Genetics exam 3 - Epigenetic gene regulation and non-coding RNAs

how can a molecular change be epigenetic

must be passed from cell to cell

epigenetic regulation via transcription factor or noncoding RNA

TF or noncoding RNA recognizes sequence and binds

recruits other proteins like DNA methyltransferase and histone modifying enzymes

changes are maintained in subsequent divisions

cis-epigenetic changes

maintained in cell division

euchromatin

chromosomal regions not stained during interphase; loop domains not packed

transcriptionally active

central in nucleus

heterochromatin

more compact

round peripheral of nucleus

inhibits transcription, prevents movement of TEs, and prevents viral proliferation

Constitutive heterochromatin

regions that are heterochromatic at the same location in all cell types

close to centromere or telomere

highly methylated on cytosines

H3K9me2 common jistone modification in yeast and animals

facultative heterochromatin

heterochromatin that varies in its location among different cell types

gene within usually silenced

methylation at CpG islands in gene regulatory regions which silences genes

what posttranslational modifications do amino tails of histones go through

reader domains help proteins bind to particular PTMs in nucleosome

writer domains can add PTMs and eraser domains remove them

recruitment domains recruit other proteins like chromatin remodelers

what are the features of higher-order structural features

• closer more stable contacts of nucleosomes with each other via HP1

  • HP1 recognizes H3K9me3 and bridges nucleosomes

• forms closer loop domains

• binds to nuclear lamina

  • helps organize into chromatin territories

• may undergo liquid-liquid separation

  • formation of liquid-like compartments that are formed by macromolecules that become concentrated in a given location and come out of solution

Phases of Heterochromatin formation

1. nucleation - chromosomal site bound by chromatin-modifying enzymes and chromatin-remodeling complexes

2. spreading - adjacent euchromatin is turned into heterochromatin

3. barrier - spreading stops when it reaches a barrier

faculative heterochromatin is passed from cell to cell after forming in embryonic development and is maintained by DNA methylation, histone modifications, and DNA polymerase

genomic imprinting

form of gene regulation where offspring expresses a copy of gene from one parent but not both

Igf2 gene is de novo methylated in sperm formation

methylation occurs at imprinting control region and differentially methylated region

this inihibit binding of CTC binding factor which allows sitmulation of gene by na enhacer

X- chromosome inactivation (XCI)

happens during embryogenesis in female mammels

X-inactivation center (Xic) has the genes Xist and Tsix

before XCI, Tsix gene is expressed on both chromosome

in inactivation, an X chromosome begins to express Xist gene which binds to Xic and spread forming a barr body

pioneer factors

can recognize and bind to DNA sequences exposed on the surface of a nucleosome

recruit chromatin-remodeling complexes and histone modifying enzymes to carry out epigenetic changes

involved in activation and silencing in genes

can decrease DNA methylation

What kind of environmental agents cause epigenetic changes

dietary effect and toxins in the environments

agouti gene in mice

promotes synthesis of yellow fur pigment

in one strain a TE with a promoter is inserted before Agouti gene called A^vy allele

when fed diet promoting DNA methylation, offspring had darker fur

bees and royal jelly

Worker bees: small, sterile, typically live only for weeks

• Nurse bees: produce a secretion called royal jelly

• Bees that eat royal jelly into adulthood become queens

Larvae injected with a DNA methyltransferase inhibitor became queen bees

non-coding RNAs

do not code polypeptides

what kind of molecules can ncRNAs bind to

DNA - via complementary base pairing

• Other RNAs

• Via complementary base pairing

• Proteins

• ncRNA molecules can form stem-loop structures, which may bind to pockets on the surface of proteins

what kinds of functions can ncRNAs perform

• scaffold - binding to group of proteins

• guide - guides protein to site in cell

• alteration of protein function or stability

• ribozyme

• blocker - blocks cell process

• decoy - ncRNA recognizes another ncRNA and sequesters it

HOTAIR

ncRNA that alters chromatin structure

on chromosome 12 in cluster of HoxC genes

Acts as a scaffold that guides two histone-modifying complexes to their target genes

polycomb repressive complex 2 (PRC2) binds to 5’ end of HOTAIR

LSD1 binds to 3’ end

binds to GA rich region by target gene and trimethylation occurs to inhibit transcription

how can ncRNAs affect the ability of mRNAs

translate or degrade mRNA

Hoe did flower color in petunias show RNA silencing

inserted cloned genes involved in synthesis of flower pigment

flower pigmentation did not deepen in some cases and showed variegation

concluded that additional copies of a gene can sometimes supress the expression of both itself and endogenous counterpart

production of double-stranded RNA involved in lowering mRNA levels

does antisense RNA inhibit or activate mRNA translation

inhibit

sense RNA inhibits as well

Fire and mellos experiment

used a nematode worm

injected them with either mex-3 antisense or mix of mex-3 sense and antisense RNA

mix had no mex-3 production , just anitsense had a little

showed double stranded RNA is more potent inhibitor - names RNA interference

RNA interference (RNAi)

double stradned RNA causes silencing of mRNA

mediated by microRNAs and small interfering RNAs

microRNA (miRNA)

transcribed by endogenous eukaryotic genes

made from pri-miRNA via transcription

small interfering RNA (siRNA)

transcribed from exogenous sources

by viruses or experimentally injected

RNA-Induced Silencing Complex (RISC)

• binds to mRNA

• inhibits translation without degrading the mRNA

  • common for miRNA

• degradation of the mRNA through cleavage

  • common with siRNA

functions and benefits of RNA interference

production of miRNA silences expression of specific mRNAs

RNAi provides defense against viruses

can inhibit transcription by causing chromatin modifications

mechanism of RNA interference

1. pri-miRNA folds into hairpin and is processed into smaller size

2. binds to expotin 5

3. pre-miRNA or pre-siRNA recognized and cut by dicers into double stranded RNA

4. RISC cis formed, one strand is degraded

5. RISC recognizes specific mRNA and target it for silencing by degradation or p-body formation

CRISPR-Cas system

prokaryotic system that defends against foreign invaders

PIWI-interacting RNA

defense system in animals

interacts with ncRNA to inhibit movement of TE

CRISPR locus

series of repeated sequences containing segments from bacteriophage DNA

Defense mechanism of CRISPR-Cas system

1. Adaption (spacer aquisition)

   1. Cas1 and Cas2 protein complex cleaves bacteriophage DNA

   2. A piece is inserted into Crispr gene

   3. creates a spacer which is passed to daughter cells

2. Expression Phase

   1. After adaptation exposure will cause gene expression

   2. region of tracrRNA complementary to repeats in pre-crRNA

   3. pre-crRNA is cleaved into crRNA

   4. crRNA-tracrRNA complex binds to Cas9

3. Interference phase

   1. crRNA acts as a guide that causes tracrRNA-crRNA-Cas 9 complex to bind

   2. phage proliferation is inhibited