Imprinted transcriptional units in humans and mice. Modified from http://igc.otago.ac.nz/home.html.

Location Transcriptional unit Functional Imprinting status Expressed ICR Protein RNA b Human (Mouse) Human (Mouse) component Human Mouse allele methylation Name or description Description

1p36 (4 E2) TP73 (Trp73) I NR M Tumour related protein 1p31 DIRAS3 PD NO P Ras homolog 2p12 (6 C3) LRRTM1 (Lrrtm1) I NR leucine rich repeat transmembrane 2p15 (11 A3) COMMD1 (Commd1) NI I M Copper metabolism gene Murr1 (Zrsr1) NO I P M U2 small nuclear RNP auxiliary factor 4q22.1 (6 C1) NAP1L5 (Nap1l5) I I P M Nucleosome assembly protein 6p11 (1 B) PRIM2 (Prim2) I NR M Primase, polypeptide 2 6q24 (10 A1) HYMAI (Hymai) I PD P M misc RNA PLAGL1 (Plagl1) I I P Zinc finger protein 6q25 (17 A1) IGF2R (Igf2r) PI? I M Insulin-like growth factor receptor 2 (Air) NO I P M Igf2r AS SLC22A2 (Slc22a2) PI? I M Organic cation transporter SLC22A3 (Slc22a3) PI? I M Organic cation transporter 7p12 (11 A1) DDC (Ddc) Exon1a transcript NR I P Dopa decarboxylase GRB10 (Grb10) I I M(P)c M Growth factor receptor-bound protein 7q21 (6 A1) CALCR (Calcr) PD I M Calcitonin receptor 7q21.3 (6 A1) SGCE (Sgce) I I P Sarcoglycan, epsilon PEG10 (Peg10) I I P M Retroviral gag pol homologue PPP1R9A (Ppp1r9a) I I M Protein phosphatase inhibitor PON1 (Pon1) PD NI P Paraoxonase 1 PON3 (Pon3) NR PD M Paraoxonase 3 PON2 (Pon2) NR PD M Paraoxonase 2 ASB4 (Asb4) NR I M Ankyrin repeat and SOCS box 7q32.2 (6 A3) CPA4 (Cpa4) I NR M Carboxypeptidase MEST (Mest) I I P M Alpha/beta hydrolase fold family MIRN335 (Mirn335) NR I microRNA MESTIT1 I NO P MEST AS COPG2IT1 (Copgas2) I I P COPG2 AS COPG2 (Copg2) CD I P(M)d Coatomer protein complex subunit KLF14 (Klf14) I I M Krüppel-like factor 14 8p23 (8 A1.1) DLGAP2 I ND P Membrane associated guanylate kinase 8q24.3 (15 D3) KCNK9 (Kcnk9) I I M Potassium channel (Peg13) NO I P M misc RNA 10p14 (2 A1) SFMBT2 (Sfmbt2) NR I P M Scm-like with 4 mbt domains 10q22 (10 B4) CTNNA3 (Ctnna3) PD NR M Catenin, alpha 3 10q26.11 (7 F3) INPP5F_V2 (Inpp5f_v2) V2 isoform only I I P M misc RNA (retro) 11p15 (7 F5) H19 (H19) I I M P miRNA host miR-625 I I M miRNA IGF2 (Igf2) I I P Insulin-like growth factor 2 IGF2AS (Igf2as) I I P IGF2 AS INS (Ins2) I I P Insulin TH (Th) NR I M ? Tyrosine hydroxylase ASCL2 (Ascl2) CD I M HLH transcription factor TSPAN32 (Tspan32) NI I M Tetraspanin 32 CD81 (Cd81) NI I M Transmembrane 4 superfamily TSSC4 (Tssc4) NI I M Tumor suppressing candidate KCNQ1 (Kcnq1) I I M Voltage-gated potassium channel KCNQ1OT1 (Kcnq1ot1) I I P M KCNQ1 AS KCNQ1DN I NO M BWRT protein CDKN1C (Cdkn1c) I I M Cyclin-dependent kinase inhibitor (Msuit1, AF313042) NO I M misc RNA SLC22A18AS PD NO M SLC22A18AS putative protein SLC22A18 (Slc22a18) I I M Organic cation transporter PHLDA2 (Phlda2) I I M Pleckstrin homology-like domain NAP1L4 (Nap1l4) NR I M Nucleosome assembly protein (Tnfrsf23) NO I M TNF receptor superfamily OSBPL5 (Osbpl5) I I M Oxysterol binding protein-like 5 ZNF215 PD NO M Zinc finger protein 11p15.4 (7 E3) AMPD3 (Ampd3) NI I M AMP deaminase (isoform E) 11p13 (2 E) WT1-Alt transcript (Wt1) I NR P Zinc finger protein WT1AS (Wt1as) PD NI P WT1 AS 11q13.4 (7 F5) DHCR7 (Dhcr7) NI I M 7-dehydrocholesterol reductase 11q23 (9 A5) SDHD (Sdhd) CD NR P Succinate dehydrogenase, subunit 12q13 (15 F1) SLC38A4 (Slc38a4) NR I P Amino acid transporter 12q21 (10 C3) DCN (Dcn) NI PD M Proteoglycan 13q14 (14 D2) HTR2A (Htr2a) NI/CD I M Serotonin receptor 14q32 (12 F1) BEGAIN [BEGAIN - sheep] NR NR P brain-enriched guanylate kinase-associated (imprinted in sheep) DLK1 (Dlk1) I I P Delta-like 1 homolog DLK1 downstream transcripts NR I P misc RNA (Mico1) NR I M Circadian oscillating (Mico1os) NR I M Circadian oscillating MEG3 (Meg3) I I M P misc RNA miR-337 NR I M miRNA RTL1 (Rtl1) NR I P Retrotransposon-like 1 Anti-PEG11 (anti-Rtl1) anti-Rtl1 NR I M Rtl1-AS miR-431 NR I M miRNA miR-433 NR PD M miRNA miR-127 NR I M miRNA miR-434 NR PD M miRNA miR-432 NR PD M miRNA miR-136 NR I M miRNA MEG8 (Rian) MEG8 (Rian) NR I M snoRNA host miR-370 NR I M miRNA (MBII-78) NO I M snoRNA (MBII-19) NO I M snoRNA 14q(0) NR I M snoRNA 14q(I) (MBII-48) NR I M snoRNA (MBII-49) NO I M snoRNA (MBII-426) NO I M snoRNA 14q(II) (MBII-343) NR I M snoRNA [RBII-36-rat] NO NO ? snoRNA 86 copies (Mirg) (Mirg) NR I M miRNA host miR-411 NR I M miRNA miR-380 NR I M miRNA miR-376b NR I M miRNA miR-376 NR I M miRNA miR-134 NR I M miRNA miR-154 NR I M miRNA miR-410 NR I M miRNA DIO3 (Dio3) NR I P Deiodinase, iodothyronine type III Location Transcriptional unit Functional Imprinting status Expressed ICR Protein RNA b Human (Mouse) Human (Mouse) component Human Mouse allele methylation Name or description Description

15q11-q12 (7C-B5) (Peg12) NO I P Gsk-3-binding protein family MKRN3 (Mkrn3) I I P Makorin, ring finger protein ZNF127AS (Zfp127as) NR I P MKRN3 AS MAGEL2 (Magel2) I I P MAGE-like protein NDN (Ndn) I I P Necdin, neuronal growth suppressor (AK014392) NR PD P Ndn AS BM117114 NO I P EST (Pec2) NR I P LINE-rich intergenic (BB077283) NO I P EST (Pec3) NR I P LINE-rich intergenic (Nccr) NR I P ?miRNA host PWRN1 I ?NO NK misc RNA C15ORF2 I NO NK 1156 aa intron-less gene in primates only SNURF-SNRPN SNURF (Snurf) I I P SNRPN upstream reading frame SNRPN (Snrpn) I I P M Small nuclear ribonucleoprotein SNORD107 (MBII-436) I I P snoRNA SNORD64 (MBII-13) I I P snoRNA SNORD108 I NO P snoRNA SNORD109A I NO P snoRNA SNORD116@ I I P snoRNA cluster SNORD115@ I I P snoRNA cluster SNORD109B I NO P snoRNA UBE3A-AS I I P UBE3A AS UBE3A (Ube3a) I I M Ubiquitin protein ligase ATP10A (Atp10a) I CD M ATPase, Class V GABRB3 (Gabrb3) CD NI P Gamma-aminobutyric acid receptor GABRA5 (Gabra5) CD NI P Gamma-aminobutyric acid receptor GABRG3 (Gabrg3) CD NI P Gamma-aminobutyric acid receptor 15q21 (2 E5) GATM (Gatm) NI I M Glycine amidinotransferase 15q24 (9 E3.1) MIRN184 (Mirn184) NR I P microRNA (AS4) NR I P misc RNA (4930524O08Rik, A19) NO I P misc RNA RASGRF1 (Rasgrf1) NR I P P Guanine nucleotide exchange factor 16p13 (16 A1) ZNF597 (Zfp597) I NR M Zinc finger protein 18q11 (18A2-B2) IMPACT (Impact) NI I P Imprinted and ancient 18q21.1 TCEB3C I NO M Transcription elongation factor 19q13.41 ZNF331 PD NO M Zinc finger protein 19q13.43 (7A2-B1) ZIM2 (Zim2) I I P(M)d Zinc-finger protein (Zim1) NO I M Zinc-finger protein PEG3 (Peg3) I I P M Zinc-finger protein ITUP1/MIMT1 (Usp29) I NO P Imprinted transcript variant1 USP29 (Usp29)e NR I P Ubiquitin-specific protease ZIM3 (Zim3) NR I M Zinc-finger protein (human) No ORF (mouse) ZNF264 (Zfp264) NR I P Zinc-finger protein (human) No ORF (mouse) 20q11.21 (2 H1) MCTS2 (Mcts2) I I P M RNA binding protein HM13 (H13) NR I M Signal peptide peptidase 20q11.23 (2 H1) NNAT (Nnat) I I P M Neuronatin 20q13 (2 H3) L3MBTL (L3mbtl) I NI P DMR Polycomb group 20q13 (2 E1-H3) GNAS (Gnas) NESP55 I I M Neuroendocrine secretory protein 55 GNASXL I I P M Large isoform of GS-a (F7) NO PD M Hypothetical protein (Mm.125770) Exon-1A I I P M misc RNA GS-alpha I I M Stimulatory G-protein, alpha subunit SANG (Nespas) I I P GNAS AS (X) All genes affected by X inactivation NI I M All genes affected by X inactivation Xq13 (X D) XIST (Xist) NI I P X-inactive specific transcript TSIX (Tsix) NI I M XIST antisense (X A7) (Xlr3b) NO I M X linked lymphocyte regulated (Xlr4b) NO I M X linked lymphocyte regulated (Xlr4c) NO I M X linked lymphocyte regulated

Abbreviations. AS, antisense transcript; miRNA, microRNA; misc RNA, RNA of unknown function CD, conflicting data; I, reported to be imprinted; ICR, Imprint control region; ND, not detected; NI, reported to be not imprinted; NO, no orthologue known; NR, no reports of imprinting status; M, maternal; P, paternal; PD, provisional data; PI, polymorphic imprinting bNoncoding RNAs only cImprinting is isoform dependent. dZIM2 and COPG2 are reported to be oppositely imprinted in human and mouse. e Mouse Usp29 appears to split into two genes in human and cow (ie MIMT1 and Usp29).

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APPENDIX 2: THE EVOLTION OF IMPRINTING: CHROMOSOMAL MAPPING OF ORTHOLOGUES OF MAMMALIAN IMPRINTED DOMAINS IN MONOTREME AND MARSUPIAL MAMMALS

The following appendix is a publication from mid-2007 which examines the evolution of genomic imprinting and X inactivation through localisation of orthologues of imprinted genes in marsupials and monotremes.

Edwards CA, Rens W, Clarke O, Mungall AJ, Hore TA , et al. (2007) The evolution of imprinting: chromosomal mapping of orthologues of mammalian imprinted domains in monotreme and marsupial mammals. BMC Evol Biol 7: 157.

Although my final contribution to this publication was relatively minor, the time taken generating my results consumed a reasonable proportion of my early PhD project. I isolated three imprinted gene orthologues in platypus ( UBE3A , GNAS and DLK1 ) and determined their localisation on the challenging platypus chromosomes. My results were later independently confirmed by co-authors.

As discussed in Chapter 6, this publication significantly contributed to our understanding of the evolution of genomic imprinting and imprinted loci. Specifically, it disproved the hypothesis that imprinted genes evolved mono-allelic gene expression at one chromosomal region (such as the sex chromosomes) and then transferred this to other genomic regions upon rearrangement.

159 BMC Evolutionary Biology BioMed Central

Research article Open Access The evolution of imprinting: chromosomal mapping of orthologues of mammalian imprinted domains in monotreme and marsupial mammals Carol A Edwards†1, Willem Rens†2, Oliver Clarke2, Andrew J Mungall3, Timothy Hore4, Jennifer A Marshall Graves4, Ian Dunham3, Anne C Ferguson-Smith*1 and Malcolm A Ferguson-Smith2

Address: 1Department of Physiology, Development and Neuroscience, University of Cambridge, Downing Street, Cambridge CB2 3DY, UK, 2Cambridge Resource Centre for Comparative Genomics, Department of Veterinary Medicine, University of Cambridge, Cambridge CB3 OES, UK, 3Wellcome Trust Sanger Institute, Wellcome Trust Genome Campus, Hinxton, Cambridge CB10 1SA, UK and 4Research School of Biological Sciences, The Australian National University, Canberra, Australia Email: Carol A Edwards - [email protected]; Willem Rens - [email protected]; Oliver Clarke - [email protected]; Andrew J Mungall - [email protected]; Timothy Hore - [email protected]; Jennifer A Marshall Graves - [email protected]; Ian Dunham - [email protected]; Anne C Ferguson-Smith* - [email protected]; Malcolm A Ferguson-Smith - [email protected] * Corresponding author †Equal contributors

Published: 6 September 2007 Received: 13 March 2007 Accepted: 6 September 2007 BMC Evolutionary Biology 2007, 7:157 doi:10.1186/1471-2148-7-157 This article is available from: http://www.biomedcentral.com/1471-2148/7/157 © 2007 Edwards et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Abstract Background: The evolution of genomic imprinting, the parental-origin specific expression of genes, is the subject of much debate. There are several theories to account for how the mechanism evolved including the hypothesis that it was driven by the evolution of X-inactivation, or that it arose from an ancestrally imprinted chromosome. Results: Here we demonstrate that mammalian orthologues of imprinted genes are dispersed amongst autosomes in both monotreme and marsupial karyotypes. Conclusion: These data, along with the similar distribution seen in birds, suggest that imprinted genes were not located on an ancestrally imprinted chromosome or associated with a sex chromosome. Our results suggest imprinting evolution was a stepwise, adaptive process, with each gene/cluster independently becoming imprinted as the need arose.

Background imprinting in placental mammals over 20 years ago there Genomic imprinting is an epigenetic phenomenon that has been much speculation about how the mechanism has been well-characterised in eutherian mammals. has evolved. Despite this, the range of mammalian species Imprinted genes are expressed from one of the two paren- tested for imprinting is limited and very few non-mam- tally inherited chromosome homologues and repressed malian vertebrates have been experimentally assessed. on the other. The mechanism of parental-origin specific Mammals that diverged early from the lineage of euthe- gene expression is associated with heritable differential rian mammals are ideally suited for investigating imprint- modifications to the DNA and chromatin that are pro- ing evolution by comparing epigenetic mechanisms grammed during gametogenesis [1]. Since the discovery of within mammalian species. Such comparative analysis

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has wider implications for our understanding of the evo- perspective, the evolution of placentation exerted selective lution of the epigenetic control of genome function. To pressure to imprint growth-related genes present on both date, based on investigations of eutherian imprinted the X and the autosomes. The basis of this model is the orthologues, imprinting has been demonstrated at some suggestion that genes imprinted in the placenta utilise a loci in marsupials (both Macropus eugenii {tammar wal- non-coding RNA mechanism that parallels the function of laby} and Monodelphis domestica {grey short-tailed opos- the Xist non-coding RNA essential for X inactivation in sum} which diverged from each other approximately 70 placental mammals. Most recently, data have emerged million years ago) but not in monotremes (platypus and proving that marsupial imprinted X-inactivation and plat- echidna) [2-5]. This suggests that, if imprinting arose only ypus sex chromosome dosage compensation occur via a once in mammals, it evolved somewhere between the mechanism that is independent of the XIST-mediated divergence of monotremes (prototherians) from therian mechanism occurring in mouse and man [18,19]. This mammals around 166 million years ago (MYA) [6] and finding is not consistent with either of the two proposed the divergence of marsupials (metatherians) from euthe- models linking X inactivation to autosomal imprinting. rian mammals approximately 147 MYA. Another theory postulates that imprinted domains The egg-laying monotreme is an important link between evolved through chromosomal duplication and that birds and viviparous mammals, and is therefore of inter- imprinted genes were originally located on one (or a few) est for studies on the evolution of imprinting. In addition, ancestral pre-imprinted chromosome region(s) and then the platypus has been shown to possess 10 sex chromo- dispersed in mammalian genomes through recombina- somes, 5 Xs and 5 Ys [7,8]. In male meiosis these 10 chro- tion or transposition events [20]. Duplication of a set of mosomes form a multivalent chain consisting of genes may have led to random monoallelic expression as alternating X and Y chromosomes [7]. The 5Y and 5X a means of dosage compensation and, subsequently, chromosomes segregate alternately from a translocation imprinting (parental-origin specific gene activity/repres- chain to form male (5Y) and female (5X) determining sion) following divergence of the paralogues. If imprinted sperm. Dosage compensation mechanisms have not been genes were found to be located on one or two platypus elucidated in monotremes. Parallels have been drawn autosomes this would constitute some evidence for this between epigenetic mechanisms associated with genomic hypothesis. Alternatively, given the large number of plat- imprinting and X chromosome dosage compensation in ypus sex chromosomes that may have epigenetically regu- female eutherian mammals. Hence determining the pres- lated dosage compensation mechanisms, it is possible ence, organisation and location of imprinted orthologues that autosomal imprinted domains might have arisen in the monotreme can provide a useful framework for through translocation of sex chromosome-linked genes comparative mechanistic and evolutionary studies. onto autosomes carrying with them vestiges of the regula- tory sequences required for parental origin specific sex Recently, different views on the evolution of imprinting chromosome dosage compensation. It is relevant to note mechanisms have been expressed. Two views are based on however, that the platypus sex chromosome system bears the similarities between X chromosome inactivation no relationship to the XY system in viviparous mammals (XCI) and autosomal genomic imprinting that have long (Rens et al. submitted for publication). been noted [9]. Since both have a number of features in common, such as the association with non-coding and In order to understand the emergence of imprinting after anti-sense RNA and some related patterns of histone mod- the divergence of monotremes from the mammalian line- ifications, it has been suggested that X-inactivation was age we have isolated platypus (Ornithorhynchus anatinus) the 'driving force' behind the evolution of imprinting and tammar wallaby (Macropus eugenii) bacterial artificial [10]. This idea has grown from the finding that, in marsu- chromosome (BAC) clones that contain orthologues of pials, XCI is an imprinted event with the paternal X being mouse and human imprinted domains and investigated preferentially inactivated in all tissues [11,12]. In Mus their localisation on tammar wallaby and platypus chro- musculus (mouse) and Bos taurus (cow), imprinted XCI is mosomes. We have determined the chromosomal loca- an early event confined to extra-embryonic tissues [13,14] tion of 8 imprinted gene orthologues in the platypus, and occurring prior to the reprogramming of the X in the representing 7 different clusters of imprinted genes in the epiblast which leads to random XCI in embryonic deriva- mouse or human (the IGF2 imprinted domain, IGF2R, the tives [15,16]. Once inactivation was fixed on the X chro- DLK1/DIO3 imprinted domain, GRB10, the GNAS com- mosome in ancestral mammals, it has been suggested that plex, a gene from the Prader-Willi/Angelman Syndrome these mechanisms were adopted by autosomes to estab- complex and SLC38A4). In addition 8 imprinted gene lish genomic imprinting[10]. An alternative to the 'driving orthologues were mapped in the tammar wallaby – a force' hypothesis is the view that imprinting and X-inacti- ninth was mapped previously. Three of these genes belong vation co-evolved when the placenta emerged [17]. In this to the Beckwith-Wiedemann Syndrome (BWS) ortholo-

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gous region and two to the DLK1/DIO3 region. The genes investigated here represent the best-characterised imprinted domains known in the mammalian genome and can be considered in the context of the information available on their imprinting status. Our analysis contrib- utes to the identification of regions of syntenic homology across a range of vertebrates including chicken and the prototherian, metatherian and eutherian mammals.

Results Identification of platypus and tammar wallaby BACs containing imprinting orthologues Each of the imprinted genes described in this report have been mapped by fluorescent in situ hybridisation (FISH) of BAC clones to metaphase chromosomes of platypus and wallaby cells in culture to determine their regional position and in some instances, to confirm retention of clustering across the cluster (Figures 1 & 2).

The orthologues of both the insulin like growth factor 2 (IGF2) and one of its receptors (M6P/IGF2R), have previ- ously been characterised in the platypus [Gen- bank:AY552324 and Genbank:AF151172] [3,21]. IGF2 is a paternally expressed imprinted gene in both eutherian and marsupial mammals but has been shown not to be imprinted in birds and monotremes [2,4,22]. In mouse and human it forms part of a large imprinted cluster that can be divided into two imprinted subdomains – one con- taining the IGF2 and H19 genes, and the other containing CDKN1C and several genes showing tissue-specific imprinting in the mouse placenta including CD81. These two contiguous subdomains map to chromosome FISHconFigutain rmeai ng1pping orth onolog platypues usof mimprintedetaphase genes chromosomes of BACs 11p15.5 in humans (BWS critical region) and mouse dis- FISH mapping on platypus metaphase chromosomes tal chromosome 7. A fragment of IGF2 was amplified of BACs containing orthologues of imprinted genes. from platypus DNA using primers from the highly con- (A) DIO3, (B) DLK1, (C) IGF2R, (D) SLC38A4, (E) IGF2, (F) served second coding exon C2 in platypus. This was used GRB10, (G) GNAS (and platypus 8 paint in green) and (H) as a probe to screen platypus and wallaby BAC libraries. UBE3A. Scale bar is 10 !m. M6P/IGF2R is a large gene consisting of 48 exons which encodes a protein of 2482 amino acid residues in mouse. It is expressed from the maternally inherited chromosome is an intronless gene that codes for type III iodothyronine in mice [23] and has also been shown to be imprinted in deiodinase (D3), a 278 amino acid selenoprotein in the opossum Didelphis virginiana[3]. This gene is bialleli- human. It is a predominantly paternally expressed gene cally expressed in monotremes and also lacks IGF2 bind- which is part of the DLK1/DIO3 cluster which is found at ing properties in these species [3]. To screen the wallaby 14q32 in humans and distal chromosome 12 in mice. BAC library, a probe was designed to Macropus rufogriseus DLK1 is a Delta-like protein member of the Notch family (red-necked wallaby) IGF2R mRNA [Genbank:AF339159]. of signalling molecules and is found in all vertebrates. Despite this DLK1 is not as conserved as the other The other genes/regions chosen for this study had not pre- imprinted genes in this study so in order to produce viously been characterised in monotremes or marsupials. probes to screen the libraries, the trace archives from The CD81 gene encodes a member of the transmembrane NCBI were searched with DLK1 sequences from other spe- 4 superfamily which is preferentially expressed from the cies. By searching the Monodelphis domestica trace maternal allele in mouse placentas [24]. CD81 is approx- archive with human DLK1 [Genbank:NM_003836], imately 240 kb downstream of IGF2 in human. A probe of TI_395847291 was identified and a probe designed to the entire human CD81 coding sequence was used to the most conserved regions between the two sequences screen the wallaby BACs and 5 positives were found. DIO3 was used to screen the wallaby library. Chicken DLK1

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large cluster that spans 4 Mb in human and includes the ubiquitin protein ligase E3A gene (UBE3A) that is expressed from the maternally inherited chromosome. This gene has previously been assigned to wallaby chro- mosome 5 [25,26]. Finally, solute carrier family 38, mem- ber 4 (SLC38A4 also called ATA3) is located on human 12q13 and mouse distal chromosome 15. It is found in a gene cluster with two other solute carriers of which it is the only imprinted one, being repressed on the maternally inherited chromosome. Probes were designed to highly conserved regions in each of these genes and used to screen platypus and tammar wallaby BAC libraries.

Further information on all probes used for library screens, the sequences they were designed against and the number of BACs identified can be found in [see Additional file 1].

FISH mapping of Platypus BACs The platypus karyotype (2 n = 52) consists of 21 auto- somes and 10 sex chromosomes (5X's and 5Y's in male and 5 X-pairs in female). One positive BAC for each gene was chosen for FISH analysis. The BACs were labelled with biotin using a standard nick translation protocol and localised on platypus chromosomes by FISH on male platypus metaphase preparations. Fig 1a shows the local- ization of DIO3 to a site distal to the centromere of platy- pus chromosome 1. DLK1 maps close to DIO3 in platypus (Fig 1b) IGF2R and SLC38A4 both localise to platypus chromosome 2, IGF2R to a position close to the centro- mere of chromosome 2, and SLC38A4 to distal 2q (Fig 1c and 1d). IGF2 maps to distal platypus chromosome 3p contaFIiSgHu rmieninga 2pp iorthologuesng on tamm aofr mimpetarpinhtedase geneschromosomes of BACs (Fig 1e). GRB10 is positioned near the centromere of plat- FISH mapping on tammar metaphase chromosomes ypus 4 (Fig 1f). Fig 1g shows GNAS on platypus chromo- of BACs containing orthologues of imprinted genes. some 8 as confirmed by FISH using a chromosome 8 (A) DIO3 (green) and DLK1 (red), (B) GNAS, (C) IGF2, (D) specific paint. A fainter signal was also observed on platy- CD81, (E) IGF2R, (F) GRB10, (G) MRPL23, and (H) SLC38A4 pus X5. UBE3A is found on platypus chromosome 18 (Fig (red) with chromosome 3 in green. Scale bar is 10 !m. 1h). All gene locations are shown on the platypus G- banded karyotype (Fig 3). sequence [Genbank:XM_421369] identified the platypus FISH mapping of Tammar Wallaby BACs trace file TI_752207707 to which a probe was designed to The tammar wallaby karyotype (2 n = 16) consists of 7 screen the platypus library. The growth factor receptor- autosomes and the two sex chromosomes. The tiny Y bound protein 10 gene (GRB10) is expressed from the chromosome is not shown in Figure 3. The genes were paternally inherited chromosome in both mouse and localised on male tammar wallaby metaphase chromo- human brain. In other organs, it is maternally expressed in somes using FISH with labelled BAC DNA (as above). mouse and biallelically expressed in the human. It DIO3 and DLK1 (Fig 2a) were mapped to tammar wallaby appears to be a solitary imprinted gene which is located chromosome 1q about one third distal from the centro- on human 7p12 and mouse proximal 11. The GNAS com- mere. GNAS also was mapped to chromosome 1q but plex is located on human 20q13.3 and mouse distal 2. considerably more distal from the centromere (Fig 2b). This is a complex domain with a number of differentially IGF2, CD81, and MRLP23 were mapped to the same imprinted, alternatively spliced transcripts. The guanine cytogenetic region on tammar wallaby chromosome 2p nucleotide binding protein, alpha stimulating gene (Fig 2c, 2d and 2g). GRB10 localised to tammar wallaby (GNAS) is highly conserved in vertebrates. The Prader- 3p (Fig 2f).IGF2R was mapped to 2q, half way down that Willi/Angelman Syndrome cluster is located at human arm (Fig 2e). SLC38A4 was mapped to tammar wallaby 15q11–13 and mouse central chromosome 7. This is a

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FLoigcuatrieo n3 of orthologues of mammalian imprinted genes on the karyotypes of platypus (A) and tammar wallaby (B) in red Location of orthologues of mammalian imprinted genes on the karyotypes of platypus (A) and tammar wallaby (B) in red. Gene names in black are those previously mapped genes from other studies, (reviewed in [25, 44]). The position of the orthol- ogous genes in human are shown on the left.

chromosome 3p, as confirmed by chromosome painting Lambda clones containing IGF2 have previously been with a chromosome 3 specific paint (Fig. 2h). mapped to tammar wallaby chromosome 2p [27]. In order to confirm this location and see if synteny was con- Conservation of synteny served in this species, BACs containing 2 other genes from Dlk1 and Dio3 encompass a 1 MB region in the mouse. In this region were isolated. CD81 is preferentially expressed order to ascertain whether synteny is conserved within the from the maternally inherited allele in mouse placentas. DLK1/DIO3 domain, DLK1 containing BACs were identi- MRPL23 is located 175 kb upstream of IGF2 in humans fied in both species. One BAC from each species was used and it encodes the mitochondrial ribosomal protein L23. for FISH analysis. DIO3 (Fig 1a) and DLK1 (Fig 1b) This gene does not appear to be imprinted in mammals. mapped to a site on the long arm 1/3 the arm length from Hence the genes selected here fall into three different the centromere of platypus chromosome 1. In tammar functional and regulatory categories which may not have wallaby DLK1 and DIO3 also mapped to the same loca- conserved ancestral linkage. For example the two different tion as shown by FISH analysis with the probes labelled in imprinted subdomains might be separated from each two different colours (Fig 2a). other and/or the unrelated mitochondrial protein. One

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Table 1: Summary of chromosomal locations of genes studied in human, mouse, wallaby, opossum, platypus and chicken

Gene Human location Mouse location Wallaby Opossum Platypus Chicken location location location location

DIO3 14q Distal 12 1q 1 1q 5 DLK1 14q Distal 12 1q 1 1q 5 GNAS 20q Distal 2 1q 1 8p 20 GRB10 7p Proximal 11 3p 6 4p 2 IGF2 11p Distal 7 2p 5q [35] 3p 5 CD81 11p Distal 7 2p Unplaced - 5 MRPL23 11p Distal 7 2p Unplaced - 5 IGF2R 6q Proximal 17 2q 2 Centric 2 3 SLC38A4 12q Distal 15 3q 8 2q 1 UBE3A 15q Central 7 5 [23] 7 18p 1 SNRPB 20p 2 1q [23] 1 - 20

Chicken and opossum locations are taken from the UCSC genome browser except for opossum IGF2 [35] positive BAC for each of these genes was used for FISH instead are replaced by regions homologous to regions which showed that IGF2,CD81 and MRPL23 do indeed other then human 14q13.2 and chicken 5. map together on tammar wallaby chromosome 2p (Fig 2c, 2d and 2g). GNAS is located on platypus 8p in contig16 together with 31 other genes (4 unidentified) all of which have ortho- The location of imprinted orthologues in the chicken by logues on human 20q13, chicken 20, and opossum 1. in-silico methods has been recently published [28]. We Only one gene (Fam38A) is located on human 16q and have also performed an in-silico analysis to identify the chicken 11 and is probably a mistake in this contig assem- chromosomal locations of the imprinted genes in the bly. GRB10 was found on platypus 4p in contig107, which opossum using the UCSC genome browser[29,30]. The contains 13 other genes. All of the genes have orthologues results of this analysis and the FISH mapping are summa- on chicken 2. Three of them have orthologues on human rised in Table 1. 7q36.1 and the other eight are on human 7p12 (4 genes are unidentified). An inversion in the eutherian lineage In silico identification of orthologues and chromosomal separated these genes from each other. In the marsupial locations of genes contiguous with imprinted genes Monodelphis domestica these two gene clusters are not syn- The transcripts of the human imprinted genes were tenic but are localized on different chromosomes (chro- aligned by BLAST to find orthologues within the Ensembl mosome 8, 6 and 3 respectively, Ensembl Opossum Platypus Ornithorhynchus anatinus database release 5. The release 4). IGFR2 and SLC38A4 are found in small contigs platypus contigs in the database contain several predicted with a limited number of genes. genes, which were then identified by blasting to find alignments with the NCBI human genome database. UBE3A is located on platypus 18p in contig121 together Orthologues of these genes were subsequently localised in with 15 other genes (3 unidentified). All of the genes have chicken and opossum by BLAST alignment in Ensembl. orthologues on chicken 1. However, UBE3A is localized The results are shown in Table 1 and Table 2. on human 15q. Two other genes are on human 2q and the remaining genes in this contig are on human Xp21.2 or DLK1 is located on platypus 1q in ultracontig378 which Xp11.4. As these genes are syntenic in platypus and contains 49 predicted genes, most of them with ortho- chicken, this contig represents the ancestral configuration. logues on human 14q, chicken 5, and opossum 1. The Before the marsupial-eutherian split, one fission sepa- three genes that have orthologues elsewhere might be rated the human Xp region from the human 15q and mistakes in the ultracontig assembly. Genes that are human 2q regions; the latter two regions are still together present on either side of DIO3 on human chromosome in opossum. A subsequent fission in the eutherian lineage 14q are mapped in the same platypus ultracontig378. On separated the human 15q and human 2q regions. Unfor- opossum chromosome 1 DLK1 and DIO3 are 1.6 Mb tunately, IGF2, CD81, MRPL23 and SNRPB are not yet rec- apart according to Ensembl-opossum. The platypus ognised in the Ensembl Platypus Ornithorhynchus anatinus ultracontig378 does not correspond to a continuous database 5. region in opossum. The predicted genes between KIAA1622 and GSC are not identified in opossum but This approach identified conserved synteny at the major- ity of extended loci examined. We identified one large

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Table 2: In silico identification of orthologues and chromosomal locations of genes contiguous with imprinted genes.

Platypus Contig Predicted gene Human orthologue Human location Chicken assignment Opossum assignment

1q Ultracontig378 Ox_plat42681 LGMN 14q32.12 5 1 Ox_plat497848 High E-value - - - Nov499641/Nov5433 GOLGA5 14q32.12 5 1 Nov5434 CHGA 14q32.12 5 1 Ox_plat52244 ITPK1 14q32.12 5 1 Ox_plat7773 MOAP1 14q32.13 5 - Ox_plat1058 C14orf130 14q32.13 5 1 Nov0444/Ox_plat88666 BTBD7 14q32.13 5 1 Ox-plat454053/Nov11241/Ox_plat35285 KIAA1409 14q32.13 5 1 Ox_plat548259 High E-value - - - Nov11242/Ox_plat367667 ASB2 14q32.13 5 1 Nov11243 High E-value 14q32.13 - - Nov11244/Ox_plat4560 OTUB2 14q32.13 5 1 Nov11245 KIAA1622 14q32.13 5 1 Ox_plat116474 KIAA1622 14q32.13 5 1 insertion Nov11246/Ox_plat42680 High E-value - - - insertion Nov12040/Ox_plat411980 SERPINA11 14q32.13 5 - insertion Nov12038/Ox_plat468381/Nov12037 High E-value - - - insertion Nov12036 GSC 14q32.13 5 1 insertion Nov12034/Ox_plat475598 DICER1 14q32.13 5 1 Nov12033 CLMN 14q32.13 5 - Nov9154 C14orf49 14q32.13 5 1 Ox_plat409095 High E-value - - - Nov65921/Nov6591 BDKRB2 14q32.2 5 1 Nov65905/Ox_plat42182/Nov6589;6588 C14orf103 14q32.2 5 1 Nov6588 C14orf129 14q32.2 5 1 Nov13812/Ox_plat6298 PAPOLA 14q32.2 5 - Nov12141/Ox_plat6576 VRK1 14q32.2 5 1 Nov14770 BCL11B 14q32.2 5 1 Nov14773 KRT19 17q21.2 27 2 Nov14774/Nov3153/Ox_plat403179 SETD3 14q32.2 5 1 Nov2410 KIAA1822 14q32.2 5 1 Nov7692 CYP46A1 14q32.2 5 - Nov7691 LOC91461 14q32.2 5 - Nov4045/Ox_plat43367 EML1 14q32.2 5 1 Nov12424/Ox_plat123106 DEGS2 14q32.2 5 1 Nov5723/Ox_plat43391 YY1 14q32.2 5 1 Nov5724 SLC25A29 14q32.2 5 1 Nov5726 C14orf68 14q32.2 5 1 Nov5727/Ox_plat494845 WARS 14q32.2 5 1 Nov5730 High E-value - - - * Nov5731 DLK1 14q32.2 5 1 Nov5732/Ox_plat43477 DNAH1 3p21.1 12 6 Nov5733/Ox_plat522828 DYNC1H1 14q32.32 5 1 Nov5734/Ox_plat6302 HSP90AA1 14q32.32 3 - Nov5735/Ox_plat461283 WDR20 14q32.32 5 1 Nov5736/Ox_plat549051/Ox_plat5738/ RAGE 14q32.32 5 1 Ox_plat456837 Nov5740/Ox_plat526165 KIAA0329 14q32.32 5 1 Nov5741 ANKRD9 14q32.32 5 1 Nov9609 KIAA1446 14q32.2 5 - 8p Contig16 Ox_plat6649 CYP24A 20q13.2 20 1 Ox_plat6759 PFDN 20q13.2 20 - Ox_plat44306 DOK 20q13.2 20 - Ox_plat1864 CBLN4 20q13.31 20 1 Ox_plat373291 High E-value - - - Ox_plat6760 CSTF1 20q13.31 20 1 Ox_plat485472 C20orf32 20q13.31 20 1 Ox_plat509072 C20orf43 20q13.31 20 1 Ox_plat21038/Ox_plat49662 High E-value - - - Ox_plat50570 BMP7 20q13.31 20 1 Ox_plat6664 SPO11 20q13.32 20 1 Ox_plat6762 RAE1 20q13.32 20 1 Ox_plat501210 RBM30 20q13.32 20 - Ox_plat21169 CTCFL 20q13.32 20 1 Ox_plat364767 PCK1 20q13.32 20 1 Ox_plat50577 TMEPAI 20q13.32 20 1 Nov 9262 TMEPAI 20q13.32 20 1 Ox_plat21326 C20orf80 20q13.32 20 - Ox_plat21104 RAB22A 20q13.32 20 1 Ox_plat21317 C20orf80 20q13.32 20 - Ox_plat69044 PPP4R1L 20q13.32 20 1

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Table 2: In silico identification of orthologues and chromosomal locations of genes contiguous with imprinted genes. (Continued)

Ox_plat50567 VAPB 20q13.32 20 1 Ox_plat499610 STX16 20q13.32 20 - Nov9272 Fam38A 16q24.3 11 1 Ox_plat499488 C20orf45 20q13.32 20 1 * Nov9275 GNAS 20q13.32 20 1 Ox_plat6777 TUBB1 20q13.32 - 1 Ox_plat21080 High E-value 20q13.32 - - Nov9282 C20orf174 20q13.32 20 1 Ox_plat4456 PHACTR3 20q13.33 20 1 Ox_plat454388 CDH26 20q13.33 - 1 4p Contig107 Ox_plat400863 CUL1 7q36.1 2 8 Ox_plat10731 EZH2 7q36.1 2 3 Nov0280 PDIA4 7q36.1 2 8 Nov0281 High E-value - - - Ox_plat49897 COBL 7p12.1 2 6 * Ox_plat451754/Nov0283 GRB10 7p12.1 2 6 Nov0284 DDC 7p12.2 2 6 Nov0285 FIGNL1 7p12.2 - 6 Ox_plat403769/Nov0286 IKZF1 7p12.2 2 6 Nov0287 ZPBP 7p12.2 2 6 Nov0288 High E-value - - - Ox_plat4844817/Ox_plat467096 High E-value - - - 2 Contig1301 Nov7295 SLC22A2 6q25.3 3 2 * Q9N1T1 IGF2R 6q25.3 3 2 2q Contig538 Nov6345/Nov6346/Ox_plat486528 SLC38A1 12q13.11 1 8 * Nov6348 SLC38A4 12q13.11 1 8 18p Contig121 Ox_plat15397 UBE3A 15q11.2 1 7 Ox_plat498667 MGC26733 2q11.2 1 - Ox_plat3315 TMEM131 2q11.2 1 7 Ox_plat59493 TMEM47 Xp21.2 1 4 Nov7105/Nov7106/Ox_plat390388 High E-value - 1 Ox_plat85743 CXorf22 Xp21.2 1 4 Ox_plat472396 PRRG1 Xp21.2 1 4 Ox_plat1731 XK Xp11.4 1 4 Nov7111 CYBB Xp11.4 1 4 Ox_plat85753 DYNLT3 Xp11.4 1 4 Ox_plat514733 SYTL5 Xp11.4 1 4 Ox_plat7251 SRPX Xp11.4 1 4 Ox_plat375200 RPGR Xp11.4 1 4 Ox_plat1440 OTC Xp11.4 1 4

inversion, and potential errors in the platypus contig X- and five Y chromosomes. Furthermore, X5 carries the assembly. Finally, we determined that the UBE3A region DMRT1 orthologue present on the avian Z and thought to on platypus chromosome 18 and chicken chromosome 1 be sex determining. Platypus X1 was previously thought to represent an ancestral configuration of 15 genes which show homology with the human X (see for example ref during eutherian evolution has undergone fission placing 29), but this is not confirmed by the draft platypus several of them on two regions on the human X chromo- sequence (Ensembl release 44) that instead shows homol- some. ogy to chicken chromosome 3, 11, 12, 13, and Z and human chromosome 2 and 5 (Rens et al submitted). The- Discussion rian X-linked genes mapped to date are predominantly Studies that consider the chromosomal relationships localised to platypus chromosome 6 [31]. The results indi- between autosomal imprinting and dosage compensation cate that the monotreme sex chromosome system is unre- mechanisms in the range of mammals that include lated to the XY sex chromosome system of other monotremes, marsupials, mouse and man are likely to mammals which must have arisen after the divergence of provide insights into the evolution of the mechanisms monotremes 166 MYA. This intriguing system combined involved. In a wider context, this will aid in understanding with an apparent absence of genomic imprinting makes it the evolution of epigenetic controls regulating genome important to localize imprinted genes on platypus chro- function. mosomes in order to consider the evolution of epigeneti- cally regulated dosage compensation systems. These Monotremes, due to their early offshoot from the other localizations also serve to define regions of syntenic mammalian species, are an ideal class for various kinds of homology between vertebrates including monotremes genetic, cytogenetic and epigenetic research. Whereas and eutherian mammals. In addition, the placement of most male mammals have an XY complement and female such genes on the cytogenetic map will contribute to birds have a ZW complement, the male platypus has five

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anchoring the platypus genomic sequence currently being the mechanism of dosage compensation remains to be generated. determined in platypus, the lack of imprinted orthologues on sex chromosomes does not favour the idea that Here we mapped the chromosomal location of imprinted imprinted genes arose as duplications from the X. genes in the platypus and tammar wallaby. Eight imprinted gene orthologues (representing six different This, and the absence of imprinting in the platypus to imprinted mouse/human clusters) localized to 6 different date, suggests that monotreme X chromosome dosage autosomes in the platypus as shown in Fig. 1a–h. In tam- compensation preceded genomic imprinting which sub- mar wallaby eight imprinted gene orthologues (three sequently adopted the same mechanism, or that sex chro- belonging to the BWS region) representing the same six mosomes dosage compensation in monotremes is an imprinted domains were mapped to 5 of the 7 different unrelated event. The latter is more likely since monotreme autosomes. First, the results will be discussed in relation sex chromosomes share no homology with the human X to other genes mapped in platypus and tammar wallaby. (Rens et al submitted). The position of orthologues of Second, the imprinted gene orthologue localization will imprinted genes provides no insight regarding the be discussed in relation to imprinting evolution. hypothesis of co-evolution of X-inactivation and imprint- ing in mammals being associated with placentation [17]. Comparative gene mapping Gene mapping is one of the tools used to define regions The results show that the selected imprinted gene clusters that are conserved between different species. The localiza- are scattered among autosomes in the platypus and tam- tion of orthologues of imprinted genes (red) on platypus mar wallaby karyotypes; the clusters do not group chromosomes is presented in Figure 3a with genes together in either species. Data from comparison of the mapped previously in black [31-34]. Gene mapping data distribution of the imprinted gene orthologues in platy- are still limited in platypus, hence mapping the ortho- pus and tammar wallaby with their locations in the logues of imprinted genes will anchor contigs to specific human karyotype reflects the high number of rearrange- chromosomes and aid in constructing a platypus-human ments that occurred in the lineages of either the homology map. monotremes or placental mammals. The position of genes on the prototherian ancestor will be more relevant The localization of orthologues of imprinted regions in to evaluating the imprinting duplication hypothesis and tammar wallaby is presented in Fig 3b. We show that IGF2 comparing it with data generated here. However, the pro- is located at the telomere of tammar chromosome 2. A totherian ancestral karyotype remains to be determined recent paper placed the M. domestica orthologue of IGF2 and will be assisted by the establishment of a genome on 5q [35] a region that was previously shown to be wide comparison between monotremes/marsupials and equivalent to 6p in tammar [36]. This discrepancy might an outgroup species. be due to the poor resolution of chromosome paints at the telomeres and suggests that there may be a small The SNRPN gene in the PWS/AS cluster arose from a tan- region at the tip of M. domestica 5q which is homologous dem duplication of the SNRPB gene so its syntenic rela- to 2p in the tammar wallaby. It is interesting that GNAS tionship with imprinted GNAS is of interest. The SNRPB and SNRPB are close on tammar wallaby 1p (our results duplication had already occurred when the marsupials and Rapkins et al[26]), which is part of a region that is diverged from the eutherian line as SNRPB and SNRPN conserved in a large set of marsupial species[37]. GNAS is are tandemly arranged in both tammar and opossum. In located on human distal 20p and SNRPB on distal 20q. silico analysis of this region in the chicken shows that there Human chromosome 20 is a chromosome that is con- is only one copy of SNRPB and that it is only 166.9 kb served in all eutherian mammals, the mapping of GNAS away from GNAS on chromosome 20 implying that these and SNRPB indicates that it is conserved in marsupials as genes were close in the ancestral mammalian karyotype. well. The four homologous regions mapped in this report add to the complexity of the rearrangements that have In-silico analysis reveals that SNRPB and GNAS are 36.6 occurred during chromosome evolution between human Mb apart in the opossum and 54.5 Mb apart in human. and tammar wallaby. For instance, tammar wallaby chro- Therefore although these two genes are located on the mosome 1 has regions homologous to human 5, 7, 9, 10, same chromosome they have become separated by one or 14, 16, 20 and X (our results and Alsop et al.[25]) more inversions. Furthermore, in opossum, tammar, plat- ypus, chicken and zebrafish, the PWS/AS genes SNRPN Imprinted gene orthologue localization and UBE3A are on separate chromosomes and are The overall conclusion made from the mapping data is expressed biallelically in tammar [26]. Together these that the orthologues of these imprinted genes are not findings suggest that imprinted regulation was acquired found on sex chromosomes in either species. Although after the loss of close synteny with GNAS and a major rear-

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rangement that united SNRPN and UBE3A. However, it ments were cloned into pCR® 2.1-TOPO® (Invitrogen) also remains theoretically possible that the SNRPN and using the manufacturers protocol. DNA from the plas- UBE3A genes lost imprinting in the macropodid lineage mids was prepared using GeneElute™ Plasmid Miniprep and their imprinting state is ancestral for therians. Kit (Sigma) then sequenced to confirm its identity.

Conclusion BAC Isolation The combined data of chicken, marsupial and platypus The OA_Bb Platypus BAC library (Clemson University gene position suggest that imprinted gene orthologues Genomics Institute, South Carolina, USA) and the ME have existed on separate chromosomes since before _KBa Tammar wallaby BAC library (Arizona Genomics imprinting evolved. This makes the hypothesis, that there Institute, USA) were screened with [#-32P] dCTP (Amer- was a single or small number of ancestrally imprinted sham Pharmacia Biotech) labelled PCR products. Label- chromosomes, unlikely. The observation that some ling was performed under the following conditions 94°C imprinted domains in mouse and human are not for 5 minutes, 25 cycles of 93°C for 30 sec, 50°C for 30 imprinted in marsupials, suggests that imprinting was a sec 72°C for 30 sec and 1 cycle of 72°C for 5 min. Probes step wise process during evolution beginning after the were denatured at 99°C for 5 min and snap chilled before evolution of viviparity and continuing convergently in the hybridisation. The library membranes were hybridised marsupial and eutherian lineages. Thus the evolution of and washed at low stringency (55°C). They were then imprinting has most likely been a long process with each exposed to X-ray film at -70°C overnight. BACs were cluster independently evolving or indeed losing, its streaked to single colony and tested by PCR with their imprinting mechanisms as the need arose. This suggests identifying primers to ensure they contained the correct an element of adaptation in the process of imprinting evo- gene. lution. Preparation of BAC Probes Methods BAC DNA was isolated using the protocol described at the Amplification and sequence analysis Wellcome Trust Sanger Institute methods website [42]. The published coding sequences of the genes of interest The DNA probes were labelled by nick translation with were obtained from as many species as possible from Ent- Biotin-16-dUTP using a standard protocol. rez Gene at the NCBI webpage [38] [Additional File 1]. Sequences were then aligned to each other using the Clus- Localization of DNA probes talW program [39,40] and PCR Primers designed to the Chromosome specific DNA was prepared from flow- regions of greatest homology within the same exon. sorted platypus chromosomes and fluorescence in situ hybridization was performed according to protocols Platypus genomic DNA (gDNA) was extracted from pri- described previously [7,43]. The labelled DNA probes mary fibroblasts using standard protocols [41]. IGF2, (and chromosome paints for chromosome identification) DIO3 and SLC38A4 were amplified in a 15 !l reaction were hybridized to male platypus and wallaby chromo- containing 1× NEB buffer [42], 500 !M dNTPs, 2.5 !g BSA some preparations and detected with Cy3-avidin. (Sigma), 0.067% v/v "-mercaptoethanol, 0.6 U Taq polymerase (Applied Biosystems), 0.75 !M of each primer Image analysis and 50 ng gDNA. GNAS, GRB10 and IGF2R were ampli- Images were captured using the Leica QFISH software fied in a 25 !l reaction containing 1× PCR Buffer (Bio- (Leica Microsystems) and a cooled CCD camera (Photo- line), 1.5 mM MgCl, 250 !M dNTPs, 1.5 U Taq metrics Sensys) mounted on a Leica DMRXA microscope polymerase (Bioline), 0.3 !M of each primer and 50 ng equipped with an automated filter wheel, DAPI, FITC, and gDNA. PCR cycling was, 94°C for 5 min, 35 cycles at 94°C Cy3 specific filter sets and a 63×, 1.3 NA objective or for 30 sec, annealing temperature (specific for each primer 100×, 1.4 NA objective. see table 1) for 30 sec, 72°C for 30 sec and 5 min at 72°C. UBE3A was amplified in a 25 !l reaction containing 1× Abbreviations PCR Buffer (KOD Hot Start, Novagen), 300 !M dNTPs, 1 MYA – Million Years Ago mM MgSO4, 0.5 U Hot Start KOD polymerase, 0.6 !M of each primer and 50 ng gDNA. PCR cycling was 94°C for 2 XCI – X Chromosome Inactivation min, 31 cycles of 94°C for 15 sec, 60°C for 30 sec, 68°C for 30 sec, then 5 minute at 68°C. BAC – Bacterial Artificial Chromosome

The PCR products were separated by electrophoresis and FISH – Fluorescent In-situ Hybridisation the appropriately sized fragments were excised and cleaned (Qiaquick Gel Extraction Kit; Qiagen). These frag-

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Authors' contributions 13. Takagi N, Sasaki M: Preferential inactivation of the paternally derived X chromosome in the extraembryonic membranes CAE and AM conducted BAC library screening and probe of the mouse. Nature 1975, 256:640-642. characterisation, WR and OC conducted FISH experi- 14. Xue F, Tian XC, Du F, Kubota C, Taneja M, Dinnyes A, Dai Y, Levine ments, and TH conducted gene characterisation. CAE and H, Pereira LV, Yang X: Aberrant patterns of X chromosome inactivation in bovine clones. Nat Genet 2002, 31:216-220. WR carried out in silico analysis. CAE, WR, AFS drafted the 15. Okamoto I, Otte AP, Allis CD, Reinberg D, Heard E: Epigenetic manuscript, ID, MAFS and JMG contributed reagents and dynamics of imprinted X inactivation during early mouse provided input to the manuscript, AFS and MAFS con- development. Science 2004, 303:644-649. 16. Mak W, Nesterova TB, de Napoles M, Appanah R, Yamanaka S, Otte ceived, designed and coordinated the study. AP, Brockdorff N: Reactivation of the paternal X chromosome in early mouse embryos. Science 2004, 303:666-669. 17. Reik W, Lewis A: Co-evolution of X-chromosome inactivation Additional material and imprinting in mammals. Nat Rev Genet 2005, 6:403-410. 18. Duret L, Chureau C, Samain S, Weissenbach J, Avner P: The Xist RNA gene evolved in eutherians by pseudogenization of a Additional file 1 protein-coding gene. Science 2006, 312:1653-1655. Probes used for BAC library screening, shows the number of BACs identi- 19. Hore T, Koina E, Wakefield M, Graves JAM: XIST is absent from the X chromosome, and its flanking region is disrupted in fied by each probe. non-placental mammals. Chromosome Res 2007. Click here for file 20. Walter J, Paulsen M: The potential role of gene duplications in [http://www.biomedcentral.com/content/supplementary/1471- the evolution of imprinting mechanisms. Hum Mol Genet 2003, 2148-7-157-S1.doc] 12 Spec No 2:R215-20. 21. Weidman JR, Murphy SK, Nolan CM, Dietrich FS, Jirtle RL: Phyloge- netic footprint analysis of IGF2 in extant mammals. Genome Res 2004, 14:1726-1732. 22. Nolan CM, Killian JK, Petitte JN, Jirtle RL: Imprint status of M6P/ IGF2R and IGF2 in chickens. Dev Genes Evol 2001, 211:179-183. Acknowledgements 23. Barlow DP, Stoger R, Herrmann BG, Saito K, Schweifer N: The CAE is funded by an MRC studentship. AFS is an associate member of the mouse insulin-like growth factor type-2 receptor is FP6 Epigenome Network of Excellence. We are grateful to the other mem- imprinted and closely linked to the Tme locus. Nature 1991, bers of the SAVOIR consortium, including Wolf Reik and Gavin Kelsey for 349:84-87. 24. Lewis A, Mitsuya K, Umlauf D, Smith P, Dean W, Walter J, Higgins M, helpful discussions during the course of this work. The research was sup- Feil R, Reik W: Imprinting on distal chromosome 7 in the pla- ported by a grant from the Wellcome Trust to the Cambridge Resource centa involves repressive histone methylation independent Centre for Comparative Genomics. of DNA methylation. Nat Genet 2004, 36:1291-1295. 25. Alsop AE, Miethke P, Rofe R, Koina E, Sankovic N, Deakin JE, Haines H, Rapkins RW, Marshall Graves JA: Characterizing the chromo- References somes of the Australian model marsupial Macropus eugenii 1. Da Rocha ST, Ferguson-Smith AC: Genomic imprinting. Curr Biol (tammar wallaby). Chromosome Res 2005, 13:627-636. 2004, 14:R646-9. 26. Rapkins RW, Hore T, Smithwick M, Ager E, Pask AJ, Renfree MB, 2. Killian JK, Nolan CM, Stewart N, Munday BL, Andersen NA, Nicol S, Kohn M, Hameister H, Nicholls RD, Deakin JE, Graves JA: Recent Jirtle RL: Monotreme IGF2 expression and ancestral origin of assembly of an imprinted domain from non-imprinted com- genomic imprinting. J Exp Zool 2001, 291:205-212. ponents. PLoS Genet 2006, 2:e182. 3. Killian JK, Byrd JC, Jirtle JV, Munday BL, Stoskopf MK, MacDonald RG, 27. Toder R, Wilcox SA, Smithwick M, Graves JA: The human/mouse Jirtle RL: M6P/IGF2R imprinting evolution in mammals. Mol imprinted genes IGF2, H19, SNRPN and ZNF127 map to Cell 2000, 5:707-716. two conserved autosomal clusters in a marsupial. Chromo- 4. O'Neill MJ, Ingram RS, Vrana PB, Tilghman SM: Allelic expression some Res 1996, 4:295-300. of IGF2 in marsupials and birds. Dev Genes Evol 2000, 210:18-20. 28. Dunzinger U, Nanda I, Schmid M, Haaf T, Zechner U: Chicken 5. Suzuki S, Renfree MB, Pask AJ, Shaw G, Kobayashi S, Kohda T, orthologues of mammalian imprinted genes are clustered on Kaneko-Ishino T, Ishino F: Genomic imprinting of IGF2, macrochromosomes and replicate asynchronously. Trends p57(KIP2) and PEG1/MEST in a marsupial, the tammar wal- Genet 2005, 21:488-492. laby. Mech Dev 2005, 122:213-222. 29. UCSC Genome Browser [http://genome.ucsc.edu/] 6. Bininda-Emonds OR, Cardillo M, Jones KE, MacPhee RD, Beck RM, 30. Karolchik D, Baertsch R, Diekhans M, Furey TS, Hinrichs A, Lu YT, Grenyer R, Price SA, Vos RA, Gittleman JL, Purvis A: The delayed Roskin KM, Schwartz M, Sugnet CW, Thomas DJ, Weber RJ, Haussler rise of present-day mammals. Nature 2007, 446:507-512. D, Kent WJ: The UCSC Genome Browser Database. Nucleic 7. Rens W, Grutzner F, O'Brien P C, Fairclough H, Graves JA, Ferguson- Acids Res 2003, 31:51-54. Smith MA: Resolution and evolution of the duck-billed platy- 31. Waters PD, Delbridge ML, Deakin JE, El-Mogharbel N, Kirby PJ, Car- pus karyotype with an X1Y1X2Y2X3Y3X4Y4X5Y5 male sex valho-Silva DR, Graves JA: Autosomal location of genes from chromosome constitution. Proc Natl Acad Sci U S A 2004, the conserved mammalian X in the platypus (Ornithorhyn- 101:16257-16261. chus anatinus): implications for mammalian sex chromo- 8. Grutzner F, Rens W, Tsend-Ayush E, El-Mogharbel N, O'Brien PC, some evolution. Chromosome Res 2005, 13:401-410. Jones RC, Ferguson-Smith MA, Marshall Graves JA: In the platypus 32. Wallis MC, Delbridge ML, Pask AJ, Alsop AE, Grutzner F, O'Brien PC, a meiotic chain of ten sex chromosomes shares genes with Rens W, Ferguson-Smith MA, Graves JA: Mapping platypus SOX the bird Z and mammal X chromosomes. Nature 2004, genes; autosomal location of SOX9 excludes it from sex 432:913-917. determining role. Cytogenet Genome Res 2007, 116:232-234. 9. Cattanach BM, Beechey CV: Autosomal and X-chromosome 33. Delbridge ML, Wallis MC, Kirby PJ, Alsop AE, Grutzner F, Graves JA: imprinting. Dev Suppl 1990:63-72. Assignment of SOX1 to platypus chromosome 20q by fluo- 10. Lee JT: Molecular links between X-inactivation and auto- rescence in situ hybridization. Cytogenet Genome Res 2006, somal imprinting: X-inactivation as a driving force for the 112:342L. evolution of imprinting? Curr Biol 2003, 13:R242-54. 34. Kirby PJ, Waters PD, Delbridge M, Svartman M, Stewart AN, Nagai 11. Sharman GB: Late DNA replication in the paternally derived K, Graves JA: Cloning and mapping of platypus SOX2 and X chromosome of female kangaroos. Nature 1971, SOX14: insights into SOX group B evolution. Cytogenet 230:231-232. Genome Res 2002, 98:96-100. 12. Richardson BJ, Czuppon AB, Sharman GB: Inheritance of glucose- 6-phosphate dehydrogenase variation in kangaroos. Nat New Biol 1971, 230:154-155.

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35. Lawton BR, Obergfell C, O'Neill RJ, O'Neill MJ: Physical mapping of the IGF2 locus in the South American opossum Monodel- phis domestica. Cytogenet Genome Res 2007, 116:130-131. 36. Rens W, O'Brien PC, Yang F, Solanky N, Perelman P, Graphodatsky AS, Ferguson MW, Svartman M, De Leo AA, Graves JA, Ferguson- Smith MA: Karyotype relationships between distantly related marsupials from South America and Australia. Chromosome Res 2001, 9:301-308. 37. Rens W, O'Brien PC, Fairclough H, Harman L, Graves JA, Ferguson- Smith MA: Reversal and convergence in marsupial chromo- some evolution. Cytogenet Genome Res 2003, 102:282-290. 38. NCBI Entrez Gene [http://www.ncbi.nlm.nih.gov/sites/ent rez?db=gene] 39. ClustalW [http://www.ebi.ac.uk/clustalw/index.html] 40. Thompson JD, Higgins DG, Gibson TJ: CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice. Nucleic Acids Res 1994, 22:4673-4680. 41. Sambrook J, MacCallum P, Russell D: Molecular Cloning: A Labo- ratory Manual. Third edition. , CSHL Press; 2001. 42. Wellcome Trust Sanger Institute - Human Genome Meth- ods [http://www.sanger.ac.uk/HGP/methods/] 43. Rens W, O'Brien PC, Yang F, Graves JA, Ferguson-Smith MA: Kary- otype relationships between four distantly related marsupi- als revealed by reciprocal chromosome painting. Chromosome Res 1999, 7:461-474. 44. Grutzner F, Deakin J, Rens W, El-Mogharbel N, Marshall Graves JA: The monotreme genome: a patchwork of reptile, mammal and unique features? Comp Biochem Physiol A Mol Integr Physiol 2003, 136:867-881.

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APPENDIX 3. THE STATUS OF DOSAGE COMPENSATION IN THE MULTIPLE X CHROMOSOMES OF THE PLATYPUS

The following appendix is a publication from mid-2008 that reports for the first time that female platypus do undergo dosage compensation of genes borne on the five X chromosomes, and that this involves an incomplete form of random X inactivation.

Deakin JE, Hore TA, Koina EK, Graves JAM (2008) The status of dosage compensation in the multiple X chromosomes of the platypus. PLoS Genet 4(7):e1000140.

My contribution to this publication consisted of the identification of single nucleotide polymorphisms from multiple X chromosomes of the platypus individual (‘Glennie’) that had its entire genome sequenced (Warren et al., 2008). These polymorphisms were critical for RT-PCR experiments performed by Janine Deakin to show that the X inactivation observed on platypus sex chromosomes is random (ie. no allelic bias at the tissue level) as opposed to imprinted or non-random.

173 The Status of Dosage Compensation in the Multiple X Chromosomes of the Platypus

Janine E. Deakin*, Timothy A. Hore, Edda Koina, Jennifer A. Marshall Graves Research School of Biological Sciences, The Australian National University, Canberra, Australia

Abstract Dosage compensation has been thought to be a ubiquitous property of sex chromosomes that are represented differently in males and females. The expression of most X-borne genes is equalized between XX females and XY males in therian mammals (marsupials and ‘‘placentals’’) by inactivating one X chromosome in female somatic cells. However, compensation seems not to be strictly required to equalize the expression of most Z-borne genes between ZZ male and ZW female birds. Whether dosage compensation operates in the third mammal lineage, the egg-laying monotremes, is of considerable interest, since the platypus has a complex sex chromosome system in which five X and five Y chromosomes share considerable genetic homology with the chicken ZW sex chromosome pair, but not with therian XY chromosomes. The assignment of genes to four platypus X chromosomes allowed us to examine X dosage compensation in this unique species. Quantitative PCR showed a range of compensation, but SNP analysis of several X-borne genes showed that both alleles are transcribed in a heterozygous female. Transcription of 14 BACs representing 19 X-borne genes was examined by RNA-FISH in female and male fibroblasts. An autosomal control gene was expressed from both alleles in nearly all nuclei, and four pseudoautosomal BACs were usually expressed from both alleles in male as well as female nuclei, showing that their Y loci are active. However, nine X-specific BACs were usually transcribed from only one allele. This suggests that while some genes on the platypus X are not dosage compensated, other genes do show some form of compensation via stochastic transcriptional inhibition, perhaps representing an ancestral system that evolved to be more tightly controlled in placental mammals such as human and mouse.

Citation: Deakin JE, Hore TA, Koina E, Graves JAM (2008) The Status of Dosage Compensation in the Multiple X Chromosomes of the Platypus. PLoS Genet 4(7): e1000140. doi:10.1371/journal.pgen.1000140 Editor: Jeannie T. Lee, Massachusetts General Hospital, Howard Hughes Medical Institute, United States of America Received March 31, 2008; Accepted June 24, 2008; Published July 25, 2008 Copyright: ß 2008 Deakin et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This work was supported by a block grant from The Research School of Biological Sciences at Australian National University. Competing Interests: The authors have declared that no competing interests exist. * E-mail: [email protected]

Introduction The sex chromosomes of therian mammals are remarkably conserved. The X chromosomes of all placental mammals have Monotremes are unique mammals that exhibit a mix of reptilian virtually identical gene contents, and the marsupial X chromo- and mammalian features, as they lay eggs, yet have fur and some shares two thirds of the human X, defining it as the ancient produce milk for their young. Represented only by the fabled X conserved region [8]. The largest platypus X was also thought platypus and four species of echidna, they are distantly related to to share this ancient region [9]. However, comparisons of the gene humans and other eutherian (‘placental’) mammals, having contents of platypus, human and marsupial sex chromosomes diverged from therian mammals (eutherians and marsupials) 166 reveal that the ancient region of the therian X is entirely million years ago (MYA) [1]. homologous to platypus chromosome 6 [6]. Instead, platypus X Monotreme genomes also show a curious mixture of reptilian and chromosomes share considerable homology with the chicken Z mammalian characteristics. They have a smaller genome than chromosome, including DMRT1, a dosage-sensitive gene that is a therian mammals [2], and their karyotype comprises a few large candidate for bird sex determination [6,10]. chromosomes, and many small ones, somewhat reminiscent of The monotreme sex chromosome complex is proposed to have chicken macro and microchromosomes. Most curious of all is the sex evolved by repeated autosome translocation onto an original bird- chromosome system of monotremes. Although monotremes, like like ZW pair [5,11]. The possession of a chain of nine sex other mammals, subscribe to an XY system of male heterogamety, chromosomes by the echidna, seven of which are shared with they have multiple X and Y chromosomes [3] which form a platypus [12], means that the chain is at least 30 M years old. How multivalent translocation chain during meiosis [4]. Platypus a ZW system of female heterogamety was transformed into an XY (Ornithorhynchus anatinus) have ten sex chromosomes; males have five system of male heterogamety has been vigorously debated [13]. X chromosomes (X1X2X3X4X5) and five Y chromosomes Mammalian Y chromosomes are much smaller and more (Y1Y2Y3Y4Y5), and females five pairs of X chromosomes [5]. variable than their X chromosome partners, but share homology During male meiosis, X and Y chromosomes pair within terminal within pseudoautosomal regions, and also between coding genes pseudoautosomal regions [6], forming a chain of alternating X and Y on the X and Y. This supports the theory that heteromorphic sex chromosomes (numbered by their order in the chain X1–Y1–X2–Y2– chromosomes evolved from a pair of homologous autosomes in a X3–Y3–X4–Y4–X5–Y5) which segregate into five X-bearing (female- mammal ancestor after one member of the pair acquired a sex determining) and five Y-bearing (male-determining) sperm [7]. determining locus, which lead to suppression of recombination

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Author Summary array of epigenetic mechanisms, including binding with variant histones [31], histone modifications [32,33] and differential DNA Dosage compensation equalizes the expression of genes methylation [34,35], contribute to the transcriptional silencing of the found on sex chromosomes so that they are equally X-borne genes. An accumulation of LINE1 elements may provide expressed in females and males. In placental and marsupial ‘‘booster stations’’ for the propagation of silencing signal along the mammals, this is accomplished by silencing one of the two chromosome [36]. The molecular mechanism of X inactivation X chromosomes in female cells. In birds, dosage compen- seems to be much simpler in marsupials. The region homologous to sation seems not to be strictly required to balance the the XIC in eutherians is disrupted in marsupials and monotremes expression of most genes on the Z chromosome between and no evidence of XIST has been found in the regions that ZZ males and ZW females. Whether dosage compensation juxtapose flanking markers [37–39]. XIST may have evolved in exists in the third group of mammals, the egg-laying eutherians from relics of an ancient protein-coding gene [40]. monotremes, is of considerable interest, particularly since the platypus has five different X and five different Y Molecular mechanisms shared between marsupial and eutherian chromosomes. As part of the platypus genome project, inactivation so far have been limited to late replication [41] and genes have now been assigned to four of the five X histone underacetylation of the inactive X [42]; DNA methylation chromosomes. We have shown that there is some does not seem to be involved in marsupial X inactivation [43]. evidence for dosage compensation, but it is variable It was suggested that marsupial X inactivation might represent an between genes. Most interesting are our results showing ancestral form of paternally imprinted X inactivation [26,44], and that there is a difference in the probability of expression this hypothesis is supported by imprinted inactivation in mouse for X-specific genes, with about 50% of female cells having extra-embryonic tissues [45], which, like marsupial X inactivation, is two active copies of an X gene while the remainder have less stable and incomplete, and does not involve DNA methylation only one. This means that, although the platypus has the [46]. However, unlike marsupials, this imprinted X inactivation in variable compensation characteristic of birds, it also has mice requires Xist [47,48]. The XIC, along with an accumulation of some level of inactivation, which is characteristic of dosage LINE1 elements on the X, may control random inactivation in compensation in other mammals. eutherians and its absence correlates to the absence of XIST and LINE1 accumulation on the marsupial X [49]. and ultimately resulted in differentiation between members of the The dosage difference for Z-borne genes between ZZ male and pair (reviewed in [14,15]). A similar scenario is proposed for the ZW female birds is equally as extreme as for the mammal X. Yet evolution of the bird Z and W from an ancient autosomal pair [16]. birds do not appear to achieve dosage compensation by silencing Comparative gene mapping between the mammal X and bird Z one Z chromosome in males, since both alleles can be [17,18] shows that they arose from different autosomal pairs. demonstrated to be active by RNA-FISH and SNP analysis Although they are non-homologous, the XY of therians and ZW [50,51]. Quantitative PCR showed that nine of ten Z-borne genes of birds do possess similar general properties. The bird Z, like the have a male-female ratio close to 1:1 [52], but in microarrays, 40 mammal X, is highly conserved between species [18], whereas the W zebrafinch and 964 chicken Z-borne genes showed a range of male is degraded to different extents in different bird groups. Also, the bird to female ratios from 2:1 (,10% of genes) to 1:1 (,10% of genes), Z and the mammal X are large chromosomes carrying many genes, with a mode in the middle [53]. In chicken embryos, the mean and are well conserved between species, whereas the heterogametic male to female ratio is 1.4–1.6 for Z-linked genes, consistent with chromosome (W and Y) is small, heterochromatic and varies greatly an absence of complete dosage compensation [54]. This in size and gene content. The X and Z chromosomes both appear to incomplete dosage compensation suggests that differences in gene have sex-biased gene content. For example, the human X dosage may be critical for only a few genes on the bird Z chromosome is enriched with genes involved in brain function, sex compared to the mammal X. and reproduction [19–21], and in male (but not female) specific The molecular mechanisms behind bird dosage compensation genes [22], and the chicken Z is enriched with genes involved in male are yet to be elucidated. Differences in male to female ratios (but not female) reproduction [23]. between Z linked genes suggest that at least some are regulated at Despite these similarities between the mammal XY and the bird the transcriptional level. A region on the short arm of the Z ZW sex chromosome systems, the extent to which genes on the X chromosome containing over 200 copies of a 2.2 kb repetitive and Z are dosage compensated is remarkably different. X sequence called MHM (male hypermethylated), is hypermethy- chromosome inactivation overcomes differences in gene dosage lated on the Z chromosomes in male embryos, but hypomethy- between XX females and XY males in therian mammals. In lated on the Z in females [55]. MHM is transcribed only in females somatic cells of female humans and mice, genes on one X become and accumulates as non-coding RNA near the DMRT1 locus in genetically inactive [24] and transcriptionally silenced [25] early in the nucleus. A higher proportion of genes subject to dosage embryogenesis, a state that is somatically heritable. In marsupials, compensation are clustered in this MHM region [56]. This too, genes on one X chromosome are inactivated [26]. suggests that dosage compensation in birds is via upregulation of X inactivation mechanisms in eutherians and marsupials differ gene expression in females, controlled by MHM [57]. in a number of important aspects. In somatic cells of eutherians, The platypus presents a fascinating system in which to study inactivation is random between maternally and paternally derived dosage compensation. The need for such a system would appear to X chromosomes, whereas in marsupials only the paternal X is be acute, since the five X chromosomes of the complex account for silenced. X inactivation in eutherians is more stable and complete 15% of the haploid genome, and are mostly unpaired by the five Y than in marsupials [26], although it was recently discovered that chromosomes, which together account for only 6%, and are at between 5% [27] and 15% [28] of genes on the human X escape least half heterochromatic. Thus 12% of the genome is subject to inactivation, mostly on the region added recently to the X in the 1:2 dosage differences. The homology of the platypus sex eutherian lineage [28]. chromosomes with the bird Z, and lack of homology with the At the molecular level, eutherian X inactivation results from a mammal X, raises questions of whether dosage compensation is complex process controlled by a master locus (the X inactivation incomplete and bird-like, or related to the mammal X inactivation centre XIC), which includes the non-coding XIST gene [29,30]. An system–or is completely different from both.

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There are almost no studies of dosage compensation in However, for three X-specific genes, the ratio was near 0.5, which monotremes, and none using any molecular techniques. Early would be expected if the genes were not compensated between XY studies of replication timing of platypus X1 found no asynchronous males and XX females. Two genes had intermediate ratios (,0.7), replication of the unpaired region of this chromosome [58]. This suggesting partial dosage compensation (Table 2). Statistical tests suggests that if the platypus does compensate for gene dosage, it is of the null hypothesis that there is no difference in expression levels unlikely to do so by X inactivation. Determining whether the between males and females, were compromised by the high platypus X chromosomes are dosage compensated has previously variability between individuals, which resulted in p-values been difficult in the absence of knowledge of the genes on platypus supporting the null hypothesis (p = 0.05) for all X-specific genes. X chromosomes. This variation could not be attributed to particular cell lines The assignment of genes to four of the five X chromosomes as consistently showing higher or lower expression for the different part of the platypus genome project now presents an opportunity genes tested (see Figure S1). The trend towards a higher level of to investigate dosage compensation in this species. We used three expression in females than in males for X-specific genes suggests different approaches to determine activity of genes located on four that different genes may be incompletely compensated to different of the five platypus X chromosomes, and present evidence of extents. significant transcriptional silencing of platypus X-borne genes. SNP Identification and Expression Results We used a bioinformatics approach to identify SNPs in genes on We used quantitative real-time RT-PCR, SNPs (Single four of the five platypus X chromosomes (details in Materials and Nucleotide Polymorphisms) and RNA fluorescence in situ hybrid- Methods). We searched the Ensembl database for exonic sequence ization (RNA-FISH) to examine dosage compensation in the from predicted genes on platypus chromosomes X1, X2, X3 and platypus. First we gained an overall assessment of the level of X5 and compared these to platypus whole genome traces. Within dosage compensation by comparing the amounts of transcript these alignments we searched for single nucleotide mismatches from X-specific, autosomal and pseudoautosomal genes in males appearing more than once at the same site. Possible SNPs were and females using quantitative real-time RT-PCR. We then identified SNPs within the sequence of X-borne genes to Table 1. Genes contained within BACs mapped to X determine if they are expressed from both alleles, or only one, as chromosomes as part of genome sequencing project. would be expected from imprinted X inactivation. Finally, we used RNA-FISH to examine the probability of transcription from the two alleles in female and male cells. BAC Chromosome Gene Expression

Determination of Male:Female Expression Ratios by qRT- 636L7 X1/Y1 CRIM1 + PCR 286H10 X1/Y1 CAMK2A + We determined male to female gene expression ratios for two SLC6A7 + autosomal genes and 19 genes on platypus X1, X2, X3 and X5, 10 CDX1 + of which are X-specific and nine pseudoautosomal (shared with EN022941 + the Y chromosomes adjacent in the meiotic translocation chain). # 4D21 X1 Ox_plat_124086 + Genes chosen were from BAC (Bacterial Artificial Chromosome) 271I19 X /Y JARID2 + clones mapped to platypus X chromosomes as part of the genome 2 2 project [6], as this localization indicated directly whether genes DTNBP1 + were X-specific or pseudoautosomal. BAC-end sequences from 650K19 X2/Y2 GMDS +

mapped BACs were aligned to the genome to reveal the genomic 158M16 X3 APC + sequence contained within each BAC. Genes within BACs were 165F5 X3/Y2 IRX1 + identified using the platypus genome Ensembl database (http://www. 830M18 X EN149971 + ensembl.org/Ornithorhynchus_anatinus/index.html) (Oana5.0). 5 The presence of these genes within the BACs was confirmed OaBb_24M14 X5 DMRT2 + by PCR and sequencing, and expression of these genes in DMRT3* 2 fibroblasts was determined (Table 1). We used RNA isolated DMRT1* 2

from independently derived primary fibroblast cell lines 54B19 X5 FBXO10 + representing 16 different individuals (eight males and eight 22O3 X5 SHB + females). Expression of these genes was normalized to the expression levels of the housekeeping gene ACTB, an autosomal 752F12 X5 SEMA6A + gene located on platypus chromosome 2. 271G4 X5 SLC1A1 + Male to female ratios were calculated for the normalized data 236A5 X5 ZNF474 + for each gene. The ratio was near 1 for both autosomal control LOX + genes (G6PD and HPRT1) on platypus chromosome 6. We also measured expression levels for nine pseudoautosomal genes with Expression detected in fibroblasts is indicated (+ expressed in fibroblasts; 2 indicates no detectable expression in fibroblasts). Ensembl gene identifiers have copies on X and Y. The expression ratios of seven genes were high been provided for genes not named in the Ensembl gene build (Jan. 2007). (0.86–1.49), indicating that the Y-borne, as well as the X-borne, Unless otherwise stated, BAC clones are from the CHORI-236 female platypus alleles are active. However, two pseudoautosomal genes (CDX1 BAC library. # and GMDS) had ratios of about 0.5, suggesting that the Y locus is Identifier assigned by the Oxford Functional Genomics group gene build. *Expression data from [10] not active. 1These gene names have been abbreviated from the Ensembl gene build For five of the ten X-specific genes, ratios were high (0.81–0.99), designations ENSOANG00000002294, and ENSOANG00000004997. as would be expected if genes were largely or fully compensated. doi:10.1371/journal.pgen.1000140.t001

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Table 2. Male:female ratio for expression of platypus X genes Table 3. Genes with SNPs, identified from the genome in fibroblast cells normalized to the autosomal ACTB sequence and validated by PCR and sequencing. housekeeping gene.

Gene Chromosome SNP Expressed in Fibroblasts Gene Chromosome Male:Female Ratio p-value CCNG1 X1/Y1 C/T + Autosomal GABRB2 X1/Y1 C/A + G6PD 6 1.12 0.21 SYNPO X1/Y1 C/T + HPRT1 6 0.97 0.96 GMDS X2/Y2 C/T + Pseudoautosomal ADAMTS16 X3 C/T 2 CRIM1 X /Y 1.15 0.50 1 1 FRMPD1 X5 C/T 2 CAMK2A X /Y 1.39 0.11 1 1 ACO1 X5 G/T 2 CDX1 X /Y 0.50 0.01 1 1 FBXO10 X5 A/C + EN02294 X /Y 1.48 0.57 1 1 EN14997 X5 G/T + SLC6A7 X /Y 1.49 0.06 1 1 SHB X5 A/G +

DTNBP1 X2/Y2 0.86 0.47 + indicates expression detected in fibroblasts; 2 indicates no detectable JARID2 X2/Y2 0.93 0.88 expression in fibroblasts.

GMDS X2/Y2 0.48 0.05 doi:10.1371/journal.pgen.1000140.t003

IRX1 X3/Y2 0.98 0.85 X-specific pseudoautosomal gene GMDS and the X specific genes. No significant difference from a 1:1 ratio was observed, implying the Ox_plat_124086 X1 0.91 0.45 absence of imprinting (Table 4 and Figure S2). Biallelic expression APC X3 0.85 0.76 with equivalent expression from alternate alleles for the three X- SHB X 0.81 0.43 5 specific genes eliminates the possibility that genes on platypus X5 LOX X5 0.94 0.88 are subjected to complete paternal inactivation (as is observed in

EN14997 X5 0.71 0.18 marsupials), and directed our approaches to examining the probability of transcription from the two loci by RNA-FISH. FBXO10 X5 0.73 0.32 SLC1A1 X 0.36 0.07 5 RNA-FISH Detection of Primary Transcripts ZNF474 X 0.99 0.69 5 RNA-FISH detects the sites of primary transcription in DMRT2 X5 0.49 0.10 interphase cells by hybridization with large intronic sequences SEMA6A X5 0.55 0.14 that are spliced from cytoplasmic mRNA. Thus large genomic probes were required for the genes of interest. doi:10.1371/journal.pgen.1000140.t002 BAC clones mapped to platypus X chromosomes as part of the genome project and found to contain genes expressed in fibroblast, found in the platypus genome sequence within 57 genes on were used for RNA-FISH experiments (Table 1). These included platypus chromosomes X1 (29), X2 (6), X3 (6) and X5 (16). We the four clones discussed above (one from X2 and three from X5). validated a subset of these SNPs by sequencing PCR products We also included BAC OaBb_24M14 (GenBank Accession derived from genomic DNA isolated from the same female animal No. AC152941) containing DMRT2, which had been fully (‘‘Glennie’’) used for the genome sequencing project and tested sequenced previously and whose expression had been confirmed expression of these genes in fibroblast RNA isolated from this same in fibroblast cell lines [10]. A BAC containing the HPRT1 gene individual. Of ten genes tested, seven were found to be expressed located on chromosome 6, OaBb_405M2 (GenBank Accession in fibroblasts (ss76901227–ss76901236) (Table 3). No. AC148426), was used as an autosomal control. HPRT1 was BAC clones for these seven potentially X-specific SNP- detected in the platypus fibroblast EST library sequenced as part containing genes were isolated, by using sequence up to 100 kb of the genome project (GenBank Accession No. EG341684). The either side of the gene to search the platypus trace archive for 14 BACs together contained 19 genes; two pseudoautosomal BAC-end sequences. We confirmed that BACs contained the BACs contained four and two genes respectively and one X- gene(s) of interest by PCR and direct sequencing. BACs were specific BAC contained two genes (Table S1). mapped by DNA-FISH to male metaphase chromosomes to Transcription of the 14 BACs described above was initially confirm their location on an X and determine whether they have examined by RNA-FISH in female and male fibroblasts (Figure 2). Y homologues (data not shown). Three genes with validated SNPs As a control, RNA-FISH was followed by DNA-FISH to ensure on X1 were found to be pseudoautosomal, and based on genome that RNA signals were located near one (X-specific genes in males) assembly co-ordinates, all other unvalidated X1 SNPs are or both of the alleles (X-specific genes in females, autosomal and predicted to likewise fall within the pseudoautosomal region. pseudoautosomal genes). Only those cells with two DNA-FISH Similarly, the SNP on X2 was shown to have a homologue on Y2 signals per nucleus (or one signal for X-specific genes in males) by FISH. However, the three X5 genes containing SNPs are X- were included in analysis. Data from the male RNA-FISH specific. experiments was used to determine the efficiency of detection for Sequencing of X-specific SNPs revealed that all genes were each gene which was then used to extrapolate the expected biallelically expressed (Figure 1), as were the pseudoautosomal percent of nuclei with biallelic expression in females, which is SNPs (data not shown). Allele specific real-time PCR was used to expected if there is no X inactivation (Table 5 - refer to Table S2 determine if alleles were expressed to the same extent for the for complete RNA-FISH dataset).

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HPRT1, an autosomal control gene located on chromosome 6, was expressed from both alleles in 96–97% of nuclei (Figure 3A). Genes within four pseudoautosomal BACs on X1, X2 (including GMDS) and X3 were also expressed from both alleles in most female nuclei (77–84%), as well as in most male nuclei (62–92%), showing that the Y, as well as the X, alleles are active (Figure 3B). Two pseudoautosomal BACs used for RNA-FISH contain more than one gene, so it remains possible that not all genes within these BACs have an active Y copy. We obtained quite different results from the BAC containing CRIM1, a X1-Y1 pseudoautosomal gene which was expressed from only one allele in most male (81%) and female cells (71%) (Figure 3C). Except for this locus, we conclude that for the pseudoautosomal loci we tested, both X alleles are active in females, and both X and Y alleles are active in males. We then tested transcription from nine X-specific BACs on platypus X1, X3 and X5. Transcription from both alleles was observed on average in only 45% of nuclei (Figure 3D). Different genes showed a range of transcription of both alleles, from 20% (SEMA6A) to 53% (Ox_plat_124086). These X-specific genes were therefore expressed very differently from the autosomal and pseudoautosomal genes, and significantly different to that expected for biallelic expression, indicating some level of transcriptional inactivation for these genes. Two colour RNA FISH was performed with genes FBXO10 and SHB, located within 500 kb of each other. Co-location of the two RNA signals showed the same X in all of the 51% of cells expressing from only one allele. (Figure 4). A few cells (12%) displayed biallelic expression from SHB with monoallelic expres- sion of FBXO10, and in 37% of nuclei, both genes were expressed from both alleles. As a control, this experiment was performed on male nuclei showing that RNA-FISH signals co-located in all nuclei in which genes were expressed. This experiment was carried out only for two genes lying close together, as results from genes situated further apart (and hence with a gap between signals expressed from the same chromosome) would make results from cells expressing only one of each gene, difficult to interpret. RNA-FISH results were validated for a subset of genes (HPRT1, CRIM1, GMDS, SEMA6A and DMRT2) on four other independently derived primary fibroblast cell lines from different individuals (one male and three females). Results for each cell line are shown in Table S3. As observed (Figure 2), the autosomal gene HPRT was expressed from both alleles in most nuclei (88% male and 83–90% female), as was the pseudoautosomal gene GMDS (86%, 85–90%). The pseudoautosomal gene CRIM1, as before, was expressed from both X chromosomes in only 24–56% of female nuclei and X and Y in only 24% of male nuclei. As observed (Figure 2), both X-specific Figure 1. Biallelic expression of three X-specific genes. SNPs genes (DMRT2 and SEMA6A) were expressed from the single X in (marked by boxes) were identified in the genome sequence demon- strated by sequencing fibroblast cDNA from the sequenced animal 99% of male nuclei, and both X chromosomes in half of female (‘‘Glennie’’). nuclei (45–60% and 38–43% respectively). Although there was some doi:10.1371/journal.pgen.1000140.g001 variation between individuals, overall results were similar between all six cell lines tested in this study. Statistical analysis revealed that only the two X-specific genes had a significant difference between the Table 4. Relative allele expression determined by allele- males and females for the number of nuclei expressing only one allele specific real-time RT-PCR. (p = 0.0006 and 0.0008 respectively).

Discussion Gene Allele A Allele B The very large proportion of the genome (,12%) that is X- FBXO10 (A/C) 0.47 0.53 specific in the platypus, and the homology of the multiple platypus EN14997 (G/T) 0.51 0.49 X chromosomes to the chicken Z but not the therian X SHB (A/G) 0.50 0.50 chromosome, makes them a most interesting species for exploring GMDS (C/T) 0.52 0.48 the origins of dosage compensation in mammals. We therefore tested the transcription of genes on platypus X1, X2, doi:10.1371/journal.pgen.1000140.t004 X3 and X5 in order to search for evidence of random X inactivation

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Figure 2. Summary of RNA-FISH results in platypus cells. Frequency of cells in which transcription of no (yellow), one (red) or two (blue) alleles is detected by RNA-FISH in male and female interphase nuclei. Autosomal control, pseudoautosomal and X-specific genes are grouped, with a distinct difference observed between the X-specific genes and the autosomal and pseudoautosomal genes. doi:10.1371/journal.pgen.1000140.g002

(as in eutherian mammals), paternally imprinted X inactivation (as in for mammals, but this is incomplete and variable between genes. marsupials), or incomplete and variable dosage compensation (as in Our demonstration that genes were expressed equally from both chickens). Random inactivation would be manifested as dosage alleles suggests that paternal inactivation and imprinted partial equality between males and females, expression from both SNP expression is unlikely. Our demonstration that both alleles are variants overall but only a single allele per nucleus detected by RNA- expressed in about half the nuclei rules out complete X FISH. Paternal inactivation would be manifest by dosage equality, inactivation (random or imprinted), as is also seen for many but expression of only one SNP variant, and only one allele per partially escaping genes in eutherians and marsupials. nucleus would be detected by RNA-FISH. Bird-like incomplete The variability in overall expression between different X-borne dosage compensation would be manifest as a wide range of dosage genes resembles the range of expression of genes on the bird Z in relationships between males and females, expression of both SNP males and females that indicates a more relaxed, or more variable, variants and expression from both alleles in each nucleus. dosage compensation system [53]. Biallelic expression of Z-borne Our results are not strictly consistent with any of the above genes was also found by examining expression of different alleles of predictions. Quantitative RT-PCR showed male:female expres- two genes from fibroblast cultures established from single cells sion ratios near 0.5 or 1.0 for different genes, although both SNP [51]. These results taken together suggest that bird dosage alleles were expressed for all genes at an equal level in a compensation is partial and differs between genes on the Z. heterozygote. Our examination of transcription of X-specific Thus dosage compensation of X-borne genes occurs to some platypus genes by RNA-FISH revealed that about half of female extent in the platypus, and has features of both bird-like and cells expressed only one allele. The RNA-FISH results showed a mammal-like sex chromosome dosage compensation. clear difference between the transcription of X-specific loci compared with pseudoautosomal and autosomal loci. Is Partial Inactivation Ancestral? These data imply that genes from platypus X-specific regions Together, our findings have parallels in observations of some show some form of compensation via transcriptional inhibition, as genes on the marsupial X and the mouse X in extra-embyronic

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Table 5. Expected vs observed frequency of nuclei with biallelic expression in females.

Efficiency (p) Female Biallelic Frequency Expected Observed P-value % %

Autosomal HPRT1 0.98 96 97 0.96 Pseudoautosomal CRIM1 0.60 36 26 ,0.01 CAMK2A, SLC6A7, CDX1, 0.84 71 77 0.16 EN02294 JARID2, DNTBP1 0.96 92 80 ,0.01 GMDS 0.92 85 84 0.70 IRX1 0.77 59 83 ,0.01 X-specific Ox_plat_124086 0.98 96 53 ,0.01 APC 0.97 94 52 ,0.01 SEMA6A 0.99 98 20 ,0.01 ZNF474, LOX 0.99 98 43 ,0.01 DMRT2 0.98 96 51 ,0.01 SHB 0.90 81 49 ,0.01 FBXO10 0.99 98 52 ,0.01 SLC1A1 0.99 98 39 ,0.01 EN14997 0.99 98 46 ,0.01

Efficiency (p) of RNA-FISH hybridisation was determined from the results obtained in male fibroblasts and extrapolated to determine the expected frequency of nuclei with two signals, one signal and no signal per cell using the formula p2+2pq+q2 = 1, where p2 is the number of nuclei with two signals, 2pq represents nuclei with one signal and q2 is the number with no signal. P-values were determined by a x2 test with 2 degrees of freedom. doi:10.1371/journal.pgen.1000140.t005

tissues, whose paternal alleles are partially inactive, or ‘‘escaper’’ genes on the recently added region of the human and mouse X, which are partially expressed from the inactive X. The observations of partial inactivation in all three major mammalian lineages suggests that partial inactivation observed here in platypus represents a basic form of mammalian X inactivation, which has come under tighter control during therian Figure 3. Co-localization of transcripts (RNA - green) and their evolution, ultimately resulting in the highly stable and complex corresponding gene loci (DNA - red). (A) The autosomal control HPRT1 is expressed from both loci in both sexes since two signals are form of inactivation typical of most eutherian X-borne genes. detected for both RNA and DNA-FISH in both males and females. (B) Partial inactivation has been documented for two marsupial Pseudoautosomal BAC 286H10 is expressed from both X chromosomes in genes (out of a total of five) in some tissues. PGK1 isozyme variants females and the X and Y in males, since two signals are detected for both showed strong expression from the maternal allele and weaker RNA and DNA-FISH in males and females. (C) Pseudoautosomal CRIM1 expression from the paternal allele in cells from heterozygous located on X1 is expressed from only one X in females and only one of the female kangaroos, even in single clones [59], and G6PD from X and Y alleles in males, since two DNA signals but only one RNA signal is detected in both males and females. (D) X-specific SEMA6A located on X5 hybrid marsupials showed a heteropolymer band, diagnostic of is expressed from only one of the two X chromosomes in females, as well expression from both alleles in a single cell [60]. Differences as from the single X in males, showing one RNA and DNA signal in males between species, tissues and even between genes make it difficult to but two DNA signals and only one RNA signal in females. generalize about the nature of marsupial X inactivation, and these doi:10.1371/journal.pgen.1000140.g003 experiments could not distinguish whether partial expression from the paternal X is due to low expression from paternal X involve all genes on the X chromosome, but in recent years it was chromosomes in every cell, or to a mixture of two X-active and found that 5% to as many as 15% of human genes escape one X-active cells. RNA-FISH was used to show that the tammar inactivation in lymphoblastoid [27] and fibroblast cell lines [28] wallaby X-borne gene SLC16A2 was expressed from only one respectively. Remarkably, transcription of some of these genes in allele in most fibroblast cells [61]. fibroblasts varies between individuals, as seems to be the case for The partial silencing displayed for platypus X-specific genes also platypus. Partial expression of genes on the inactive X has also has some parallels to genes on the human X that escape been observed in other eutherians, including the mouse, cow and inactivation. X inactivation in humans was initially thought to mole [62,63]. Typically, these escaper genes are fully expressed

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Figure 4. Two-colour RNA-FISH of neighbouring genes FBXO10 (red) and SHB (green). (A) Male nucleus expresses both genes from the single X. (B) Female nucleus expresses both genes from the same single X chromosome. (C) Female DNA-FISH showing that loci are located together. (D) Diagram depicting the region and the location of BACs used for RNA-FISH. Grey boxes indicate genes located between these two BACs. doi:10.1371/journal.pgen.1000140.g004 from the active X and partially expressed from the inactive X level of biallelic expression. Unlike the hypothesis put forward by [28,64]. Ohlsson et al [64], this type of expression is not limited to those We propose that partial inactivation was the mechanism for with low levels of expressions. compensating differences in gene dosage in an ancestral mammal. Is partial expression in therian mammals explained by stochastic expression? Data on partial expression of genes on the paternal X Partial X Inactivation and the Probability of Transcription in marsupials are equivocal; the partial expression of the maternal To date, it has been difficult to differentiate between the PGK1 allele in clones, and the fainter paternal isozyme alternative hypotheses that partial inactivation is due to a lowered heteropolymer band for G6PD are explained equally well by both rate of transcription in all cells, or from a lowered probability of hypotheses. The few data that would distinguish these hypotheses expression per cell in the population. Ohlsson et al [65] argued for escapers on the inactive human X do not conclusively that genes transcribed at a low level show a low probability of eliminate either hypothesis. Assays of the partially expressed transcription in the cell population, rather than a uniformly low human X-borne gene CHM (REP1) in single cells showed that transcription level. They propose that genomic imprinting and X CHM was expressed from the inactive X in most (70%) but not all chromosome inactivation evolved by regulating, not the activity of cells from one cell line, and in only seven out of ten hybrid cell each locus, but the probability that it is expressed, and making this lines carrying an inactive X [67]. More recently, a study on dosage parent specific [65]. compensation in human lymphoblastoid cell lines found that genes This radical hypothesis is supported by our RNA-FISH data, escaping X inactivation were not subject to the higher levels of which show that platypus genes differ in the frequency of nuclei in variation found for fibroblast cell lines, suggesting that the which one or both alleles are transcribed, giving an overall partial expression of the escaper genes is not stochastic but subject to dosage compensation that differs from gene to gene. The data tight regulation [27]. RNA-FISH performed on both fibroblasts from the bird Z is equivocal; the variability between genes is and lymphoblastoid cells for these escaper genes would conclu- thought to reflect differences in the rate of transcription, but could sively rule out stochastic expression. equally well reflect differences in the probability that a locus is It is important to note the difference in the number of genes in transcribed. RNA-FISH of five chicken genes shows that most are human which escape inactivation between fibroblast cell lines, transcribed from both alleles in most cells [50]; however, the low where 15% of genes are said to escape inactivation [28] and efficiency of signal detection (about a quarter of nuclei had no lymphoblastoid cell lines where only 5% of genes escape [27]. signals), and the different tissues used makes this hard to interpret. Similarly in marsupials, differences have been found in the Efficient RNA-FISH on the chicken Z genes for which we have inactivation status of genes between tissues [26]. Our study has data in platypus would test the hypothesis that partial inactivation only used fibroblast cell lines due to the difficultly in obtaining of the Z in male birds operates by altering the probability of tissue samples in large enough sample sizes, as the platypus is listed transcription, rather than uniformly downregulating transcription. as a ‘‘vulnerable’’ species. A comparison of results for other tissues Our finding that two genes located 500 kb apart are expressed may show different results. from the same chromosome implies that the stochastic expression Several human X-borne genes that escape from inactivation of X-specific genes is coordinated in cis. Furthermore, a recent have a widely expressed Y homologue, and some others have study has shown that this type of probabilistic expression is homology to a Y-borne pseudogene that represents a recently widespread on human autosomes, with their data suggesting that inactivated partner on the Y. The Y homologue of an X/Y pair as many as 1000 human genes are subject to stochastic monoallelic often has a lower level of expression than its partner on the X expression [66]. Around 80% of these genes also showed some (reviewed in [68]), similar to the lower level of expression exhibited

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by alleles on the inactive X in females. However, the presence of a seem to be the case in birds. Dosage compensation for the 964 Y homologue does not necessarily negate the need for dosage genes on the bird Z chromosome extends over a range from compensation, as some Y alleles have evidently taken on functions complete compensation (,10% genes) to no compensation (,10% different from those of their X homologue. Nearly all escaper genes) with most falling between these extremes [53]. This suggests genes are part of the region added to the eutherian X chromosome either that the necessity for strict dosage compensation has been and only recently recruited to the inactivation system, suggesting over-emphasized, or that genes on the bird Z chromosome are that their partial escape from X inactivation correlates with much more tolerant of dosage differences than genes on the progressive assimilation of genes into the X inactivation systems therian X [71]. once the Y paralogue has degenerated. By no means are all genes dosage sensitive [71]. For instance, many protein products, such as enzymes, are controlled at Pseudoautosomal Genes and Inactivation different levels in the cell, so transcriptional control is not essential. In eutherian mammals, small terminal regions of the X and Y For some genes, a dosage difference may even be essential for are homologous, and pair and recombine at male meiosis. These function; for instance, a 2:1 dosage of DMRT1 has been suggested pseudoautosomal regions (PARs) are relics of the X added region to define male versus female development in birds [72]. that have not yet degraded [15]. Genes within the PAR have no One gene that does not display equal expression between males need of dosage compensation. and females and may even be hypertranscribed in females of both There are two PARs on the human X. PAR1 on the short arm platypus and zebrafinch is SEMA6A, a gene on platypus X5 and represents a relic of ancient XY homology, and contains genes that the avian Z. From our data, platypus SEMA6A appears not be are expressed from the Y, and not inactivated on the X [69]. The subject to dosage compensation by real-time RT-PCR, yet RNA- smaller PAR2 was added very recently to the long arm of the Y FISH results show that it predominantly has only one allele active from the long arm of the X, but two genes in the region (SYBL1 per cell. In zebrafinch liver, SEMA6A is expressed more than two- and SPRY3) are subject to inactivation, not only on the inactive X, fold more in females with just one copy than males with two copies but also on the Y [70]. [53]. Although these results were obtained from different cell types We observed that seven of the nine platypus genes from the in the different species, it is intriguing that in both cases there is pseudoautosomal regions displayed as much or more expression some evidence of hypertranscription in females. from males than females, as assessed by quantitative RT-PCR, It is therefore likely that only a minority of genes on the suggesting that they are expressed from Y as well as the X alleles. mammalian X really need to be dosage compensated. The RNA-FISH of these genes showed that both alleles were expressed difference in the level of control of sex chromosome activity may in most cells in females (two X alleles) and males (X and Y alleles). therefore be a side-effect of the mechanism used for dosage Two of these BACs contained multiple genes, so detection of compensation. Eutherian mammals subscribe to a whole-X predominantly two signals per cell does not necessarily mean that mechanism in which inactivation spreads along the X. The bird all genes are active on both chromosomes; however, expression Z, however, seems to have a piecemeal dosage compensation analysis of transcripts from each of these BACs confirms that most system in which different genes appear to show different levels of of these genes (3/4 in BAC 286H10 and 2/2 BAC 271I19) have compensation, and compensated genes are clustered [56]. active Y homologues. Two pseudoautosomal genes CDX1 and The alternative is that the genes on the bird Z and therian X GMDS had male:female expression ratios near 0.5 but an almost evolved under different selective pressures. We know that the gene equal probability of expression, suggesting that either both alleles content of these chromosomes is different, having originated from are downregulated in males, or alternatively, the Y allele sequence two different pairs of autosomes, and we also know that the gene has sufficiently diverged from that of the X homologue, leaving it content of sex chromosomes is biased toward sex-specific expression. unable to be amplified by our primers. The human X is enriched for genes involved in brain function, and A fifth platypus pseudoautosomal gene showed a completely sex and (particularly male) reproduction [19–22]. The chicken Z different expression pattern. CRIM1 (cysteine rich transmembrane chromosome gene content is male-biased yet noticeably deficient in BMP regulator 1), located on platypus X1-Y1, had equivalent female-biased genes [23]. Commenting on the finding that dosage expression in males and females, but was usually expressed from compensation in birds is much less tightly controlled than in therian only one allele in both males (81% of nuclei) and females (69%). mammals, Graves and Disteche [71] suggested that expression There are two possible explanations. Firstly, the Y homologue may differences in Z-borne genes between males and females may have have evolved a new male-specific function like many genes on the been selected for to control sex-specific characters. Since platypus sex human Y [15], and be testis specific, so silencing of one X in chromosomes show considerable homology to the bird Z, the females evolved to equalize expression of the X homologue. functions of platypus X-borne genes are likely to be equivalent to Alternatively, inactivation of both X and Y could be equivalent to those on the chicken Z. the silencing of PAR2 genes on the long arm of the human X. Perhaps, then, partial and variable silencing in the platypus SYBL1 and SPRY3 undergo silencing on both the X and Y, the dosage compensates some essential genes, leaves some genes product of their evolutionary history as a block transposed from uncompensated where dosage differences are essential for sex- the X (where it was subject to inactivation) to the Y, where it was specific function, and partially compensates most genes in dosage compensated to match the X [70]. proportion to their dosage-sensitivity, as is evidently the case for Thus for most pseudoautosomal genes there is no need for birds. dosage compensation on the X because the Y allele is active, and no dosage compensation is observed. Conclusions We found that genes on the multiple platypus X chromosomes Is More Tightly Controlled Dosage Compensation Linked show partial and variable dosage compensation. This is very similar to Gene Function? to the partial and variable dosage relationships of genes on the The chromosome-wide X inactivation in mouse and human has chicken Z chromosome, with which the platypus X chromosomes given rise to the expectation that dosage compensation for genes share considerable homology. However, unlike birds, platypus on sex chromosomes is critical for life. However, this does not dosage compensation involves transcription from only one of the

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182 Dosage Compensation in Platypus two alleles in a proportion of cells and is coordinated at least on a Quantitative Real-Time RT-PCR regional level. Transcriptional inhibition is a property shared by X Total RNA was extracted from eight different male and eight chromosome inactivation in therian mammals. Thus, platypus different female fibroblast (toe web) cell lines (at passage 6 to 8) to dosage compensation has features shared with dosage compensation represent a total of 16 individuals. First-strand cDNA was of the bird Z and the mammal X. synthesized by oligo (dT) priming using Superscript III (Invitro- gen). Primers for each gene were designed using the Plexor Materials and Methods program (Promega) (Table S4). PCR reactions were carried out using Quantitect SYBR Green PCR kit (Qiagen) according to the Identification of Expressed Genes within BACs Mapping manufacturer’s instructions. Amplifications were performed and to the X Chromosomes detected in a Rotorgene 3000 cycler (Corbett Research). To BAC-end sequences from CHORI-236 BAC clones (http:// determine the detection range, linearity and real-time PCR bacpac.chori.org), mapped to platypus X chromosomes as part of amplification efficiency for each primer pair, standard curves the genome project, were aligned against the genome sequence. were calculated over a 10-fold serial dilution of fibroblast cDNA. A Genes within the genomic region contained between the BAC-end series of two-fold serial dilutions were also carried out to confirm sequences were identified by using the Ensembl database (http:// the ability of the PCR conditions to detect this level of difference in www.ensembl.org/Ornithorhynchus_anatinus/index.html). An expression. All dilutions and samples were run in triplicate. additional four BACs were chosen because they span genes with Cycling conditions consisted of an initial hold cycle of 95uC for SNPs that were potentially X-specific. These BACs were identified 15 min, 40 cycles of 94uC for 15 sec, annealing at the appropriate by searching the platypus sequence trace archives containing temperature listed in Table S4 for 15 sec and extension at 72uC BAC-end sequence data (http://www.ncbi.nlm.nih.gov/Traces) for 20 sec for data acquisition. Melting curves were constructed with genomic sequence from 100 kb up and downstream of the from 45uC–95uC to confirm the purity of the PCR products and gene of interest. direct sequencing of products was performed to confirm their PCR was performed on the BACs to confirm that the genes identity. Relative expression of each gene was determined by predicted to be contained within the BAC were present. The PCR normalization to ACTB expression using the formula where the cycling conditions for all primers were as follows: an initial CtRef CtTarget ratio of ACTB to target = (1+ERef) /(1+ETarget) [74]. denaturing step of 94uC for 2 min, 30 cycles of 94uC for 30 sec, Statistical significance was assessed, for the null hypothesis that annealing for 30 sec at the appropriate temperature (Table S4), there was no difference between male and female expression levels, 72uC for 1 min and a final extension at 72uC for 10 min. using an unrelated samples 2-tailed t test with unequal variance. To determine whether genes within BACs were expressed in fibroblasts, total RNA was extracted from female and male Bioinformatic Prediction of Expressed Single Nucleotide fibroblast cell lines using Gene Elute Mammalian Total RNA Miniprep extraction kit (Sigma). RNA was treated with DNA-free Polymorphisms (SNPs) in Platypus (Ambion) to remove any contaminating DNA and Superscript III Exonic sequence from predicted genes on platypus chromo- (Invitrogen) was used to generate cDNA using random hexamers somes X1, X2, X3 and X5 were extracted from the Ensembl 46 as primers for first strand synthesis. To ensure there was no database, using the Biomart tool (http://www.ensembl.org/ genomic DNA contamination in the cDNA sample, a RT-negative biomart/martview). These sequences were compared to the control was made by excluding the Superscript III enzyme from platypus whole genome shotgun sequence traces (‘‘Ornithorhynchus the first strand synthesis reaction and was used as a negative anatinus WGS’’) deposited on the trace archive at NCBI (http:// control in all RT-PCR experiments. Where possible, primers were www.ncbi.nlm.nih.gov/Traces), using MegaBLAST [75]. Poten- designed to span introns. Primers, annealing temperatures and tial single-nucleotide polymorphisms (SNPs) were discovered by product sizes are listed in Table S4. PCR was carried out using the manually searching within the BLAST output for single nucleotide same cycling conditions described above. Each set of primers was mismatches occurring in approximately 50% of target traces. The tested on female and male RT-positive and RT-negative samples chromatogram files containing a potential SNP were extracted TM as well as genomic DNA. PCR products were gel purified using a from the trace archive and assembled using Sequencher 4.7 QIAquick Gel Extraction kit (Qiagen) and directly sequenced by (Gene Codes Corporation, Michigan). This assembled sequence AGRF (Brisbane). (including surrounding intronic sequence) was tested for unique- ness within the platypus genome using BLAT [76] on the UCSC DNA-FISH on Metaphase Chromosomes test browser (http://genome-test.cse.ucsc.edu). For the four BACs not previously mapped, 1 mg of DNA from these BACs was labeled by nick translation with digoxigenin –11- Allele Specific Real-Time PCR dUTP (Roche Diagnostics), Spectrum-Orange or Spectrum-Green To validate identified SNPs and test expression in fibroblasts, (Vysis). Unincorporated nucleotides were removed from Spec- DNA was extracted from the ‘‘Glennie’’ fibroblast cell line using the trum-Orange and Spectrum-Green labeled probes using Probe- Dneasy Blood and Tissue kit (Qiagen) and RNA was extracted as Quant G50 micro columns (GE Healthcare). Probes were described above. First strand synthesis was performed on RNA using precipitated with 1 mg platypus C0t1 DNA and hybridized to the Supercript III First-Strand Synthesis System for RT-PCR kit male and/or female platypus metaphase chromosomes and (Invitrogen) according to manufacturer’s instructions. PCR and RT- fluorescent signals for digoxigenin labeled probes were detected PCR was carried out using the primers listed in Table S4. using the protocol described by Alsop et al [73]. A Zeiss Axioplan2 To quantify the expression level of SNPs for three X-specific epifluorescence microscope was used to visualize fluorescent SNPs and one pseudoautosomal gene, allele-specific real-time signals. Images for DAPI-stained metaphase chromosomes and PCR was carried out. Allele specific primers were designed with fluorescent signals were captured on a SPOT RT Monochrome the 39end base of either the forward or reverse primer CCD (charge-coupled device) camera (Diagnostic Instruments corresponding to the specific allele (refer to Table S5 for primer Inc., Sterling Heights) and merged using IP Lab imaging software sequences and corresponding annealing temperatures). The (Scanalytics Inc., Fairfax, VA, USA). different alleles were amplified in separate tubes. Real-time PCR

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was performed using Quantitect SYBR Green PCR kit (Qiagen) genes were not due to the inability of the technique to detect both with amplifications performed and detected in a Rotorgene 3000 transcripts, an experiment where BACs containing SEMA6A and cycler (Corbett Research). Cycling conditions are the same for CRIM1 were labeled with either Spectrum Green or Spectrum those described in the quantitative PCR section with all samples Orange (Vysis) or with biotin-16-dUTP (Roche Diagnostics) was run in triplicate. Genomic DNA for ‘‘Glennie’’ was included as a performed. Biotin-labeled probes were detected with avidin-FITC control since the allele frequency ratio should be 1:1, permitting (Vector Laboratories Inc.), with FITC signals amplified by allele-specific amplification bias to be detected and corrected. additional layers of biotinylated anti-avidin (Vector) and avidin- Known homozygous cDNA samples and pooled homozygous FITC. No differences between direct labeling and biotin labeling samples with varying ratios of each allele (0.2, 0.4, 0.6, 0.8) were followed by amplification were detected. included to ensure the technique was sensitive enough to detect small differences. Allele relative expression levels were calculated Supporting Information using the formula: frequency of allele A = 1/(2EDCt+1) [77], where Figure S1 Real-time results for X-specific genes. Each point is a DCt = (AcDNA2BcDNA)2(AgDNA2BgDNA) and converted to a ratio of allele A to allele B. PCR products were sequenced to confirm different cell line (shown in the same order in each graph). Male the identity of products. cell lines are shown in blue, female cell lines in red. Expression has been normalised to ACTB. RNA/DNA-FISH on Interphase Nuclei Found at: doi:10.1371/journal.pgen.1000140.s001 (0.15 MB PDF) Male and female fibroblast cells (from toe web) were cultured on Figure S2 Allele-specific real-time RT-PCR results for EN14997. gelatin-coated coverslips in AminoMax C100 medium (Invitrogen) Standards for each allele are shown in red or green and ‘‘Glennie’’ at 30uC in an atmosphere of 5% CO2. Cells on coverslips were cDNA in pink. cDNA from homozygous individual for the opposite washed with PBS, permeabilized for 7 minutes on ice using CSK allele in each case is in dark grey, showing that the primers do not buffer plus Triton X (100 mM NaCl, 300mM sucrose, 3 mM amplify both alleles. No template control is light grey. MgCl2, 10 mM PIPES pH 6.8, 2 mm Vanadyl Ribonucleoside Found at: doi:10.1371/journal.pgen.1000140.s002 (0.09 MB PDF) Complex (VRC), 0.5% Triton X) and fixed in 3% paraformal- Table S1 Ensembl Identifiers, genome co-ordinates and corre- dehyde for 10 minutes. Coverslips were dehydrated via a series of sponding location in human and chicken for genes found within ethanol washes (70%, 80%, 95%, 100%), air-dried and denatured. BACs used for RNA FISH. Probes were labeled as described in the DNA-FISH on metaphase Found at: doi:10.1371/journal.pgen.1000140.s003 (0.04 MB chromosomes section. Hybridization buffer (4 SSC, 40% dextran 6 DOC) sulphate, 2 mg/ml BSA, 10 mM VRC) was added to each probe. Probes were denatured at 75uC for 7 min and allowed to Table S2 RNA-FISH dataset. preanneal for 20 min. 10 ml of probe was added to each coverslip Found at: doi:10.1371/journal.pgen.1000140.s004 (0.03 MB and hybridized overnight in a humid chamber at 37uC. Coverslips DOC) were washed in 0.46SSC with 0.3% Tween 20 at 60uC for Table S3 RNA-FISH results for three additional female and one 2 minutes followed by a wash in 26SSC with 0.1% Tween 20 for male cell lines. 1 min at room temperature. Coverslips were fixed in 3% Found at: doi:10.1371/journal.pgen.1000140.s005 (0.03 MB paraformaldehyde for 10 minutes, treated with 0.1 mg/ml RNase DOC) for 1 hour at 37uC and subjected to DNA-FISH following the same hybridization protocol described for DNA-FISH on Table S4 List of primers used for SNP validation (SNP), metaphase chromosomes. Nuclei were viewed under a fluores- confirmation of expression in fibroblasts (Expression), BAC cence microscope in several different focal planes, with 100 nuclei confirmation (BAC) and qRT-PCR. examined for each probe for both males and females. Found at: doi:10.1371/journal.pgen.1000140.s006 (0.06 MB Efficiency (p) of RNA-FISH hybridisation was determined from DOC) the results obtained in male fibroblasts and extrapolated to Table S5 Primers used for allele-specific real-time PCR. determine the expected frequency of nuclei with two signals, one Found at: doi:10.1371/journal.pgen.1000140.s007 (0.03 MB 2 2 signal and no signal per cell using the formula p +2pq+q = 1, where DOC) p2 is the number of nuclei with two signals, 2pq (q = 12p) represents 2 nuclei with one signal and q is the number with no signal. P-values Acknowledgments were determined by a x2 test with two degrees of freedom. Inconsistencies between RNA-FISH results in previous exper- We thank Colin L. Kremitzki for providing BAC clones. iments examining transcription have been attributed to the inability to detect weak signals, which could be overcome by, Author Contributions not only using a combination of RNA and DNA-FISH, but also by Conceived and designed the experiments: JED JAMG. Performed the amplifying the RNA-FISH signal [78]. In order to ensure that the experiments: JED TAH. Analyzed the data: JED TAH EK JAMG. Wrote differences between autosomal, pseudoautosomal and X-specific the paper: JED JAMG.

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186 APPENDIX 4: GENOME OF THE MARSUPIAL MONODELPHIS DOMESTICA REVEALS INNOVATION IN NON-CODING SEQUENCES

The following appendix is a publication from early 2007 that reports the genomic sequence of the gray short-tailed opossum (Monodelphis domestica), the first marsupial to have its genetic makeup fully characterised.

Mikkelsen TS, Wakefield MJ, Aken B, Amemiya CT, Chang JL, Duke S, Garber M, Gentles AJ, Goodstadt L, Heger A, Jurka J, Kamal M, Mauceli E, Searle SM, Sharpe T, Baker ML, Batzer MA, Benos PV, Belov K, Clamp M, Cook A, Cuff J, Das R, Davidow L, Deakin JE, Fazzari MJ, Glass JL, Grabherr M, Greally JM, Gu W, Hore TA, et al. (2007) Genome of the marsupial Monodelphis domestica reveals innovation in non-coding sequences. Nature 447: 167-177.

My contribution to this 164-author publication was a number of localisation experiments and bioinformatic analyses, most of which were reported in Chapter 2. My demonstration that marsupials do not possess a homologue of XIST, and have rearrangements in the region homologous to the X inactivation centre, was critical to the section of this paper discussing X inactivation. As discussed in Chapter 6, this paper predicts that the evolution of XIST within the eutherian mammals imposed restrictions upon evolution of the X chromosome, suppressing genomic rearrangement and enriching the insertion of LINE1 repeat elements.

187 Pages 188-199 cannot be shown as they derive from the copyrighted publication:

Mikkelsen TS, Wakefield MJ, Aken B, Amemiya CT, Chang JL, Duke S, Garber M, Gentles AJ, Goodstadt L, Heger A, Jurka J, Kamal M, Mauceli E, Searle SM, Sharpe T, Baker ML, Batzer MA, Benos PV, Belov K, Clamp M, Cook A, Cuff J, Das R, Davidow L, Deakin JE, Fazzari MJ, Glass JL, Grabherr M, Greally JM, Gu W, Hore TA, et al. (2007) Genome of the marsupial Monodelphis domestica reveals innovation in non-coding sequences. Nature 447: 167-177. doi:10.1038/nature05805

This publication can be retrieved from: http://dx.doi.org/10.1038/nature05805