoter activity or transcription rates directly.

4. RNA Sequencing (RNA-seq) (For Genome-Wide Expression Profiling):
   Principle: RNA-seq provides a global view of gene expression changes, including the PUMA gene, in response to p53 activation.

How it works: After inducing p53 activity (via overexpression or stress), isolate RNA from cells, and perform high-throughput sequencing to quantify gene expression. You can analyze whether PUMA mRNA levels increase among other p53 target genes.

Why it’s suited: RNA-seq allows you to see global effects of p53 on gene expression, including but not limited to PUMA. This method is useful for broader questions about p53’s regulatory role.

Advantages:

Genome-wide, unbiased analysis of gene expression.
Identifies new p53 target genes.
Limitations:

Expensive and requires complex data analysis.
Doesn’t show direct interaction between p53 and the promoter or transcriptional activity.

Method Selection Summary:
Reporter Gene Assays are ideal for directly testing if p53 activates transcription from the PUMA promoter, providing quantitative data on promoter activity.

ChIP-qPCR/ChIP-seq would confirm whether p53 binds directly to the PUMA promoter, establishing mechanistic evidence of p53 binding.

qRT-PCR would allow measurement of the endogenous mRNA levels of PUMA, confirming whether the promoter activity translates to increased transcription in a natural cellular context.

Experimental Design Consideration:

If you want direct functional evidence of p53’s effect on the PUMA promoter, start with a reporter gene assay.
Follow up with ChIP to confirm p53 binding at the promoter.
Use qRT-PCR to confirm whether this binding leads to increased endogenous PUMA transcription.
Each method provides complementary information, so combining them would give you a comprehensive understanding of p53’s role in activating the PUMA promoter.

Question 8

You have a hypothesis that a protein of interest binds to a response element (cis-regulatory sequence) in the promoter of the BAXgene. What method could you use to determine this?

Answer 8

To test your hypothesis that a protein of interest binds to a response element (cis-regulatory sequence) in the promoter of the BAX gene, the best method would be Chromatin Immunoprecipitation (ChIP), followed by either qPCR (ChIP-qPCR) or ChIP-sequencing (ChIP-seq). This technique allows you to investigate protein-DNA interactions in living cells and is the most direct way to determine whether your protein of interest binds to a specific DNA sequence in the promoter of the BAX gene.

Why ChIP is the Best Method:
Direct Measurement of Protein-DNA Binding: ChIP enables you to detect the binding of specific proteins to specific DNA regions within their native chromatin environment. You can test whether your protein of interest is associated with the cis-regulatory sequence in the BAX promoter.

Native Context: This method captures interactions in live cells, which means you are observing the protein-DNA interaction as it occurs under physiological conditions, including the influence of chromatin structure and other regulatory factors.

Specificity: ChIP uses antibodies specific to your protein of interest to immunoprecipitate DNA-protein complexes. You can use qPCR or sequencing to precisely detect the enrichment of the response element in the pulled-down DNA.

Steps of ChIP for Testing Protein Binding to the BAX Promoter:

1. Crosslinking: Treat cells with a crosslinking agent like formaldehyde, which covalently links proteins to the DNA they are bound to, preserving the protein-DNA interactions.
2. Chromatin Fragmentation: Sonicate the chromatin to shear the DNA into small fragments (typically around 200-500 bp). This helps isolate the specific DNA regions bound to proteins.
3. Immunoprecipitation: Use an antibody specific to your protein of interest to immunoprecipitate the protein-DNA complexes from the chromatin. Other chromatin not bound by the protein will be washed away.
4. Reversal of Crosslinks: Reverse the crosslinks by heating the samples, which releases the DNA from the protein-DNA complexes.
5. Detection:

ChIP-qPCR: Use PCR with primers flanking the BAX promoter response element to test whether this specific sequence is enriched in the immunoprecipitated DNA.

ChIP-seq: For a broader approach, you can sequence all DNA fragments that were co-immunoprecipitated, allowing you to determine the exact binding sites of your protein across the genome, including the BAX promoter.

Why ChIP-qPCR vs. ChIP-seq:
ChIP-qPCR: If you are focused specifically on the BAX promoter and want to test the binding of your protein to a particular response element (cis-regulatory sequence), ChIP-qPCR is ideal. It is more targeted, faster, and less expensive. You can design primers to amplify only the region of the BAX promoter containing the response element.

ChIP-seq: If you’re interested in a broader view of where your protein binds across the genome, including the BAX promoter, ChIP-seq will allow you to map all potential binding sites of your protein. This method is more expensive and requires more complex data analysis but gives you genome-wide insights.

Alternative Methods (Less Ideal):
Electrophoretic Mobility Shift Assay (EMSA): While this method allows you to test in vitro protein-DNA binding, it doesn’t reflect the in vivo chromatin context and can’t confirm binding in living cells.

DNA Footprinting: Another method for in vitro studies that shows where a protein protects DNA from cleavage, indicating binding sites. However, like EMSA, it lacks in vivo context.

Conclusion:
ChIP-qPCR or ChIP-seq is the best method to determine if your protein of interest binds to the response element in the BAX promoter. It allows for direct, specific detection of protein-DNA interactions within living cells, ensuring that you are studying this interaction in its native biological context.