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The noncoding RNA IPW regulates the imprinted DLK1-DIO3 locus in an induced pluripotent stem cell model of Prader-Willi syndrome

Nature Genetics volume 46, pages 551–557 (2014)Cite this article

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Abstract

Parental imprinting is a form of epigenetic regulation that results in parent-of-origin differential gene expression. To study Prader-Willi syndrome (PWS), a developmental imprinting disorder, we generated case-derived induced pluripotent stem cells (iPSCs) harboring distinct aberrations in the affected region on chromosome 15. In studying PWS-iPSCs and human parthenogenetic iPSCs, we unexpectedly found substantial upregulation of virtually all maternally expressed genes (MEGs) in the imprinted DLK1-DIO3 locus on chromosome 14. Subsequently, we determined that IPW, a long noncoding RNA in the critical region of the PWS locus, is a regulator of the DLK1-DIO3 region, as its overexpression in PWS and parthenogenetic iPSCs resulted in downregulation of MEGs in this locus. We further show that gene expression changes in the DLK1-DIO3 region coincide with chromatin modifications rather than DNA methylation levels. Our results suggest that a subset of PWS phenotypes may arise from dysregulation of an imprinted locus distinct from the PWS region.

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Figure 1: Genome-wide gene expression analysis in PWS-iPSCs.
Figure 2: Characterization of the DLK1-DIO3 locus in PWS.
Figure 3: IPW regulates gene expression at the DLK1-DIO3 locus.
Figure 4: IPW affects chromatin modifications at the DLK1-DIO3 locus.
Figure 5: Model summarizing the effect of IPW depletion on the expression of MEGs in the DLK1-DIO3 region during early development.

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Acknowledgements

Fibroblasts obtained from an individual with PWS harboring complete mUPD of chromosome 15 were kindly donated by V. Gross-Tsur (Multidisciplinary Prader-Willi Syndrome Clinic, Child Neurology Unit, Shaare Zedek Medical Center). This research was partially funded by the Israel Science Foundation–Morasha Foundation (grant 1252/12) and by the Israel Ministry of Science and Technology Infrastructure (grant 3-9693).

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Authors and Affiliations

  1. Department of Genetics, Stem Cell Unit, Institute of Life Sciences, The Hebrew University, Jerusalem, Israel

    Yonatan Stelzer, Ido Sagi, Ofra Yanuka & Nissim Benvenisty

  2. Stem Cell Research Laboratory, Shaare Zedek Medical Center, The Hebrew University, Jerusalem, Israel

    Rachel Eiges

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  1. Yonatan Stelzer
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Contributions

Y.S. contributed to the conception and design of the study, the collection and assembly of data, data analysis and interpretation, and manuscript writing. I.S. contributed to the collection and assembly of data and graphic design. O.Y. and R.E. contributed to the collection and assembly of data. N.B. contributed to the conception and design of the study, financial support, data analysis and interpretation, and manuscript writing.

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Correspondence to Nissim Benvenisty.

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The authors declare no competing financial interests.

Integrated supplementary information

Supplementary Figure 1 Characterization of PWS-iPSCs.

(a) G-band karyotyping of representative PWS-iPSC lines, demonstrating a diploid genome, excluding chromosome 15 aberrations. (b) Representative alkaline phosphatase staining for PWS-iPSC lines. Scale bar, 200 μm. (c) Mean expression levels ± s.d. of representative pluripotency markers in four PWS-iPSC lines (PWS-iPSC-1-A and B and PWS-iPSC-2-A and B) compared with five control (WT) PSCs (two iPSC and three ESC lines). (d) Gene expression analysis of 20-d-old embryoid bodies (EBs) from the two PWS-iPSC lines, demonstrating differentiation in vitro into all three embryonic germ layers. DES, NCAM1 and AFP represent markers for mesoderm, ectoderm and endoderm, respectively (mean expression levels ± s.d.; two biological replicates). (e) Mean expression levels ± s.d. of representative potential targets of PEGs in the PWS locus, in PWS and control (WT) fibroblasts.

Supplementary Figure 2 Genome-wide gene expression analysis in PWS-iPSCs derived from a patient harboring a maternal UPD of chromosome 15.

(a) Establishment of PWS-iPSCs with maternal UPD in the PWS region. (b,c) Analysis of gene expression by moving average plot along chromosome 15q (b, logarithmic scale) and 14q (c) in PWS-iPSC-3 relative to normal (WT) PSCs. Vertical dashed lines mark the genomic regions of the PWS (b) and DLK1-DIO3 (c) loci.

Supplementary Figure 3 Analysis of known imprinted genes in PWS-iPSCs.

(a) RTL1 expression ratio (in RPKM, logarithmic scale, two biological replicates), demonstrating its marked downregulation in Pg-iPSCs relative to normal PSCs. (b) qRT-PCR of the mean relative fold change ± s.d. (three biological replicates each) of RTL1, MEG3, miR-370 and miR-409 in normal PSCs before and after knockdown of RTL1 (+siRNA). (c) qRT-PCR of the mean relative fold change ± s.d. of miR-371-3 in normal PSCs, Pg-iPSCs and PWS-iPSCs (two biological replicates each), demonstrating comparable expression levels of miR-371-3 in PWS-iPSCs and normal PSCs. (d) Mean expression levels ± s.d. of representative known PEGs in distinct loci. Analyses were conducted on three PWS-iPSC lines (PWS-iPSC-1-A and B and PWS-iPSC-2-A), three Pg-iPSC lines (Pg-iPSC-A-11, 20 and 26) and five control PSCs (two iPSC and three ESC lines). WT-PSCs, normal PSCs.

Supplementary Figure 4 Gene expression analyses in PWS cell lines.

(a) Analysis of gene expression by moving average plot along chromosome 14q in PWS parental fibroblasts relative to control fibroblasts. Vertical dashed lines mark the genomic boundaries of the DLK1-DIO3 region. (b) qRT-PCR of the mean relative fold change ± s.d. (three biological replicates each) in MEG3 in mature PWS cells derived from three affected individuals, showing negligible expression levels in patient-derived B cells. N.E., not expressed. (c) Expression analysis of all MEGs represented in the expression arrays in the parental fibroblasts (left panel) and undifferentiated iPSCs (right panel). WT, control.

Supplementary Figure 5 Proper monoallelic expression of imprinted genes in the DLK1-DIO3 locus in PWS-iPSCs.

Direct sequencing of the transcribed region of MEG3 and RTL1 in genomic DNA (gDNA) and complementary DNA (cDNA) in PWS-iPSCs derived from three patients. Note that both MEG3 and RTL1 are expressed in a monoallelic fashion.

Supplementary Figure 6 Chromatin regulation in PWS-iPSCs.

(a) qRT-PCR of the mean relative fold change ± s.d. (three biological replicates each) in HOTAIR in PWS-iPSCs before and after transfection with an overexpression plasmid. (b) qRT-PCR of the mean relative fold change ± s.d. (three biological replicates each) in MEG3 in PWS-iPSCs before and after transfection with the HOTAIR overexpression plasmid, relative to control (WT) PSCs. (c) Allelic sequence analysis following ChIP for H3K9me3 reveals the association of this histone modification with both methylated and unmethylated alleles of the IG-DMR in normal PSCs. (d) qRT-PCR of the mean relative fold change ± s.d. in ZFP57 in Pg-iPSCs (three biological replicates each) and PWS-iPSCs (three biological replicates each) following introduction of IPW, relative to control (WT) iPSCs.

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Stelzer, Y., Sagi, I., Yanuka, O. et al. The noncoding RNA IPW regulates the imprinted DLK1-DIO3 locus in an induced pluripotent stem cell model of Prader-Willi syndrome. Nat Genet 46, 551–557 (2014). https://doi.org/10.1038/ng.2968

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