Paper 14: "X-Inactivation, Imprinting, and Long Noncoding RNAs in Health and Disease"

Regulating Gene Dosage

every organism faces the challenge of regulatin gene dosage - in diploids, genes are generally assumed to be expressed from both alleles, but in mammals, several classes of genes are expressed from only one allele per cell

Gene Dosage and Disease

X-linked and imprinted diseases are among the most common congential human disorders, such as Rett, fragile X, PWS/AS and Beckwith-Wiedemann Syndrome

Genomic Imprinting

mammals require both maternal and paternal genomic contribution to develop into healthy, viable organisms

Genomic Imprinting in Mice

approximately 150 imprinted genes which are typically located in clusters of 3-12 genes that are spread over 20-3,700kb of DNA

Imprinting Control Region (ICR)

each well-studied cluster has a discrete imprinting control region (insulation) that exhhibits parent-of-origin specific epigenetic modifications → methylation has been shown to be imposed at a precise time in cells

Gametes and Imprints

in gamets, imprints are wiped and then respectively restored to the maternally and paternally imprinting pattern on both alleles

Barr Body

sex of cat cells could be deduced by a subnuclear structure, today called the “Barr Body” → condensed X chromosome

Lyonization

Mary Lyon discovered that the condensed X is the result of whole-genome silencing → X-chromosome inactivation = Lyonization

Purpose of X Chromosome Inactivation

compensates for dosage differences between males and females by rendering all cells functionally monosomic for the X chromosome

Coordination of XCI

coordinated by an X-inactivation center which controls X chromosome counting, random X chromosome choice and initiation of silencing along 1,000 genes of the X

X-Inactivation in Mice

steps are copmleted in the peri-implantation embryo within the 10-20 cell epiblast lineage - once established, the pattern of XCI is stably propagated in the soma, with conservation of the inactivated X

Primordial Germ Cells

Male: XaY, Female: XaXa

Meoisis

Xp is inactive in sperm and will remain inactive until cell enters blastocyst stage, Xm is active in ora

Blastocyst Stage - Epiblast Lineage

Xp is reactived for the epiblast lineage (will become somatic tissue), after random X-inactivation then occurs in the embryo

Blastocyst Stage - Extracellular Tissue

maintain the inactive parental X (Xp)

Random X-Inactivation

only embryo has random X-inactivation, other tissues preferentially inactivate paternal X

X-Inactivation in Marsupials

Paternal X is imprited to undergo silencing preferentially, fixed choice since paternal X will always be turned off → can be active in second generation

Cis-Acting Control Regions - Mechanisms

both, XCI and genomic imprinting are regulated by cis-acting master control regions

Cis Control Regions

“cis” = on same chromosome

Xic in X-Chromosome Inactivation

identified a single Xic spreading across a 100-500kb region - if deleted, no silencing anymore, and if added at an ectopic location (autosome) results in couting, choosing, and silencing of the autosome → Xic is necessary and sufficient for XCI

Cis-Acting Control Regions and Genomic Imprinting

regulated by cis-acting ICRs that influence allele expression across long distances - nearly all imprinted clusters contain at least one each of maternally and paternally expressed genes

Imprinting Control Regions (ICRs)

just a few kb long → ICR deletions result in the loss of imprinting of multiple genes within a cluster

CTCF

insulator binding protein - blocks enhancer from activating IGF2

ICR

insulator, CTCF binds to ICR

IGF2 Expression

CTCF is not bounded to ICR due to mehtylation and therefore enhancer can turn on IGF2 production

IGF2

stimulates growth

H19

halds growth

Prader-Willi Syndrom (PWS)

involves loss of function of a number of genes on chromosome 15, inlduing SNORD116 - leads to obsetiy, reduced muscle tone and mental ability

Angelman Syndrome (AS)

complex disorder of the nervous system that arises from loss of function of UBE3A → delayed development, intellectual disability, severe speech impariment

Beckwith-Wiedemann Syndrom (BWS)

overgrowth disorder due to mutations in IGF2 and H19

Sillver Russel Syndrome (SRS)

undergrowth and asymmetry disorder that also arises from mutations in IGF2/H19

Xist

the X-inactive specific transcript (Xist) produces a 17-20kb RNA that decorates the X chromosmome during the initiation of XCI

Expression of Xist

Xist is only expressed from the Xi and is requried for the whole-chromsome silencing

Tsix

on active X, Tsix is expressed and therefore Xist is repressed - originally on both chromosomes

Jpx

lncRNA expression activates Xist gene (cis and trans) randomly on onl your chromosome

PRC2

Xist RNA bidns to the “Polycomb Repressive Complex 2” and the YY1 transcription factor, which will then bind to the nucleation center and achonring the repressive complex

Lysine 27

Xist/PRC2/YY1 complexes spread along the X, inactivating it by trimehtlyation of Lysine 27 on Hsistone 3 (= H3K27me3)

H3K27me3

recruits PRC1 which blocks RNA Pol II activity by clustering nuecleosomes

Active X - Tsix

on active sex, Tsix recruits Dnmt3a to methylate Xist promoter, pairs with it and blocks any Xist RNA from Xa → antisense action

Tsix Coordination - Way 1

Tsix coordinates C chromosome pairing to generate epigenetic assymetric to determine the selection of the future Xa and Xi

Tisx Coordination - Way 2

Tsix recruits DNA methyltransferase (Dnmt3a) to silence Cist

Tisx Coordination - Way 3

blocks recruitment for PRC2 to Xist, potentially binding PRC2 → duplexes with Xist/RepA and possibly serves as a decoy for PRC2 recruitment

Random Choice

Tsix determines allelic choice by repressing Xist transcription on one allele - only first choice is random, after choice will be conserved in all cells