Molecular Biology 8 : Alternative splicing and disease transcriptional regulation, role of histones, chromatin modelling and DNA methylation

Question 1

What are the steps of the splicing of the ß-globin gene ?

Answer 1

• 3’ cleavage and addition of poly(a) tail

- intron cleave (between 31-32 and 105-106), exon ligation = beta-globin mRNA 1-147

Question 2

What are beta thalassemias ?

Answer 2

β thalassemias are a group of inherited blood disorders. They are forms of thalassemia caused by reduced or absent synthesis of the beta chains of hemoglobin that result in variable outcomes ranging from severe anemia to clinically asymptomatic individuals.

Question 3

How can incorrect exon alternative splicing be implicated in diseases like beta thalassemia ?

Answer 3

• normal adult B-globin primary RNA transcript = 3 exos and 2 introns
• some single-nt changes that destroy a normal splice site cause exon skipping –> mRNA missing an exon
• some single-nt changes that destroy normal splice sites activate cryptic splice sites (part of an intron is not removed) –> mRNA w/ extended exon
• some single-nt changes that create new splice sites cause new exons to be incorporated –> mRNA w/ extra exon inserted between exon 2 and exon 3

Question 4

Where do transcriptional control and RNA processing happen in the cell ?

Answer 4

The nucleus.

Question 5

Where does the control of :
- RNA transport and localization
- mRNA degradation
- translation
- protein activity
happen ?

Answer 5

All in the cytosol.

Question 6

How is transcription regulated ?

Answer 6

• Initiation of transcription : assembly of general transcription factors at promoter - basal transcription machinery.
• Activation of transcription : transcription factors bind promoter-proximal regulatory elements and stimulate the rate of transcription.
• Activation of transcription at a distance : enhancers, insulators, silencers, locus control region.
• Tissue-specific control : operates through promoter- proximal elements and distal enhancers.

Question 7

How can enhancers interact w/ TFs to regulate transcription ?

Answer 7

Activator proteins can bind enhancers (distal binding sites for activators).
This triggers the binding of general TFs, RNA Pol II, mediator, chromatin remodeling complexes and histone-modifying enzymes (e.g. histone acetylases)
Bending of the DNA then allow the enhancer to interact w/ this complex via the mediator to initiate trancription.

Question 8

explain why regulation of transcription in eukaryotes can be done by multiple regulatory sequences (elements).

Answer 8

Bacteria : general one regulatory sequence right upstream of promoter
Yeast : general have many regulator sequences upstream of promoter
Human : even more regulatory sequences bot up and downstream, w/ enhances that can be found at a distance upstream of the promoter

Question 9

What does transcription initiation require in vivo ?

Answer 9

Many proteins, including activators, the mediator complex, RNA Pol, general TFs, chromatin remodelers and HAT (histone actyltransferase).

Question 10

What are enhancers ?

Answer 10

• Enhancers = sequences that activate transcription from a distance.
• Stimulate transcription at considerable distance (e.g. > 50 kb).
• Upstream or downstream of promoters. Orientation-independent.
• Binding sites for transcription activators.

Question 11

What are transcription factors ?

Answer 11

• TFs = proteins that bind regulatory sequences to activate transcription
• Have an activation domain and a DNA-binding domain
• Eukaryotic TFs have modular structure

Question 12

How does the glucocorticoid receptor (GR) work ?

Answer 12

GR = AD-DBD-LBD (Activator Domain - DNA-Binding Domain - Ligand-Binding Domain)
Resting state : LBD bound to dimeric inhibitor
When hormones enters the cell, it binds LBD and displaces the inhibitor
GR then enters the nucleus and activation gene transcription via the DBD

Question 13

In the mouse genome, what activators of the transthyretin (TTR) gene are expressed in hepatocytes ?
What about other cells ?

Answer 13

HNF1 and HNF3 (located in the promoter-proximal regions) are expressed only in hepatocytes
C/EBP, HNF4 and AP1, located both in the promoter-proximal and enhancer regions, are expressed in all cells

Question 14

What is the function of chromatin remodeling complexes (CRCs)?

Answer 14

• native chromatin cannot be transcribed : the DNA binding sites are inaccessible
• CRCs perturb or re-position nucleosomes, allowing access to DNA binding sites
• transcriptional activator protein (TAP) binds w/ its atrget site in DNA
• TAP recruits TFIID and TBP to the TATA box
• RNA Pol holoenzyme can join the transcription complex

Question 15

What are examples of histone modifications of the nucleosome core particle ?

Answer 15

Lysine acetylation on H3, H4, H2B and H2A
Arginine methylation on H3 and H4
Lysine methylation on H3 and H4
Serine phosphorylation H3 and H4
Lysine ubiquitination on H2A and H2B C-term

Question 16

How does histone acetylation work ?

Answer 16

• +ely charged tails of nucleosomal histone proteins probably nteract w/ the -ely charged phosphate group of DNA
• actylation of the tails weakens their interactions w/ DNA and may permit some TFs to bind DNA

Question 17

What are specific example of DNA methylation ?

Where are these methylations observed ?

Answer 17

• Addition of methyl groups at CpG sites on the DNA.
5’-CpG-3’
3’-GpC-5’
• CpG islands : A higher frequency of CpG as compared across the vertebrate genome, found mainly near transcription start sites.
• Cytosine methylation found in mammalian and plant DNA, but not in worms, flies, or yeast.

Question 18

What is associated w/ hypermethylation ? - hypomethylation ?

Answer 18

Hypermethylation acts to silence genes.

Hypomethylation is associated with active genes.

Question 19

What residues are methylated in eukaryotes ?

Which residues are deaminated ?

Answer 19

• Approximately 5% of all cytosine residues are methylated
• Most common at CpG sequences
• Non random distribution of CpG throughout the genome
• Deamination of cytosine and methylcytosine

Question 20

What are CpG islands ?

Answer 20

• An increased number of CpG (i.e. 5’-CG-3’) in 5’ region of genes
• Long stretches of DNA typically 1-2kb
• CpG islands have an average G-C content of 60% as compared to the rest of the genome which has 20%
• CpG islands are generally unmethylated
• 29,00 CpG islands in the human genome
• All constitutively expressed housekeeping genes have CpG islands
• These account for about half the islands in humans
• Remaining islands are found at promoters of tissue specific genes
• CpG islands are unmethylated in tissue specific promoters
• The presence of unmethylated CpG islands may be necessary but not sufficient for gene transcription.