Expression of Gene Control Flashcards
What are the eukaryotic DNA control elements?
TATA box
Promoter proximal elements
Enhancers
Describe the TATA box
25-35bp upstream of transcription start site
determines site of transcription initiation
Directs binding protein of RNA pol I (where general transcription factors bind)
Describe promoter proximal element
within 200bp upstream of transcription start site
~20bp long
regulates transcription -> bound by cell specific factors
Describe enhancers
Multiple control elements, 8-20 bp long each=> up to 100-200 bp long total
200 tens of kbp upstream or downstream from promotor, or last exon of gene, also within intron
Regulate transcription in cell specific way
Describe thalassemia β
inherited anemia caused by insufficient production of β-globin protein by erythroid cells
Caused by many mutations, including at promoter-> reduces amound of β-globin mRNA -> decreases protein production (clinically mild)
Describe thalassemia γδβ
caused by deletion of locus control region (LCR) of β-globin gene cluster
Hemophilia B Leyden
X linked clotting disorder
1% of normal factor IX until puberty in males (result of mutation in promoter => prevents binding of transcriptional activators )
After puberty up to 60% of normal factor IX due to androgen receptor activation that has overlapping binding sites at promoter
Describe fragile X syndrome
Mental retardation, dimorphic facial features, macroorchidism (abn large testes) in 1/1,500 males
CGG repeat at 5’ region of FMR I gene-> methylation of cytosine residues in CpG-> transcriptional inactivation of FMR I gene
Normal = 6-50 repeats, Fragile x = > 200 repeat -> increased transcriptional silencing
What is the role of transcriptional activators and repressors?
They are proteins that are encoded by one gene but act on other genes (Trans) regulating (increasing or decreasing) transcription
What are the 2 classes of sequence activators and repressors?
Sequence specific binding proteins
Cofactors
How do sequence specific binding proteins work?
Binds promoter or enhancer in target genes to reg. transcription
Usually binds element 6-8bp long
Bind by inserting α Helices into major groove of DNA -> creates contact between amino acid side chains of protein and based of DNA
How do cofactors work?
Do not bind DNA directly
bind sequence specific DNA binding proteins -> affect translation
Cause sequence specific DNA binding proteins to work more or less efficiently
What are the domains of sequence specific DNA binding proteins
They have 2 (modular)
DNA binding domain - reads DNA and binds with high specificity – highly conserved
Activation/ repressor domain - binds cofactors, on outside, bind general transcription factors, not highly conserved
Describe helix turn helix sequence specific DNA binding proteins
Homeodomain act on simillar genes Important for development HOX family, Pit 1, MSX 60 aa domain 2 alpha helicies -> motif
Describe zinc finger sequence specific DNA binding proteins
tetrahedral organizaiton created by Zn residue
Bind nuclear receptors for Estrogen, androgen, retinoic acid
Describe the basic helix loop helix sequence specific DNA binding proteins
zipper with looped region
myoD, myogenin, Myf5
homo and hetero dimers
Describe the basic leucine zipper
dimerized through alpha-helical zipper domains
no loop
homo and heterodimerization
c-fos, c-jun
Describe androgen insensitivity syndrome
decreased androgen response due to mutation in AR DNA binding domain or ligand binding domain <- mutation in binding domain or ligand binding domain (zinc finger)
Describe waardenburg syndrome type II
Mutation of MITF gene renders it nonfunctional -> this affects transcription of genes important in pigmentation and hearing
protein made but does not function -> cannot bind to DNA to activate Genes (basic helix loop helix)
Describe Msx2 and craniosynostosis
Proline to histidine substitution-> causes tighter binding to DNA -> excessive expression of gene-> premature fusion of skull
How is combinatorial control a mechanism for controlling gene expression?
Increases number of potential sequences where family or sequence specific transcription factors can bind -> new DNA binding specificity
Available to zinc finger, bZIP, bHLH
How do transcriptional activators and repressors control transcription once they are bound to DNA?
by regulating assembly of initiation complexes
regulating rate of initiation of transcription
regulating changes in chromatin structure (ability of transcription factors to bind promoters)
What are the 2 classes of chromatin remodeling factors? How do they work?
DNA dependent ATP-ases
- SWI/SNFs
- disrupt histone octamers and DNA
HATs and HDAC
-reversibly modify histones via acetylation
What are HATs and HDACs and how does their activity influence transcription?
HATs: histone acetyltransferases
- Acetylate n-terminal of histone
- Neutralizes positively charged ends -> decrease DNA interaction?
- Acetylation of lysine -> specific transcription factor binding -> pattern of Acetylation = code to recruit factors & influence binding
- Co-activators
HDACs: Histone deacyetylases
- Keep, return + charge -> interaction with – DNA?
- Acetylation of lysine (pattern) = code for transcription factor binding
- Co-repressors
Trans acting factors can work as activators or repressors by recruiting HATs or HDACs
Give an example of a disease where histone acytlation has been altered, describe the defect
Rubinstein-Taybi Syndrome
- Rare 1/125,000
- Growth retardation, mental retardation, craniofacial dysmorphism, abnormally broad thumbs and great toes
- Mutation of CREB binding protein (CBP) -> co-activator of many transcription factors
- Transcription of MANY genes does not take place or is insufficient
Leukemia
- Hematopoietic malignancy
- Gain of function of fusion-proteins -> altered activity of regulators
How do activators/repressors modulate transcription?
interact with general transcription factors/ Pol II proteins
Change initiation or elongation of transcript
Influence rate of initiation
How do activators/repressors modulate transcriptional machinery?
Influence rate of assembly
How do activators/repressors modulate chromatin?
change transcription factor’s ability to bind (accessibility)
How are transcriptional regulation and specificity achieved?
regulation
-depends on DNA-protein and protein-protein interaction-> change DNA conformation or chromatin structure and initiation complex forms
specificity
-depends on binding of transcriptional activators or repressors to DNA control elements
How does protein-DNA interaction contribute to transcriptional control?
control is combinatorial
- several thousand transcriptional activators/repressors can differentially change expression of genes in different cells in response to different stimuli
What is Euchromatin?
Where genes reside in accessible form of chromatin
What is heterochromatin?
Repressed because DNA is inaccessible
-on centromeres, telomeres and internal chromosome positions
What is the difference between constitutive and facultative heterochromatin?
Constitutive - always inaccessible, contains satellite DNA
ex. centromeric DNA
Facultative - can be euchromatin, depends on cell type, developmental stage and enriched LINE sequences
ex. X-inactivation
How are sequence specific DNA binding proteins regulated?
regulation of conformation of DNA binding protein changes due to ligand binding (i.e. zinc finger)
Regulation of entry of DNA binding proteins into nucleus (nuclear localization region- NLR)
Regulation of amount of transcription factor in cell ([concentration])
Regulation of DNA binding
Phosphorylation of DNA binding protein changes protein degradation, recruitment of co-activators and DNA binding
How is the activity of nuclear hormone receptors controlled?
In nuclear receptors binding of hormones affect receptor and recruitment of co-activators/ repressors and translocation into nuclies
i.e estrogen receptor activation, glucocorticoid receptor activation
How does Tamoxifen act in breast cancer therapy?
Normal: estrogen induces transcriptional activation through estrogen receptor (causes Dimerization of ER -> histone acetylation-> pol II recruitment -> CTD Phosphorylation -> ER/p160 release)
In pathology (ER + breast cancer): Tamoxifen binds ER, prevents recruitment of HATs -> no acetylation of histones -> no recruitment of pol II (may recruit depressors)
How does NFkb pathway work? What drug changes its pathway, and how does it work?
Held in cytoplasm by binding to inhibitor (IkB) which masks nuclear localization signal
- If Ikb phosphorylated -> degraded -> NFkB released and moves to nucleus
- In nucleus NFkB turns on genes involved in inflammatory response
Asprin is anti-inflammatory in part b/c inhibits phosphorylation -> IkB not degraded and NFkB cannot enter nucleus to activate inflammatory genes
How does NF-AT pathway work? What drug changes this pathway and how does it work?
- Phosphorylated in cytoplasm, cannot enter nucleus
- High intracellular Ca -> activates calcineurin’s phosphatase activity -> NF-AT dephosphorylated -> nuclear localization sequence exposed -> goes to nucleus
- In nucleus NF-AT affects transcription of immune response genes & heart function
Immunosuppresants (cyclosporine and FK506) -> inhibit calcineurin => inhibit NF-AT
Describe the β-Catenin pathway and how it can be changed
Typically phosphorylated by APC, GSK3 and Axin (due to absence of Wnt signaling)
-if Wnt signaling occurs -> β-Catenin no longer phosphorylated (targeted for ubiquitination) b/c APC and complexing proteins destabilized -> cytoplasm concentration increases
- up [β-Catenin] -> some enters nucleus -> interacts with TCF family transcription factors -> promotes expresson of Wnt responsive genes
Positive feedback loop
What type of cancer is APC mutation involved in?
Colon cancer
How does p53 work normally?
Guardian of Genome
active in growth arrest, DNA repair, apoptosis
down regulated by MDM2 binding (masks activation domain targets for destruction by ubiquitin- proteosome pathway
present in >50% of cancers
Describe ubiquitin and its function
ubiquitous (highly conserved)
yeast = human
Covalently linked to protein by ATP dependent pathways of E1, E2 & E3 enzymes
Once ubiquinated, proteins taken to 26s proteosome for degradation
Describe how DNA binding activity of sequence specific DNA binding protein can be inhibited
Id proteins
Heterodimerize with helix loop helix domains
-prevent DNA binding through loss of basic domain
Describe a protein modification that can alter activity of a sequence specific DNA binding protein
Phosphorylation affects activity of trans acting factors
- CREB (Cyclic AMP response element binding protein) -> phosphorylated due to events triggered by ligand binding to guanine nucleotide binding protein coupled receptor
- CREB in active until phosphorylated -> if phosphorylated recruits histone acytly transferase, CBP (with HAT activity -> RNa pol II) => => activation of gene
Rubinstein-Taybi Syndrome
- Rare 1/125,000
- Growth retardation, mental retardation, craniofacial dysmorphism, abnormally broad thumbs and great toes
- Mutation of CREB binding protein (CBP) -> co-activator of many transcription factors
- Transcription of MANY genes does not take place or is insufficient
Beyond transcriptional regulation, how can gene expression be controlled?
control of mRNA export from nucleus
control of mRNA degradation
control of efficiency of translation
control of protein degradation
