Transcriptional Regulation 2
2 gene regulatory proteins that activate Eve transcription
1. Bicoid and Hunchback 2. Presence of both required for stripe 2 module activation
What are the specific patterns of expression of each of the 4 regulatory proteins?
1. Bicoid and Hunchback: anterior 2. Giant: posterior and anterior 3. Krüppel: 1 stripes in middle
How were DNA binding sites for these 4 gene regulatory proteins determined? What is their relationship?
1. DNA footprinting 2. In some cases, binding sites overlap; proteins compete for binding
2 gene regulatory proteins that repress Even transcription
1. Krüppel and Giant 2. Presence of either turns off stripe 2 module
What causes nuclear receptor activation?
1. Ligand binding: conformation change 2. Inhibitory protein dissociates, coactivator proteins bind to TAD, DBD binds to DNA - increases transcription
How would you show phosphorylation is required for TF activation?
1. Make AB for phosphorylated protein, attach to bead, incubate with nuclear extract, and centrifuge 2. Map location of protein phosphorylation via site-directed mutagenesis 3. Fusion protein: Gal4 DBD, WT TF TAD. 4. Control: same as above with TF TAD with replaced amino acid 5. Incubate each fusion protein w/ and w/o cAMP and a plasmid (Gal 4 recognition sequence, promoter, luc. reporter gene) and compare luc. expression
2 reasons that signal molecules generate specific responses
1. Only certain cells have receptors for it 2. Each cell type contains diff. combination of other gene regulatory proteins that collaborate w/ activated receptor to influence transcription of specific genes
How would you detect TF activation by binding to a substrate?
1. Plasmid: TF binding sequence, promoter, luc. reporter gene 2. Incubate with TF 3. Compare luc. expression with and without addition of ligand
What are 7 ways that gene regulatory proteins are regulated in eucaryotic cells?
1. Protein synthesis 2. Ligand binding 3. Covalent modification, e.g. phosphorylation 4. Addition of 2nd subunit 5. Unmasking 6. Stimulation of nuclear entry 7. Release from membrane
How would you demonstrate the modular construction of the Eve gene regulatory region?
1. Remove a regulatory module (e.g. for stripe 2), place in front of a test promoter and a reporter gene, and reinsert in Dros. genome 2. After development of embryo, reporter gene is expressed in that stripe
CREB protein
1. cAMP response element-binding protein: a TF that binds to DNA sequences called cAMP response elements (CRE) 2. Increases or decreases transcription 3. Regulates somatostatin
What does the amount of TF present depend on?
Amount of gene activation
In what form do all nuclear receptors bind to DNA?
As homodimers or heterodimers
Addition of a 2nd subunit
Complex between DNA-binding and activation subunits
3 steroids that Zn finger proteins bind to
Cortisol, estradiol, testosterone
Ligand binding
E.g. steroid receptors to steroids; receptors then bind DNA and initiate transcription
Drosophila Even-skipped (Eve) gene
Expression patterns regulated by specific gene regulatory proteins in cytoplasm distributed unevenly along length of embryo - expressed in 7 stripes with separate nuclei
What happens in flies deficient in Bicoid and Hunchback?
Fail to express Eve efficiently in stripe 2
What other effect can ligand-binding have on the nuclear receptor?
Inactivation (e.g. thyroid)
What is an inactive receptor protein bound to?
Inhibitory proteins
2 roles of nuclear receptors
Intracellular receptors and effectors for signal
Nuclear receptors
Ligand-modulated gene regulatory proteins 1. Activated by hydrophobic signal molecules that diffuse directly across plasma membrane 2. Alters their ability to control transcription of specific genes
How else can activity of a nuclear receptor be controlled?
Localization 1. Inactive: in cytoplasm 2. Activation via ligand: entry into nucleus
Unmasking
Phosphorylation of an inhibitor protein
Release from membrane
Release of gene regulatory protein from a membrane bilayer by regulated proteolysis
Stimulation of nuclear entry
Removal of an inhibitory protein that keeps the regulatory protein from entering the nucleus
How are the nuclear receptors related?
Same DNA-binding domain: Zn fingers
What determines the specificity of nuclear receptors?
Size and orientation of repeats in DNA sequence (direct repeat, inverted repeat, etc.)
How were distributions of these 5 gene regulatory proteins determined?
Staining a developing embryo with ABs directed against each one
What happens to flies deficient in either Giant or Krüppel?
Stripe 2 expands anteriorly or posteriorly, respectively
Domains in a nuclear receptor
TAD, DBD, ligand-binding domain
Protein synthesis
TF synthesized only when needed, rapidly degraded by proteolysis to prevent accumulation
2 non-steroid that Zn finger proteins bind to
Vitamin D3, thyroxine, retinoic acid
