Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2024 Sep 28;17(1):29.
doi: 10.1186/s13072-024-00552-8.

PRKACB is a novel imprinted gene in marsupials

Trent Newman  1 , Donna M Bond  2 , Teruhito Ishihara  1   3 , Phoebe Rizzoli  1 , Quentin Gouil  4 , Timothy A Hore  2 , Geoff Shaw  1 , Marilyn B Renfree  5
Affiliations

PRKACB is a novel imprinted gene in marsupials

Trent Newman et al. Epigenetics Chromatin. .

Abstract

Background: Genomic imprinting results in parent-of-origin-specific gene expression and, among vertebrates, is found only in therian mammals: marsupials and eutherians. A differentially methylated region (DMR), in which the methylation status of CpG dinucleotides differs between the two alleles, can mark the parental identity of imprinted genes. We developed a computational pipeline that detected CpG islands (CGIs) marked by both methylated and unmethylated signals in whole genome bisulfite sequencing data. This approach identified candidate marsupial DMRs in a publicly available koala methylome. One of these candidate DMRs was associated with PRKACB, a gene encoding the protein kinase A catalytic subunit beta. Nothing is known about the imprinting status of PRKACB in eutherian mammals although mutations of this gene are associated with endocrine neoplasia and other developmental disorders.

Results: In the tammar wallaby and brushtail possum there was parent-of-origin-specific DNA methylation in the PRKACB DMR in which the maternal allele was methylated and the paternal allele was unmethylated. There were multiple RNAs transcribed from this locus. Allele-specific expression analysis identified paternal expression of a PRKACB lncRNA and an mRNA isoform. Comparison of the PRKACB gene start site between marsupials and eutherians demonstrated that the CGI is longer in marsupials. The PRKACB gene product functions in the same signalling pathway as the guanine nucleotide-binding protein alpha subunit encoded at the GNAS locus, a known eutherian imprinted gene. In a mouse methylome Gnas had three differentially methylated CGIs, while in the koala methylome the GNAS locus had two unmethylated CGIs.

Conclusions: We conclude that PRKACB is a novel, DMR-associated marsupial imprinted gene. Imprinting of PRKACB in marsupials and GNAS in eutherians may indicate a conserved selection pressure for imprinting of the protein kinase A signalling pathway in therians with the two lineages adapting by imprinting different genes.

Keywords: GNAS; PRKACB; Convergent evolution; Differential methylation; Genomic imprinting.

PubMed Disclaimer

Conflict of interest statement

The authors declare no competing interests.

Figures

Fig. 1
Fig. 1
Pipeline for candidate DMR detection identifies PRKACB. A Schematic of the processes involved in the bioinformatic DMR candidate detection pipeline. The steps indicated by the yellow boxes were achieved using custom scripts. B, C, E CGIs were assessed for methylation in terms of the percent of WGBS reads that were methylated and the standard deviation (STD) of the methylation across the CpG sites in the CGI. B Known mouse imprinted genes have differentially methylated CGIs in a mouse brain methylome. C Known marsupial imprinted genes had differentially methylated CGIs in a koala brain methylome, the koala methylome highlighted an unmethylated IGF2R promoter CGI and a differentially methylated intragenic CGI. D Illustration of the koala IGF2R locus, exons are arrowheads indicating the direction of transcription, introns are thinner shaded regions (red indicates maternal expression). CpG density is plotted as a percentage averaged over 500 bp windows. Methylated (purple) and unmethylated (green) WGBS read counts and the percent methylation are plotted over the region. E Grey boxes indicate the selection gates for koala DMR candidates, the PRKACB candidate DMR is indicated. Note, plots C and E contain the same data but highlight different genes of interest. Species silhouettes are Murinae and Phascolarctos cinereus, courtesy of PhyloPic.org
Fig. 2
Fig. 2
Differential methylation of koala but not mouse PRKACB CGI. Scale illustration of the A mouse Prkacb locus and the B koala PRKACB locus, exons are arrowheads indicating the direction of transcription, introns are thinner shaded regions. The structures of two Prkacb isoforms are indicated for the mouse. CpG density is plotted as a percentage averaged over 500 bp windows. Methylated (purple) and unmethylated (green) WGBS read counts and the percent methylation are plotted over the region. Species silhouettes are Mus musculus and Phascolarctos cinereus, courtesy of PhyloPic.org
Fig. 3
Fig. 3
Differential methylation of marsupial PRKACB CGI. A The PRKACB locus in tammar, the position of the bisulfite primers used to clone the CGI region for DNA methylation analysis are indicated. B The position of the CpG sites and SNP within the assessed amplicon. Sanger genotyping of the SNP used for allele-phasing of methylation signals. C Allele-specific methylation analysis of tammar tail tissue showing methylated (purple) and unmethylated (green) CpG sites on the paternal (left) and maternal (right) allele for one of four animals. D The PRKACB locus in brushtail possum, the position of the bisulfite primers used for DNA methylation by amplicon sequencing. E The position of the CpG sites and SNPs within the two assessed amplicons. Illumina genotyping of the SNPs used for allelic-phasing of methylation signals in PCR amplicons 1 and 2. F Allele-specific methylation analysis in the brushtail possum liver showing methylated (purple) and unmethylated (green) reads at 15 CpG sites (assessed by 2 bisulfite amplicons) on the paternal (left) and maternal (right) allele for one of two animals. Species silhouettes are Phascolarctos cinereus, Notamacropus (Macropus) eugenii and Trichosurus vulpecula, courtesy of PhyloPic.org
Fig. 4
Fig. 4
Transcription from the PRKACB locus. A The brushtail possum PRKACB locus illustrating selected transcript structures detected in short read transcriptomes from PY muscle, spleen and liver and the estimated region containing a low-level lncRNA signal in muscle. Below, the coverage tracks of forward (brown) and reverse (yellow) transcript mapped reads for the different tissues, “sashimi” lines reflect junction-spanning reads with the width of the lines a function of the number of reads. B The tammar PRKACB locus illustrating selected transcript structures detected across short read adult gonad transcriptomes and long read transcriptomes from a term fetus and PY gonads. C The structure, compressed horizontally relative to B, and expression level of all of the merged Stringtie transcript stuctures in the tammar. The expression level for each transcript is reported as FPKM, and given a colour to reflect this value, for the different tammar tissues. D RT-PCR gels showing the presence of the lncRNA at different stages of ovary (top) and testis (bottom) development. Asterisks highlight the difference between the ovary and testis in terms of expression at the adult stage. The position of the amplicon is indicated in C. E The opossum PRKACB locus illustrating selected transcript structures and annotated transcripts detected in short read transcriptomes from ovary, testis and muscle. Species silhouettes are Trichosurus vulpecula, Notamacropus (Macropus) eugenii and Monodelphis domestica, courtesy of PhyloPic.org
Fig. 5
Fig. 5
Allele-specific expression from the PRKACB locus. A The tammar lncPRKACB locus indicating the location of the primers used for allele-specific expression analysis. The blue transcript colour indicates paternal expression, the position of the CGI is provided for reference. B Allele-specific expression of lncPRKACB in tammar liver (top) and muscle (bottom), the horizontal line distinguishes two sets of mother-young. Informative homozygous genotype in a matched maternal sample (left), heterozygous SNP in PY genomic DNA (centre) and expression of lncPRKACB from the paternal allele (right). C The rest of the tammar PRKACB locus indicating coding transcripts, PRKACB_iso1 and PRKACB_iso2. The location of isoform-specific and nested primers used for allele-specific expression is indicated. D Allele-specific expression of PRKACB is transcript-specific in tammar. The blue transcript colour indicates paternal expression, white transcript colour indicates biallelic expression. Informative homozygous genotype in a matched maternal sample (left), heterozygous SNP in PY genomic DNA (centre left) and expression of PRKACB_iso1 (centre right) and PRKACB_iso2 (right). E The brushtail possum lncPRKACB locus indicating the location of the SNPs used for allele-specific expression analysis. The exon positions are aligned from the tammar lncPRKACB. The grey transcript colour indicates unknown parent-of-origin-specific expression, the position of the CGI is provided for reference. F Monoallelic expression of the lncPRKACB across the three SNP sites in amplicon sequencing from brushtail possum PY muscle tissue. G The rest of the brushtail possum PRKACB locus indicating coding transcripts, PRKACB_iso1 and PRKACB_iso2. The blue transcript colour indicates paternal expression, white transcript colour indicates biallelic expression. The location of the SNP used for allele-specific expression in the shared 3′ region is indicated. F Biallelic expression of PRKACB in a brushtail possum PY liver and monoallelic paternal expression in PY muscle
Fig. 6
Fig. 6
Marsupial-specific PRKACB CGI structure. A Cladogram indicating the phylogenetic relationship of the monotreme, marsupial and eutherian species analysed. Monotremes diverged before the separation of therians but are positioned (dotted line) between eutherians and marsupials to facilitate comparison. B Synteny comparison of the region containing the TTLL7, PRKACB and SAMD13 genes. The annotated gene regions are shown as white arrowheads, CpG density is shown below, averaged over 500 bp windows. Dark grey links indicate pairwise alignments between two species, orange links indicate inverted alignments. The green shaded region highlights the PRKACB start site. C Repeat element composition and CpG density over the CGI region at the PRKACB start site of different animals. A 50 kb region is plotted for each animal, centered on the gene start site, with CpG density averaged over 100 bp windows. The location of the tammar lncPRKACB transcript is indicated. LTRs are pink, LINE/L2 elements are cyan, other LINEs are dark blue, SINEs are green and other transposons are yellow. Below the transposable elements, tandem repeats are indicated as grey lines. The mouse, elephant, platypus, agile gracile opossum, grey short-tailed opossum, brushtail possum regions were reversed to correct for larger inversions or a different chromosome orientation relative to the other species. Species silhouettes are Homo sapiens, Mus musculus, Loxodonta africana, Tachyglossus aculeatus, Ornithorhynchus anatinus, Gracilinanus, Monodelphis domestica, Phascolarctos cinereus, Notamacropus (Macropus) eugenii and Trichosurus vulpecula, courtesy of PhyloPic.org
Fig. 7
Fig. 7
Marsupial-specific GNAS CGI structure. Scale illustration of the A mouse Gnas locus and the B koala GNAS locus, exons are arrowheads indicating the direction of transcription, introns are thinner shaded regions. CpG density is plotted as a percentage averaged over 500 bp windows. Methylated (purple) and unmethylated (green) WGBS read counts and the percent methylation are plotted over the region
Fig. 8
Fig. 8
Lineage-specific imprinting of the cAMP/PKA signaling pathway. Simplified diagram of the cAMP/PKA signaling pathway indicating steps of interest subjected to lineage-specific imprinting. In eutherians (left), the GNAS gene is imprinted and Gsα (G subunit alpha) has expression from the maternal allele (red). In marsupials (right), the PRKACB gene is imprinted and is expressed from the paternal allele (blue). Dark grey indicates an unconfirmed or unknown imprinting status of a particular pathway step in the lineages
See this image and copyright information in PMC

Similar articles

See all similar articles

References

    1. Adhami HA, Evano B, Digarcher AL, Gueydan C, Dubois E, Parrinello H, et al. A systems-level approach to parental genomic imprinting: the imprinted gene network includes extracellular matrix genes and regulates cell cycle exit and differentiation. Genome Res. 2015;25:353–67. - PMC - PubMed
    1. Babak T, DeVeale B, Tsang EK, Zhou Y, Li X, Smith KS, et al. Genetic conflict reflected in tissue-specific maps of genomic imprinting in human and mouse. Nat Genet. 2015;47:544–9. - PMC - PubMed
    1. Bartolomei MS, Ferguson-Smith AC. Mammalian genomic imprinting. Cold Spring Harb Perspect Biol. 2011;3:a002592. - PMC - PubMed
    1. Bathgate RAD, Siebel AL, Tovote P, Claasz A, Macris M, Parry LJ. Purification and characterization of relaxin from the tammar wallaby (Macropus eugenii): bioactivity and expression in the corpus luteum. Biol Reprod. 2002;67:293–300. - PubMed
    1. Bennetts L, Lin M, Aitken RJ. Cyclic AMP-dependent tyrosine phosphorylation in tammar wallaby (Macropus eugenii) spermatozoa. J Exp Zool. 2004;301A:118–30. - PubMed

Substances

LinkOut - more resources