BACKGROUND
genomic imprinting is an epigenetic process that affects a small subset of genes resulting in their expression/repression in a parent of origin dependent fashion.
one set of imprinted genes is expressed only from the paternally inherited allele, while another set is expressed only from the maternally inherited chromosome.
the imprint is reset at each generation when the two haploid genomes are separate, either during gametogenesis or immediately after fertilization, when the two genomes are physically separated in their own pronuclei.
imprinted genes generally reside in clusters around a cis acting element called an imprinting control region  that exerts its effects over a large chromosomal domain  .
imprinted domains can contain genes that are biallelic, paternally expressed or maternally expressed.
monoallelic expression can be universal , or limited to a subset of tissues.
the most common type of tissue specific limitation is to the extraembryonic lineages, exemplified by the placenta and the yolk sac.
this latter observation has provided strong support for the idea that the evolutionary origins of imprinting are rooted in extraembryonic tissue biology.
in mouse uniparental embryos, the tissue most severely affected is the trophoblast, the precursor of several placental cell types.
gynogenetic/parthenogenetic embryos have almost none by midgestation, whereas androgenetic embryos have hyperplastic trophoblast.
moreover, derivation of trophoblast stem cells from parthenogenetic mouse blastocysts is extremely inefficient, and is accompanied by selective loss of imprinting of several genes .
these observations led us to hypothesize that a gene critical for trophoblast establishment/function in blastocysts is expressed from a paternally-inherited chromosome, and is therefore missing from gynogenetic/parthenogenetic embryos.
a microarray comparison of the transcriptomes of androgenetic and gynogenetic blastocysts revealed that the pcg gene sfmbt <dig> is expressed almost exclusively from the paternal allele starting at the blastocyst stage  <cit> .
monoallelic expression in all tissues is preserved up to e <dig> , after which a high level monoallelic expression is preserved in extraembryonic tissues, while significantly reduced, but biallelic, expression in somatic tissues can be observed.
these observations place sfmbt <dig> in the class of imprinted genes that are specific to the extraembryonic tissues.
sfmbt <dig> maps to the proximal region of chromosome  <dig>  this region was mapped as imprinted through the chromosome translocation studies of cattanach and colleagues  <cit> , but no imprinted gene had been identified until our study  <cit> .
here we extend our analysis of the domain surrounding sfmbt <dig>  and the search for a potential icr.
our results indicate that sfmbt <dig> is the only gene within a  <dig> mb region that is imprinted, and that we find no evidence of a classical icr displaying robust germline specific dna methylation that is preserved after fertilization.
we also show that imprinting appears to be unique to mice and rats, and is associated with the acquisition of a block of mirnas in one of the introns.
our results suggest that we have caught a gene "in the act" of becoming imprinted.
RESULTS
sfmbt <dig> is the only imprinted gene in this domain
the largest imprinted cluster, the snrpn locus, is  <dig>  mb.
we therefore assessed the allelic expression of  <dig> annotated protein coding genes, including sfmbt <dig>  within a  <dig>  mb domain, from gata <dig> to mrc <dig>  several unannotated genes were also tested for allelic expression, but these will not be discussed further.
gata <dig> lies  <dig> kb proximal to sfmbt2; the next most proximal gene, cugbp <dig>  is  <dig>  mb upstream of gata <dig>  indicating the presence of a gene desert.
prkcq, immediately telomeric to sfmbt <dig>  is not expressed in extraembryonic tissues .
with the exception of sfmbt <dig>  none of the genes assayed displayed monoallelic expression in domesticus x castaneus or castaneus x domesticus placenta or yolk sac .
interestingly, a recent study of e <dig>  hybrid embryos, using a massively parallel sequencing approach, revealed several new imprinted genes.
however, none were found in the proximal region of chromosome  <dig>  including sfmbt <dig>  <cit> .
this is consistent with our observation that sfmbt <dig> becomes biallelic in embryo-derived somatic tissues.
none of the neighbouring genes is imprinted in embryo.
together with our findings, these results indicate that sfmbt <dig> is the only imprinted gene within a  <dig>  mb region of proximal chromosome  <dig>  and only in early embryo and extraembryonic tissues.
sfmbt <dig> has multiple transcriptional starts, stops and differential splicing .
the two transcriptional starts sites  are utilized differently; the more 5' start is specific to extraembryonic tissues, while the 3' start is ubiquitous  <cit> .
there is a non-coding antisense transcript that starts in the first common intron downstream of the two sfmbt <dig> tss.
the putative promoter for this antisense transcript embodies part of a cpg island .
in placenta, this transcript is also imprinted and like sfmbt <dig> is expressed from the paternal allele .
this result is in contrast with other imprinted gene domains that possess antisense non-coding transcripts, for example the kcnq1ot <dig> and airn transcripts in the kcnq <dig> and igf2r loci, respectively .
in these latter cases, the antisense transcript is expressed from the opposite allele as the sense transcript.
oocyte dmr is sparse and largely asymmetric
fifteen imprinted domains are regulated by germline differentially methylated regions ; twelve are methylated on the maternal chromosome in somatic tissues and in oocytes, while three are methylated on the paternal allele in somatic tissues and in sperm  <cit> .
several other dmrs have been identified, but not tested for icr function.
in addition, many imprinted genes display a characteristic chromatin signature  <cit> .
a search of the broad institute chip-seq database revealed strong peaks of h3k4me <dig> and h3k27me <dig> binding across the sfmbt <dig> sense and antisense tss and coinciding with a cpg island; however, in contrast with other germline dmrs, there was no discernible h3k9me <dig> or h4k20me <dig> binding in es cells  <cit> .
there is a large cpg cluster of approximately  <dig>  kb that spans the tss for sfmbt <dig>  including the start site that is used only in extraembryonic tissues , and extends  <dig> kb downstream of the ubiquitous tss .
the tss for the non coding  antisense rna also resides within the cpg island.
five amplicons from this region were queried for methylation by bisulfite mutagenesis in oocyte and sperm genomic dna .
two of the five amplicons, me <dig> and cg <dig>  contained polymorphisms that allowed us to distinguish parental alleles after bisulite mutagenesis and sequencing; these two amplicons were analysed in placental dna from c57bl <dig> x castaneus f <dig> placentae.
unlike other dmrs from well conserved imprinted domains, the cpg cluster spanning the promoter region of sfmbt <dig> did not contain a block of methylated cpgs; methylated cytosines were distributed sparsely throughout the cpg cluster .
the amplicon spanning the promoter  <dig> start site  is not noticeably methylated in either sperm or oocyte, although there is a slight increase in methylation on the maternal allele in both oocyte and placenta.
methylation of me <dig> in fetal brain is similarly sparse, and does not display any allelic bias .
four of the amplicons contained cpg sites that were methylated in oocyte but not sperm; however, in two cases this observation was confined to a single site, and in one case to four out of  <dig> potential sites.
interestingly, three of these four amplicons contained methylated asymmetric cytosines in oocyte dna, a conclusion that was drawn from the fact that all unique clones contained cs at these positions.
following this observation, the other strand of cg <dig> was subjected to bisulfite mutagenesis and sequencing to assess the symmetry of the four cpg sites identified in the initial analysis.
all four proved to be asymmetrically methylated, and an additional five asymmetric sites were identified.
this observation strongly suggests that dna methylation may be a secondary consequence of ordered chromatin structure in oocytes.
none of the maternal methylated cytosines in cg <dig> was conserved in placental dna.
rat sfmbt <dig> is imprinted, but bovine sfmbt <dig> is biallelic
the human sfmbt <dig> gene is not imprinted .
in examining the genomic sequences within and near mouse and human genes, it became apparent that the mouse gene differs from the human gene in two respects:
<dig>  the syntenic region of the mouse and human domains is quite different.
a comparison of human, mouse, rat and cow syntenic regions indicates that the rat domain is likely ancestral; the human gene has undergone one small translocation; the mouse and bovine domains are rearranged significantly .
<dig>  rat and mouse genes possess a large block of mirnas in intron  <dig>  while all other mammals with well annotated genomes, including human and bovine, do not have this block of mirnas .
the mirnas fall within the mir- <dig> family.
these observations raised the possibility of testing whether chromosomal arrangements are important for acquisition of the imprint in the mouse gene, or whether the block of mirnas is the critical feature driving imprinting at this locus.
if the rat gene is biallelic, then some aspect of the chromosomal rearrangement in the mouse may have led to the acquisition of monoallelic expression through some kind of position effect.
on the other hand, if the rat gene is imprinted, then recent insertion of the block of mirnas may play a pivotal role in the establishment of a new imprinted domain.
we found a polymorphism in the 3' utr of the rat sfmbt <dig> gene in f <dig> and sprague dawley rats, and assayed allelic expression in mid-gestation placenta, yolk sac and brain from  f <dig> and  f <dig> fetuses.
as with mice, placenta and yolk sac expressed predominantly the paternal allele, while brain showed clear biallelic expression .
the bovine genomic region encompassing sfmbt <dig> has undergone significant rearrangements in comparison with rat, mouse and human .
polymorphisms between two species of cattle, bos taurus and plains bison, were used to assess allelic expression in f <dig> blastocysts.
unlike mouse blastocysts, which display monoalleic expression from the paternal allele, bovine blastocysts are clearly biallelic .
thus, chromosomal rearrangements are not associated with the acquisition of imprinting, at least not in cattle.
peromyscus sfmbt <dig> lacks a large block of mirnas and is biallelic
the observation that the old world rodent sfmbt <dig> gene is imprinted, while other mammalian genes are not, prompted us to investigate the gene in other rodents.
rabbits, guinea pigs, squirrels and kangaroo rats are represented in the genome database.
a blast search of intron  <dig> sequences against mirbase revealed that only kangaroo rats possessed a single potential mir- <dig> family mirna sequence that folds appropriately with rnafold .
in the rodent phylogeny, kangaroo rats are within the mouse-related clade, although at a distance from mus and rattus  <cit> .
peromyscus, or deer mice, are indigenous to north america, and are evolutionarily more distant from old world rodents , than rats and mice are from each other , but are closer than the other rodents with genomic sequence in the public database.
they are represented by several species that occupy separate ranges, with clearly defined hybrid zones, where interbreeding is limited.
there is evidence that some of the hybrid dysgenesis may be a function of incompatible genomic imprinting  <cit> .
the peromyscus genome is represented in the public database by  <dig>  fold coverage of unassembled shotgun sequence traces.
we used mouse exonic plus flanking intronic sequences to blast search the peromyscus sequence database, and were able to assemble most of the sfmbt <dig> coding sequence by analysing cdna derived from p. maniculatus placenta.
primers from the exons flanking intron  <dig> were used to amplify a  <dig> kb sequence from p. maniculatus genomic dna.
sequence analysis of this intron revealed the presence of three potential mir- <dig> sequences with good folding properties , although none of the putative pre-mirnas resembled mouse or rat mir- <dig> outside of the mature mirna sequence.
we next examined the allelic expression of sfmbt <dig> in peromyscus placenta.
analysis of placenta cdna from reciprocal crosses of two sister species, p. polionotus and p. maniculatus, revealed that the peromyscus sfmbt <dig> gene is not imprinted in either species of deer mice .
discussion
the sfmbt <dig> gene represented an entrée into a new imprinted domain, whose extent was unknown.
our analysis has revealed that it comprises a single coding gene, with a spliced antisense transcript that is transcribed from the first common intron and is also imprinted; this latter is likely a lincrna  <cit> , and its imprinted expression may reflect open/closed chromatin states of the parental alleles.
no other genes tested within  <dig>  mb of sfmbt <dig> display monoalleic expression in placenta, and published data from another study indicates that no genes in this domain are imprinted in e <dig>  somatic tissues  <cit> .
a recent computational analysis supported placental imprinting of sfmbt <dig>  using criteria heavily dependent on the two histone marks, h3k4me <dig> and h3k27me <dig>  mentioned above  <cit> .
no other genes within the domain examined in our study passed the computational test in this study, although one could argue that the criteria chosen for the machine learning exercise may have been biased.
the cpg island that spans the tsss for the various sfmbt <dig> transcripts is not likely to be regulated by dna methylation since very little methylation exists at this locus in both placental and somatic tissues as measured by bisulfite sequencing.
only seven cpg sites  display consistent methylation in oocyte genomic dna.
the four maternal cpgs from cg <dig> that were queried in placental dna did not remain methylated, suggesting that dna methylation is a secondary consequence of silencing mediated by some other mechanism.
indeed, methylation was largely asymmetric; of  <dig> cytosines that survived bisulfite mutagenesis in oocyte genomic dna,  <dig> were asymmetric, and four of these resided within the context of a cpg dinucleotide, raising a question about whether reports of cpg methylation based on sequence analysis of only one strand are indeed symmetric as is assumed by most investigators.
a recent report of extraembryonic tissue development in the absence of dnmt <dig>  3a and 3b supports the notion that dna methylation has little if any function in placenta and yolk sac  <cit> .
it is possible that asymmetric methylation of oocyte genomic dna, perhaps mediated by dnmt3a/dnmt3l/dnmt3b, may drive establishment of a heritable chromatin structure that does not depend on continued dna methylation.
there are three additional cpg islands within and near sfmbt2; one in intron  <dig>  one in intron  <dig> and another between sfmbt <dig> and the next telomeric gene prkcq .
none of these cpg islands is conserved between rats and mice, the intron  <dig> cpg island is methylated on both alleles in mouse placenta , and the other two cpg islands, which are part of a recently expanded retrotransposon family, are not present in mus castaneus .
the tss cpg island is therefore the most likely regulatory region for sfmbt <dig>
single gene imprinted domains comprise a small subset of imprinted genes.
of six reported genes , five have well documented methylation analyses .
four of these display methylation of the silenced  tss  <cit> , while no dmr could be found near gatm  <cit> , similar to our observations for sfmbt <dig>
the broad institute chip-seq database indicates that there is very strong association of the cpg island with ring1b, ezh <dig>  suz <dig>  h3k <dig> me2/ <dig> and h3k27me <dig>  but not h3k <dig> me <dig> or me <dig>  nor h4k20me <dig> in es cells.
interestingly, the human gene also displays high h3k4me <dig> and h3k27me <dig> binding, and measurable although reduced ezh <dig> and ring1b association in es cells.
moreover, the human gene is associated with two antisense lincrnas, one at each end of the sfmbt <dig> primary transcript.
this is interesting because the human gene is not imprinted.
some other aspect of chromatin structure may be important for imprinted regulation of sfmbt <dig> in extraembryonic tissues.
rodents from the mus and rattus genuses imprint their sfmbt <dig> gene, while other mammals do not, including rodents from the peromyscine genus.
the acquisition of a large block of mirnas within an intron of the rodent gene strongly suggests that some aspect of mirna biology is driving silencing of this locus from the maternal allele in early embryos and extraembryonic tissues.
a survey of mirbase reveals that there are approximately  <dig> mirna loci, some of which are clusters, in the mouse genome, which is roughly equivalent to  <dig> % of protein coding genes.
of  <dig> imprinted genes,  <dig>  contain mirnas, while 20% contain some kind of small ncrna including mirnas; 84% of these gene arrangements are conserved in humans.
the numbers represent a significant enrichment among imprinted genes for co-regulation by some kind of ncrna.
one could speculate that local expansion of ncrnas might flag a gene for silencing in one of the parental germlines, and that selection would fix the silencing, perhaps by insertion of a more efficient dmr, thus obviating the need for retention of the ncrna cluster.
the annotated mirnas in both the rat and mouse arrays are from the mir- <dig> family.
however, it is clear that there are likely more mirnas in each block than have been annotated.
dot plots of mouse and rat introns against both themselves and each other reveal significant repetitive sequence homology, even after the annotated mirnas are removed .
in addition, both mouse and rat arrays can be organized into tandem repeats of approximately  <dig> kb , although the rat repeats suffer from gaps in the sequence database.
in contrast, no other mammalian introns examined contain repetitive sequences.
there may be more mirnas, or at least mirna-like sequences, in both mouse and rat introns that remain to be annotated.
for example, a small random section of the rat intron was subjected to analysis with mipred  <cit>  to reveal the presence of two additional predicted pre-mirna sequences from the mir- <dig> family .
kangaroo rats and deer mice fall within the mouse related clade of rodents  <cit> .
both possess potential mir- <dig> mirna sequences in intron  <dig>  although these do not resemble the mir- <dig> pre-mirna sequences in rats and mice, apart from the putative mature mirna.
it is unclear whether these potential mirnas in kangaroo rats and peromyscus are evolutionary precursors or remnants.
several of the mirnas from the murine sfmbt <dig> locus are expressed and processed in placenta , not surprising given that they are part of the primary transcript for sfmbt <dig>  which is robustly expressed in placenta.
recent analysis of small rna libraries with massively parallel sequencing strategies have indicated that several of the mirnas from the cluster are expressed in es cells  <cit> ; interestingly, the number of reads does not correlate with the frequency of the mirna within the block of repeats, suggesting that the primary transcript likely folds in a complex manner that only allows processing of a subset of the potential mirnas.
whether these mirnas mediate any functions in placental biology remains an open question.
there are mir- <dig> family members in other mammalian genomes, notably human and pig, although in far fewer numbers than in rodents.
moreover, there are several mmu-mir- <dig> at other locations in the mouse genome.
any of the mirnas processed from the sfmbt <dig> primary transcript would be imprinted, although testing this is problematical given the highly repetitive nature of the array itself, and the presence of other family members at other locations in the genome.
genomic imprinting is an epigenetic mechanism that is restricted to mammals and angiosperm plants.
there is mounting evidence that it is mediated by maternal factors, possibly as a means of controlling extraembryonic tissues that parasitize the maternal reproductive tract  <cit> .
studies of the evolution of imprinting indicate that it seems to have emerged at different times at different loci  <cit> .
interestingly, several imprinted clusters contain mirnas or snords, and some of these have been demonstrated to be imprinted themselves.
the role of ncrnas in imprinting also includes regulatory functions, such as that exerted by the kcnq1ot <dig> antisense transcript at the kcnq <dig> locus, and the airn transcript at the igf2r locus  <cit> .
rnai biology is providing interesting new insights into gene regulation.
studies in plants and yeast have revealed a wide array of effects, from post transcriptional gene silencing involving both mrna degradation and translational control, to heterochromatin formation, particularly at centromeres.
a recent report of the ncrna clusters at the dlk <dig> and snrpn imprinted loci has revealed that transcripts containing precursors of the mirnas or snords from these loci are retained by the nucleus  <cit> .
this raises the interesting notion that the function of the ncrnas at these loci may be more similar to the regulation of centromeres in yeast than to "traditional" cytoplasmic rnai.
such a role is also implied by the phenotype of both dicer and ago <dig> conditional null alleles in mouse oocytes, which fail to complete the first meiotic division due to misaligned chromosomes  <cit> .
interestingly, maternal loss of dgcr <dig>  a component of the mirna processing pathway, is without effect on development, even though the profile of misexpressed mirnas in maternal dicer -/- and dgcr <dig> -/- zygotes is the same  <cit> .
it is tempting to speculate that expression of a subset of the mirnas from the sfmbt <dig> cluster provides the rnai transcriptional silencing biochemistry with the molecular substrates that allow it to target the repeated sequences at the sfmbt <dig> intron for transcriptional epigenetic silencing on the maternal allele.
the evolution of mirnas is highly dynamic, and is characterized by expansions and contractions, partly through segmental duplication/deletion events.
the mir- <dig> family was acquired by amniotes approximately  <dig> mya  <cit> , and has remained relatively small; humans possess one copy on chromosome  <dig>  and another on the x chromosome, while pigs and orangutans possess only the x linked gene.
mice have acquired, in addition to the large cluster at sfmbt <dig>  several other copies on chromosomes  <dig>   <dig>   <dig>   <dig> and  <dig>  although current mirna annotation and archiving may be incomplete for this family.
interestingly, a large cluster of mirnas in a different family expanded in primates after divergence from the rodent lineage, and is expressed exclusively in placenta  <cit> .
this cluster maps to chromosome 19q <dig> , and is near several loci that display either imprinted expression or parent of origin effects in humans .
a recent report demonstrated paternal expression of the primary transcript encoding this mirna cluster in human placenta  <cit> .
the primary transcript is associated with a classic germline dmr; this locus is however much older than the cluster at the rodent sfmbt <dig> gene by at least  <dig> million years.
as mentioned above, two other imprinted loci, the dlk <dig> domain and the snrpn region, also contain large clusters of non-coding rnas .
they also possess classic germline dmrs.
the lack of any noticeable germline methylation at sfmbt <dig> may reflect its youth as an imprinted domain, suggesting that imprinted regulation precedes establishment of differential methylation.
this raises the possibility that one general mechanism by which genes or gene domains become imprinted is through local expansion of mirnas  <cit> , and that the rodent sfmbt <dig> locus is a window on an actively evolving imprinted domain.
how these clusters of ncrnas mediate gene silencing at the transcriptional level will be an interesting puzzle to solve.
CONCLUSIONS
the very recent insertion of a large block of mirnas into the intron of the pcg gene sfmbt <dig> in old world rodents coincides with the acquisition of placental imprinting at this locus.
the transcriptional start site cgi does not display heritable differential methylation, suggesting that maternal silencing by other chromatin mechanisms mediates imprinting at this locus.

 genomic imprinting is an epigenetic process that affects a small subset of genes resulting in their expression/repression in a parent of origin dependent fashion. one set of imprinted genes is expressed only from the paternally inherited allele, while another set is expressed only from the maternally inherited chromosome. the imprint is reset at each generation when the two haploid genomes are separate, either during gametogenesis or immediately after fertilization, when the two genomes are physically separated in their own pronuclei. imprinted genes generally reside in clusters around a cis acting element called an imprinting control region that exerts its effects over a large chromosomal domain. imprinted domains can contain genes that are biallelic, paternally expressed or maternally expressed. monoallelic expression can be universal, or limited to a subset of tissues. the most common type of tissue specific limitation is to the extraembryonic lineages, exemplified by the placenta and the yolk sac. this latter observation has provided strong support for the idea that the evolutionary origins of imprinting are rooted in extraembryonic tissue biology. in mouse uniparental embryos, the tissue most severely affected is the trophoblast, the precursor of several placental cell types. gynogenetic/parthenogenetic embryos have almost none by midgestation, whereas androgenetic embryos have hyperplastic trophoblast. moreover, derivation of trophoblast stem cells from parthenogenetic mouse blastocysts is extremely inefficient, and is accompanied by selective loss of imprinting of several genes. these observations led us to hypothesize that a gene critical for trophoblast establishment/function in blastocysts is expressed from a paternally-inherited chromosome, and is therefore missing from gynogenetic/parthenogenetic embryos. a microarray comparison of the transcriptomes of androgenetic and gynogenetic blastocysts revealed that the pcg gene sfmbt is expressed almost exclusively from the paternal allele starting at the blastocyst stage. monoallelic expression in all tissues is preserved up to e, after which a high level monoallelic expression is preserved in extraembryonic tissues, while significantly reduced, but biallelic, expression in somatic tissues can be observed. these observations place sfmbt in the class of imprinted genes that are specific to the extraembryonic tissues. sfmbt maps to the proximal region of chromosome this region was mapped as imprinted through the chromosome translocation studies of cattanach and colleagues, but no imprinted gene had been identified until our study. here we extend our analysis of the domain surrounding sfmbt and the search for a potential icr. our results indicate that sfmbt is the only gene within a mb region that is imprinted, and that we find no evidence of a classical icr displaying robust germline specific dna methylation that is preserved after fertilization. we also show that imprinting appears to be unique to mice and rats, and is associated with the acquisition of a block of mirnas in one of the introns. our results suggest that we have caught a gene "in the act" of becoming imprinted.  sfmbt is the only imprinted gene in this domain the largest imprinted cluster, the snrpn locus, is mb. we therefore assessed the allelic expression of annotated protein coding genes, including sfmbt within a mb domain, from gata to mrc several unannotated genes were also tested for allelic expression, but these will not be discussed further. gata lies kb proximal to sfmbt2; the next most proximal gene, cugbp is mb upstream of gata indicating the presence of a gene desert. prkcq, immediately telomeric to sfmbt is not expressed in extraembryonic tissues. with the exception of sfmbt none of the genes assayed displayed monoallelic expression in domesticus x castaneus or castaneus x domesticus placenta or yolk sac. interestingly, a recent study of e hybrid embryos, using a massively parallel sequencing approach, revealed several new imprinted genes. however, none were found in the proximal region of chromosome including sfmbt. this is consistent with our observation that sfmbt becomes biallelic in embryo-derived somatic tissues. none of the neighbouring genes is imprinted in embryo. together with our findings, these results indicate that sfmbt is the only imprinted gene within a mb region of proximal chromosome and only in early embryo and extraembryonic tissues. sfmbt has multiple transcriptional starts, stops and differential splicing. the two transcriptional starts sites are utilized differently; the more 5' start is specific to extraembryonic tissues, while the 3' start is ubiquitous. there is a non-coding antisense transcript that starts in the first common intron downstream of the two sfmbt tss. the putative promoter for this antisense transcript embodies part of a cpg island. in placenta, this transcript is also imprinted and like sfmbt is expressed from the paternal allele. this result is in contrast with other imprinted gene domains that possess antisense non-coding transcripts, for example the kcnq1ot and airn transcripts in the kcnq and igf2r loci, respectively. in these latter cases, the antisense transcript is expressed from the opposite allele as the sense transcript. oocyte dmr is sparse and largely asymmetric fifteen imprinted domains are regulated by germline differentially methylated regions; twelve are methylated on the maternal chromosome in somatic tissues and in oocytes, while three are methylated on the paternal allele in somatic tissues and in sperm. several other dmrs have been identified, but not tested for icr function. in addition, many imprinted genes display a characteristic chromatin signature. a search of the broad institute chip-seq database revealed strong peaks of h3k4me and h3k27me binding across the sfmbt sense and antisense tss and coinciding with a cpg island; however, in contrast with other germline dmrs, there was no discernible h3k9me or h4k20me binding in es cells. there is a large cpg cluster of approximately kb that spans the tss for sfmbt including the start site that is used only in extraembryonic tissues, and extends kb downstream of the ubiquitous tss. the tss for the non coding antisense rna also resides within the cpg island. five amplicons from this region were queried for methylation by bisulfite mutagenesis in oocyte and sperm genomic dna. two of the five amplicons, me and cg contained polymorphisms that allowed us to distinguish parental alleles after bisulite mutagenesis and sequencing; these two amplicons were analysed in placental dna from c57bl x castaneus f placentae. unlike other dmrs from well conserved imprinted domains, the cpg cluster spanning the promoter region of sfmbt did not contain a block of methylated cpgs; methylated cytosines were distributed sparsely throughout the cpg cluster. the amplicon spanning the promoter start site is not noticeably methylated in either sperm or oocyte, although there is a slight increase in methylation on the maternal allele in both oocyte and placenta. methylation of me in fetal brain is similarly sparse, and does not display any allelic bias. four of the amplicons contained cpg sites that were methylated in oocyte but not sperm; however, in two cases this observation was confined to a single site, and in one case to four out of potential sites. interestingly, three of these four amplicons contained methylated asymmetric cytosines in oocyte dna, a conclusion that was drawn from the fact that all unique clones contained cs at these positions. following this observation, the other strand of cg was subjected to bisulfite mutagenesis and sequencing to assess the symmetry of the four cpg sites identified in the initial analysis. all four proved to be asymmetrically methylated, and an additional five asymmetric sites were identified. this observation strongly suggests that dna methylation may be a secondary consequence of ordered chromatin structure in oocytes. none of the maternal methylated cytosines in cg was conserved in placental dna. rat sfmbt is imprinted, but bovine sfmbt is biallelic the human sfmbt gene is not imprinted. in examining the genomic sequences within and near mouse and human genes, it became apparent that the mouse gene differs from the human gene in two respects: the syntenic region of the mouse and human domains is quite different. a comparison of human, mouse, rat and cow syntenic regions indicates that the rat domain is likely ancestral; the human gene has undergone one small translocation; the mouse and bovine domains are rearranged significantly. rat and mouse genes possess a large block of mirnas in intron while all other mammals with well annotated genomes, including human and bovine, do not have this block of mirnas. the mirnas fall within the mir- family. these observations raised the possibility of testing whether chromosomal arrangements are important for acquisition of the imprint in the mouse gene, or whether the block of mirnas is the critical feature driving imprinting at this locus. if the rat gene is biallelic, then some aspect of the chromosomal rearrangement in the mouse may have led to the acquisition of monoallelic expression through some kind of position effect. on the other hand, if the rat gene is imprinted, then recent insertion of the block of mirnas may play a pivotal role in the establishment of a new imprinted domain. we found a polymorphism in the 3' utr of the rat sfmbt gene in f and sprague dawley rats, and assayed allelic expression in mid-gestation placenta, yolk sac and brain from f and f fetuses. as with mice, placenta and yolk sac expressed predominantly the paternal allele, while brain showed clear biallelic expression. the bovine genomic region encompassing sfmbt has undergone significant rearrangements in comparison with rat, mouse and human. polymorphisms between two species of cattle, bos taurus and plains bison, were used to assess allelic expression in f blastocysts. unlike mouse blastocysts, which display monoalleic expression from the paternal allele, bovine blastocysts are clearly biallelic. thus, chromosomal rearrangements are not associated with the acquisition of imprinting, at least not in cattle. peromyscus sfmbt lacks a large block of mirnas and is biallelic the observation that the old world rodent sfmbt gene is imprinted, while other mammalian genes are not, prompted us to investigate the gene in other rodents. rabbits, guinea pigs, squirrels and kangaroo rats are represented in the genome database. a blast search of intron sequences against mirbase revealed that only kangaroo rats possessed a single potential mir- family mirna sequence that folds appropriately with rnafold. in the rodent phylogeny, kangaroo rats are within the mouse-related clade, although at a distance from mus and rattus. peromyscus, or deer mice, are indigenous to north america, and are evolutionarily more distant from old world rodents, than rats and mice are from each other, but are closer than the other rodents with genomic sequence in the public database. they are represented by several species that occupy separate ranges, with clearly defined hybrid zones, where interbreeding is limited. there is evidence that some of the hybrid dysgenesis may be a function of incompatible genomic imprinting. the peromyscus genome is represented in the public database by fold coverage of unassembled shotgun sequence traces. we used mouse exonic plus flanking intronic sequences to blast search the peromyscus sequence database, and were able to assemble most of the sfmbt coding sequence by analysing cdna derived from p. maniculatus placenta. primers from the exons flanking intron were used to amplify a kb sequence from p. maniculatus genomic dna. sequence analysis of this intron revealed the presence of three potential mir- sequences with good folding properties, although none of the putative pre-mirnas resembled mouse or rat mir- outside of the mature mirna sequence. we next examined the allelic expression of sfmbt in peromyscus placenta. analysis of placenta cdna from reciprocal crosses of two sister species, p. polionotus and p. maniculatus, revealed that the peromyscus sfmbt gene is not imprinted in either species of deer mice. discussion the sfmbt gene represented an entrée into a new imprinted domain, whose extent was unknown. our analysis has revealed that it comprises a single coding gene, with a spliced antisense transcript that is transcribed from the first common intron and is also imprinted; this latter is likely a lincrna, and its imprinted expression may reflect open/closed chromatin states of the parental alleles. no other genes tested within mb of sfmbt display monoalleic expression in placenta, and published data from another study indicates that no genes in this domain are imprinted in e somatic tissues. a recent computational analysis supported placental imprinting of sfmbt using criteria heavily dependent on the two histone marks, h3k4me and h3k27me mentioned above. no other genes within the domain examined in our study passed the computational test in this study, although one could argue that the criteria chosen for the machine learning exercise may have been biased. the cpg island that spans the tsss for the various sfmbt transcripts is not likely to be regulated by dna methylation since very little methylation exists at this locus in both placental and somatic tissues as measured by bisulfite sequencing. only seven cpg sites display consistent methylation in oocyte genomic dna. the four maternal cpgs from cg that were queried in placental dna did not remain methylated, suggesting that dna methylation is a secondary consequence of silencing mediated by some other mechanism. indeed, methylation was largely asymmetric; of cytosines that survived bisulfite mutagenesis in oocyte genomic dna, were asymmetric, and four of these resided within the context of a cpg dinucleotide, raising a question about whether reports of cpg methylation based on sequence analysis of only one strand are indeed symmetric as is assumed by most investigators. a recent report of extraembryonic tissue development in the absence of dnmt 3a and 3b supports the notion that dna methylation has little if any function in placenta and yolk sac. it is possible that asymmetric methylation of oocyte genomic dna, perhaps mediated by dnmt3a/dnmt3l/dnmt3b, may drive establishment of a heritable chromatin structure that does not depend on continued dna methylation. there are three additional cpg islands within and near sfmbt2; one in intron one in intron and another between sfmbt and the next telomeric gene prkcq. none of these cpg islands is conserved between rats and mice, the intron cpg island is methylated on both alleles in mouse placenta, and the other two cpg islands, which are part of a recently expanded retrotransposon family, are not present in mus castaneus. the tss cpg island is therefore the most likely regulatory region for sfmbt single gene imprinted domains comprise a small subset of imprinted genes. of six reported genes, five have well documented methylation analyses. four of these display methylation of the silenced tss, while no dmr could be found near gatm, similar to our observations for sfmbt the broad institute chip-seq database indicates that there is very strong association of the cpg island with ring1b, ezh suz h3k me2/ and h3k27me but not h3k me or me nor h4k20me in es cells. interestingly, the human gene also displays high h3k4me and h3k27me binding, and measurable although reduced ezh and ring1b association in es cells. moreover, the human gene is associated with two antisense lincrnas, one at each end of the sfmbt primary transcript. this is interesting because the human gene is not imprinted. some other aspect of chromatin structure may be important for imprinted regulation of sfmbt in extraembryonic tissues. rodents from the mus and rattus genuses imprint their sfmbt gene, while other mammals do not, including rodents from the peromyscine genus. the acquisition of a large block of mirnas within an intron of the rodent gene strongly suggests that some aspect of mirna biology is driving silencing of this locus from the maternal allele in early embryos and extraembryonic tissues. a survey of mirbase reveals that there are approximately mirna loci, some of which are clusters, in the mouse genome, which is roughly equivalent to % of protein coding genes. of imprinted genes, contain mirnas, while 20% contain some kind of small ncrna including mirnas; 84% of these gene arrangements are conserved in humans. the numbers represent a significant enrichment among imprinted genes for co-regulation by some kind of ncrna. one could speculate that local expansion of ncrnas might flag a gene for silencing in one of the parental germlines, and that selection would fix the silencing, perhaps by insertion of a more efficient dmr, thus obviating the need for retention of the ncrna cluster. the annotated mirnas in both the rat and mouse arrays are from the mir- family. however, it is clear that there are likely more mirnas in each block than have been annotated. dot plots of mouse and rat introns against both themselves and each other reveal significant repetitive sequence homology, even after the annotated mirnas are removed. in addition, both mouse and rat arrays can be organized into tandem repeats of approximately kb, although the rat repeats suffer from gaps in the sequence database. in contrast, no other mammalian introns examined contain repetitive sequences. there may be more mirnas, or at least mirna-like sequences, in both mouse and rat introns that remain to be annotated. for example, a small random section of the rat intron was subjected to analysis with mipred to reveal the presence of two additional predicted pre-mirna sequences from the mir- family. kangaroo rats and deer mice fall within the mouse related clade of rodents. both possess potential mir- mirna sequences in intron although these do not resemble the mir- pre-mirna sequences in rats and mice, apart from the putative mature mirna. it is unclear whether these potential mirnas in kangaroo rats and peromyscus are evolutionary precursors or remnants. several of the mirnas from the murine sfmbt locus are expressed and processed in placenta, not surprising given that they are part of the primary transcript for sfmbt which is robustly expressed in placenta. recent analysis of small rna libraries with massively parallel sequencing strategies have indicated that several of the mirnas from the cluster are expressed in es cells; interestingly, the number of reads does not correlate with the frequency of the mirna within the block of repeats, suggesting that the primary transcript likely folds in a complex manner that only allows processing of a subset of the potential mirnas. whether these mirnas mediate any functions in placental biology remains an open question. there are mir- family members in other mammalian genomes, notably human and pig, although in far fewer numbers than in rodents. moreover, there are several mmu-mir- at other locations in the mouse genome. any of the mirnas processed from the sfmbt primary transcript would be imprinted, although testing this is problematical given the highly repetitive nature of the array itself, and the presence of other family members at other locations in the genome. genomic imprinting is an epigenetic mechanism that is restricted to mammals and angiosperm plants. there is mounting evidence that it is mediated by maternal factors, possibly as a means of controlling extraembryonic tissues that parasitize the maternal reproductive tract. studies of the evolution of imprinting indicate that it seems to have emerged at different times at different loci. interestingly, several imprinted clusters contain mirnas or snords, and some of these have been demonstrated to be imprinted themselves. the role of ncrnas in imprinting also includes regulatory functions, such as that exerted by the kcnq1ot antisense transcript at the kcnq locus, and the airn transcript at the igf2r locus. rnai biology is providing interesting new insights into gene regulation. studies in plants and yeast have revealed a wide array of effects, from post transcriptional gene silencing involving both mrna degradation and translational control, to heterochromatin formation, particularly at centromeres. a recent report of the ncrna clusters at the dlk and snrpn imprinted loci has revealed that transcripts containing precursors of the mirnas or snords from these loci are retained by the nucleus. this raises the interesting notion that the function of the ncrnas at these loci may be more similar to the regulation of centromeres in yeast than to "traditional" cytoplasmic rnai. such a role is also implied by the phenotype of both dicer and ago conditional null alleles in mouse oocytes, which fail to complete the first meiotic division due to misaligned chromosomes. interestingly, maternal loss of dgcr a component of the mirna processing pathway, is without effect on development, even though the profile of misexpressed mirnas in maternal dicer -/- and dgcr -/- zygotes is the same. it is tempting to speculate that expression of a subset of the mirnas from the sfmbt cluster provides the rnai transcriptional silencing biochemistry with the molecular substrates that allow it to target the repeated sequences at the sfmbt intron for transcriptional epigenetic silencing on the maternal allele. the evolution of mirnas is highly dynamic, and is characterized by expansions and contractions, partly through segmental duplication/deletion events. the mir- family was acquired by amniotes approximately mya, and has remained relatively small; humans possess one copy on chromosome and another on the x chromosome, while pigs and orangutans possess only the x linked gene. mice have acquired, in addition to the large cluster at sfmbt several other copies on chromosomes and although current mirna annotation and archiving may be incomplete for this family. interestingly, a large cluster of mirnas in a different family expanded in primates after divergence from the rodent lineage, and is expressed exclusively in placenta. this cluster maps to chromosome 19q, and is near several loci that display either imprinted expression or parent of origin effects in humans. a recent report demonstrated paternal expression of the primary transcript encoding this mirna cluster in human placenta. the primary transcript is associated with a classic germline dmr; this locus is however much older than the cluster at the rodent sfmbt gene by at least million years. as mentioned above, two other imprinted loci, the dlk domain and the snrpn region, also contain large clusters of non-coding rnas. they also possess classic germline dmrs. the lack of any noticeable germline methylation at sfmbt may reflect its youth as an imprinted domain, suggesting that imprinted regulation precedes establishment of differential methylation. this raises the possibility that one general mechanism by which genes or gene domains become imprinted is through local expansion of mirnas, and that the rodent sfmbt locus is a window on an actively evolving imprinted domain. how these clusters of ncrnas mediate gene silencing at the transcriptional level will be an interesting puzzle to solve.  the very recent insertion of a large block of mirnas into the intron of the pcg gene sfmbt in old world rodents coincides with the acquisition of placental imprinting at this locus. the transcriptional start site cgi does not display heritable differential methylation, suggesting that maternal silencing by other chromatin mechanisms mediates imprinting at this locus. 
an interesting feature of both mouse and rat sfmbt genes is the presence of a large block of mirnas in intron other mammals, including the bovine, lack this block of mirnas. no imprinted gene had been identified from this region until our study demonstrated that the pcg gene sfmbt is expressed from the paternally inherited allele in early embryos and extraembryonic tissues. our results are discussed in the context of ncrnas at other imprinted loci. the proximal region of murine chr has long been known to harbour one or more imprinted genes from classic genetic studies involving reciprocal translocations. we analyzed allelic expression of approximately genes within a mb domain and found that sfmbt and an overlapping non-coding antisense transcript are the only imprinted genes in this region. we also demonstrate that rat sfmbt is imprinted in extraembryonic tissues.
 the proximal region of murine chr has long been known to harbour one or more imprinted genes from classic genetic studies involving reciprocal translocations. no imprinted gene had been identified from this region until our study demonstrated that the pcg gene sfmbt is expressed from the paternally inherited allele in early embryos and extraembryonic tissues. imprinted genes generally reside in clusters near elements termed imprinting control regions, suggesting that sfmbt might represent an anchor for a new imprinted domain.  we analyzed allelic expression of approximately genes within a mb domain and found that sfmbt and an overlapping non-coding antisense transcript are the only imprinted genes in this region. these transcripts represent a very narrow imprinted gene locus. we also demonstrate that rat sfmbt is imprinted in extraembryonic tissues. an interesting feature of both mouse and rat sfmbt genes is the presence of a large block of mirnas in intron other mammals, including the bovine, lack this block of mirnas. consistent with this association, we show that human and bovine sfmbt are biallelic. other evidence indicates that pig sfmbt is also not imprinted. further strengthening the argument for recent evolution of sfmbt is our demonstration that a more distant muroid rodent, peromyscus also lacks imprinting and the block of mirnas.  these observations are consistent with the hypothesis that the block of mirnas are driving imprinting at this locus. our results are discussed in the context of ncrnas at other imprinted loci. accession numbers for peromyscus cdna and intron genomic dna are, respectively. 