601 - epigenetics

Conrad waddington

coined the term epigenetics as the branch of biology which studies the causal interactions between genes and their products which bring the phenotype into being

epigenetics

regulation and persistence of gene activity or cell state without a change in the underlying DNA sequence

biological outcomes of epigenetics

cell type specific expression and stable cellular identity

mitotic inheritance

epigenetic states may persist through mitosis without DNA sequence changes (epigenetic memory)

cell identity

daughter cells can retain lineage-specific expression programs

not DNA alone

histone marks and transcription factors also reinforce memory

disease relevance

aberrant silencing can persist in a tumor cell lineage

in cancer, promoter hypermethylation of certain tumor suppressor genes may be maintained through repeated cell divisions, contributing to stable gene silencing within a tumor clone

DNMT1 mediated DNA methylation maintenance

the pattern of methylation remains through replication

epigenetic mechanisms

RNA interference

histone modifications

DNA methylation (imprinting)

genetic and environmental influences

DNA sequence variants, SNPs, CpG alteration TF binding sites

exposures and signaling

natural roles of DNA methylation

imprinting

x chromosome inactivation

heterochromatin maintenance

developmental controls

tissue specific expression controls

CpG

cytosine-phosphate-guanine

CpG islands

rich CpG rich regions frequently associated with promoters and are usually unmethylated in normal cells

CpG methylation

DNA methyltransferase (DNMT) transfers a methyl group to carbon 5 of the cytosine base, often at CpG sites

genomic context matters

the relationship between CpG methylation and transcription depends on where the CpG is located

promoter CpG islands

often unmethylated in active/permissive promoters

gain of methylation often accompanies stable gene repression

enhancers

variable; highly cell type and state dependent

higher methylation can accompany reduced enhancer activity

gene bodies

frequently methylated in expressed genes

often correlates positively with transcription; functions vary

repeats/transposons

generally highly methylated

helps repress repetitive elements and maintain genome stability

DNMT1

most abudnant DNA methyltransferase

recognizes hemimethylated DNA

therefore serves as the maintenance DNMT

DNMT3a/b

de novo methylation of unmethylated CpG

MBD (methyl binding domain proteins) modulation

methylated DNA can block transcription directly

methyl binding proteins also block areas of transcription factors

methyl binding domain proteins

bind to DNA with methyl groups

MeCP2- on the x chromosome and subjected to X inactivation, mutation results in Rett Syndrome

MBD1/2/3/4 and Kaiso

may work in concert with each other

recruit additional transcriptional repressors

can bind to specific sequences that are not methylated

may recruit DNMTs or HDACs

passive demethylation

during cell division due to inactive/absent DNMTs

active demethylation

controlled demethylation in differentiated cells:

genome wide active demethylation in zygotes

locus specific active demethylation has been observed in somatic cells such as neurons

generate 5hydroxymethylC and other intermediate products

Chromatin

exisits in two states, heterochromatin and euchromatin

wrapped around 4 core histone to make a nucleosome

forms an octomer

H1 is a linker histone

Histone tails

~25% of the mass of each histone is an exteriorized N-terminus accessible to enzymatic modification

acetylation, methylation, phosphorylation, and deamination

at least 60 residues are available for modification

writers and erasers

enzymes add or remove chemical tags on histone tails including HATs HDACs and methyltransferase demethylases

readers

specialized protein domains recognize specific modifications and recruit machinery to activate or silence genes

Coffin-Lowry syndrome

ID

head and face abnormalities

mutations in RSK2 gene - histone phosphorylation

XLD

Rubenstein Taybi syndrome

short stature

moderate to severe ID

distinctive facial features

mutations in CREB binding protein - histone acetylation

Crosstalk

DNA and histones rarely change without the other

X inactivation

combined DNA and histone changes

begins with transcription of the non-coding RNA Xist

Xist RNA coats the X chromosome, recruiting polycomb complex silencing elements

LINE elements help in the silencing by serving as “booster elements” or “way station”

CpG methylation and chromatin modifications maintain silencing

measure epigenetic changes

differentiated methylated vs unmethylated cytosines: bisulfite/enzymatic treatment

identify genomic regions modified by chromatin structures

bisulfite treatment

sodium bisulfite conversion of DNA, convert unmethylated cytosines to uracils

PCR, methylated cytosines appear as C unmethylated cytosines appear as T

CHIP

chromatin immunoprecipitation