Gene Expression regulation

Question 1

What are the different eukaryotic DNA control elements?

Answer 1

DNA elements that act locally:

1. TATA box,
2. promotor proximal elements,
3. ehancers

Question 2

What is the TATA box

Answer 2

a sequence “TATAAA” about 30 bp upstream of start site. This is where TFs bind, it determines txn initiation site and directs RNA pol II binding

Question 3

What are promoter proximal elements

Answer 3

Generally located within 200 bps upstream of transcription start site and are ~ 20 bps long. Promoter proximal elements help to regulate transcription, and can bound by factors in a cell type specific manner.

Question 4

What are enhancers?

Answer 4

Contain multiple control elements, each 8-20 bps long. enhancer can be 100-200 bps long. An enhancer can be 200-tens of kilobases upstream or downstream from the promoter or the last exon of the gene, or within an intron. enhancers may help to regulate transcription in a cell type specific manner

Question 5

Diseases that arise from mutations in DNA control elements

Answer 5

Thalassemias, Hemophelia B. leyden, fragile X syndrome

Question 6

What are the roles of transcriptional activators and repressors?

Answer 6

Proteins than diffuse and affect transcription of genes, either through activation or repression

Question 7

What are the two classes of activators and repressors

Answer 7

1. sequence specific DNA binding proteins that bind to promoter or enhancer elements usually 6-8 base pairs long. Usually bind DNA by inserting their alpha-helices into the major groove of DNA, making contacts between the amino acid side chains of the protein and the bases in the DNA.
2. co factors. Bind to sequence specific DNA binding (not directly to DNA)

Question 8

What are the 4 basic types of DNA binding domain motifs and what characterizes them?

Answer 8

1. Homeodomain (helix-turn-helix): there are 3 helices, two are parallel, the other is perpendicular to those.
2. Zinc finger: 3 loops of protein, each of which has one alpha helix associated with it. these loops are held together by four cysteins binding to a zinc ion.
3. Basic leucine zipper: dimer of two linearly shaped proteins that “zip” together on either side of the DNA. they have small basic AA tails to recognize and bind DNA
4. Helix loop Helix: Similar to Bzip, but it has a out loop in the middle.

Question 9

Describe the domains of a sequence specific DNA binding protein.

Answer 9

They have a DNA binding domain and protein binding (activation or repression) domain

Question 10

What are the two classes of Chromatin remodelling factors and how to they work.

Answer 10

1. DNA dependent ATP-ase (SWI/SNF): disrupt histone octomers and DNA, reposition nucleosomes on DNA
2. Histone modifiers, though acetylation or deacytlation (and methylation)

Question 11

what is a HAT and how does it work?

Answer 11

Histone acetyl transferase, coactivators- acetylate N-termini of histones. acetylating the N-temini of histones would neutralize the positively charged ends and eliminate electrostatic interactions with DNA phosphates (thus opening up nucleosomal DNA for general transcription factors/Pol II transcriptional apparatus). A currently more favored model is that the acetylation of lysines allows for the binding of specific transcription factors

Question 12

What is HDAC and how does it work?

Answer 12

Histone deacetylases, corepressors, When HDAC is active, histones retain positive charge at N-terminal ends (HDACS remove acetyl groups from histones), The original thought was that this would maintain the interaction with DNA and prevent access of transcription factors to promoter. A currently more favored model is that the acetylation of lysines allows for the binding of specific transcription factors

Question 13

What is a disease where histone acetylation is altered?

Answer 13

Rubinstein-Taybi Syndrome, and leukemia

Question 14

How do activators/repressors modulate transcription via their interaction with general transcriptional machinery vs. with chromatin.

Answer 14

General transcription factors can recruit HATs or HDACs, which then acetylate and alter the structure chromatin/histones to affect gene expression. Some TFs can switch being activators or repressors depending on whether they recruit HAT or HDAC

Question 15

What is combinatorial control as applies to Transcription factors?

Answer 15

Multiple transcription factors act in different combinations in different cell types, in response to different stimuli, and at different times to provide more fine tuned control of gene expression

Question 16

How are sequence specific DNA binding proteins regulated?

Answer 16

(5 ways)

1. Ligand binding to alter conformation
2. regulation of entry into the nucleus
3. amount of TF in cell can be regulated
4. DNA binding regulation,
5. phosphorylation (can affect many things such as degradation, DNA binding, and recruitment)

Question 17

Describe how the activity of nuclear hormone receptors is controlled,

Answer 17

The activity of nuclear hormone receptors is controlled by the presence of their respective hormone. Most of these have zinc finger domains. For estrogen receptor, for example, estrogen binding causes it to dimerize and activates its transcriptional activation function

Question 18

How does tamoxifen act in breast cancer therapy?

Answer 18

Tamoxifen binds to estrogen receptor and recruits co repressors. it also prevents recruitment of HATs. This prevents the estrogen’s function of inducing cell proliferation.

Question 19

Give 2 example of a sequence specific DNA binding protein regulated by nuclear entry.

Answer 19

NF kappa B and NFAT

Question 20

Tell me how NF kappa B is regulated by nuclear entry.

Answer 20

1. NF kappa B: is normally held in the cytoplasm by its binding to its inhibitor, IkappaB which masks the nuclear localization signal. In response to stimuli, IkappaB can be phosphorylated, targeting it for degradation. NFkappaB is then released and moves to the nucleus, where it turns on a number of target genes, including those involved in inflammation
   * Note- the anti-inflammatory, aspirin, works in part by inhibiting the phosphorylation, and thus degradation of IkappaB.

Question 21

Tell me how NFAT is regulated by nuclear entry.

Answer 21

High intracellular calcium (triggered by the binding of a ligand to a cell membrane receptor) activates calcineurin’s phosphatase activity- which dephosphorylates cytoplasmic NF-AT. This exposes the nuclear localization sequence, allowing NF-AT to enter the nucleus where it affects transcription of genes involved in the immune response and in heart function.
*Note- two commonly used immunosuppresants (cyclosporin and FK506) act by inhibiting calcineurin, thereby inhibiting NF-AT action.

Question 22

Describe how the amount of an activator/repressor can be regulated within the cell (2 examples)

Answer 22

1. Beta Catenin: In the absence of Wnt signaling, cytoplasmic Beta-catenin is targeted for degradation through the ubiquitin-proteasome pathway via phosphorylation that occurs through the action of glycogen synthase 3 (GSK3) in a complex with Axin and APC. In the presence of Wnt signaling, the Axin-APC-GSK3 complex is destabilized, preventing phosphorylation of beta-catenin and leading to an increase in the cytoplasmic protein. This increase allows some of the Beta-catenin to move to the nucleus, where it interacts with the TCF family of transcription factors and promotes the expression of Wnt responsive genes.
   * Note importance of mutations in APC for colon cancer.
2. p53- is downregulated by binding to the MDM2 protein which not only masks its activation domain, but also targets it for destruction by the ubiquitin-proteasome pathway.
   * Note importance of p53 in human cancers.

Question 23

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

Answer 23

The Id family members negatively regulate DNA binding by heterodimerizing with other HLH proteins through their HLH domains, but preventing DNA binding due to their lack of a basic domain.

Question 24

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 24

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

Question 25

Aside from transcriptional regulation, list 4 additional mechanisms to control levels of gene expression.

Answer 25

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