BIOL 4003 Module 9: Understanding Epigenetics and Gene Regulation

How can epigenetic changes be targeted by transcription factors or noncoding RNAs?

Transcription factors can recognize the sequence of a specific gene, bind to that gene, and recruit other proteins that modify histones or methylate the DNA

ncRNA bind to specific complementary gene sequences and recruit proteins too

Constitutive vs faculative heterochromatin

Similarities: both heterochromatin, more compact than euchromatin, have repeat sequences, more DNA methylation, specific histone modifications

Constitutive: Found close to centromere or telomere, more short tandemly repeated sequences, ALWAYS heterochromatin in all cell types

Facultative: Reversible based on development or cell type, between centromere and telomere, LINE repeats (transposable elements)

Function of reader domains

To bind the protein with that domain to histone tails with specific matching posttranslational modifications

Higher order structure of heterochromatin in cell nucleus

-closer, more stable contact between nucleosomes

-closer loop domains

-Binding to nuclear lamina

HP1 protein

Forms a dimer between modified H3 histones to bridge heterochromatin loop domains to make them more compact

Phases of heterochromatin formation

nucleation (one site is bound by chromatin-modifying enzymes & chromatin remodeling complexes)

spreading (adjacent chromatin is converted)

barrier (interphase chromosomes have barriers to stop heterochromatin)

Molecular mechanisms to maintain heterochromatin during cell division

-DNA methylation -> maintenance methylation

-Histones recruiting chromatin-modifying enzymes to daughter chromatids

-DNA polymerase recruiting chromatin-modifying complexes

-Local higher-order structures favoring reformation of heterochromatin

How can genomic imprinting (Igf2) occur during development?

Igf2 gene is de-novo methylated during sperm formation, NOT egg formation

Methylation at ICR and DMR inhibits CTC-binding factor from forming loop, allows nearby enhancer to work

How can X-chromosome inactivation occur during development?

During embryogenesis, X chromosomes pair up, pluripotency factors shift to one X signalling it to remain active, other expresses Xist

Xist RNA is tethered to Xic, Xist RNAs bind to each other and coat chromatin, Xist RNA recruits proteins that silence targeted X and promote compaction

How can the formation of specific cell types occur during development?

Very complicated

Trithorax group of protein complexes activates genes

Polycomb group represses genes

PRE-binding protein binds to polycomb response element, PRC2 recruited, PCR2 methylates H3 in gene to be silenced, recruits PRC1 and gene is silenced

How can epigenetic changes occur via environmental factors?

Avy mice -> diet can increase DNA methylation of CpG islands and inhibit Avy gene, lead to darker fur

Honeybees -> royal jelly inhibits DNA methylation, queens have access to more genetic material

Vernalization -> to flower, FLC must be turned off, cold temperatures activate protein complexes that repress FLC

Ability of ncRNA to bind to other molecules

ncRNA-DNA via complementary base pairing

ncRNA-RNA via complementary base pairing

ncRNA-proteins via stem loop structures binding to pockets on protein surfaces

6 general functions of ncRNA

Scaffold - ncRNA binds group of proteins

Guide - ncRNA binds to protein, guides it to specific site in cell through complementary base pairing

Alteration of protein function - ncRNA binds to protein, alters its structure/function (catalyst activity, binding, stability)

Ribozyme - ncRNA has catalytic functions

Blocker - ncRNA physically prevents cellular process from happening

Decoy - ncRNA recognizes another ncRNA and sequesters it

How does HOTAIR inhibit transcription?

HOTAIR has scaffold and guide regions, binds PCR2 and LSD1 then guides them to GA-rich region next to target gene, they modify the histones through methylation/demethylation and that inhibits transcription

Fire and Mello results

When injected embryo with mix of sense and antisense RNA, only partially inhibited mRNA

When injected embryo with double stranded RNA, almost entirely inhibited mRNA

Basis for RNA interference theory

Steps in RNA interference

Mediated by microRNA or small interfering RNA

mi/siRNA folded into hairpin, exported from nucleus, trimmed to 20-25 bp double strand

Protein recognizes mi/siRNA, binds and degrades one strand, forming RISC (RNA-induced silencing complex)

RISC complex binds to specific cellular mRNA

miRNA -> not perfect binding, complex is quarantined

siRNA -> perfect binding, complex is degraded

How does CRISPR-cas provide bacteria with defense against bacteriophages?

Cas1/2 cleaves phage DNA, inserted into Crispr gene and becomes spacer

tracrRNAs and pre-crRNA are transcribed, tracrRNA bind to crRNA because of complementarity at repeats

Cas9-tracrRNA-crRNA complex forms

crRNA part of complex binds to phage DNA, Cas9 cleaves phage DNA into pieces and deactivates it so it can't proliferate