Flashcards in Chromatin and epigenetics Deck (91):
How can histones be removed?
Adding high concentration of salt (2M NaCl). Leaves the nuclear scaffold and DNA loops.
What is the structure of histones?
H3/H4 tetramer + 2 H2a/H2b dimers with DNA wrapped around makes a nucleosome
Histones are small, positively charged (for DNA binding) proteins. Each core histone dimer has 6 DNA binding surfaces that organise 3 DNA turns - the histone octamer organises 14bp of DNA in 1.75 helical turns of DNA.
N terminal and C terminal extensions can contact other nucleosomes (aids condensing of DNA) and be modified.
What is a chromatosome?
Core histone octamer plus one linker histone (e.g. H1) and 2 full turns of DNA. The linker histone tightens the angles of the entry/exit of the DNA
What is the nucleosome structure of chromatin fibres?
Positively charged N termini of histone binds DNA on neighbouring nucleosomes (and other histones)
High levels of histone H1
Solenoid structure (30nm fibre)
What is heterochromatin?
Generally repressive. DNA is highly condensed, inactive. Found near centromeres and telomeres. Replicates late.
What is euchromatin?
Contains active genes. Extended structure, is digested first by DNase, replicates early.
How can chromatin be derepressed?
Remodelling factors (ATP dependent)
Histone modifying enzymes
Facilitators of elongation
Note: not being activated, just being dereppresed. Chromatin environment can prevent transcription. In vitro, naked DNA + basal transcription factors and RNA pol II gives efficient transcription - chromatin represses this in vivo.
What is FACT?
Facilitates Chromatin Transcription. A protein that moves with the polymerase and loosens nucleosomes in front of it, replacing once transcription has occurred.
How was FACT identified?
Chromatin was assembled on a promoter template in vitro (forms a random array on template)
An activator was added to allow remodelling (positions histones for good transcription)
The remodelled template was purified by gel filtration
The components/nuclear were added back and checked for initiation and elongation.
Found that the purified system could initiate on a chromatin template, but pol II stalled early on. This was overcome by FACT, which doesn't require ATP.
How can histone modifications be studied?
ChIP-seq (chromatin immunoprecipitation). Live cells are fixed with formaldehyde and sonicated/digested.. Antibodies to histone modifications e.g. H3K9 pull down bits of DNA interested in, cross links are reversed and DNAD is sequenced and mapped.
What does acetylation of lysine on histones do?
Correlates with gene activity. Acetylation neutralises the positive charge on lysine, opening up chromatin into a relaxed state. Acetylated by HATs (contain bromodomains to bind acetylated histones), removed by HDACs. Acetylation also provides a binding platform.
What residues can be methylated on histones?
Lysine and arginines (and probably others). Can be mono, di or trimethylated.
What do methylation codes result in?
Di or trimethylation of H3K9 and trimethylation of H3K27 are inactivating
Di or trimethylation of H2K4 is activating
Can have 'poised' genes in some cells (e.g. ES cells) where there is H3K4me3 and H3K27me3 for rapid switching on/off of genes as cells change lineage.
What does scSet do?
scSet 1 binds phosphorylated serine 5 of the CTD of RNA pol II and trimethylates H3K4 during transcription (activates)
scSet 2 binds phosphorylated serine 2 of CTD and trimethylates H3K36
How can histone methylation be erased?
Amine oxidation - mono and di methylated lysine. Requires a protonated N in the substrate (so can't act on trimethylated)
Radical attack - probably for all methylated forms. Can remove me3, though not all enzymes of this class do this
Deimination - for monomethylated arginines. Causes formation of citrulline (a non-encoded amino acid). Unsure if this can be reversed.
How does LSD1 function?
A demethylase. Can be an activator or a repressor, depending on the complex formed. If in complex with Human Co-Rest, get H3K4 demethylation (repressing gene expression). If form a complex with Human AR get demethylation of H3K9 (activating gene expression)
How does heterochromatin form?
K9 is deacetylated, K4 is demethylated
K9 is methylated
HP1 binds via the chromodomain at K9me2/3. This brings in more proteins for heterochromatin formation.
How are histone modifying enzymes recruited?
Directly recognising a histone modification e.g. bromodomain recognising lysine acetylation
Specific transcription factor being required for recruitment and/or activity
Chromatin modifying enzymes interacting with RNA pol II complex
Non coding RNA recruiting histone modifying enzymes
How could lncRNAs affect transcription in cis?
Transcription in cis of lncRNAs displaces DNA bound factors that inhibit/activate transcription of a neighbouring gene
Nascent ncRNA transcripts tether chromatin modifying complexes/transcriptional regulators either activating or repressing transcription.
How could lncRNAs affect transcription in trans?
Trans-acting ncRNAs could be a platform for assembling protein complexes. Target sites are specified by DNA binding proteins
Trans ncRNAs specify binding sites by forming hybrids with complementary DNA sequences then recruit chromatin modifiers etc
lncRNAs could modulate the activity of protein complexes through conformational changes.
What is CLIP?
A method of identifying RNAs bound to proteins.
Isolate RNA-protein complexes with an antibody to RNA binding proteins. Digest ends and add alkaline phosphatase. Ligate 3' linkers and radioactively label. Resolve on a gel and digest bound protein. Ligate a 5' adapter and sequence (RT-PCR)
What is ChIRP?
Chromatin isolation by RNA purification. Like ChIP, but use biotinylated oligonucleotides that are complementary to ncRNAs for the pull down. Can identify ncRNA by RT-PCR, DNA sequence and RNA binding proteins (western blot/mass spec)
What factors can effect transgene expression?
Enhancers (up regulating expression/expressing in a location not intended)
Silencers (down regulation)
Heterochromatin (down regulation)
DNA methylation (down regulation)
What is position effect variegation?
A variegation caused by the inactivation of a gene in one cells through its abnormal juxtaposition with heterochromatin
What are enhancer elements?
Regions of DNA which contain binding sites for transcription factors to up regulate transcription of a gene. Are orientation independent, can be upstream or down store, proximal or distal. Transcription factors and coactivators (such as HATs) bind. Marked by histone modifications such as H3K4me1 and H3K27ac and enhancer RNAs.
What is the difference between typical enhancers and super enhancers?
Typical enhancers regulate housekeeping genes and have moderate levels of enhancer RNAs
Super enhancers regulate cell specific/inducible genes and have high levels of enhancer RNAs and H3K4me1, H3K27ac and a bromodomain protein BRD4 that brings in a kinase to phosphorylate RNA pol II for transcription
How do super enhancers activate?
Experiment with endothelial cells and TNF-alpha. When TNF-alpha stimulus is applied, de novo super enhancers form (detected by ChIP with BRD4) and other enhancers are de-comissioned to drive rapid inflammatory responses.
What is I-BET?
An inhibitor of super enhancers. Binds bromodomain on BRD4 and is in trials for some leukaemias.
What are silencers?
Can be short or long. Long DNA elements are associated with CpG silencing and telomeres in yeast. Cause formation of heterochromatin over many kilo bases.
What are classical silencers?
Orientation and position independent. Can be overcome by enhancers in certain tissues (e.g. h-thyrotropin-beta is normally silenced but switched on by an enhancer in the thyroid).
What are locus control regions?
Sections of DNA which controls expression of genes. They often have DNase hypersensitivity sites (HSs). At the human gene CD2, the HS region 3 in the locus control region is necessary to maintain open chromatin. Form a loop of transcriptional competence, anchored with CTCF and cohesin
What are HSs?
Hypersensitivity sites to DNAse. Have a core region of 200-300bp where they bind multiple transcription factors. Some act as enhancers but not all.
How is PEV involved in LCRs?
Position effect variegation can occur if regions of an LCR that open chromatin are deleted. This results in the random spreading of heterochromatin across the gene in some promoters but not all - stochastic silencing. If the gene is in euchromatin and regions of the LCR are deleted, there will be no effect.
How can genes be tested to find whether they contain an LCR?
Integrate the gene into a mouse. If it contains an LCR, there will be no position effect variegation, and expression will be copy number dependent - the LCR will open up chromatin in the areas that the gene integrates into heterochromatin if it is present.
How can areas of matrix or scaffold attachment regions be identified?
No DNAse hypersensitive sites
Do immunoprecipitation with nuclear scaffold and isolate DNA that remains associated
Find an AT rich sequence which has structural and functional roles.
What is the major scaffold attachment protein?
Sc1 - topoisomerase II, involved in supercoiling.
What are insulating elements?
Insulate genes from position effects e.g. protect from encroachment of heterochromatin
Insulate genes from enhancers without affecting basal activity
Include imprinting control regions
What does CTCF do?
A protein that acts at insulating elements to block enhancer action at imprinted genes. Binds where there is no DNA methylation.
What is Hi-C?
A method of looking at looping in chromatin. Cross link DNA, cut with restriction enzyme, fill ends and biotinylate. Ligate with a blunt end ligature, purify using the tag and there. Sequence.
What are TADs?
Topologically associating domains. Regions of the genome that have hing frequency of local interactions. Are separated by borders that limit interactions between adjacent TADs which have many architectural proteins (at TAD borders, not in the TADs)
What is the link between birth weight and metabolic syndromes?
The Hertfordshire data shows that the lighter the birth weight, the greater the risk of diabetes and impaired glucose tolerance. Odds ratio shows a large increase in risk. Also an increased risk of diabetes (and cardiovascular disease, insulin resistance and obesity) if there is a high birth weight due to maternal obesity and diabetes. A U shaped curve results.
What is the foetal insulin hypothesis?
Genetically determined insulin resistance or defects in insulin secretion result in impaired foetal growth (low levels of insulin-mediated growth) and susceptibility to diabetes in adulthood.
What is the programming hypothesis relating to birth weight and diabetes in adulthood?
A stimulus/insult applied at a critical or sensitive period of development results in long term or permanent changes in the structure and function of the organism. Poor maternal nutrition and/or placental dysfunction leads to foetal malnutrition and altered organ structure/function. When the foetus is born into conditions of adequate nutrition this leads to organ malfunction, type 2 diabetes and hypertension and metabolic syndromes later in life
What is the evidence from human studies for the foetal insulin hypothesis?
Individuals with mutations in the glucokinase (senses glucose levels in beta cells and stimulates insulin release) gene have a rare form of diabetes (monogenic) and a reduced birth weight (500g compared to siblings)
GWAS found 15% of birth weight variance was explained by foetal genetic variation - 9 of the loci found were also associated with type 2 diabetes. Effect on size is small (but many loci)
What is the evidence from human studies for intrauterine programming to link birth weight to diabetes in adulthood?
Looking at Danish twin registry - found differences in development of diabetes in monozygotic and dizygotic twins, suggesting an environmental factor.
Studied individuals who were exposed to the famine of the dutch hunger winter during gestation - in mid/late gestation offspring had reduced birth weight and poor glucose tolerance in adulthood
Look at siblings born pre and post maternal bariatric surgery - found lower glucose tolerance and higher body fat pre surgery. Suggests not just passing on obesity genes.
What are the structural effects of intrauterine programming relating to birth weight and diabetes in animal models?
Reduced beta cell mass - laid down in utero and fixed in number. Affected by reduced maternal protein
Reduced nephron number
Nature/number of neuronal projections - can't regulate energy balance and food intake
What are the mechanisms for intrauterine programming relating birth weight and diabetes?
Permanent structural changes of organs
Accelerated cellular ageing
Epigenetic programming of gene expression
What are the accelerated cellular ageing effects of intrauterine programming relating to birth weight and diabetes in animal models?
Telomere shortening (shorten duce to cell division and oxidative stress - islets in pancreas are susceptible to oxidative stress). More short telomeres are seen at 3 months in a mouse model in which the mother was malnourished.
More mRNAs associated with ageing are found in 3 month old mice with affected mothers
What are the effects of epigenetic transcriptional programming of gene expression in intrauterine programming relating to birth weight and diabetes in animal models?
Candidate genes are transcription factors - master regulators
Found changes in programming in the glucocorticoid receptor, PPARalpha, PDX1.
HNF4alpha: mutations in this are sufficient to cause diabetes; expressed early in life; islets from type 2 diabetics have 50% less HNF4alpha. Reduction in mRNA and protein in malnourished mothers, but only small difference in methylation in islets cells. In the enhancer region there are large differences in histone modifications resulting in lower interaction rates.
What are the effects of epigenetic post-transcriptional programming of gene expression in intrauterine programming relating to birth weight and diabetes in animal models?
Insulin signalling proteins such as IRS-1 and p110beta have lower expression levels in malnourished mothers (mouse model), although the mRNA level is the same. Suggested effect of miRNAs or other post transcriptional modifications.
How has the evidence from animal models for the effects of intrauterine programming relating birth weight to diabetes transferred over to human studies?
Biopsies of adipose tissue shows that there are reduced levels of insulin signalling proteins such as GLUT4, p85, p110beta and IRS-1. No reduction in the insulin receptor and others. Animal model is a good reflection.
Mechanisms of IRS-1 and p110beta appear to be post transcriptional - no difference in mRNA level.
What are the different cytosine modifications?
5mC - 5 methyl cytosine ('5th DNA base')
5hmC - 5 hydroxymethyl cytosine ('6th base')
5fC - 5 formyl cytosine ('7th base')
5caC - 5 carboxyl cytosine ('8th base')
How did DNA methylation evolve?
From prokaryotic restriction/modification systems against parasitic elements - methylate viral genome
Present in major eukaryotic groups (plants, animals, fungi)
Evolutionarily volatile - lost in C. elegans and some yeast.
What epigenetic phenomena are associated with DNA methylation?
Genomic stabilit and control of transposon activity
Cancer cell biology
Transcriptional regulation and long term cellular memory
What DNA sequences are found to be methylated?
Symmetric - CpG. Most abundant in mammals.
Asymmetric - non CpG e.g. CpA, CpT. Not maintained upon cell division; reestablished de novo by DNMT1
Where is DNA methylation found in the genome?
Majority of CpG pairs are methylated in mammals including gene bodies, endogenous repeats and transposons (to silence)
Methylated sequences are punctuated by non-methylated sequences called CpG islands. Overlap the promoter regions of many human genes
What are CpG islands?
Regions of non-methylated sequences with elevated GC content. Overlap promoter regions of 60-70% of human genes
How does DNA methylation degrade?
5mC deaminated to thymine spontaneously - results in under representation of CpG
How does DNA methylation aid X inactivation?
CpG islands are methylated de novo on the inactive X chromosome
How is IGF2 imprinting regulated through epigenetics?
IGF2 is paternally expressed (growth factor), the maternal chromosome expresses H19 - a ncRNA. A DMR (differentially methylated region) is methylated on the paternal chromosome to allow the enhancer to act on the IGF2 gene promoter, whilst the maternal chromosome is non-methylated. This allows binding of the CTCF insulator protein, causing the enhancer to not be able to 'reach' the promoter of the IGF2 gene, instead activating transcription of H19.
How is DNA methylation implicated in cancer cells?
Tumour suppressor genes are hypermethylated in cancer cells
What does DNMT1 do?
A maintenance methyl transferase. During semi-conservative DNA methylation, sees hemimethylated CpG islands and methylates the other strand to make symmetrical.
How does DNA methylation aid lineage determination in embryos?
In embryonic stem cells - Elf5 is hypermethylated in cells to become extra-embryonic tissue, whilst it is hypomethylated in embryonic tissue. When DNMT1 is deleted, Elf5 is active in the trophoblast
How is DNA demethylated in the blastocyst of mice?
Active: Rapid loss of DNA methylation in a single cell cycle suggests active removal of 5mC by enzymes
Passive: DNMT1 is excluded from the nucleus in the 1-8 cell stage resulting in passive loss of mCpG
What does Dnmt3 A/B do?
A DNA methyl transferase that is required for de novo methylation
What does Dnmt3 L do?
Doesn't have active methyltransferase activity, is a regulator of Dnmt3a and Dnmt3b. Required for establishment of maternal imprints in growing oocytes and establishment of methylation at retrotransposons in non-dividing prospermatogonia.
What is the effect of Dnmt1 knock out?
Reduction of CG methylation to 5-30% of wild type. Retrotransposon expression in reactivated. Defects in X inactivation and genomic imprinting. Lethal in embryogenesis
How does Dnmt1 maintain DNA methylation?
Is recruited by PCNA and Np95 during DNA replication to methylate hemimethylated daughter strands.
What is the effect of Dnmt3a and Dnmt3b knock out?
Double knock out is lethal in embryogenesis. Genome wide loss of DNA methylation. Point mutations in Dnmt3b cause the human disease ICF syndrome - an immune deficiency; no methylation at specific points e.g. satellite DNA
How does methylated DNA induce silencing?
Repulses transcription factors (masks binding sites)
Attracts repressors/repulsion of activators e.g. MeCP2 binds and recruits other repressors.
What is the MDB family?
A family of methyl-CpG binding protein. Includes MeCP2. Are members of multi protein complexes that function in transcriptional repression. Thought to associate with histone deacetylase activity and establish silent chromatin
What are the methods for active demethylation?
Enzymatic removal of the methyl group from 5mC
Base excision repair through direct excision of o5mC
Deamination of 5mC to T
Nucleotide excision repair
Radical S-adenosylmethionine based de-methylation
What does the TET protein do?
Oxidates 5mC to 5hmC, then 5hmC to 5fC, then 5fC to 5caC
What is the role of 5hmC in DNA demethylation?
5hmC is eventually converted to 5caC/5fC by TET proteins. Then TGD converts these to an abasic group which is then repaired to cytosine by base excision repair.
How is DNA methylation targeted?
Direct inhibition by intrinsic sequence properties
Targeted by a DNA demethylatioon mechanism
Transcription factors preventing binding of Dnmt's; Dnmt3's can't bind H3K4me3 marked chromatin
How are transcriptionally active sites protected from DNA methylation?
The CpG islands are protected by transcription factors, nucleosome exclusion, H3K4 methyltransferases, DNA-nascent RNA helices inducing R loops of ssDNA, enzymes associated with DNA demethylation
How are promoter regions silenced?
Repressive transcription factors recruit chromatin remodeller LSH, linker histone H1, heterochromatin protein HP1, H3K9 methyltransfeases and de novo methyltransferases, often in that order
How can DNA methylation be mapped?
Bisulfite treatment: Treating DNA with bisulfite changes unmethylated C's to U. Then look for a C-> T transition after PCR amplification. however, can't distinguish between 5hmC and 5mC.
Can also do immunoprecipitation followed by arrays
What is the evidence for 5mCpG being a cause of transcriptional silencing?
In vitro, methylation reduces promoter activity
Demethylation (by inhibiting methylators) increases expression of a reporter gene
Does promoter methylation always correlate with gene silencing?
No. At CpG poor promoters, methylation still allows transcription.
How does the environment influence epigenetics?
Signalling pathways, involving DNA methylation, histone modification and chromatin remodelling to change the phenotype.
Examples are vernalisation in plants (epigenetic responses to cold and long term memory), royal jelly in honey bees (nutrition controls reproductive status by DNA methylation) and epigenetic studies in monozygous twins (shows diverging disease susceptibility)
What is the genetic evidence from mChr2 for imprinting?
Mice with a paternal duplication have broad flat backs and are hyperkinetic
Mice with a maternal duplication have narrow bodies and fail to suckle and are hypo kinetic
Indicates imprinted genes on the disomic segment
How were imprinted genes mapped?
By analysing uniparental disomies for most chromosomes
How was Igf2 imprinting discovered?
Paternal knock out and double knock out had the same phenotype, whilst a maternal knock out had a normal birth weight. Suggested gene was expressed from paternal chromosome only.
What is the lifecycle of an imprinted genes?
Imprinting is established in gamete formation, maintained through embryogenesis (survives epigenetic reprogramming) and then read in life. In the gonads, imprinting is erased then reestablished to start over again
How is imprinting maintained through early development?
Paternal allele is protected against de novo methylation though H3K4me3 markers
Maternal allele maintains DNA methylation through imprint specific factor Zfp57 - a zinc finger protein that binds to imprint control regions and recruits Dnmt1 and other methylators.
How can viable mice be generated from bi-maternal embryos?
Engineer one of the chromosomes to imprinting control regions to prevent repression.
What molecular changes cause human imprinting disorders?
Chromosomal rearrangements (deletions, duplications, translocations)
Epimutations (aberrant methylation of a differentially methylated region without alteration of the genomic DNA sequence. Could be primary - no DNA sequence change or secondary - occurs as a result of a DNA mutation acting in cis or trans)
What does the imprinting control region ICR1 do?
At the Igf2/H19 imprinting region. Hypomethylation of ICR1 on the maternal chromosome allows binding of CTCF which is a barrier to enhancer looping, thus preventing production of the Igf2 gene, instead inducing expression of the H19 ncRNA
Structure of the gene region:
Igf2 gene --- ICR1 --- H19 --- enhancer x2
What do disturbances in Igf2 imprinting result in?
Over expression of Igf2 - beckwith-Wiedemann syndrome. Over growth. ICR is methylated on maternal chromosome preventing CTCF binding and allowing enhancer looping
Under expression of Igf2 - Silver-Russel syndrome. ICR is hypomethylated on paternal chromosome, allowing CTCF binding and preventing enhancer looping