Evolution of the CDKN1C-KCNQ1 imprinted domain

Abstract

Background: Genomic imprinting occurs in both marsupial and eutherian mammals. The CDKN1C and IGF2 genes are both imprinted and syntenic in the mouse and human, but in marsupials only IGF2 is imprinted. This study examines the evolution of features that, in eutherians, regulate CDKN1C imprinting.

Results: Despite the absence of imprinting, CDKN1C protein was present in the tammar wallaby placenta. Genomic analysis of the tammar region confirmed that CDKN1C is syntenic with IGF2. However, there are fewer LTR and DNA elements in the region and in intron 9 of KCNQ1. In addition there are fewer LINEs in the tammar compared with human and mouse. While the CpG island in intron 10 of KCNQ1 and promoter elements could not be detected, the antisense transcript KCNQ1OT1 that regulates CDKN1C imprinting in human and mouse is still expressed.

Conclusion: CDKN1C has a conserved function, likely antagonistic to IGF2, in the mammalian placenta that preceded its acquisition of imprinting. CDKN1C resides in synteny with IGF2, demonstrating that imprinting of the two genes did not occur concurrently to balance maternal and paternal influences on the growth of the placenta. The expression of KCNQ1OT1 in the absence of CDKN1C imprinting suggests that antisense transcription at this locus preceded imprinting of this domain. These findings demonstrate the stepwise accumulation of control mechanisms within imprinted domains and show that CDKN1C imprinting cannot be due to its synteny with IGF2 or with its placental expression in mammals.

Figure 1

Immunohistochemical localisation of p57^KIP2 in the tammar yolk sac. Haematoxylin and eosin stained bilaminar yolk sac (BYS, A.) and trilaminar yolk sac (TYS, B.) showing trophoblast cells (Tr), yolk sac endodermal cells (En), and, in the trilaminar yolk sac only, mesenchymal cells (Me) and vitelline vessels (VV). The trophoblast lies adjacent to the maternal endometrium (Endo). p57^KIP2 was found in the cytoplasm and nucleus of most trophoblast and endodermal cells in the bilaminar (C.) and trilaminar (D.) yolk sac placenta. Although mesenchymal cells were also stained, fewer were positive. IgG antibody controls also showed weak cytoplasmic, but not nuclear staining (bilaminar, E; trilaminar, F). Day 25 of gestation stages are shown, but staining did not change notably over the stages examined (Days 19 to 26).

Figure 2

CDKN1C expression in the bilaminar (BYS-diamonds) and trilaminar (TYS-squares) yolk sac placenta. Stages examined; 19–21 (n = 8), 22–24 (n = 7), and 25–26 (n = 6). CDKN1C mRNA levels were higher in the TYS between days 19 and 24 than the BYS, but most notable was the significant increase in expression, in both regions of the yolk sac, after day 24 (days 25–26). Means sharing the same superscripted letters are not significantly different (P > 0.05). Means with different superscripts are significantly different (P ≤ 0.05).

Figure 3

Structure of the IGF2 to CDKN1C region. Tammar BAC clones from GenBank (NCBI) were used to derive the tammar sequence (A). A multiple PIP alignment of mouse and tammar sequence against human (B). Conserved regions (red) and regions of homology (green) occur mostly in gene-rich regions (black boxes), but also in the intergenic regions. The KCNQ1OT1 region is highly conserved between mouse and human, but divergent in tammar (indicated by white). IGF2 to CDKN1C in the tammar (C, top), human (C, middle), and mouse (C, bottom). In human and mouse the region includes two imprinted domains regulated the paternally methylated DMR1 and the maternally methylated DMR2 (blue and pink lollipops respectively). Mouse and human regions have been modified from published figures [31, 33, 74, 75]. Gene order is conserved between human, mouse, and tammar. The imprint status of eight of the fourteen genes are conserved between mouse and human (paternally expressed, blue; maternally expressed, pink; biallelic, black). In the tammar, IGF2 and INS are also paternally expressed, while CDKN1C is biallelic. The imprint status of the remaining genes has not been determined (grey). CpG islands and the distribution of repetitive elements are similar between mouse and human and tammar. However, the CpG island associated with DMR2 is absent in the tammar. There are similar numbers of LINE/SINE elements (blue bars) and simple repeats (green) in all species, but fewer DNA elements and LTR elements (pink and purple bars respectively) in the tammar.

Figure 4

The KCNQ1 region in human, tammar, mouse and chicken. Exons 1a/b to 15 are shown as vertical lines and intronic distances by horizontal lines. The KCNQ1OT1 transcript (blue line) is transcribed from a promoter within the maternally methylated CpG island (red box with pink lollipops). A multiple PIP alignment of mouse, tammar and chicken against human KCNQ1 (A.). Conserved (red) and homologous regions (green) are common between mouse and human, especially in intron 10 (spanning the majority of KCNQ1OT1). Both tammar and chicken are divergent from human (divergence indicated by white) in this, and other intronic regions, with only exons showing high homology. The exon-intron structure of human and tammar is highly conserved, with the notable exception of intron 9, which is markedly reduced in the tammar (B). Human, mouse and chicken have at least one CpG island in the KCNQ1 region, while tammar has no CpG islands. There is a notable reduction in the number of repetitive elements in chicken compared to the mammalian species. DNA elements (pink), LTR elements (purple), simple repeats (green) and non-LTR (LINE/SINEs, blue). A conserved region of highly repetitive sequence in mouse and human in intron 9 is indicated by a red dashed box.

Figure 5

A multiple PIP alignment of mouse, tammar, and chicken intron 10 against human (A). There are many areas of high conservation (red) and homology (green) between human and mouse, but little with tammar and chicken. The transcription start site (TSS) of KCNQ1OT1 is indicated by a blue arrow. Although the TSS is not highly conserved between mouse and human, the upstream promoter region is, as is the position of the CpG island relative to the TSS (B.). Although mouse and human have a similar numbers and types of repetitive elements, only the L1MB element upstream of the TSS may be conserved. In tammar, as in other regions, there are fewer DNA elements (pink) and LTR elements (purple) compared to human and mouse. Simple repeats (green) and non-LTR (LINE/SINEs, blue) are similar in human, mouse and tammar, but significantly fewer in chicken.

Figure 6

Expression analysis of the KCNQ1OT1. Primers designed from intron 10 of KCNQ1 were used to determine expression of the KCNQ1OT1 anti-sense RNA. Primers yield a single 400 bp band as confirmed by genomic DNA PCR (result not shown). Expression was only detected in the trilaminar portion of the placenta (TYS) and not the bilaminar (BYS) and only in the final stages of pregnancy. RT+ denotes samples that have been reverse transcribed. RT- denotes DNased RNA, also used in the PCR reaction to ensure no DNA carryover. -VE represents the negative control reaction in which template was omitted and M, indicates DNA marker.

Figure 7

The sequence coverage of repetitive elements in sequences from human, mouse, and tammar. A box plot showing the percent of total sequence masked by SINEs (dark blue), LINEs (light blue), LTR elements (purple), DNA elements (pink), simple repeats (teal), and low complexity regions (pale green) in intron 1, 1b, 9, 10, and 14 of KCNQ1 (A). The percent sequence coverage for each element over the entire region from IGF2-CDKN1C is shown by open circles. There was significantly less sequence masked by LTR and DNA elements in tammar compared to both mouse and human, while there was significantly more sequence occupied by low complexity regions (*). There was significantly less sequence occupied by LINEs in mouse compared to human (**). There was a large range in the sequence covered by LINEs in all species, but particularly in human and mouse. The percent sequence occupied by different types of repetitive elements in the region from IGF2-CDKN1C, in all of KCNQ1, and in introns 9, 10, 1, 1b, and 14 assessed separately (B). The relative percentage of sequence occupied by LINEs is noticeably more than the percentage occupied by LINEs in any other intron from KCNQ1 and this increase was significant for mouse (*). No other significant differences in the relative amounts of sequence covered by different types of elements was seen between regions within species. However, there was also a noticeable increase in the relative amount of simple repeats in intron 9 of tammar compared to other KCNQ1 introns in the tammar.