Lecture 10: Non Coding Variation

Question 1

Explain: Vanin-1 (VNN1) & The San Antonio Family Heart Study = 4

Answer 1

1. Major project of the Genetics Department at Texas Biomedical
   Research Institute to FIND GENES INVOLVED IN HEART DISEASE

• 1,400 individuals - 40 large MEXICAN AMERICAN FAMILIES with HIGH INCIDENCE OF HEART DISEASE AND DIABETES.

• The largest family contains 100 examined individuals

• Vanin-1 (VNN1) IDENTIFIED AS CARDIOVASCULAR DISEASE GENE.

Question 2

VNN1 functional variants?

Answer 2

VNN1 is gene associated with cardiovascular diseases.

113 Genetic Variants in VNN1- which is functional?

Question 3

Be able to describe the “gold-standard” tests for assessing regulatory
variation = 2.

Answer 3

1. test for DIFFERENTIALLY INTERACTING REGULATORY PROTEINS
   - electrophoretic mobility shift assay (EMSA) & others
2. test variants in FUNCTIONAL ASSAYS IN VITRO
   - reporter gene assay & others

Question 4

Understanding Electrophoretic Mobility Shift Assay (EMSA) : what you need?

Answer 4

1. Radiolabeled DNA probe
   ~30 nt around the SNP
2. Cell protein extract
   What cell type to use?

Steps:
1. Binding reaction:
- allow proteins to bind DNA probe (Transcription factors)

2. Electrophoresis:
   - size separation.
   - DNA probes bound with protein will
   be large

Question 5

Using EMSA we can determine if there is an allelic difference in TF binding of VNN1:

Answer 5

Diagram - yes?

Question 6

Issues with EMSAs: 3

Answer 6

1. probe-limiting effects – higher order complexes
2. cell type issues
3. in vitro only (no chromatin context?)

Question 7

Reporter gene vs transfection vs cells

Report gene assay

Answer 7

1. Reporter Gene:
   Luciferase (bioluminescence)
   GFP (fluorescence)
2. Transfection:
   Transient
   Stable
3. Cells:
   Different cell types
   Different cell stimuli

Question 8

Reporter Gene assays diagram

Answer 8

On gene

… ref…Minimal promoter … luciferase…light(small)

…alt…minimal promoter…luciferase … light(big)

On histogram
Alt is bigger than ref for luciferase activity

Question 9

Understanding Massively Parallel Reporter Gene Assays

Answer 9

1. Systematically identify CASUAL VARIANTS
2. Synthetic DNA libraries
3. Reporter Gene containing a 3’UTR unique barcode (BC)

How is looks like
…regulatory elements C … reporter…Barcode C

Question 10

Issues with Reporter Gene Assay : 3

Answer 10

1. Cell-specific effects
2. Inducible effects – stimulation etc
3. Experimental effects/errors
   (dirty DNA, normalisation, many others)

Question 11

Effect of cell type on -308 Variant Function:

Answer 11

Purpose: Illustrates how NF-kB activity varies across different cell types under various conditions

Measurement: Relative Luciferase Units (indicates reporter gene expression level)

Key Findings:
NF-kB activity differs significantly depending on cell type and stimuli

Highlights the importance of considering cell type and experimental conditions in reporter gene assays

Question 12

Solutions to the issues with reporter Gene assays.

Answer 12

1. Develop a CREDIBLE MODELof how the GENETIC VARIATION AFFECTS FUNCTION
2. Develop in VIVO ASSAYS THAT ASSESS ALLELE-SPECIFIC CHROMATIN ARRANGEMENT
3. Develop IN VIVO REPORTER GENE ASSAYS IN DIFFERENT ‘cellular contexts’.
4. GENOME EDITING (CRISPR of course!)

Question 13

In vivo Functional Tests:

Chromatin accessibility Assay by sequencing: CHA-seq

Answer 13

To assess chromatin accessibility, which is crucial for understanding DNA-protein interactions and gene regulation.

Question 14

CHA- seq process

Answer 14

1. Assigns relative value of accessibility
2. Map DNA binding proteins to a specific region
3. Value indicates localised chromatin arrangement (and implied transcriptional activity)

This method is valuable for studying gene expression and regulatory mechanisms by revealing how chromatin structure influences transcriptional activity.

Question 15

Understanding Chromatin States

Answer 15

Chromatin States:
Inactive/Closed Chromatin (Heterochromatin):

DNA is tightly wrapped around histones.
Less accessible for transcription.

Active/Open Chromatin (Euchromatin):
DNA is more loosely wrapped around histones.
More accessible for transcription.

Visual Representation:
Illustration A:
Depicts DNA tightly wrapped around histones (blue), indicating inactive or closed chromatin (heterochromatin).

Illustration B:
Shows DNA more spread out with fewer histones, indicating active or open chromatin (euchromatin).

Question 16

CHA-seq diagram understanding

Answer 16

1. Single Nucleotide Polymorphism (SNP):
   A variation at a single position in a DNA sequence among individuals.
   Example: One sequence has a cytosine © and another has a thymine (T) at the same position.
2. Sequencing Process:
   DNA sequences are analyzed to identify SNPs.
   A nuclease enzyme is used to cut the DNA.
   The resulting fragments are sequenced to determine the nucleotide order.
3. Output:
   The sequencing results show the differences in the DNA sequences.
   These differences help identify the SNPs.

Question 17

The future? 4

Answer 17

1. Need large collections of true positive (functional) and true negative (neutral) variants:
   - Essential for understanding the impact of genetic variations.
2. Need Whole Genome Sequence for large populations:
   - Comprehensive data to capture genetic diversity.
   - Enables population-wide studies and personalized medicine.
3. Genome-wide assays will provide useful data on function:
   - High-throughput techniques to assess gene function and regulation.
   - Crucial for identifying functional elements in the genome.
4. Prediction of Functional variation (add to ENCODE) - 10 years?
   - Integrating functional predictions into the ENCODE project.
   - Aiming for accurate prediction of functional variants within the next decade.