Lec 4: overview of epigenetic mechanisms of gene regulation Flashcards
Gene X environment =
phenotype
X= chromatin
what did Conrad Hal Waddington do??
coined the term epigenetics to mean above or in addition to genetics to explain differentiation
- wondering how to get multiple cells from single cells and get differences
- interested in local-environmental factors and how impact cells that had potential to grow
how do diff adult stem cells know their fate?? (waddington)
- Myoblasts can only form muscle cells
- Keratinocytes only form skin cells
- Hematopoetic cells only become blood cells
But all have identical DNA sequences.
what is epigenetics?
describes phenomena in which genetically identical cells or organisms express their genomes differently, causing phenotypic differences.
- Information coded beyond the DNA sequence (e.g. covalent modifications to the DNA or modifications to the chromatin structure)
- the study of HERITABLE changes in gene expression or cellular phenotype caused by mechanisms other than changes in the underlying DNA sequence
what is epigenomics?
the sum of genome-wide epigenetic patterns, distinguishes and defines one tissue from another, stem cells from somatic cells, and aged from young cells’
- multiple targets
- look for changes inherited across and within organisms
- e.g. studies of psychiatric patients
genetic vs epigenetics
genetics
- mutations
- static (genes)
- genotype
- germ cell inheritance
epigenetics
- alterations
- dynamic (chromatin)
- phenotype
- somatic epigenetic inheritance
describe pathway for environmental gene programming
- (extracellular) epigenator = differentiation signals, temp variations
- GF vind to rec, activate cell signalling pathways, result in periphery activation in cell= cellular response in cytoplasm which enter nucleus and recruit epigenetic modifying agents
- epigenetic initation= DNA binding factor, non-coding RNA
- epigentic maintainer= hostone/DNA modifiers, histone variants
cells are genetically _____ and epigenetically ____
genetically identical
epigenetically variable
- body generates diff cell types into diff tissues and express diff proteins (epigenetically variable)
- variations from RNA interference, histone modification, DNA methylatin
what are histone modifications?
- covalent attachments to AA of histones (using side chain groups)
- affect the affinity (binding) of histones to both DNA and to other proteins which can interact with histones.
- affect nucleosome and higher order DNA structures.
- predominately added to the extended
histone tail structures.
histone modifications are ____ - ____ modifications
post-translational
name 4 types of histone modifications
1) acetylation (lysine)
2) methylation (lysine, arginine)
3) phosphoyrlation (serine, threonine)
4) ubiquitination (lysine)
- others= sumoylation (lys), glycosylation, biotinylation
evidence of the histone code
- Specific modifications correlate with specific processes.
- Specific modification patterns and sequences.
- Interactions of specific modified residues with classes of protein domains.
epigenetic code includes 4 characteristics
- Stable (not transient) through the cell cycle
- Heritable (through cell generations)
- Consistent (always assoc. with defines chromatin behaviour)
- Predictive (i.e., upon discovery of a new modification)
what is ChIP?
chromatin immunoprecipitation
function of ChIP?
- evaluate change in histone modification and levels of histone phosphorylation, methylation or Tc binding to region of interest on gene
- distinguish protein interacting with DNA
7 steps to ChIP
1) cross link protein to DNA in living cells with formadehyde
2) break open cells, shear DNA
3) add pre-blocked protein G Agarose Beads (charged)
4) add primary antibody of interest
5) immunoprecipitate to ENRICH for fragments bound by protein of interest
6) reverse cross-link, treat with proteinase K
7) detect and quantify precipitate DNA through PCR and hybridization methods
what is PCR?
enrichment
amplify for specific region of DNA
what s array based ChIP on chip?
- looking across genome
- on array have tag
- take cells, fix them, do immuno-precipitation, wash to remove unbound non-specific and compare
- antibody is specific to one type of histone modification
- can label sequences using colour/radioactivity and hybridize against micro-array
more sequences= ore intense colour at specific location (DNA)
localized: gene or region-specific histone modifications (3)
1) Transcription= H3-K14ac; H3-K4me
2) silencing= H3-K9me; H4-
K20me
3)DNA repair H2Ag-S129ph
broad: regional or genomic histone modifications (3)
1) chromosome condensation= H3-S10ph
2) apoptosis= H2B-S14ph
3) DNA repair = core histone SUMO
methylation is involved in….
- where does it occur?
involved in activation, depending on modification and repression of histones
- occur more than once at lysine sites
H3K4me3= promoter, activation
acetylation is mainly involved in what process??
activation
nucleosomes in promoter region
AcH3/H4= gene activation
name 2 enzymes that catalyze histone tail modification
HDAC
HAT
what is HDAC?
histone deaceylases
function of HDAC
- Family of 11 enzymes -
- remove acetyl groups from lysine amino
acid on histone proteins, - allowing the histones to wrap the DNA more tightly.
- Classified into 4 classes;
- Part of multi-protein complexes, e.g., Sin3a, NuRD; associated with transcriptional repression.
HDAC recruited how
Recruited to their target promoters through a physical interaction with a sequence- specific transcription factor.
what is HAT?
histone acetyltransferases
what is function of HAT?
- Enzymes that acetylate conserved lysine amino acids on histone proteins.
- Acetylation of lysine neutralizes the positively-charge histone, reducing the affinity between the negatively charged DNA, which renders DNA more accessible to transcription factors.
- Divided into 5 families; e.g., CREB-binding protein (CBP).
- Part of multi-protein complexes, e.g., TFTC complexes; associated with transcriptional activation.
how is HAT recruited?
Recruited to their target promoters through a physical interaction with a sequence- specific transcription factor.
mechanism of histone acetylation
- repressing factor (RF) recruit machienry/HDAC to condense chromatin and maintain heterochromatin
- activating transcription factor= HAT, maintain euchroamtin acetylated nucleosome
- pos charge becomes neutral
- histone tail will relax, DNA will become available to Tc and can unwind
in condense solenoid (chromatin), what is function of nucleosomes?
block binding of RNA polymerase II to promoter
what happens when acetyl groups are added to histone tails?
remodel the solenoid so DNA is accessible for transciption
which factors affect the activation of histone acetylation??
- dietary factors (e.g. alcohol, caffeine, calorie restriction, high-Na)
- drugs (HDAC, SIRT inhibitor)
- metabolism: inflammation, immune activity, infection, hypoia, UV light, smoking, exercise, glucose
which drug is an HDAC inhibitor?
valproate
which factors affect inhibiton of histone acetylation
- dietary factors (biotin, niacin, garcinol, copper, chronium, nickel)
- drugs (HAT inhibitor= promoter deacetylation, acetyl-coA analog)
- metabolism- estrogens, high glucose
