Chromatin remodelling and modification
( week 9)

Role of chromatin structure in differential gene expression

Mechanisms of regulation of transcription by histone acetylation vs histone methylation

Role of DNA methylation in transcription control

Key proteins required for activation of genes in a chromatin environment

Structure of chromatin:


  • Tightly condensed around histone proteins, forming nucleosomes.


  • Regulatory sequences, (promoters & enhancers)= hidden within condensed chromatin= inaccessible to RNA polymerase and other transcriptional proteins

Chromatin remodeling : changing the compaction of nucleosomes= allowing access to regulatory elements. Proteins and complexes facilitate chromatin remodeling by moving nucleosomes along the DNA, exposing promoters and enhancers

Histone acetylation: Acetylation of histone tails neutralizes their positive charge. Acetylated histones reduce interactions with adjacent nucleosomes and DNA, leading to a more relaxed chromatin structure. This process is reversible

Histone methylation: methylation of histone tails creates binding sites for various proteins, influencing gene expression positively or negatively. Does not alter the charge of histone tails but affects protein interactions, regulating gene expression

Histone acetylation

What:
Acetylation: addition of acetyl coA to a methyl group of lysine residues within the N terminal protruding from the histone core of the nucleosome

Key players:

  • Acetyl group from Acetyl CoA: acetylates lysine residues


  • Histone acetyltransferase(HATs): enzyme that add acetyl to the methyl

  • Eukaryotic activator proteins: direct acetyl to promoter

Results:
1) Condensed chromatin is transformed into a more relaxed structure that is associated with greater levels of gene transcription


2) More acetylation= more transcription (opp true)


3)When directed near promoters(by eukaryotic activators), chromatin structure= opened & gene expression = increased

How: acetylation removes the positive charge on the histones, thereby decreasing the interaction of the N termini of histones with the negatively charged phosphate groups of DNA

Histone de-acetylation

What: removal of acetyl group with H2O molecule. Hence removes positive charges= reducing affinity bt histones and DNA

Key players:


  • Histone deacetylase (HDAC)
  • Eukaryotic repressor proteins: direct de-acetylation to promoter

How: acetyl group is removed by one of the HDAC enzymes during deacetylation, allowing histones to interact with DNA more tightly to form compacted nucleosome assembly

Results:
Eukaryotic repressor proteins direct de-acetylation near the promoter. Leads to compression of chromatin. So DNA becomes more tightly wrapped around the histone cores, making it harder for transcription factors to bind to the DNA.


Hence, decreased levels of gene expression and is known as gene silencing

Histone methylation

What: creates binding sites for either activator or repressor proteins depending on specific residues that have been modified

Key players:


  • Lysine & arginine are methylated
  • Histone methyltransferase (HMTase)

How: up to 3 methyl groups can be added to side chain of lysine

Results:
Charge is not affected, hence remains +ve. So doesn't directly affect condensation of chromatin.


So activation or repression of gene expression can occur

What: covalent modification where methyl groups are added to cytosine nucleotides, specifically at the 5' position, forming 5-methylcytosine (5-mC)

How:
1) DNA Methyltransferases: they recognize specific sequences and add methyl groups to cytosines, often in CG dinucleotides


2) Recruitment of Methyl-CpG-Binding Proteins: Methyl-CpG-binding proteins recognize methylated CG sites


3) Recruitment of Histone Deacetylases (HDACs): Methyl-CpG-binding proteins recruit HDACs to chromatin


4) Histone Deacetylation: HDACs remove acetyl groups from histone tails in the vicinity, leading to condensed chromatin


5) Chromatin condensation: deacetylation causes nucleosomes to pack tightly, making the DNA less accessible


6) Transcriptional Repression: Condensed chromatin blocks access to transcriptional machinery, leading to gene silencing and transcriptional repression

Results:
1) CG methylation leads to inactive genes. Happens due to transcriptional repression by histone deacetylation.


2) Chromatin Condensation: Methylation recruits HDACs, which remove acetyl groups from histone tails, leading to nucleosome condensation and gene silencing.


3) Formation of CpG Islands: CG dinucleotides in promoter regions are often unmethylated, allowing active gene expression

Key players:
1) DNA Methyltransferases: enzymes responsible for adding methyl groups to cytosines


2) Methyl-CpG-binding Proteins: proteins recognizing methylated CG sites and recruiting chromatin-modifying enzymes


3) Histone Deacetylases (HDACs): proteins recruited by methyl-CpG-binding proteins; they remove acetyl groups from histone tails


4) CpG Islands: regions with unmethylated CG dinucleotides, often found in promoter regions. In evolution CG pairs disappear from anywhere other than promoter, hence u get CpG island


5) Transcription factors: proteins that regulate gene expression by binding to specific DNA sequences

Mechanism of activation of genes in a chromatin environment

B interferon enhanceosome: multiple transcription factors control expression of B interferon gene. Required for defence against viral pathogens. It is composed of multiple proteins bounded togther in a co-operative & stable binding

Histone Acetylation by GCN5: GCN5, a histone acetyltransferase (HAT), recognizes the enhanceosome and acetylates histone tails within nucleosomes near the enhancer region. Acetylation neutralizes the positive charges on histones, weakening their interaction with DNA

Recruitment of SWI/SNF Complex: Acetylated nucleosomes provide a platform for the SWI/SNF chromatin remodeling complex. This complex, powered by ATP hydrolysis, repositions nucleosomes. It moves nucleosomes along the DNA, exposing specific regions, including the TATA box within the promoter

TATA box binding: exposed TATA box within the promoter region becomes accessible for binding by the TATA box binding protein (TBP). TBP is a key component of the general transcription machinery

Recruitment of RNA Polymerase II: TBP binding marks the assembly of the pre-initiation complex. RNA polymerase II, the enzyme responsible for transcription, is recruited to the promoter. It initiates the transcription process, synthesizing RNA from the DNA template

Epigenetic inheritance

Chromatin structure inheritance: during cell division, not only is the DNA sequence replicated, but the chromatin structure (the way DNA is packaged with histone proteins) is also passed on to the next generation of cells

Histone inheritance: when a cell divides, it reuses the original histones rather than synthesizing entirely new ones. This means that the daughter cells inherit approximately half of the original histones from the mother cell

Histone modification inheritance: The old histones serve as templates for guiding the modification (such as acetylation and methylation) of new histones. Specific enzymes modify the new histones based on the modifications present on the old histones

DNA methylation inheritance: DNA methylation patterns, particularly in CpG islands, are also inherited during cell division. DNA methyltransferase enzymes recognize hemi-methylated DNA sites (where one strand is methylated, and the other is not) and replicate the methylation pattern on the newly synthesized DNA strand

Promoter activity inheritance: Epigenetic modifications, including histone acetylation, methylation, and DNA methylation, play a crucial role in regulating gene expression. The inheritance of these modifications ensures that active and inactive promoters maintain their respective states in daughter cells

How cell identity is defined by its proteome

Proteome: set of proteins produced in an organism, system or biological context. Hence, different cell types contain the same genome but they express different RNAs & proteins

Post-Translational Modifications (PTMs):
Proteins are subject to various modifications after they are synthesized. These alter a protein's structure & function.


Significance: PTMs regulate protein activity, stability, and interaction with other molecules. This fine-tuning is essential for a cell to adapt to its environment and perform specific tasks

Cell Differentiation:
During development, cells undergo differentiation, a process where they become specialized into specific cell types (e.g., muscle cells, blood cells). This differentiation involves changes in the proteome.
Significance: differences in protein expression and modifications lead to diverse cell types. The proteome of a muscle cell, enables it to contract, while a blood cell's proteome supports oxygen transport

Regulation is effected @ multiple levels:


Chromatin structure & methylation.
Transcription regulators.
RNA processing.
Translation.
mRNA degradation.
Protein activity.

Understanding of post translational events to explian how they affect protein activity

Protein folding

Targeted degradation

Addressing to specific sub cellular localisation

Post translational modifications

Covalent modifications

Proteolysis

Glycosylation

Some proteins fold spontaneously and others need chaperons.

Molecular chaperons:
Location: at the exit of ribosomes


Function: Recognise non native or misfolded polypeptides by their hydrophobic surfaces. And, prevent aggregation of misfolded or denatured proteins


Eg: GroEL

  • Acts as a molecular cage, encapsulating unfolded or partially folded proteins
  • ATP hydrolysis powers conformational changes in GroEL, facilitating the folding process
  • Misfolded proteins inside GroEL can make multiple folding attempts until they achieve their correct structure

Chaperones target misfolded/damaged proteins for degradation by the proteasome, which is a cellular recycling system

Proteasome:
-Recognises & binds polyubiquitinated proteins. Ubiquitination involves attaching a chain of ubiquitin molecules to the protein substrate, marking it for proteasomal recognition

  • Unfolds the proteins.
  • Degrades the target to peptides 4-25 long
  • ATP driven
  • Proteolytic activity (processive)

How cell knows where protein should be transported

Proteins can move bt compartments in diff ways: gated transport, transmembrane transport, vesicular transport


1. Gated transport: bt nucelus & cytosol
2. Transmembrane transport: bt nucleus and organelles (ie mitochondria, endoplasmic reticulum)
3. Vesicular transport: bt organelles (ie golgi to cell extracellular, golgi to early endosome to extracellular)

Signal sequencing & sorting receptors direct proteins to the correct cell address.
Import into cells: nuclear localisation sequence (NLS), chain of Lys.
Export from nucleus: Nuclear export (NES).
Import into ER: signal sequence is hydrophobic.


Protein contains a specific signals that direct their transport to particular cellular compartment. NLS guide proteins into the nucleus while other signals determine transport to the endoplasmic reticulum, Golgi apparatus or extracellular space.

Steps:

  1. Signal sequence of growing peptide is recognised by SRP to signal peptide causes a pause in translation.
  2. Translation continues and translocation begins.
  3. Binding of SRP to its receptor on the ER, and help the ribosome bind to the protein translocator.
  4. Dissociation of the ribosome from the SRP complex, and translation continues and translocator allows the polypeptide to transport across the ER lumen and folding occurs in the ER lumen.
  5. Signal peptidase is present in the ER responsible for cleaving inner peptide and releasing protein into the lumen.

Meaning: addition of sugars
Occurs in the RER & involves the addition of a common oligosaccharide.


  • Covalently attached to the side chain of an asparagine residue (N linked).
  • May be required for folding, stability and function.

Stabilised by disulfide bonds

  • Covalent bond between 2 cysteine side chains intra or inter molecular crosslinks.
  • Requires oxidative condition

Important for activation of digestive enzymes, blood group antigens and receptors, kinase substrates.


Meaning: cleavage of proteins

Molecular sinals direct addition of covalent modifications. The "fate" of protein after the code is read is:
1: Bind to proteins Y and Z
2: Move to nucleus
3: Move to proteasome for degradation
4: Move to plasma membrane

Understand how different modes of protein regulation & interpret eg of signalling cascades & transcription regulator activation

Post transcriptional activtion: signaling cascade & cancer

Oligomerisation: when multiple protein subunits come together to form a functional protein complex

Phosphorylation and Dephosphorylation:


Phosphorylation: addition of phosphate groups to amplify signals.


Dephosphorylation: removing of phosphate group to inactivate proteins & terminating the signal


Key player: protein kinases(they + or - phosphates)

GTP Binding Proteins (G Proteins): G proteins can switch between active (GTP-bound) and inactive (GDP-bound) states. Their activation or deactivation can trigger down stream signalling effects.


Eg: The Ras protein= GTPase. When activated by GTP binding, Ras initiates a cascade, leading to cell division. Mutations in Ras, preventing GTP hydrolysis, can result in continuous cell division, leading to cancer.

Transcription Regulator Activation

Protein- protein interactions:

  • Transcription factors require specific interactions to become active.


  • Interactions influenced by various proteins & co-factos allowing or preventing the transcription factor's binding to DNA.



Eg: phosphorylated MAPK kinase enters the nucelus & phosphorylates transcription factors.


Results: enable transcription factors to bind to DNA, regulating gene expression

Post translational modification: transcription regulators, including histones and other chromatin-modifying proteins, can be modified to activate or deactivate gene expression.
Eg: acetylation of histones facilitates gene activation by loosening chromatin structure. Conversely, deacetylation leads to gene repression.

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