Gene Expression Learning Goals Flashcards
1. Discuss the basics of eukaryotic transcription (DNA control elements, transcription factors) and the role they play in human disease and list the different eukaryotic DNA control elements.
DNA control elements are DNA elements that acts locally (CIS). Binding transcription factors to these elements controls the expression of the gene that the element is associated with.
These are the different Eukaryotic control elements:
A. TATA box/Initiator sequence- This element is generally 25-35 bps upstream of the transcription start site. It determines the site of transcription initiation and directs binding of RNA polymerase II. This is the site at which general transcription factors bind. *Only about 20% of the genes have a TATA box.
B. Promoter Proximal Elements- 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.
C. Enhancers- Contain multiple control elements, each 8-20 bps in length. Thus, an entire 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. Similar to promoter proximal elements, enhancers may help to regulate transcription in a cell type specific manner. Super Enhancers- many linked control elements providing greater magnitude of control.
3. Describe a disease that arises from a mutation in a DNA control element, and how the mutation leads to the disease state.
Thalassemias:
β- An inherited anemia due to deficient production of β-globin protein by erythroid cells. Can occur due to different types of mutations- one of which can occur in the β-globin promoter- reducing the amount of β-globin mRNA and thus protein produced (is usually clinically mild).
γβδ- due to deletion of the locus control region (LCR) of the B-globin gene cluster (the LCR region is essential for the transcription of all genes within the cluster)
Hemophilia B Leyden: Is an 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.
Fragile X-Syndrome: Affects 1 in ~1500 males and results in mental retardation, dysmorphic facial features, and postpubertal macroorchidism. 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 have 6-50 or so CGG repeats in this region, however, affected males have an expansion of this repeat sequence (>200 copies)- leading to increased transcriptional silencing of the FMR1 gene.
Describe the role of transcriptional activators and repressors.
Proteins encoded by one gene that act on other genes to regulate their transcription (trans). Can therefore diffuse around the nucleus and affect transcription of numerous genes. Can either activate or repress transcription.
List the two classes of activators and repressors.
- Sequence-specific DNA binding proteins
Bind to promoter or enhancer elements (DNA control elements) in their target genes to regulate transcription. The elements they bind to are usually 6-8 base pairs long. Usually bind DNA by inserting their - 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- Do not bind directly to the DNA elements but rather bind to sequence- specific DNA binding proteins and affect transcription through this contact.
Co-activators- activate transcription Co-repressors- repress transcription
Describe the domains of a sequence specific DNA binding protein.
Modularity: Sequence-specific DNA binding proteins are modular, containing two major domains:
1. DNA binding domain. The DNA binding domains (DBDs) are highly structured and evolutionarily conserved. They are folded so that they can “read” the DNA sequence and bind to their specific target DNA.
2. Activation (or repression) domain. In contrast, the activation domains are not highly conserved and are very unstructured until they bind co-factors or general transcription factors. They mediate protein-protein interaction to recruit general transcription factors.
- List the four major families of sequence specific DNA binding proteins and describe the means for categorizing the proteins into these families.
*Specificity is commonly determined by non-covalent interactions
between atoms in an alpha helix in the DNA-binding domain and
atoms on the edges of the base within a major groove in the DNA.
Families of Sequence-Specific DNA binding proteins:
More than 80% of sequence specific DNA binding proteins fall into 4 families. Each family is characterized by a conserved DNA binding domain. The domains of a particular family contain certain consensus amino acid sequences and are therefore similar in tertiary structure. They are classified based on the structure of their binding domains.
A. Homeodomain Proteins (Helix-turn-helix)
Members include Hox family, Pit1, Msx, etc.
B. Zinc-finger proteins
Members include nuclear receptors such as estrogen receptor, androgen receptor, retinoic acid receptor
C. Basic leucine zipper proteins (bZIP)
Members include c-fos and c-jun
D. Basic helix-loop-helix motif (bHLH)
Members include MyoD, myogenin, Myf5
Describe a particular human disorder that arises from a mutation in a sequence specific DNA binding protein, explaining how the mutation leads to the disorder.
Craniosynostosis- Craniosynostosis is characterized by the premature closure of one or more sutures in the skull and affects 1/3000 infants. One particular variant of craniosynostosis (Boston-type) occurs as a result of a mutation in the homeodomain protein MSX2. MSX2 is normally required for proper craniofacial development 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 amino acid substitution (proline to histidine), the protein binds DNA more strongly- giving a “gain of function” or “hypermorphic allele”. This mutated hyperactive protein then affects the transcription of other genes critical for suture closure, leading to craniosynostosis.
Describe a particular human disorder that arises from a mutation in a sequence specific DNA binding protein, explaining how the mutation leads to the disorder.
Androgen insensitivity syndrome (AIS)
Androgen insensitivity syndrome includes feminization or undermasculinization of the external genitalia at birth, abnormal secondary sexual development in puberty, and infertility. It occurs in males who are a normal karyotype (46 X,Y), but have mutations in either the DNA binding domain or the ligand binding domain of the androgen receptor (a zinc finger DNA binding protein). This makes the patients less responsive to androgens, leading to the aforementioned characteristics. Depending on the degree to which the mutation disrupts the function of the androgen receptor- varying levels of AIS can be observed (complete, partial, mild).
Describe a particular human disorder that arises from a mutation in a sequence specific DNA binding protein, explaining how the mutation leads to the disorder.
Waardenburg Syndrome type II
Is 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.
Describe combinatorial control as a mechanism for controlling gene expression.
Many sequence specific DNA binding factors bind DNA as homo or heterodimers. The Zinc finger, bZIP, and bHLH can all form heterodimers. If each monomer of the heterodimer has a different DNA binding specificity, the formation of heterodimers will increase the number of potential sequences to which that family of sequence specific transcription factors can bind. This process is referred to as Combinatorial Control. Moreover, it refers to combining different transcription binding sites allows one factor to control many different genes depending on the presence of other TF binding sites.
Describe how chromatin structure affects transcriptional control and list the two classes of chromatin remodeling factors and briefly describe how they work.
DNA isn’t free, but associates with roughly an equal mass of protein to form chromatin. The ability of transcription factors to bind DNA can be affected by chromatin structure.
The basic structural unit of chromatin is the nucleasome, which consists of a core of histone proteins around which the DNA is wound. The N-termini of histones are rich in lysine residues, which can be reversibly modified by acetylation, phosphorylation, methylation, and ubiquitination. Acetylation and methylation are associated with gene control.
There are two classes of chromatin-remodelling factors that may be involved:
1. DNA-dependent ATPases (SWI/SNF)- disrupt histone octamers and DNA.
2. Factors that reversibly modify histones through acetylation or methylation (HATs and HDACs or KMTs and KDMs)
Define HATs and HDACs and describe how their activity influences transcription.
Activators and Repressors can recruit histone acetyltransferases (HATS), deacetylases (HDACs), methyltransferases (KMTs) or demethylases (KDMs).
Histone acetyltransferases (HATS)- acetylate N-termini of histones. The original thought was that 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).
Histone deacetylases (HDACs)- In presence of HDACs, 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.
Histone Methyltransferases (KMTs)- Methylation does not alter the charge on histone lysine residues, but can be added to different degrees with 1, 2 ,or 3 methyl groups per lysine (mono-, di- or tri-methylated). The position of the histone in relation to the gene and the number of methyl groups can determine if this is stimulatory or inhibitory to transcription.
Histone Demethylases (KDMs)- Methylation can be removed by two classes of enzymes: FAD using enzymes and α-ketoglutarate using enzymes. Depending on the degree of methylation removed and location of the methylation these enzymes can be stimulatory or inhibitory to transcription.
Give an example of a disease in which histone acetylation is altered, and describe the defect that leads to altered histone acetylation.
Rubinstein-Taybi Syndrome
A rare genetic multisystem disorder (affects 1/125,000). Characterized by growth retardation, mental retardation, craniofacial dysmorphism, abnormally broad thumbs and great toes. Results from mutations in one copy of the CREB binding protein (CBP) gene. CBP is an essential transcriptional coactivator for many different transcription factors and is a histone acetyltransferase. It is normally recruited to many genes to activate transcription, and thus haploinsufficiency can result in widespread transcriptional changes.
Give an example of a disease in which histone acetylation is altered, and describe the defect that leads to altered histone acetylation.
Leukemia
A hematopoietic malignancy. Are generally the result of chromosomal translocations leading to gain of function fusion proteins- some of which involve fusions of transcriptional regulators with HATs or HDACs or KMTs, altering the activity of the regulators. Fusions function to recruit super elongation complex- altering transcription rates of different leukemia target genes.
Describe how activators/repressors modulate transcription via their interaction with general transcriptional machinery vs. with chromatin.
Two Ways Transcriptional Activators and Repressors Work:
- Interact with general transcription factors/Polymerase II associated proteins to influence initiation of elongation of the primary transcript
- Interact with chromatin to regulate accessibility of DNA to Pol II transcriptional apparatus .
Describe how the activity of nuclear hormone receptors is controlled, and how tamoxifen acts in breast cancer therapy.
Steroid hormones work by binding to the nuclear receptor family of sequence-specific Zn finger transcription factors.
Example:
Estrogen receptor binds DNA as a homodimer through a pair of Zn fingers. Estrogen Induces Transcriptional Activation Through ERby causing its dimerization.
How does tamoxifen works?
Tamoxifen antagonizes estrogen by binding to ER and preventing recruitment of HAT co-factors.
Give an example of a sequence specific DNA binding protein regulated by nuclear entry and describe the mechanism by which its entry is controlled.
NF-κB- NFκB is normally held in the cytoplasm by its binding to the inhibitor of NFκB (IκB), which masks the nuclear localization signal of NFκB. In response to a variety of stimuli, IκB can be phosphorylated, targeting it for degradation. NFκB is then released and moves to the nucleus, where it turns on a number of target genes, including those involved in inflammation (see slide).
*Note- the anti-inflammatory, aspirin, works in part by inhibiting the phosphorylation, and thus degradation of IκB. This prevents the translocation of NFκB to the nucleus, thus inhibiting the transcription of genes involved in the inflammatory response.
NF-AT- 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
Describe how the amount of an activator/repressor can be regulated within the cell.
Beta catenin- In the absence of Wnt signaling, the cytoplasmic pool of β-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 β-catenin and leading to an increase in the cytoplasmic pool of the protein. This increase allows some of the -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.
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.
Describe a mechanism by which the DNA binding activity of a sequence specific DNA binding protein can be inhibited.
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.
