Figure 1.
Identifying tammar IGF2 transcription start sites.
5′-RACE amplification was performed on (A) liver and (B) placenta RNA samples. (A) Three bands first identified in the liver (black arrows) were visible in both tissue types. (B) Additional exon 1B transcription start sites (TSS) were identified in the placenta. (C) Aligning the cloned products to the gDNA sequence identified the location of each of the non-coding exons 1A, 1B and 1C (white boxes) in relation to the coding exons 2, 3 and 4 (black boxes) followed by an untranslated region of unknown length (dashed box). TSSs are indicated by turned arrows. Two single nucleotide polymorphisms (black triangles) identified in the untranslated region were used for imprint analysis (snp1 and snp2). Diagram is not to scale.
Figure 2.
Genomic structure of tammar IGF2 including 3 TSS and comparison with mouse, human and opossum genomes.
Opossum (clone XX-223O16 ch5∶102112-82113), human (ch11∶2156597-2176597) and mouse (ch7∶149841559-149861559), IGF2 base genomes were aligned to tammar IGF2 (clone MEKBa-346C2∶62540-82539). VISTA pairwise alignments using a 100 bp sliding window was performed between each species and tammar. Pink peaks represent areas of conservation of 70% over a minimum of 100 bp between tammar and opossum and 55% over a minimum of 50 bp between tammar and human or mouse. A dotted line indicates a region with 70% conservation between P1 and P2 in both marsupials and eutherians. A schematic of each IGF2 gene is located above the VISTA plot to show relative location of the non-coding exons (open box), including the non-coding exons transcribed with the P0 TSS [12], and coding exons (black box). Location of CpG sites are represented by vertical black bars below the VISTA plot.
Figure 3.
IGF2 mRNA expression in the adult tammar.
RT-PCR expression profile after 35 cycles of each IGF2 mRNA transcript in a range of adult tammar tissues compared to GAPDH. The expression was strongest from P2, with expression of all three transcripts in the brain, eye, tongue, liver and mammary gland. No expression was detected in the No Template Control (NTC).
Figure 4.
IGF2 mRNA expression from each promoter (P1, P2 and P3) relative to a reference gene in various wallaby tissues.
(A) IGF2 mRNA expression relative to GAPDH in the Liver, (B) IGF2 mRNA expression relative to GAPDH in the brain, (C) IGF2 mRNA expression relative to B-ACTIN in the mammary gland, (D) IGF2 mRNA expression relative to B-ACTIN in the bilaminar yolk sac and (E) IGF2 mRNA expression relative to 18S in the trilaminar yolk Sac. (A, B) Expression from P2 in the pouch young (PY) is significantly higher than expression from P1 and P3 (* P<0.05) in the liver (A) and brain (B). Except for P1 in the liver, IGF2 is expressed at a higher level in the pouch young then in the adult (a, P<0.01) in both the liver and the brain (if adult hypothalamus expression is representative of total adult brain expression). (C, D, E) P2-IGF2 is also the predominantly expressed transcript in the mammary gland (* P<0.05) (C) and in the bilaminar (D) and trilaminar (E) placenta (a,b,c have significantly different means, P<0.01). There was no difference in the expression levels of any of the transcripts between non-lactating and lactating mammary glands (C).
Figure 5.
Allelic expression from the three promoters in various adult and pouch young tissues.
Direct sequencing of two single nucleotide polymorphisms (snp1 and snp2) was used to determine the allelic expression of each of the IGF2 transcripts in adult and pouch young (PY) tissues. In a 12 day old female pouch young monoallelic expression was detected from all 3 promoters in the brain and liver, except P1 in the liver was expressed from both alleles. In a 72 day old male pouch young, biased to biallelic expression was detected in the brain while the liver expression was similar to the day 12 pouch young. In the adult, both alleles were detected from all three TSS in two liver samples and monoallelic expression was detected in three mammary gland (MG) samples. Placental samples had biallelic expression in the bilaminar region (BYS) in all samples tested for P1 and P2 and 2 of the 3 samples tested for P3. Biased to monoalleleic expression was observed in the trilaminar region (TYS) in all samples tested for P1, 3 of the 4 samples for P2, and 2 of the 3 samples for P3.
Figure 6.
Methylation at the tammar IGF2 transcription start sites.
Location of CpG sites are represented by black bars below the schematic of the tammar IGF2 gene. Bisulphite sequencing of the IGF2 CpG islands (CGI) at each of the transcription start sites (CGI1, CGI2 and CGI3) and at exon 3 (CGI4) in the pouch young and adult liver, in the mammary gland, whole yolk sac placenta and in the bilaminar and trilaminar regions of the yolk sac placenta. Black circles represent a methylated CpG site and open circles represent an unmethylated CpG site. Each row represents the methylation pattern of a separate DNA fragment from the same sample. Only CGI3 and CGI4 were significantly methylated in all tissues. CGI4 appears to be differentially methylated in the placenta. However, sequence polymorphisms that might distinguish parental copies of CGI4 were not found among the available stocks of tammar placental samples.