Characterization of cis- and trans-acting elements in the imprinted human SNURF-SNRPN locus

Published online August 22, 2005 4740–4753 Nucleic Acids Research, 2005, Vol. 33, No. 15 doi:10.1093/nar/gki786 Characterization of cis- and trans-acting elements in the imprinted human SNURF-SNRPN locus Sara Rodriguez-Jato1, Robert D. Nicholls4, Daniel J. Driscoll2,3 and Thomas P. Yang1,2,3,* 1 Department of Biochemistry and Molecular Biology, 2Department of Pediatrics, 3Center for Mammalian Genetics, University of Florida College of Medicine, Gainesville, FL 32610, USA and 4Center for Neurobiology and Behavior, Department of Psychiatry, University of Pennsylvania, Philadelphia, PA 19104, USA Received April 21, 2005; Revised July 27, 2005; Accepted August 5, 2005 ABSTRACT INTRODUCTION SNURF-SNRPN (hereafter termed SNRPN) is a bicistronic imprinted gene on human chromosome 15 that encodes two polypeptides, the SmN splicing factor involved in RNA processing (1), and the SNURF (SNRPN upstream reading frame) polypeptide of unknown function (2). SNRPN also *To whom correspondence should be addressed. Tel: +1 352 392 6472; Fax: +1 352 392 2953; Email:  The Author 2005. Published by Oxford University Press. All rights reserved. The online version of this article has been published under an open access model. Users are entitled to use, reproduce, disseminate, or display the open access version of this article for non-commercial purposes provided that: the original authorship is properly and fully attributed; the Journal and Oxford University Press are attributed as the original place of publication with the correct citation details given; if an article is subsequently reproduced or disseminated not in its entirety but only in part or as a derivative work this must be clearly indicated. For commercial re-use, please contact The imprinted SNRPN locus is a complex transcriptional unit that encodes the SNURF and SmN polypeptides as well as multiple non-coding RNAs. SNRPN is located within the Prader-Willi and Angelman syndrome (PWS/AS) region that contains multiple imprinted genes, which are coordinately regulated by a bipartite imprinting center (IC). The SNRPN 50 region co-localizes with the PWS-IC and contains two DNase I hypersensitive sites, DHS1 at the SNRPN promoter, and DHS2 within intron 1, exclusively on the paternally inherited chromosome. We have examined DHS1 and DHS2 to identify cis- and trans-acting regulatory elements within the endogenous SNRPN 50 region. Analysis of DHS1 by in vivo footprinting and chromatin immunoprecipitation identified allelespecific interaction with multiple regulatory proteins, including NRF-1, which regulates genes involved in mitochondrial and metabolic functions. DHS2 acted as an enhancer of the SNRPN promoter and contained a highly conserved region that showed allele-specific interaction with unphosphorylated RNA polymerase II, YY1, Sp1 and NRF-1, further suggesting a key role for NRF-1 in regulation of the SNRPN locus. We propose that one or more of the regulatory elements identified in this study may also contribute to PWS-IC function. encodes a long (460 kb) alternatively spliced RNA transcript that contains several families of snoRNAs (3) and extends downstream to partially overlap the UBE3A gene in the anti-sense orientation. The SNRPN promoter is associated with a CpG island that is hypermethylated on the maternally inherited allele and hypomethylated on the paternally inherited allele (4). Two alternative upstream promoters and alternatively spliced non-coding exons expressed at low levels add to the complexity of the locus (5). The gene is transcribed exclusively from the paternally inherited chromosome and shows highest levels of expression in the brain and heart (2). Furthermore, SNRPN is located within an imprinted gene cluster in chromosome 15 q11–q13 that is associated with the Prader-Willi syndrome (PWS) and Angelman syndrome (AS). PWS and AS are two clinically distinct neurogenetic disorders linked to a single imprinted domain on chromosome 15 containing at least eight genes distributed across 2 Mb [reviewed in (6)]; the similarly imprinted syntenic region in the mouse is located on chromosome 7C (6). PWS arises from loss of function of genes in this region that are expressed exclusively from the paternal chromosome, while AS arises from loss of expression or mutation of the maternally expressed UBE3A gene. Multiple genetic mechanisms lead to the allele-specific loss of gene expression in AS and PWS, including deletions of the entire imprinted region, uniparental disomy (UPD), and microdeletions that encompass the 50 region of the SNRPN gene and/or a region upstream of SNRPN. High-resolution mapping of these microdeletions has led to the delineation of a bipartite imprinting control region or imprinting center (IC). All of the microdeletions associated with PWS share a 4.3 kb deleted region termed the PWS smallest region of deletion overlap (PWS-SRO), which includes the SNRPN promoter region, first exon, and part of the first intron (7). Similarly, the microdeletions associated with AS share a 0.8 kb ASSRO located 35 kb upstream from exon 1 of SNRPN (8). Thus, the IC is composed of two distinct functional components, the PWS-IC (including but not necessarily limited Nucleic Acids Research, 2005, Vol. 33, No. 15 MATERIALS AND METHODS Cell culture EBV-transformed lymphoblasts derived from PWS and AS patients were grown in RPMI medium supplemented with 10% fetal bovine serum (FBS) and 1% penicillin– streptomycin. Human SK-N-SH neuroblastoma cells were obtained from the American Type Culture Collection and grown in E-MEM supplemented with 10% FBS and 1% penicillin–streptomycin. Cells were grown at 37 C in 5% CO2. DNase I treatment of permeabilized cells for mapping of hypersensitive sites and Southern blotting Cell permeabilization and DNase I treatment was performed as described previously (20). Genomic DNA from DNase I treated cells was digested with BamHI and size-fractionated in a 0.8% agarose gel, transferred and hybridized as described previously (20). The 2.26 kb hybridization probe was isolated from plasmid pPH-B8 (21) by digestion with BamHI and EcoRI and labeled by random priming. Vector design Constructs for transient expression assays were generated from the pGL3-Basic vector (Promega). Details on vector construction are provided in Supplemental Material. Transient transfection and luciferase reporter assays SK-N-SH cells were transfected with firefly luciferase expression constructs using SuperFect (Qiagen) according to the manufacturer’s specifications (see Supplementary Material). Cells were co-transfected with pRL-TK (Promega) which contains the Renilla luciferase gene. Cells were lysed 24 h posttransfection and firefly and Renilla luciferase activities were measured (Dual-Luciferase Reporter Assay System, Promega) in a Sirius Luminometer V2.2. (Berthold Detection Systems). Firefly luciferase activity was normalized to Renilla luciferase activity. For each construct, the average and stand (...truncated)