Gene Expression

Question 1

What are the 3 DNA control elements?

Answer 1

TATA box: 25-35bp upstream of transcription start site (which it determines). Directs binding of RNA Pol II. It’s where general TFs bind
Proximal promoter: ~20bp long and ~200bp upstream of start site. Help regulate transcription
Enhancer: contains multiple control elements, each ~8-20bp (whole thing can be 100-200bp long) and can be 200-10,000s bp up- or downstream of gene, even inside intron! Similar to PPE, they regulate transcription in a cell-type specific manner.

Question 2

What 3 diseases can arise from a mutated control element?

Answer 2

Thalassemias, Hemophilia B Leyden, Fragile X Syndrome

Question 3

Thalassemias

Answer 3

(B) inherited anemia due to deficient production of b-globin protein by erythroid cells. Can occur due to mutation in the b-globin promoter, reducing the amount of b-globin mRNA & protein. GDB is due to deletion of the locus control region (LCR) of the b-globin gene cluster

Question 4

Hemophilia B Leyden

Answer 4

X-linked disorder that affects clotting. Affected males have 1% of normal factor IX active until puberty due to inherited mutations in a DNA control element in the promoter of the Factor IX gene (which prevents the binding of the appropriate transcriptional activators). Alternative transcriptional activators can bind overlapping sites in the promoter, and at puberty, when the androgen receptor becomes active- it can bind at the promoter site and increase transcription such that males after puberty make ~60% the normal amount of factor IX.

Question 5

Fragile X Syndrome

Answer 5

CGG repeat in the 5’ region of the FMR1 gene facilitates methylation of the cytosine residues in CpG islands and transcriptional inactivation of the FMR1 gene. Normal males = 6-50 CGG repeats; Affected males = 200+ copies. Result: increased transcriptional silencing of the FMR1 gene. Symptoms: mental retardation, dysmorphic facial features, and postpubertal macroorchidism.

Question 6

Describe the role of transcriptional activators and repressors.

Answer 6

Proteins encoded by one gene that act on other genes to regulate their transcription. Can therefore diffuse around the nucleus and affect transcription of numerous genes.

Question 7

List the two classes of activators and repressors.

Answer 7

1. SSDBP: bind to promoter or enhancer in their target genes to regulate transcription. Usually bind short (6-8bp) elements on DNA by inserting their a-helices into the major groove
2. Co-factors: Don’t bind directly to DNA but rather to SSDBPs and affect transcription through this contact
   NOTE: co-factors influence the RATE of transcription, while GTFs provide the pre-initiation complex needed to begin transcription.

Question 8

Describe the (2) domains of a SSDBP

Answer 8

DNA binding domain: confers sequence specificity
Activation domain: mediate protein-protein interactions that recruit the general transcription machinery and/or co-activators that modify chromatin

Question 9

List the four major families of sequence specific DNA BPs and describe the means for categorizing the proteins into these families.

Answer 9

1. Homeodomain proteins (helix-turn-helix): tend to be regulators of development and affect many genes at once. ex. Hox family, Pit1, Msx
2. Zinc-finger proteins: “finger” made up of two antiparallel beta sheets and an alpha helix, held together by a zinc ion. Finger is what binds with the DNA. The largest family of SSDBPs. Include androgen and estrogen receptors
3. bZIP: Basic leucine zipper protein. high pH region binds DNA. Chopsticks, hydrophobic residue every 7 AA. ex. c-fos & c-jun
4. bHLH: MyoD, myogenin, Myf5

Question 10

What 3 disorders arise from mutations in SSDBPs?

Answer 10

Craniosynostosis: premature closure of one or more sutures in the skull. Mutation in the homeodomain protein MSX2, which normally required for proper craniofacial dev by affecting the transcription of a number of genes important in this process. When the DNA binding domain (or homeodomain) of this protein has a one AA substitution, the protein binds DNA more strongly –> GOF or “hypermorphic allele”. Protein then affects the transcription of other genes critical for suture closure, leading to craniosynostosis.
Androgen insensitivity syndrome (AIS): feminization / undermasculinization of the external genitalia at birth, abnormal secondary sexual development in puberty, and infertility. Occurs in males who are a normal (46 X,Y), but have mutations in either the
DNA binding domain or the ligand binding domain of the androgen receptor (zinc finger). This makes the patients less responsive to androgens, leading to the aforementioned characteristics. Severity varies.
Waardenburg Syndrome type II: Characterized by deafness, pigmentation anomalies of the eyes, and
other pigmentation defects (hair, skin). Mutations in the microphthalmia-associated transcription factor
(MITF) gene (which encodes a bHLH DNA binding protein) are observed in 15-20% of the patients. This
gene encodes a transcription factor that plays a major role in the development of melanocytes.