Angelman syndrome imprinting center encodes a transcriptional promoter

Abstract

Clusters of imprinted genes are often controlled by an imprinting center that is necessary for allele-specific gene expression and to reprogram parent-of-origin information between generations. An imprinted domain at 15q11-q13 is responsible for both Angelman syndrome (AS) and Prader-Willi syndrome (PWS), two clinically distinct neurodevelopmental disorders. Angelman syndrome arises from the lack of maternal contribution from the locus, whereas Prader-Willi syndrome results from the absence of paternally expressed genes. In some rare cases of PWS and AS, small deletions may lead to incorrect parent-of-origin allele identity. DNA sequences common to these deletions define a bipartite imprinting center for the AS-PWS locus. The PWS-smallest region of deletion overlap (SRO) element of the imprinting center activates expression of genes from the paternal allele. The AS-SRO element generates maternal allele identity by epigenetically inactivating the PWS-SRO in oocytes so that paternal genes are silenced on the future maternal allele. Here we have investigated functional activities of the AS-SRO, the element necessary for maternal allele identity. We find that, in humans, the AS-SRO is an oocyte-specific promoter that generates transcripts that transit the PWS-SRO. Similar upstream promoters were detected in bovine oocytes. This result is consistent with a model in which imprinting centers become DNA methylated and acquire maternal allele identity in oocytes in response to transiting transcription.

Keywords: Angelman syndrome; imprinting; oocytes.

Fig. 1.

SNRPN upstream exon transcripts in human oocytes and brain. (A) Map of the human AS–PWS-imprinted domain. Genes shown in green are expressed from the paternal (pat) allele. UBE3A is expressed from the maternal (mat) allele. The 880-bp AS–SRO and 4.3-kb PWS–SRO are shown in red and blue, respectively. Not all products of the locus are shown, and the map is not to scale. (B) Structure of transcripts identified by 5′ RACE performed on human oocyte RNA. Exons located within the AS–SRO are shown in red. Other U exons are shown in orange. Open boxes are exonic sequences not previously documented in the UCSC Genome Browser. Numbers to the right indicate the number of clones with the indicated structure. Arrows identify the locations of RT-PCR primers used below. (C) RT-PCR analysis of U exons in whole brain and oocytes. The location of forward primers used in each lane is indicated in B above. In each case, the reverse primer was located in body exon 3. SNRPN products were identified with an exon 2 probe. Multiple bands in a lane likely represent inclusion of additional exons in the product.

Fig. 2.

RNA seq analysis of human oocyte RNA. RNA-Seq data of SNRPN upstream exon transcripts in human oocytes and brain tissues. Data are displayed as custom tracks in the UCSC Genome Browser. (A) SNURF/SNRPN gene locus, including upstream transcripts. (B) Location of the upstream exons U3–U8 and body exons 1 and 2. Exons U5 and U6 are located within the 880-bp AS–SRO. (C) RNA-Seq data for human oocytes (raw data obtained from GEO studies GSE36552 and GSE44183). (D) RNA-Seq data from human brain tissue. Tracks in C and D are scaled from 0 to 200.

Fig. 3.

SNRPN upstream exon transcripts in bovine oocytes and hypothalamus. The location of the 880-bp conserved AS–SRO sequence is shown as an open box 39.5 kb upstream of exon 1. The location of new upstream exons (red) relative to body exon 1 is indicated. SNRPN body exons are in green. (A) 5′ RACE was performed with anchor primers in SNRPN body exons 2 and 3. Most RACE products in hypothalamus and oocytes had 5′ ends at body exon 1. “H” and “O” refer to RACE products identified in the hypothalamus or oocyte, respectively. A new variably sized exon (white) located between body exons and 1 and 2 was found in several RACE products. This exon would be expected to disrupt expression of SNURF (45). (B) Southern blot of RT-PCR products amplified with forward primers in putative exons suggested by GENSCAN and reverse primer in exon 3. The blot was probed with body exon 2.