Gene Expression Regulation and Epigenetics: Repressors, Silencers, and RNA Interference, Summaries of Genetics

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B. Pierce,

Genetics: lecture notes

Gene Regulation^ II-^ Eukaryotes^ (Chapter^ 17)

• Basal transcription factors help RNA polymerase bind to promoter o Capable of minimal transcription I. Transcriptional Regulators (1st^ level of control) A. General properties o Combination of DNA-binding to specific site and activation activity o Activators- o work on local promoters to increase transcription; o 2 functional domains

1. DNA binding - bring to correct promoter
2. Transcriptional activation: interact with Basal factor like TFIID, or chromatin modification factors to activate transcription o Enhancers - o Can act at long distance (Kbp) and in any orientation-chromatin structure ▪ Position is not fixed ▪ Orientation is not fixed ▪ Can affect whole region or parts of looped chromatin where they meet ▪ Will enhance transcription of ANY adjacent gene over many thousands of bp (kb) → either stimulate or turn off basal promoter

• Example 1 : limb enhance element for sonic hedgehog (shh) gene → mutation in enhance alter expression of Shh gene ( take away enhance will turn off the gene for limb development)
• Example 2: Enhancer can be so far away that they are located in a neighboring gene. (mutation in intron of FTO gene are associated with obesity in humans because the intron of ETO contained an enhance that controlled IRX3 & IRX5 genes that are responsible for fat mutation) o Insulator - isolate the action of enhancer to a certain direction. o Repressors/Silencers o turn down gene expression at a promoter near where it binds o Different ways ▪ Actively push away RNA polymerase ▪ can act through antagonism with an activator, or independently → compete with activators for binding to a DNA site

C. Eukaryotic lessons

1. Use of the nucleus vs cytoplasm is often used in a switch in signaling
2. Multiple proteins interact - provides flexibility with shared proteins and evolutionary pathways to recruit signaling proteins
3. Promoter and enhancer elements usually bind multiple tissue-specific elements/transcription factors

• Allow promoters to integrate multiple signals;
• promoter recruit different enhancers to turn on different expression
• multiple proteins responding to different signals – bind one region – cooperative action. Recap: o Eukaryotes generally do not have operons o Both use repressors and activators, but activator very common in eukaryotes o Chromatin plays a role in control II. Chromatin remodeling (2nd^ level of control) - control of gene expression – targets of activation domains o How well they are packed together A. Nucleosome repositioning - moving nucleosomes away from promoter or binding site (like TATA box) by using transcription factor to recruits SWI/SNF nucleosome repositioning factor that moves nucleosomes down DNA so promoter DNA is exposed for transcription.

1. SWI/SNF is similar to a helicase- creates torsional stress in DNA that moves nucleosomes
2. Bound transcription factor can attract SWI/SNF to reposition neighboring nucleosomes → pushing them off promoter elements to expose bare DNA for TFIID → makes an open area of DNA that is DNAseI hypersensitive (book talks about)- a method to detect effect of opening chromatin structure. B. Histone Variant substitution

1. different versions of certain histone proteins can be substituted into a nucleosome, making that nucleosome easier to move - marks gene as easily turned on
2. Nucleosomes with histone 3.3 or histone 2B.z instead of histone 3 found near promoters of active genes will make nucleosomes with these variant looser. More easily displaced. C. Histone acetylation/deacetylation - from CH3CO- to lys and arg residues on histone tails → positive charged aa in tail domain, repulsion and weakens interaction between DNA and histone. o Specific transcription factor bind to the tightly packed DNA and recruit HAT to loosen the chromatin so TFIID can bind to TATA box.
3. Acetylation of histone tails loosen DNA binding- associated with activated gene HAT - histone acetyl transferase adds acetyl group to histones to loosen the packing and turn on gene expression. o Certain transcription factors may have acetyl transferase activity and act on surrounding nucelosomes. o Other transcription factors can bind to and recruit HAT to surrounding nucleosomes.
4. Deacetylation of histone tails tightens interaction-associated with silenced gene HDAC - histone deacetylase removes acetyl group and turn off a gene o methylation of deacetylated histones seals in silencing/compaction → really turning off the gene A drug that inhibits histone deacetylase would most likely have which effect? Answer: increase transcription
5. activators or repressors can recruit HAT or HDAC, or have activity e.g. Myc/ Max/ Mad proteins - regulate cell proliferation MAX itself can bind as homodimer (a type of complex) MAX/MAX (neutral) but binds with others as heterodimers; it can also just bind to DNA MYC/MAX heterodimer recruits HAT to activate cell proliferation genes → increase transcription: so MYC can bind MAX and HAT MAD/MAX heterodimer recruits HDAC to silence same genes → decrease transcription: so MAD can bind MAX and HDAC III. Epigenetics (3rd^ level of control) - the study of changes in organisms caused by modification of gene expression rather than alteration of the genetic code itself. - control of gene expression past just DNA sequence

• long-term silencing or activation of genes due to the combination of nucleosome packing and DNA methylation tags that keep signal to silence → silencing can be preserved through cell generations without constant control by transcription factors. Silenced genes remain intact but inactive

o No maintenance methylase will stop development Wipe of most DNA methylation B. Levels which silencing are applied to genes:

1. Turn off genes that no longer need to be expressed in differentiated cells a. Repetitive DNA satellites and transposable elements in heterochromatin b. Tissue specific genes in the “wrong” tissue turned off during development “cellular memory” c. X inactivation – Xi is heavily methylated and compacted in inactive region (methylation of DNA and histone modification).
2. Long term silencing to “regulate” genes Epigenetics as regulation of “normal” genes in tissue where they can be expressed Regulation that can last long term e.g. licking by mother rat controls glucocorticoid receptor gene expression sets up expression of gene over lifetime of baby rat Transgenerational silencing - passed through generations? Answer: it’s long term regulation but hard to study transgenerational silencing.

• Lots of examples in plants – epigenetic pass through generations
• Studies point to long-term nutritional control of gene epigenetic tags in mice: Agouti mouse model: retrotransposon sensitive to methylation changes; phenotype of mother influences mix of progeny (genetically identical). The diet of the mother effects level of methylation and differ in phenotypes. If it is coming from the father then it is transgenerational. (why?)

- Monozygotic (Identical) twins start with a lot of same methylation, as they get older, they regulate their genes differently from environmental signals and the levels of methylation differ. 3. Genomic imprinting o An epigenetic control through generations based upon sex of the parent. o silencing tags from parent passed to progeny, but differ in mother vs father o gene present but not expressed depending upon maternal or paternal homologous chromosome o seen as genes of specific sex parent is turned off in progeny ~80 genes differentially silenced – father’s copy or mother’s copy o Epigeneic control by DNA methylation in gamete o A gene is “marked” by DNA methylation so that the gene is silenced in the gamete while still in the parent o The gene retains the DNA methylation so that it continues to be silenced in the resulting child in all their somatic cells. o Each parent silences an imprinted gene differently o That gene is silenced only in the gamete from the parent of one sex. The same gene inherited from the parent of the other sex is not silenced. Produced one expressed copy of the gene in the child, although they are carrying both copies. When the child make gamete, they reprogram the imprinting (old imprints erased and make new imprints) o See deviations from Mendelian expectations: parent of origin pedigree o Gene is methylated and silenced for one parent – not expressed o Gene is active in chromosome inherited from other parent o Example: IGF2 – insulin like growth factor-2: stimulate growth of fetus o Father’s copy expressed and mother’s not ▪ If both are expressed, fetus tissues overgrow, can induce cancer ▪ If neither expressed, retarded growth of fetus and baby ▪ rIGF2 IGF receptor: soaks up IGF2 and prevents its growth stimulatory action, mother’s copy expressed, father’s copy is not. e.g Prader Willi vs Angelman syndromes - 5 adjacent genes ZNF127, NDN, SNPRN, IPW and UBE3a (do not need to know the name of genes!) Mother’s copy of ZNF127, NDN, SNPRN and and IPW are inactivated (imprinted) and UBE3 is expressed Father’s copy of UBE3a is inactivated (imprinted) and other 4 expressed Fine if normal diploid- all are expressed However small deletion in region on chromosome 15 for one parent exposes other parent’s silenced gene function - deletion in father’s chrom 15 exposes silenced ZNF127 🠚 IPW in mothers’ chromosome (only the paternal activated gene is expressed) → Prader Willi’s syndrome obesity and hypogonadism

B. Antisense RNA (sRNA) (bacteria) o Different gene makes a small RNA (sRNA) complementary to part of target mRNA. Small RNA is acting in trans o Transcription occurs, but antisense RNA binds to mRNA (base-pair with SD so SD cannot bind with ribosome) and translation is blocked.

• gene 1 produces an mRNA (e.g. OmpF) that is structural gene
• gene 2 produces a short RNA (sRNA) that is complementary to gene 1 mRNA (e.g. micF)
  • base-paring of RNA 2 to RNA 1 turns off gene 1’s translation by covering SD/AUG
  • regulation of gene 1 mRNA is by promoter on gene 2 (e.g. high osmolarity (need to preserve water) turns on micF transcription, turning off OmpF to prevent water loss → OmpF translation is regulated by MicF) - acts in trans since sRNA is diffusible factor Regulation of OmpF and ompC genes in E.coli outer membrance protein

(also hiding the sequences around the SD to prevent binding with all the SD sequences and shutting down all the expressions - → more specific) C. RNA silencing/ RNA interference (RNAi) – eukaryotes

1. RNA interference (RNAi ) o double-stranded (ds) RNA is recognized and used to degrade or block transcription or translation of other RNAs that share the same sequence o 100 nucleotide pairs of dsRNA triggers degradation of RNA transcripts containing matching sequences. o Target mRNA expression silenced: o Inhibition of transcription o Inhibition of translation o Degradation of mRNA -- dsRNA sources a. long dsRNA →RNA virus, antisense promoters siRNA - small interfering RNA - a synthetic RNA designed to specifically target a particular mRNA for degradation. b. another gene that produces a hairpin dsRNA- microRNA (miRNA) - sequence within the hairpin matches sequence in another mRNA - expression of miRNA will suppress expression of the matching mRNA - acts in trans- dsRNA is diffusible factor
2. dsRNA recognized by Dicer , cuts it into pieces 22 nt long
3. RISC binds these small pieces and uses them to hunt down matching sequence: a. perfect match to mRNA, RISC will cut up mRNA → degradation b. near-perfect match to mRNA, RISC will block translation of mRNA c. for siRNA , related protein (RITS) will find match in DNA and induce methylation and silences gene

5. Uses in cell: a. viral defense- particularly in plants b. normal silencing and heterochromatin formation in retrotransposons, satellite repeats at centromeres c. Use of miRNA in one gene for regulating another target gene

• often regulate mRNAs encoding transcription factors-miRNA would turn off target
• expression of miRNA used to fine-tune where or when transcription factor acts
• see the function of an miRNA when you mutate the miRNA gene, or change the target mRNA so it no longer matches the miRNA

6. Related RNA regulation – lncRNAs – long non-coding RNAs have more varied ways of interacting with mRNAs, miRNAs, and proteins to regulate genes sRNA (^) Single Stranded -- Blocks translation by binding to target mRNA Trans RNAi Double stranded (hairpin) miRNA (comes form organism) =Dicer+RISC siRNA (from retro transposon etc) = Dicer+RISC/RITS Trans

(RITS - > DNA-> blocks translation) (RISC - > mRNA - > blocks transcription) Riboswitch (^) Changes shape of mRNA Blocks translation Cis

DNA mutation and Repair

I. Mutation

• changes in DNA
• different levels ▪ Small change in DNA sequence ▪ Chromosomal rearrangements ▪ Source of loss of information ▪ Source of genetic variation

1. Germ line heritable/ somatic mutations just affect individual a. Heritability depends upon which cell is mutated. Somatic cell mutation (won’t pass to next generation; often mosaic) vs. germ-line mutation (earlier in development and pass through next generation). b. Somatic mutations i. One specific cell mutates and its mitotic cells have the same mutation too when replicated but the others are normal → mosaic. c. Germ-line mutations i. Animals set aside germ line early, plants do not. B. Mode of change in DNA (types of mutations and Effect on DNA molecule) 1. base substitution - A base is replaced by one of the other three bases

• transversion (purine ↔ pyrimidine), transition (within same type) → most common

2. insertion or deletion

• an extra base(s) is inserted or removed C. Effect on protein encoded in genes/exons Substitutions - depends upon genetic code. Effects of substitutions in exons on protein:

1. Silent (synonymous)- same amino acid coded (e.g. third position in triplet)
2. Missense - (nonsynonymous) changes the amino acid encoded by the codon
3. Nonsense - converts amino acid codon to a stop codon → prematured stop codon inserted

• Mutations affecting phenotype occur very rarely o Many mutations occur in noncoding regions, introns, between genes, repetitive DNA
• Many mutations even in genes have no effect on protein function. F. Causes of mutation 1. Spontaneous mutation form. a. misincorporated base during DNA replication
• wobble base pairing or tautomers of base inserts wrong base
• tautomeriz changes the shape and base pair differently than the original
• DNA editing corrects ~99% of mistakes during replication
• after proofreading: <1 error in ~1,000,000 bases… so ~ 1 misincorporation in 5 - 10 million bases. - strand slippage gives insertion or deletion e.g. microsatellite repeat - would leave mispaired DNA - locations of microsatellite repeats occurring within genes where expansion of the repeat causes genetic diseases because misregulate receptor. b. inaccurate crossing over between homologous chromosomes during meiosis
• symnaponial complex causes unequal crossing over c. Chemical Instability of DNA bases i). Depurination – loss of purine (A or G) base from sugar through hydrolysis - leaves apurinic site. After the base is lost and not repaired before replication, it will cause mutation. In bacteria, they put a A in the apurinic site and continue replicating which lead to different strand than normal. ii) Deamination of bases - NH 2 group in the base is converted to =O , can caused by weak acids or oxidants in cell - leaves mispaired base pair (constantly happening) C → U (U can be fixed because it shouldn’t be in DNA) or Cmethyl^ → T most common → thymine should be in DNA so it is hard to fix. (if they can be fixed they won’t cause mutations!)

2. Induced mutation – chemical or physical cause of DNA change a. Intercalators- insert in DNA, cause insertions or deletions

• A type of molecule that can fit between bases and distort the helix of DNA. Can stop DNA replication because polymerase can’t get pass.
• Example: aflatoxin – most potent natural mutagen made by Aspergillus fungus and can be activated by the liver to be intercalated in DNA.

b. Ionizing radiation- causes strand breakage and base oxidation 8 - oxo-G oxidized base from Guanine can base pair with C and A →cause transversion mutation. If not fixed before mitosis, it will change a GC base pair to a AT base pair later. Base excision repair will fix this. c. UV light- causes thymine dimers

• Forms bond between the two Ts on the same strand → stuck together, DNA polymerase cannot pass through. (need to fix before DNA goes into replication!) II. DNA Repair A. Proofreading during DNA replication – catches 99% of initial mutations B. Mismatch repair – mop up of DNA polymerase mistakes
• Repair soon after DNA replication to catch substitution errors.
• Recognizes old vs. new strand to repair NEW strand.
• Right after DNA replication, the new strand is not methylated, so mismatch correction proteins can recognize the unmethylated new strand and fix DNA polymerase mistakes Bacteria: mutS scans DNA for lesion by bending and testing minor groove mutation removed by removing length of one strand of DNA strand removed determined by DNA methylation- chooses the recently synthesized strand Eukaryotes/Humans: MSH2 is like MutS. Mutation in one allele leads to HNPCC cancer **C. Excision repair

1. Base excision repair**

• Specific enzyme recognizes odd base (e.g. U, oxo-G) or mispaired G-T basepair
• enzyme removes that one base (break base-sugar bond), then sugar, and allows DNA polymerase to repair
• Different protein search for and repair specific bad bases (takes out only the altered base)
• Uracil from T to U deamination – takes out U in G-U pair and replaces with C.
• Thymine – takes out T in a T-G pair and puts in a C (methyl-C to T is a common deamination)
• Can fix deamination (cause) 2. Nucleotide excision repair
• recognizes distortion in DNA, e.g. thymine dimers or modified bases

• odd numbers- 3n, 5n, 7n are infertile (one chromosome can’t be paired, so meiosis will fail →seedless)

2. In higher animals, often results in spontaneous abortion 3n about 15 - 18%, 4n about 5% of spontaneous abortions in humans Allopolyploidy - multiple genomes from different species (hybrid) hybrid is generally unable to produce a fertile offspring unless chromosome # and organization is the same in both species

• otherwise, needs to duplicate both sets of chromosomes to become fertile

1. animals- e.g. horse x donkey = mule
2. more common in plants e.g. bread wheat has 3 genomes II. Aneuploidy - loss of subset of chromosomes

• due to nondisjunction or loss of centromere A. humans- range of consequences
• X or Y viable but often sterile
• 21 Downs syndrome, 13 & 18 die within weeks of birth
• 16 & 22 in spontaneous abortions
• acrocentric chromosomes 14 &15 in eggs and pre-implant fetus
• increased miscarriage with maternal age is due to trisomy.
• male – 10x more substitution mutations increase with aging because spermatogenesis has more DNA replications → making more mistake in DNA replications. III. Chromosomal Rearrangements Causes - chromosomal breaks from radiation & DNA repair errors → chromosomal breaks induce rearrangements when repaired.
• homologous recombination at repetitive DNA (transposons, retrotransposons) → interspersed repeated sequence allows recombination between chromosomes “Balanced” change does not alter the number of genes – rearrangement “Unbalanced ” deletes or duplicates part of the DNA A. Duplications - replicates part of chromosome as an extra copy a. Tandem duplication (the copy is placed next to the sequence that was being copied)

b. Dispersed (displaced) duplication → somewhere else beside right next to c. Same or revered order.

• Changes due to imbalanced in the amount of gene products
• Phenotype from single chromosome with duplication

1. Unbalance genes e.g. Bar eyes on drosophila, human developmental and neurological a. Bar region. Duplicate the region makes the eye narrower. The more duplication, the narrower the eyes. rare more often
2. Trichromatic color vision due to duplication on X in primates

• we can detect trichromatic because we have extra copy of opsin gene
• extra copy allowed to mutate and gain new function