Regulation of Transcription 2

Lecture 2

How long is DNA?

1.5m in length so must be compacted to fit inside the nucleus

What is Chromatin comprised of?

Nucleosomes

What do nucleosomes contain?

An octomer of histone proteins 2 of each (H2A, H2B, H3 and H4)

Histones have N-terminal tails that extend outward and:

• Conntact Neighbouring Nucleosomes

• Help fold chromatin

• Serve as sites for post translational modification

What are the three types of histone tail modifications?

Acetylation, Methylation and Phosphorylation

What is acetylation?

Adding an acetyl group

• Added by histone acetyl transferases HATs

• Removed by histone deacetylases HDACs

• Occurs on lyine residues

• Neutralises positive charge which loosens the DNA-histone interaction

• Generally activating

What is Methylation?

Adding a methyl Group

• Added by HMTs - histone methyl transferases

• Removed by KDMs - lysine demethylases

• Can be activating or repressive, depending on the residue

• Methyl-lysine is recognised by chromodomain proteins

What is phosphoylation?

Addition on a phosphate group

• Key modification

• Associated with chromatin remodelling and signalling responses

What processes allow chromatin structure to regulate gene expression?

Chromatin folding and Histone Modifications

How does chromatin folding work?

Open chromatin (euchromatin) → accessible to TFs and RNA polymerase → active transcription.

Closed chromatin (heterochromatin) → tightly packed → transcriptionally silent.

How does histone modifications work?

Modified histone tails recruit co‑factors that either open or compact chromatin

Histone modification example

• Acetylation recruits bromodomain proteins → co‑activators → RNA pol II recruitment.

• Repressive methylation recruits chromodomain proteins → compaction.

What is a writer?

It adds modifications:

• HAT → adds acetyl groups

• HMT → adds methyl groups

What is a reader?

They bind modified histones

• Bromodomains → bind acetyl‑lysine

• Chromodomains → bind methyl‑lysine

What is an Eraser?

They remove modifications

• HDAC → removes acetyl groups

• KDM → removes methyl groups

These proteins interpret and modify the chromatin landscape to regulate transcription.

Activating histone marker example

• Acetyl‑lysine (H3K9ac, H3K14ac etc.)

  • Recruits bromodomain proteins

  • Opens chromatin

  • Promotes transcription

Repressive histone marker example

• Methyl‑lysine (e.g., H3K9me, H3K27me)

  • Recruits chromodomain proteins

  • Compacts chromatin

  • Silences transcription

DNA methylation - works with repressive marks

• Condenses chromatin

• ~80% of CpGs methylated in somatic cells

How does chromatin contribute to stable cell identity?

• Chromatin structure determines which genes are accessible and which remain permanently silent.

• Constitutive heterochromatin at centromeres and telomeres is always silent and highly condensed.

• Repressive histone marks + DNA methylation lock genes into inactive states.

• This prevents inappropriate gene activation (e.g., cardiomyocytes cannot be turned into neurons simply by adding retinoic acid).

• Thus, chromatin acts as a memory system that preserves cell identity across divisions.

What is a western blot?

lab technique used to detect and analyze specific proteins in a complex sample by separating them by size via gel electrophoresis

Western Blot Steps

1. SDS-PAGE

2. Transfer to membrane

3. Primary antibody incubation

4. Secondary antibody incubation

5. Detection

SDS-PAGE

• Proteins denatured by SDS

• Separated by size

Transfer to membrane

Electric current moves proteins from gel → membrane

Primary antibody incubation

• Antibody raised in rabbit/mouse

• Binds specifically to protein of interest

Secondary antibody incubation

• Recognises primary antibody

• Carries HRP enzyme

Detection

• HRP substrate produces light

• CCD camera detects signal

What is the role of a Primary Antibody?

• Binds directly to the target protein

• Provides specificity

What is the role of a Secondary antibody?

• Recognises the species of the primary antibody

• Amplifies the signal

• Carries HRP, which emits light when substrate is added

Interpret Western blot data, including band size, intensity, and loading controls.

• Band size

  • Should match expected molecular weight

• Specificity

  • One clean band = good antibody

  • Multiple bands = possible off‑target binding

• Relative protein amounts

  • Thicker/darker band = more protein

• Sample quality

  • Smearing or uneven bands indicate degradation or loading issues

What are some key controls and their functions for western blotting

• GAPDH or tubulin

  • Used as loading controls

  • Confirm equal protein loading across lanes

• Without stable loading controls, comparisons (e.g., GATA4 levels) are unreliable.

When is western blotting appropriate ?

Use Western blotting when you need to:

• Measure protein abundance

• Confirm protein size

• Assess changes in expression across conditions

• Validate antibody specificity

• Compare protein levels between cell types (e.g., GATA4 in cardiomyocytes)

What is Immunoflourescence?

powerful microscopy technique that uses fluorescently labeled antibodies to visualize specific proteins, antigens, or other molecules within cells and tissues, revealing their location, distribution, and abundance

How are antibodies used to detect proteins in cells?

• Cells/tissue are fixed to preserve structure.

• Primary antibody binds the target protein.

• Secondary antibody with a fluorescent tag binds the primary.

• Fluorescence microscopy reveals where the protein is located in the cell.

Immunofluorescence steps

1. Fix cells to preserve structure

2. Block to prevent non‑specific binding

3. Primary antibody binds target protein

4. Secondary antibody with fluorescent tag binds primary

5. DAPI staining labels DNA

6. Imaging under appropriate filters

What is a secondary only control?

Ensures fluorescence is not due to non‑specific secondary antibody binding

What is DAPI control?

Confirms nuclei are present and helps identify cell morphology

When should you use immunofluorescence?

Use immunofluorescence when you need:

• Spatial information (where the protein is in the cell)

• Single‑cell resolution

• To identify cell types using markers (MAP2, troponin C)

• To assess differentiation outcomes (e.g., BMP4 → glia)