Lecture 3 & 4- Genomic Imprinting notes PDF

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Lecture given by Dr Adele Murrell...

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Lecture 3 & 4 – Genomic imprinting and diseases

There are two copies of the genome – hence 23 PAIRS of chromosomes

There is not always equal contribution of parental genomes

Maternal

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Parthenogenesis (maternal genes only) X-linked gene expression X inactivation Mitochondrial gene inheritance Maternal age effect Maternal exposure/fetal environment Genomic imprinting

Paternal

- Androgenesis (paternal genes only) - Y-linked genes - Male specific gene expression - Paternal environmental exposure - Genomic imprinting

Mosaicism and Chimerism Chimerism – 2 genotypes from different sources mixed – results in two completely different genotypes Mosaicism – mutation/change in expression of one or more genes acquired after fertilisation – results in only some cells of the body being affected Examples – most chromosome trisomies including Down syndrome results in milder phenotypes and X-inactivation

Experiments where the pronuclei are removed and replaced are called nuclear transfer experiments

Nuclear transfer Gynogenetic and androgenetic mouse embryos failed to develop to term, suggesting both the maternal and paternal nucleus are necessary for complete development. Experiments demonstrating the developmental failure of biparental mouse embryos, gave further support for this evidence

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The genes we inherit from each of our parents do NOT contribute equally to our development

Genomic imprinting 

Why are both maternal and paternal genomes required?

Eggs and sperm undergo differential DNA modification during egg maturation and sperm maturation. Differential modification in eggs and sperm would mean that for some genes, only the maternal copy is expressed Thus, two copies of a maternal genome – parthenogenesis – would results in too much of some proteins and a complete lack of other essential proteins

Androgenetic: Hydatidiform mole

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Usually “empty ovum” fertilised by sperm which duplicated (46, XX) Or an ovum fertilised by two sperms – dispermy (79, XXX)

Model epigenetic system: Genomic imprinting

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Mono-allelic parent-of-origin gene expression Differential DNA methylation Unique set of growth-regulating genes The epigenetic mark is established in the primordial germ-line at an imprinting control region (ICR) Stable epigenetic memory system withstands somatic reprogramming

Methylation during germ line and early embryonic development

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Methylation reprogramming in the germ line and preimplantation embryo. The diagram depicts the methylation level in methylated imprinted genes and non-imprinted genetic sequences during germ cell and embryonic development

Genomic imprinting

A phenomenon whereby epigenetic gametic marks are acquired on certain genes prior to fertilisation and these marks are then maintained throughout development resulting in parent of origin specific expression

Imprint erasure during demethylation in PGCs

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Tet1 and Tet2 are expressed in PGCs Tet1−/− PGCs are reported to undergo normal demethylation But some imprinted genes have defects in Tet1−/−; Tet2−/− double knock out mice, suggesting at least a partial role of hydroxymethylation during PGC reprogramming Base excision repair proteins, such as PARP1, APE1 and XRCC1, have also been implicated in active demethylation during PGC reprogramming PGCs undergo mitosis rapidly with a doubling time of approximately 12 hours between E9.5 and E12.5 – thus passive demethylation – PGCs at this time express little to no Dnmt3a, Dnmt3b and DNMT1 is not localised to replication forks

Establishment of imprints -

New methylation imprints are established in females postnatally (B) In males, methylation imprints are established prenatally (C)  Methylation of ICRs is established in the female germline during oocyte growth and is complete by the time oocytes arrest at the metaphase II (MII) stage  Primary stage oocytes from mice at 1 day post-partum (dpp) completely lacked DNA methylation  DNA methylation at imprinted genes starts to accumulate on maternally methylated DMRs around 10 dpp

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In early stages of oocyte and sperm development – I.e. when they are still diploid, the maternal and paternal copies acquire methylation asynchronously DNMT3b is the major de novo DNMT involved in imprinting DNMT3L required for correct targeting of de novo methylation at imprinted regions

Imprinted genes occur in clusters – the importance of this, is that within such clusters there are “ICRs” Imprinting control regions that enable multiple genes to be regulated (expressed/silenced) Imprinted genes affect behaviours

Imprinted genes affect metabolism

Two imprinted loci and four congenital imprinting syndromes    

Prader-willi Syndrome Angelman Syndrome Beckwith Wiederman Syndrome Silver Russell syndrome

IGF2 imprinting cluster

Mechanisms of mono-allelic imprinted expression

Lollipop – methylation site Blue – paternal Red – maternal Closed – methylation Imprinted control regions (ICRs) Are differentially methylated regions (DMRs) – i.e are methylated on one paternal allele only ICRs acquire their DNA methylation imprint in the germ line (i.e – during gametogenesis) and they maintain these imprints after fertilisation and even in the adult organism Poised mark – both silencer and activator on the same region

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There are DMRs known as somatic DMRs, which acquire methylation after fertilisation and although these are allele-specific, these can also be tissue specific and more dynamic. They influence expression but are themselves regulated by the ICR

Beckwith Wiedemann syndrome and the IGF2 locus as a model region Two important concepts: 1) Imprint control regions 2) Epigenetic silencing and maintaining silencing at one allele

H19 – non-coding RNA which regulates an imprinted gene network 25-50% of BWS patients have biallelic rather than monoallelic expression of the IGF2 gene 50% - have an epigenetic mutation resulting in loss of imprinting of a transcript called KCNQ1OT1

CTCF – boundary factor – zinc finger IGF2 has strong promoter and can access downstream enhancers but can only do so if the ICR is methylated to prevent CTCF from binding which divides the genome into domains. IGF2 will no longer have access to the enhancers Domain 1: Contains the imprinted genes IGF2 and H19 and a DMR. The maternally expressed H19 gene encodes an apparently untranslated polII transcript and the IGF2 gene encodes a paternally expressed fetal growth factor. Upregulation of IGF2 is thought to be important in the pathogenesis of BWS and a variety of tumours. Increased expression of IGF2 may be caused by paternal chromosome duplications of chromosome 11p15, paternal uniparental disomy or alterations to differential methylation. The H19 and IGF2 genes compete for a common set of downstream enhancers located 3’ of the H19 gene. DMR1 is located 2kb upstream of the mouse H19 gene and regulates the reciprocal imprinted expression of H19 and IGF2 gene in domain 1 by functioning as a chromatin boundary element or insulator. On the maternal chromosome, DMR1 is unmethylated, permitting the binding of a zinc finger protein called CTCF. Binding of CTCF blocks access of the IGF2 promoter to the downstream enhancers. Thus, the maternal copy of H19 is activated by these enhancers and is transcribed. Methylation of the paternal copy of DMR1 and the H19 promoter are thought not only to silence the H19 promoter but also to prevent binding of the CTCF protein to DMR1. As a result, the IGF2 promoter can access the downstream enhancers and H19 is silenced. Domain 2: There are six known imprinted genes including p57KIP2, a maternally expressed gene that encodes a CDK inhibitor and negatively regulates cell proliferation. In tumours, p57KIP2 shows aberrant methylation associated with cell cycle dysregulations; however, this gene is rarely mutated in tumours. Interestingly, mutations in the gene do cause BWS and are often associated with

autosomal dominant inheritance of the syndrome. IPL is a maternally expressed gene that shows homology to Tdag51, a gene involved in Fas-mediated apoptosis. IMPT1 is a maternally expressed gene encoding a possible organic cation transporter. Mutations of this gene have been reported in breast cancer. The maternally expressed KCNQ1 gene product forms part of a potassium channel. Six known translocation sites spanning the length of this gene are strongly associated with BWS. Intron 10 of the KCNQ1 gene contains another DMR called ‘DMR2’. The paternal allele is non-methylated, permitting the paternal expression of a long transcript called KCNQ1OT1, also known as LIT1. This transcript originates near DMR2 and is transcribed in an antisense direction to the KCNQ1 gene in which it originates. Maternal methylation of DMR2 is thought to silence maternal expression of KCNQ1OT1 and to allow expression of a number of maternally expressed genes including KCNQ1 and CDKN1C. Furthermore, a targeted deletion of the paternal KCNQ1OT1 DMR2 caused diminished expression of the KCNQ1OT1 transcript and activation of expression of the KCNQ1 and CDKN1C genes. This suggests that this antisense transcript negatively regulates in cis the expression of several genes at long-range. These data suggest that the paternally-expressed KCNQ1OT1 transcript and/or DMR2 itself can function as mediators of imprinting in domain 2. Recent evidence suggests that DMR2 has insulator activity in the mouse and insulator and silencer activity in the human. ChIP experiments showed similar distribution of cohesin and CTCF, indicating that the two colocalise.

The KCNQ1OT1 lncRNA is expressed from the paternally unmethylated ICR which is methylated on the maternal allele. Recent evidence suggests that KCNQ1OT1 can directly recruit the PRC2 complex (polycomb complex) and facilitate intra-chromosomal looping to the KCNQ1 promoter

Collision model – Co-transcriptional interference

Antisense and sense transcription can result in polymerases on the same track of DNA arriving from opposite directions. Mechanistically, this could lead to physical stress since these RNA polymerases cannot pass one another without the DNA helix unwinding.

A) In cis recruitment of chromatin modifies. KCNQ1OT1, Air and Xist/Rep1 lncRNA transcribed from the coding loci are accumulated on a chromatin in cis to recruit chromatin modifies, such as histone methyltransferases, Polycomb group proteins and DNA methyltransferases. The chromatin modifiers then facilitate formation of the repressive chromatin by modification of histones and DNA. B) In trans recruitment of chromatin modifiers, the best example of this is non imprinted genes is HOTAIR, a lncRNA expressed from the HOXC locus binds to the HOXD locus in trans and

then recruits the chromatin modifiers to form the repressive chromatin, which inactivates transcription. Methylation during germ line and early embryonic development

Methylation reprogramming in the germ line and preimplantation embryo. The diagram depicts the methylation level in methylated imprinted genes (Black) and non-imprinted genetic sequences during germ cell and embryonic development. MBD3 – binds to methylated region ZFP57 – binds to maternal region and protects it STELLA – binds to both...