Reactivation of maternal SNORD116 cluster via SETDB1 knockdown in Prader-Willi syndrome iPSCs

Abstract

Prader-Willi syndrome (PWS), a disorder of genomic imprinting, is characterized by neonatal hypotonia, hypogonadism, small hands and feet, hyperphagia and obesity in adulthood. PWS results from the loss of paternal copies of the cluster of SNORD116 C/D box snoRNAs and their host transcript, 116HG, on human chromosome 15q11-q13. We have investigated the mechanism of repression of the maternal SNORD116 cluster and 116HG. Here, we report that the zinc-finger protein ZNF274, in association with the histone H3 lysine 9 (H3K9) methyltransferase SETDB1, is part of a complex that binds to the silent maternal but not the active paternal alleles. Knockdown of SETDB1 in PWS-specific induced pluripotent cells (iPSCs) causes a decrease in the accumulation of H3K9 trimethylation (H3K9me3) at 116HG and corresponding accumulation of the active chromatin mark histone H3 lysine 4 dimethylation (H3K4me2). We also show that upon knockdown of SETDB1 in PWS-specific iPSCs, expression of maternally silenced 116HG RNA is partially restored. SETDB1 knockdown in PWS iPSCs also disrupts DNA methylation at the PWS-IC where a decrease in 5-methylcytosine is observed in association with a concomitant increase in 5-hydroxymethylcytosine. This observation suggests that the ZNF274/SETDB1 complex bound to the SNORD116 cluster may protect the PWS-IC from DNA demethylation during early development. Our findings reveal novel epigenetic mechanisms that function to repress the maternal 15q11-q13 region.

Figure 1.

ZNF274 is a novel epigenetic regulator of the silent maternal allele in PWS. (A) A map of a subregion of 15q11-q13, indicating ZNF274 binding sites in SNOG1/116HGG1 within the SNORD116 cluster. Imprinted, paternally expressed genes are denoted by blue boxes. UBE3A is biallelically expressed in iPSCs. The PWS-IC is denoted by the black circle. Green lines within SNOG1/116HGG1 represent the six ZNF274 binding sites. (B) ZNF274 ChIP assays in iPSCs. Allele specificity was determined by comparing the enrichment observed on the silent maternal allele in PWS iPSCs derived from two classes of PWS patients to the active paternal allele in AS iPSCs. Enrichment at ZNF180 was used as a positive control in these assays. Quantification of ChIPs was performed and calculated as percent input for each sample. Non-specific IgG values were subtracted from percent input values. (C) ChIP assays for the repressive histone modification H3K9me3. H3K9me3 was enriched on maternal allele in iPSCs derived from all three classes of PWS patients, when compared with AS iPSCs. *P ≤ 0.1, **P ≤ 0.05, ***P ≤ 0.01.

Figure 2.

Functional role of ZNF274 complex in the silencing of the maternal allele in PWS. (A) Gene expression of ZNF274 in PWS iPSCs following transfection with control siRNAs or specific siRNAs targeting ZNF274. GADPH was used as an endogenous control. Data were normalized to PWS MIS. ChIP assays for ZNF274 (B) and H3K9me3 (C), respectively, in PWS ZNF274kd iPSCs. Enrichment at ZNF180 was used as a control in these assays. (D) Gene expression analysis of SNRPN and 116HG in PWS ZNF274kd iPSCs. GADPH was used as an endogenous control and data were normalized to PWS MIS. * P ≤ 0.1, ** P ≤ 0.05, *** P ≤ 0.01.

Figure 3.

SETDB1 associates with ZNF274 on the silent maternal allele within SNORD116. (A) Enrichment of SETDB1 within 15q11-q13 by ChIP analyses, in PWS del 1–7 and AS del 1-0 iPSCs. (B) Sequential ChIP assays in PWS del 1–7 and AS del 1-0 iPSCs within SNORD116. Enrichment at ZNF180 was used as a control in these assays. ZNF274 ChIP samples were sequentially immunoprecipitated using ZNF274 antibody followed by SETDB1 antibody. (C) Gene expression of SETDB1 in PWS 1–7 iPSCs following lentiviral transduction with control or specific shRNA constructs targeting SETDB1. GAPDH was used as an endogenous control and data were normalized to PWS MIS iPSCs. (D) ChIP analyses for enrichment of SETDB1 in PWS SETDB1kd iPSCs, relative to MIS control. Enrichment at ZNF180 was used as a control in these assays. (E) Expression of genes outside of 15q11-q13 in PWS SETDB1kd iPSCs used as negative controls. *P ≤ 0.1, **P ≤ 0.05, ***P ≤ 0.01.

Figure 4.

Reactivation of the silent maternal allele in PWS. ChIP analyses for H3K9me3 (A) and H3K4 dimethylation (H3K4me2) (B) in PWS SETDB1kd iPSCs, relative to PWS MIS iPSCs. Enrichment at ZNF180 and GAPDH was used as a control in these assays, respectively. (C) Analysis of SNRPN and 116HG expression levels in PWS SETDB1kd and PWS SD SETDB1kd iPSCs. GADPH was used as an endogenous control and data were normalized to their respective MIS controls. *P ≤ 0.1, **P ≤ 0.05, ***P ≤ 0.01.

Figure 5.

DNA methylation levels change when the silent maternal allele in PWS is reactivated. Content of 5hmC at the PWS-IC, quantified by subtraction of 5hmC from the total methylated cytosine levels, determined by HpaII sensitivity as a percentage of total cytosine in PWS del 1–7 and AS del 1-0 iPSCs (A) or PWS SETDB1kd iPSCs, relative to PWS MIS iPSCs (B). ***P ≤ 0.01. (C) Model describing the role of the ZNF274/SETDB1 complex in the establishment/maintenance of silencing of the maternal allele of 15q11-q13. Boxes represent: SNRPN (small nuclear ribonucleoprotein polypeptide N) and SNORD116 (small nucleolar RNA, C/D box 116). Filled oval represents presence of 5mC methylation at the PWS-IC. ZNF274 and SETDB1 potentially in association with other proteins recognize the tandemly repeated SNOG1 maternal alleles at early times after fertilization. Green lines within SNOG1/116HGG1 represent the six ZNF274 binding sites. Binding of the repressive complex to paternal SNOG1 alleles may be prevented by active transcription through the PWSCR at early stages of development. Binding of the complex to the maternal SNOG1 alleles results in deposition of the repressive H3K9me3 (indicated by yellow ovals) and stabilizes epigenetic silencing of the PWSCR in early development. The ZNF274/SETDB1 complex may also protect the 5mC mark at the maternal PWS-IC from oxidative modification by TET proteins that are highly expressed at the blastocyst and epiblast stages (27).