Nucleoporin 107, 62 and 153 mediate Kcnq1ot1 imprinted domain regulation in extraembryonic endoderm stem cells

Abstract

Genomic imprinting is a phenomenon that restricts transcription to predominantly one parental allele. How this transcriptional duality is regulated is poorly understood. Here we perform an RNA interference screen for epigenetic factors involved in paternal allelic silencing at the Kcnq1ot1 imprinted domain in mouse extraembryonic endoderm stem cells. Multiple factors are identified, including nucleoporin 107 (NUP107). To determine NUP107’s role and specificity in Kcnq1ot1 imprinted domain regulation, we deplete Nup107, as well as Nup62, Nup98/96 and Nup153. Nup107, Nup62 and Nup153, but not Nup98/96 depletion, reduce Kcnq1ot1 noncoding RNA volume, displace the Kcnq1ot1 domain from the nuclear periphery, reactivate a subset of normally silent paternal alleles in the domain, alter histone modifications with concomitant changes in KMT2A, EZH2 and EHMT2 occupancy, as well as reduce cohesin interactions at the Kcnq1ot1 imprinting control region. Our results establish an important role for specific nucleoporins in mediating Kcnq1ot1 imprinted domain regulation.

Conserved mechanism of nucleoporin regulation of the Kcnq1ot1 imprinted domain with divergence in embryonic and trophoblast stem cells

“…5 Here, we hypothesized that NUP107, NUP62 and NUP153, but not NUP98, play a regulatory role at the Kcnq1ot1 imprinted domain in ES and TS cells, and may contribute to the differential regulation of the Kcnq1ot1 imprinted domain between ES, TS and XEN cells. In the current study, we identified a conserved mechanism of nucleoporin regulation of the Kcnq1ot1 imprinted domain in ES and TS cells, as previously identified in XEN cells (Sachani et al, 2018). NUP107, NUP62 and NUP153 bind to the Kcnq1ot1 ICR on each side of the Kcnq1ot1 ncRNA promoter, and are required for paternal Kcnq1ot1 ncRNA expression, positioning of the paternal Kcnq1ot1 domain at the nuclear periphery, as well as recruitment of histone modifiers at the paternal Kcnq1ot1 ICR in both ES and TS cells.…”

“…Previous studies have identified roles for nucleoporins in gene regulation in yeast, Drosophila, and mammalian cells, although the mechanisms by which nucleoporins govern this regulation is not fully understood (Capelson et al, 2010;Jacinto et al, 2015; Kalverda et al, 2010; Light et al, 2013; Mendjan et al, 2006; Vaquerizas et al, 2010). Our investigation of nucleoporins, NUP107, NUP62, and NUP153, revealed a nucleoporin-dependent imprinting regulatory mechanism that mediates Kcnq1ot1 imprinted domain regulation in XEN cells, without impairment of nuclear-cytoplasmic transport function (Sachani et al, 2018). More specifically, our data showed that depletion of Nup107, Nup62 and Nup153 in mouse XEN cells led to a reduction in Kcnq1ot1 noncoding RNA expression and its volume at the paternal Kcnq1ot1 domain; a shift in positioning of paternal Kcnq1ot1 domain away from the nuclear periphery; and reactivation of a subset of normally silent paternal alleles of protein-coding genes in the domain.While DNA methylation at the Kcnq1ot1 ICR was not altered, Nup107, Nup62 and Nup153 depletion altered active and repressive histone modifications and reduced cohesin interactions at the Kcnq1ot1 ICR.…”

“…In a previous study, we investigated the role of several nucleoporins in Kcnq1ot1 imprinted domain regulation (Sachani et al, 2018). Nucleoporins comprise the nuclear pore complex, which are large aqueous transport channels embedded within the nuclear membrane.…”

“…At this early developmental time point, ES, TS and XEN stem cells have distinct imprinted expression patterns for genes in the Kcnq1ot1 imprinted domain (Golding et al, 2011; Lewis et al, 2004; Lewis et al, 2006; Terranova et al, 2008; Umlauf et al, 2004), suggesting that the Kcnq1ot1 imprinted domain may be differentially regulated even at early stages of lineage differentiation. However, the mechanisms responsible for this differential imprinted expression between the three lineages are still not clear.In a previous study, we investigated the role of several nucleoporins in Kcnq1ot1 imprinted domain regulation (Sachani et al, 2018). Nucleoporins comprise the nuclear pore complex, which are large aqueous transport channels embedded within the nuclear membrane.One key function of nuclear pore complexes is transport of cellular material between the cytoplasm and nucleus (Hoelz et al, 2011).…”

“…By comparison, no significant difference was observed between control and Nup98/96-or Nup153-depleted ES and TS cells (1.03 and 1.09; 0.75 and 0.86, respectively). To independently determine the effects of nucleoporin depletion on maternal and paternal Kcnq1ot1 ncRNA absolute transcript abundance, we used a highly sensitive, precision method of droplet digital PCR with FAM and HEX strain-specific probes (Sachani et al, 2018)…”