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| primary marker of regions of the genome that are silenced |
DNA methylation |
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mcs5109 Thu, 20 Nov 2008 04:42:36 GMT |
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| epigenetic regulation |
-inheritance of gene expression patterns
-in the absence of either mutation or the initiating signal
-Nucleosome and DNA modifications can provide the basis |
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mcs5109 Thu, 20 Nov 2008 04:42:36 GMT |
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| imprinting |
In the diploid cell, in most cases, the two alleles (one from father, the other from mother) are expressed at comparable levels
Sometime, one copy of a gene is expressed while the other is silent.
methylation is permanent
maintained across cellular generations. The hemimethylated DNA following DNA replication are restored by adding methyl groups to the newly synthesized DNA. |
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mcs5109 Thu, 20 Nov 2008 04:42:36 GMT |
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| 5-Met-cytosines |
methylated DNA |
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mcs5109 Thu, 20 Nov 2008 04:36:24 GMT |
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| MeCP2 |
-protein binds to methylated DNA
-recruit histone modifiers that completely turn off expression |
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mcs5109 Thu, 20 Nov 2008 04:36:24 GMT |
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| DNA methylation in mammalian cells |
Want to complete turn off gene
Methylated DNA (5-Met-cytosines) disrupts binding of the transcription machinery and activators
Methylated DNA can be bound by diff proteins (ex: MeCP2)
MeCP2 recruit histone modifiers that completely turn off expression |
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mcs5109 Thu, 20 Nov 2008 04:36:23 GMT |
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| Silencing in yeast |
SIR genes (Silent Information Regulator)
SIR genes (Silent Information Regulator)
Rap1 recruits Sir to the telemere
Sir2 (a part of Sir1) deacetylates the nearby nucleosomes
Unacetylated tails bind to Sir3 and Sir4
Binds many more Sir proteins
Spreading of silencing effect |
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mcs5109 Thu, 20 Nov 2008 04:31:22 GMT |
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| most common form of gene silencing |
heterochromatin
telomeres and centromeres |
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mcs5109 Thu, 20 Nov 2008 04:26:43 GMT |
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| Gene silencing |
-position effect
-not in response to a specific signal
-can switch off multiple genes
-most common form: heterochromatin
-telomeres and centromeres (typically composed of repetitive sequences and contain few protein coding genes)
If a gene is experimentally moved into these regions (e.g., by homologous recombination), that gene is typically switched off |
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mcs5109 Thu, 20 Nov 2008 04:26:43 GMT |
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| Euk transcription is regulated |
1) unmasking an activating region (23)
2) transport into and out of the nucleus (22) |
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mcs5109 Thu, 20 Nov 2008 04:23:24 GMT |
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| Signal cascade |
-cell surface receptor gets signal
-transfers to intermediate protein (e.g., kinase)
-transfer to other intermediate proteins to reach to the transcription factor for gene regulation |
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mcs5109 Thu, 20 Nov 2008 04:23:24 GMT |
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| most common way to repress the transcription in eukaryotes |
recruitment of histone modifier (remove acetyl group -> forming higher order chromatin structure) |
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mcs5109 Thu, 20 Nov 2008 04:23:24 GMT |
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| Euk repressors |
competition: Hides DNA sequence for activator binding
inhibition: Hides activating region
direct repression: Inhibit transcription initiation |
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mcs5109 Thu, 20 Nov 2008 04:23:24 GMT |
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| combinatorial control |
2 genes; each controlled by mult signals (4 in A, 3 in B)
Regulatory protein 3 acts at both genes |
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mcs5109 Thu, 20 Nov 2008 04:05:26 GMT |
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| Human b-Interferon Gene |
3 activators: (NFkB, IRF, Jun/ATF)
1 architechural DNA binding protein: (HMG)
-all bind to enhancer |
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mcs5109 Thu, 20 Nov 2008 04:05:26 GMT |
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| HO gene |
-SWI5 can bind to chromatin unaided
-SBF cannot
-SWI5recruits histone remodelling complexes
-alter nucelosome for SBF binding site |
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mcs5109 Thu, 20 Nov 2008 03:51:01 GMT |
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| Synergy |
-2 activators working together is much greater than 1 working alone
-critical for signal integration by activators
-ex: HO gene expression by SWI5 and SBF (mediator) |
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mcs5109 Thu, 20 Nov 2008 03:51:00 GMT |
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| Nucleosome Modifiers |
histone acetylase: modifies histone tails to alter packing of nucleosomes
chromatin remodeling complex: allows the transcription machinery accessing to the promoter |
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mcs5109 Thu, 20 Nov 2008 02:52:46 GMT |
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| Activator recruits Pol II indirectly |
1) Recruits trx machinery (TFIID complex/Mediator); usually recruited by >1 activator upstream
2) Recruits nucleosome modification factors; alter chromatin |
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mcs5109 Thu, 20 Nov 2008 02:52:46 GMT |
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| Pro-rich |
in mammalian
-weaker and work less universally than member of the acidic class |
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mcs5109 Thu, 20 Nov 2008 02:17:51 GMT |
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| Glu-rich |
in mammalian SP1
-weaker and work less universally than member of the acidic class |
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mcs5109 Thu, 20 Nov 2008 02:17:51 GMT |
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| acidic region in activating region |
preponderance acidic amino acids (Glu & Asp), yeast Gal4
-strong and work in any eukaryotic organism |
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mcs5109 Thu, 20 Nov 2008 02:17:51 GMT |
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| Activating regions |
-no well-defined structures
-some form A helices
-sticky surfaces
-small weak units; the more, the more, the stronger the resulting activating region.
-grouped by amino acid content:
1. Acidic region
2. Gln-rich
3. Pro-rich |
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mcs5109 Thu, 20 Nov 2008 02:17:51 GMT |
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| sticky surfaces |
-surfaces capable of interacting w/ several protein surface |
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mcs5109 Thu, 20 Nov 2008 02:17:51 GMT |
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| Basic zipper/HLH |
region of the a helix that binds DNA contains basic amino acids. |
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mcs5109 Thu, 20 Nov 2008 02:08:46 GMT |
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| Helix-Loop-Helix Proteins |
Dimer
2 helical regions: recognition A helix, flexible loop, shorter A helix |
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mcs5109 Thu, 20 Nov 2008 02:08:46 GMT |
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| Leucine Zipper Motif |
-2 long a helices grip DNA
-each a helix inserts into the major groove half a turn apart.
-Dimerization via coiled coil: 2 helices are held together by hydrophobic interactions between Leu
-homodimers and heterodimers |
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mcs5109 Thu, 20 Nov 2008 02:08:46 GMT |
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| Gal4 |
zinc cluster domain v |
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mcs5109 Thu, 20 Nov 2008 01:59:36 GMT |
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| TFIIIA |
Zinc finger protein |
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mcs5109 Thu, 20 Nov 2008 01:59:36 GMT |
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| Zinc Containing DNA-Binding Domains |
-Zinc finger protein (TFIIIA) and zinc cluster domain (Gal4)
-Zn atom interacts w/ Cys and His
-Zn serves a structural role in DNA-binding domain
-Recognition a helix inserted into the major groove.
>2 zinc finger linked: increases length DNA recognized |
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mcs5109 Thu, 20 Nov 2008 01:59:36 GMT |
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| homeodomain proteins |
-Helix-turn-helix DNA binding domain
-recognition helix for DNA
-discovered in Drosophila
-in all eukaryotes
-bind DNA as heterodimer |
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mcs5109 Thu, 20 Nov 2008 01:41:22 GMT |
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| recognition helix |
-mainly recognizes DNA sequences
-Discovered in Drosophila
-Found in all eukaryotes |
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mcs5109 Thu, 20 Nov 2008 01:41:22 GMT |
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| euk transcription factors |
-dimers
-recognize specific DNA using an a helix inserted into the major groove
-Some heterodimers
-Some are monomers |
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mcs5109 Thu, 20 Nov 2008 01:41:22 GMT |
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| bacterial transcription regulators |
-homordimers
-Each monomer inserts an a helix into DNA major groove
-Most use helix-turn-helix motif |
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mcs5109 Thu, 20 Nov 2008 01:41:22 GMT |
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| 2 Hybrid Assay |
-ID’s proteins that interact w/ each other in vivo
-Activation of gene depends protein A interacting w/ Protein B
-yeast
-finds B proteins that interact w/ a known starting protein (A: bait, B: prey) |
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mcs5109 Thu, 20 Nov 2008 01:31:20 GMT |
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| Domain swap experiment |
-Hybrid protein: (Gal4 activating region fused to the LexA DNA-binding domain)
-activated LexA DNA
-common
-Can be carried on separate polypeptides; form complex on DNA |
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mcs5109 Thu, 20 Nov 2008 01:27:07 GMT |
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| transcription factor binding sites differences in euk and pros |
-Euk transcription factors have multiple-binding sites
-only single-binding site in bacteria |
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mcs5109 Thu, 20 Nov 2008 01:27:07 GMT |
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| Gal 4 in yeast |
-binds to 4 sites upstream of Gal 1
-activates GAL1 1,000x in galactose
-Euk transcription factors have multiple-binding sites |
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mcs5109 Thu, 20 Nov 2008 01:27:07 GMT |
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| Euk activator |
-2 separate domains (DNA and activating) connected by flexible linker
-ex: Gal4 in yeast
-Euk transcription factors have multiple-binding sites |
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mcs5109 Thu, 20 Nov 2008 01:27:07 GMT |
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| Regulator binding sites |
individual regulatory protein binding site |
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mcs5109 Thu, 20 Nov 2008 01:15:14 GMT |
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| euk core promoter |
-transcriptional machinery(RNAP & GTF) binding site |
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mcs5109 Thu, 20 Nov 2008 01:15:14 GMT |
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