Gene Expression II & III

Question 1

1. List the 2 classes of chromatin remodeling factors and briefly describe how they work.

Answer 1

1. DNA-dependent ATPases (SWI/SWF): disrupt histone octamers and DNA; use ATP hydrolysis to move histone along DNA.
2. HATs & HDACs: N-termini are rich in Lys which can be modified by Acetylation, methylation, and ubiquitination. Acetylation is associated with gene control. Activators or repressors recruit HATs or HDACs

Question 2

2. Define HATs and HDACs and describe how their activity influences transcription.

Answer 2

HAT: acetylate N-term of histones. Acetyl groups neutralize the (+) charge so DNA can be freed, opening it up for TFs
HDAC: removes acetyl groups from histones; uncover (+) charge, and DNA binds back.
NEW THEORY: acetylation is a “code” to recruit TFs

Question 3

3. Give an example of a disease in which histone acetylation is altered, and describe the defect that leads to altered histone acetylation.

Answer 3

Rubinstein-Taybi Syndrome: Mutation in CREB binding protein (CBP), which is a HAT. It’s normally recruited to many genes to activate transcription, so haploinsufficiency can result in widespread transcriptional changes. Results: growth/mental retardation, canriofacial dysmorphism.
Leukemia: chromosomal translocations –> GOF fusion proteins, some are transcriptional regulators with HATs or HDACs.

Question 4

4. Describe how activators/repressors modulate transcription via their interaction with general transcriptional machinery vs. with chromatin.

Answer 4

1. Interact with general TFs/Pol II-associated proteins to influence init & elong of pri transcript
2. Interact with chromatin to regulate accessibility of DNA to Pol II

Question 5

5. Discuss the basic principles of transcriptional regulation including how specificity is achieved and how protein-DNA interactions contribute to transcriptional control.

Answer 5

1. Specificity depends on binding of transcriptional activators/repressors to DNA control elements.
2. Regulation depends on DNA-protein and protein-protein interactions
3. The interactions affect the conformation of DNA, modification of chromatin structure, formation of the transcription initiation complex.
4. Control is combinatorial, allows for several thousand transcriptional activators/repressors to differentially alter the expression of genes in different cell types and in response to different stimuli.

Question 6

List at least 4 mechanisms by which SSDBPs are regulated.

Answer 6

1. Conformation of SSDBP is altered by ligand binding
2. Regulate entry to the nucleus
3. Regulate amount of TF
4. Regulate DNA binding
5. Phosphorylation of DBP

Question 7

Describe how the activity of nuclear hormone receptors is controlled, and how tamoxifen acts in breast cancer therapy

Answer 7

Estrogen binds to nuclear hormone receptors and activates proliferative cell types (dangerous in cancer). Tamoxifen blocks by binding to estrogen receptors and prevents recruiting HATs (or recruits co-repressors)

Question 8

Give an example of a SSDBP regulated by nuclear entry and describe the mechanism by which its entry is controlled.

Answer 8

NF-κB: normally bound to IκB hiding the NLS, which holds it in the cytoplasm. When IκB is phosphorylated, it’s targeted for degradation which shows the NLS. NF-κB migrate into the nucleus and affects transcription, which causes an immune response. (Asprin blocks phosphorylation of IκB and acts as an anti-inflammative and one of the reasons low-dose aspirin is given to prevent buildup of atherosclerotic plaque).

Question 9

Describe how the amount of an activator/repressor can be regulated within the cell.

Answer 9

Beta catenin: No Wnt signaling = b-catenin is degraded by GSK3-Axin-APC.
Normal Wnt signaling = GSK3-Axin-APC is destabilized –> increase of b-catenin –> moves to nucleus –> interacts with TCF TFs
Cancer: mutated APC can’t degrade b-catenin –> upregulation of prolif. genes = colon cancer
p53: downregulated by MDM2

Question 10

Describe a mechanism by which the DNA binding activity of a sequence specific DNA binding protein can be inhibited.

Answer 10

Id protein family members downregulate DNA binding by heterodimerizing with other HLH proteins through their HLH domains, but preventing DNA binding due to their lack of a basic domain. Relative abundance of these proteins is what dictates transcription or not.

Question 11

List a protein modification that can alter the activity of a sequence specific DNA binding protein, and explain the mechanism by which the activity is altered.

Answer 11

CREB (cyclic AMP response element-binding protein)- A series of events initiated by the binding of a ligand to a guanine nucleotide binding protein coupled receptor induces the phosphorylation of the CREB protein, which, while present on the DNA, is inactive to promote transcription unless phosphorylated. Once phosphorylated, the CREB protein recruits the histone acetyl transferase, CBP, which has intrinsic HAT activity and recruits RNA Pol II- leading to transcriptional activation of the gene.

Question 12

Aside from transcriptional regulation, list at least 3 additional mechanisms to control levels of gene expression.

Answer 12

1. Control of mRNA export from the nucleus
2. Control of mRNA degradation
3. Control of efficiency of translation 4. Control of protein degradation