Gene Expression and Chromatin

Question 1

What are the different eukaryotic DNA control elements?

Answer 1

TATA Boxes (initiator sequences), promoter proximal element, enhancers

Question 2

What is a TATA Box (initiator sequence)?

Answer 2

The site at which transcription factors bind, 25-35 base pairs upstream from the transcription start site. It determines the site of transcription initiation and directs the binding of RNA pol II.

Question 3

What is a promoter proximal element?

Answer 3

A 20 base pair sequence that is approximately 200 base pairs upstream from the transcription start site. Depending on the cell type, specific factors might bind to it.

Question 4

What is an enhancer?

Answer 4

A series of nucleotides, upstream or downstream, consisting of multiple control elements totaling 100-200 bps. Each control element is 8-20 bps in length. Can be very far away.

Question 5

What are three diseases that arise from mutations in DNA control elements?

Answer 5

ThalassemiasHemophilia B LeydenFragile X Syndrome

Question 6

Describe the mutation that leads to thalassemia and its clinical presentation.

Answer 6

A mutation in the promoter of the ß-globulin gene. This results in less ß-globulin protein. A patient would present with clinically mild” symptoms including tiredness, fatigue, and shortness of breath

Question 7

Describe the mutation that leads to Hemophilia B Leyden and its clinical presentation.

Answer 7

A mutation in the promoter of the Factor IX gene. Less Factor IX is produced (1% when young). This negatively affects the ability of the body to form blood clots. X-linked and effects young males most.

Question 8

Describe the mutation that leads to Fragile X Syndrome and its clinical presentation.

Answer 8

Caused by excess CG repeats in the 5’ region of the FMR1 gene which leads to transcriptional silence of gene. Causes mental retardation, dysmorphic facial figures, post pubertal macro orchidism.

Question 9

What roles do transcriptional activators and repressors perform?

Answer 9

Activation and repression of transcription!But you should know that these are proteins made from other genes.

Question 10

What are the two classes of activators and repressors?

Answer 10

Sequence specific DNA binding proteinsCo-factors

Question 11

How do sequence specific DNA binding proteins work?

Answer 11

They bind to promoter enhancer elements that are 6-8 base pairs long on the target genes. They bind DNA by inserting alpha helices into the major groove. AA R groups contact base pairs of DNA to regulate transcription.

Question 12

How do co-factors work?

Answer 12

They bind to sequence specific DNA binding proteins (not DNA) and affect transcription through this contact.

Question 13

What are the two domains of a sequence specific DNA binding protein?

Answer 13

DNA binding domain: confers sequence specificityActivation domain: mediates protein-protein interactions and recruits general transcription machinery/co-factors

Question 14

What are the four major families of sequence specific DNA binding proteins?

Answer 14

Homeodomain (helix-turn-helix),Zinc Finger,Basic Leucine Zipper (bzip),Helix-Loop-Helix

Question 15

How does one go about categorizing sequence specific DNA binding proteins?

Answer 15

Based on the structures of their DNA binding domain!

Question 16

What are three diseases that can arise from a mutation in a sequence specific DNA binding protein?

Answer 16

CraniosynostosisAndrogen Insensitivity SyndromeWaardenburg Syndrome Type II

Question 17

Describe the mutation that leads to craniosynostosis and its clinical manifestations.

Answer 17

A mutation in homeodomain MSX2 causes it to bind too strongly to DNA. This affects the transcription of other genes critical for suture closure leading to craniosynostosis (pre mature fusion of forehead).

Question 18

Describe the mutation leading to androgen insensitivity syndrome and its clinical manifestations.

Answer 18

Mutations in the either the DNA binding domain or activation domain in the androgen receptor protein leads to feminization and under masculinization.

Question 19

Describe the mutation that leads to Waardenburg Syndrome Type II and its clinical manifestations.

Answer 19

Mutation in the MITF gene that encodes for a transcription factor that plays a major role in the development of melanocytes leads to deafness and pigmentation anomalies of hair and skin.

Question 20

What is combinatorial control?

Answer 20

Thousand of transcription factors can come together in different ways to regulate thousands of genes differentially.

Question 21

List the two classes of chromatin remodeling factors and describe how they work.

Answer 21

DNA dependent ATPases that disrupt histone octomers and DNA.Factors that reversibly modify histones through acetylation via histone actetyltransferases (HAT) + histone deacetylases (HDAC)

Question 22

How does HAT activity influence translation?

Answer 22

Acetylation of lysine allows for the binding of specific transcription factors and once histone is acetylated it recruits transcription factors. HATS are co-activators.

Question 23

How do HDACs influence transcription?

Answer 23

In the presence of HDACs histones retain positive charge and this maintains its interaction with DNA which prevents access of transcription factors to promoters. HDACs are co-repressors.

Question 24

What are two diseases in which histone acetylation is altered?

Answer 24

Rubinstein-Taybi SyndromeLeukemia

Question 25

What mutation leads to Rubinstein-Taybi Syndrome and what are its clinical manifestations?

Answer 25

A mutation in the Creb Binding Protein, a HAT (co-activator), leads to widespread transcriptional changes during development.Growth retardation + mental retardation + craniofacial dysmorphism.

Question 26

How do activators/repressors modulate transcription...On polymerase II + transcription factors?Chromatin?

Answer 26

Interact with pol II + transcription factors to initiate/block elongation of primary transcript.Interact with chromatin to regulate accessibility of DNA to pol II.

Question 27

What are the basic principles of transcriptional regulation (4)?

Answer 27

1. Specificity of activators/repressors to DNA control elements.2. DNA-protein, protein-protein regulating interactions.3. Interactions affecting conformation of DNA (HAT/HDAC).4. Control is combinatorial.

Question 28

What are the 5 mechanisms in which DNA binding proteins are regulated?

Answer 28

1. The conformation of the DNA-binding protein can be altered by ligand binding2. Entry into the nucleus can be regulated3. The amount of transcription factor in the cell can be regulated4. DNA binding can be regulated5. Phosphorylation of the DNA-binding protein can alter various properties

Question 29

How is the activity of nuclear hormone receptors regulated? How does tamoxifen work?

Answer 29

Estrogen binds to nuclear hormone receptors and activates proliferative cell types. This can be dangerous in cancer. Tamoxifen antagonizes estrogen by binding to estrogen receptors and preventing recruitment of HAT cofactors.  Or, it can recruit co-repressors in some cell types.

Question 30

What is an example of a sequence specific DNA binding protein (SSDBP) being regulated by nuclear entry?What is the mechanism?

Answer 30

In the stomach, Nf-Kb is a SSDBP that enters the nucleus to turn on an inflammatory response when needed.Normally it is sequestered in another protein, IkB which gets ubiquinated and degraded to free nf-kb.  Nonpathogenic salmonella can interfere with the ubiquitination of Ikb and thus blocks an inflammatory response. 

Question 31

How can the amount of an activator/repressor be regulated in a cell?

Answer 31

Consider Beta catenin (BC).  In the absence of WNT signaling, it is to be degraded in a ubiquitin-proteosome pathway. In the presence of WNT signaling, it is not degraded, its [cytoplasmic] rises, and it goes to the nucleus and triggers expression of WNT responsive genes. 

Question 32

How can DNA binding of a SSDBP be inhibited?

Answer 32

 An example - Id proteins: 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.  Relative abundance of these proteins is what dictates transcription or not.

Question 33

What is an example of a protein modification that can alter the activity of a SSDBP?What is the mechanism?

Answer 33

When the CREB protein is interacting with DNA, it needs to be phosphorylated to promote transcription.  It becomes phosphorylated when a ligand binds its  guanine nucleotide binding protein. Once phosphorylated, it recruits a HAT, Creb Binding protein, and RNA pol II. 

Question 34

What are four other mechanisms (besides transcriptional regulation) to control levels of gene expression?

Answer 34

1. Control of mRNA export from the nucleus2. Control of mRNA degradation3. Control of efficiency of translation4. Control of protein degradation