12.2 Cell Nucleus and Gene Expression Flashcards
(PEPCK) gene
- Gluconeogenic enzyme
- Converts pyruvate to glucose
- Enzyme synthesized when glucose levels are low
- Gluc low so up regulate guconeogesis need to up regulate PEPCK
Regulation of gluconeogesis includes 3
- DNA regulatory sequences
- Specific transcription factors
- Signal transduction pathway that activates expression (e.g. hormones)
PEPCK Regulatory Region
-GRE = glucocorticoid response element- binds to glucocorticoid receptor
-TRE = thyryoid receptor element – binds to thyroid hormone receptor
-IRE = insulin receptor element- binds insulin hormone
-TATA is a general binding site, then the rest are very specific
multiple binding site and TF
responce element = DNA sewuence
Deletion Analysis/Mapping
Identifies regions of gene responsible for binding transcription factors
if you cut out 75-40, doesn’t do much at all
But if you cut out some or all of the TATA, GC box and CAAT box it does go dow to 14 or 50% so these regions are NB
now #6 if you cut it out it goes up? That’s odd. Need to get away from the idea that DNA is a linear molecule. If you cut this out you effect the architecture and you make it more able to bind other TF since you altered the architecture, you increased the activity.
Cis-regulatory elements
cis = on same stretch on DNA, on same as DNA
trans regulatory elements
trans = included TF. Proteins that come from somewhere else and binds to the DNA
DNA Footprinting Assay
Transcription factors binds to DNA protect DNA from digestion by nucleases (creates footprint)
- Identifies regions of gene responsible for binding transcription factors
- DNA can be isolated from protein and sequenced
-foot printing means it]’ll leave a footprint of the sequence.
-allow to bind. then hit with DNase1, it’ll randomly cut in into little fragments, but some areas will bounce off and can’t get access to the sequence.
-DNA can be used to “fish” transcription factor out of nuclear extract so that protein can be characterized.
-no fragments in the middle makes a footprint because the proteins binds to that area
only fragments you see are the ones with the end labels
Genome Wide Location Analysis
-This can get you much more info: more modern
1.
treat with formaldehyde: holds then in place, binds them there
-Then isolate the chromatin (DNA + protein) then cut them into fragments
-the dots/squares are TF inducing trascipton in dif areas of the DNA
-Which genes are turned on in what conditions?
2. -What genes turned on by the triangle? need antibody for TF (the Y) this is immunopercipitation
3A. antibody treatment includes precipitation of chromatin fragments containing bound TF
3b. while leaving all unbound chromatid frag in solution
4. reverse cross links and purify DNA
5. Where in the genome are these TB binding sites
5a. sequence DNA ChIP-seq
5b. amplify DNA fragments with floresently labeled nucleotides. can amplify with nuclotides, denature then and hybridize them to microarray for intergeneric DNA (promoter regions)
6. hybridize to DNA microarray containing intergentic regions
7. spot containing known intergentic region
Activation by Steroid Hormone
1) Hormone enters cell from extracellular fluid
2) Diffuses through cytoplasm
3) Hormone binds to glucocorticoid receptor protein
4) changes conformation and translocate to nuc where it acts as TF and binds glucocorticoid receptor element of the DNA
5) Glucocorticoid receptor binds to GRE which activates trasciption of DNA
6) lwading to syntheses of specific proteins in cytoplasm
–upregulate gluctoneogenic pathway for more glucose and you get more cortisol which can easily go across PM into cytoplasm onto glucocorticoid receptor, into nuc, and dimerized in nun and acts as TF and binds to respocne element
2 classes of transciptional Activators
- Interact with basal transcription machinery
- Act on chromatin to change state
- Remodeling complex, Histone modification complex
Histone Modifications
- epigenetic mod need a chemical mod: acetylation or methylation
- TSS= trasciption start site-Acetylation localized primarily in the promoter region of active genes
- H3K36 methylation occurs mostly in the transcribed region
- Methylated HK36 recruits Rpd3 enzyme that deacetylates lysine residues
- Deacetylated lysine residues prevents initiation of transcription within coding region
Transcriptional Activator (via acetylation of histones) 5 steps
- DNA in repressed state (deacetylated)
- GR binds and recruits CPB coactivator (histone acetyltransferase) - Lysine residues acetylated
- Acetylated histones recruit SW1/SNF remodeling complex
- TFIID binds to open region of DNA- TAF 250 and TBP acetylate additional nucleosomes
- RNA Pol II binds to promoter
4 alternative actions of Chromatin Remodeling Complex SW1/SNF
- remodelling complex remodel the nucleotide core so that it’s accessible to the RNA pol?
- at step some RNApol can’t gain access to TATA because DNA is wound around Nucleosome core.
1) Sliding exposes TATA site
2) Reorganization of histone octamer provides access to promoter
3) Histone variants exchanged for H2A/H2B
4) Histone octamer disassembled
Nucleosomal Landscape of
Yeast Genes
- 5’ end of gene has highly defined chromatin structure
- Nucleosome free region (NFR) at transcription start site
- -1 nucleosome undergoes most extensive modifications upon transcriptional activation
Paused Polymerases
RNA polymerase II held downstream of promoter
Held in paused state by inhibitory factors DSIF and NELF (sitting on gene but not trascribining)
Inhibition relieved by
Phosphorylation
Elongation factors (ELL). the pausing can be relived and pol activated by specific activation (kinease) and by elongation factors
Transcription factors may act at level of transcription elongation
Permits rapid activation of genes
Transcriptional Repression
-Histone methyltransferase methylates histones
Lys 9 of histone H3
-binds to core repressor which recites HDAC which starts deacetylation then that’s followed by methylation which makes it inactive (inducees heterchromatization)
-loss of acetyl group and addition of a methyl lead to chromatin inactivation and gene silencing
DNA Methylation: Overview and low it changes with age
-Epigenetic control of gene expression by methylation of promoters
-Maintains inactive state
-Methylation is reversible
-Methylation at “CG rich islands” (symmetrical recognition site)
-methylation is mark of inactivation
fully differentiated cells are highly methylated
-Methylation levels change during development
-Blastocyst has low level of methylation
-Adult somatic cells highly methylated
