Trisomic Expression of Nuclear Receptor Interacting Protein (NRIP1) in Down Syndrome Trophoblasts Dampens Estrogen Signaling and Disrupts Cell Fusion

Deirdre M. Logsdon¹, Isabela Pereira², Kindyll Wetta¹, Lynae Smith¹, Cameron Hebert¹, John Rinn², and Justin J. Brumbaugh¹

¹Molecular, Cellular, and Developmental Biology, University of Colorado at Boulder, Boulder, USA

²Biofrontiers Institute, University of Colorado at Boulder, Boulder, USA


Abstract Text:

Down Syndrome (T21) is the most common aneuploidy disorder in humans and is characterized by a full or partial trisomy of chromosome 21. Individuals with T21 display fusion defects within their placental syncytiotrophoblasts (STBs) that act as the primary barrier between mother and fetus, however the etiology of such fusion defects is unknown. Here, we aimed to 1) characterize fusion defects in T21 vs. euploid trophoblast-like cells and 2) identify a potential mechanism responsible for T21 fusion defects. We utilized 6 pairs of age- and sex- matched iPS cell lines differentiated with BMP4, a TGFb inhibitor (A83-01), and a fibroblast growth factor receptor inhibitor (PD173074, BAP) into trophoblast-like cells and demonstrate that T21 trophoblast-like cells exhibit significantly reduced cell fusion compared to euploid controls following BAP differentiation (Pair 1:p < 0.05, Pair 2:p < 0.0001, n=3 for both pairs). We then performed bulk RNA-sequencing of iPS cell lines, BAP-differentiated and undifferentiated, to characterize transcriptional differences in T21 trophoblast-like cells compared to euploid controls. Following DESeq2 and Gene Set Enrichment Analysis, we found that estrogen-responsive gene sets were significantly enriched in euploid lines compared to T21 (p < 0.05). To measure estrogen-responsiveness, we treated age- and sex-matched iPS cell lines (n=3 for 2 separate pairs) with 1mM 17-b-estradiol, 1mM of an estrogen receptor inhibitor (Tamoxifen), or vehicle control during BAP differentiation. We then used immunofluorescence microscopy to calculate fusion indices ((number of nuclei within syncytial plaques-number of syncytial plaques)/total number of nuclei) and performed quantitative RT-PCR to measure relative expression of STB marker transcripts (CGB and ERVW-1). In agreement with previous reports, euploid cells increased fusion and expression of STB markers when supplemented with estradiol, whereas treatment with Tamoxifen significantly decreased fusion and STB-related transcripts (p < 0.05). However, T21 lines exhibited a strikingly dampened response to estradiol supplementation and resembled euploid Tamoxifen-treated levels of fusion and STB transcript abundance. We then hypothesized that trisomic expression of nuclear receptor interacting protein 1 (NRIP1), a gene on chromosome 21, is in-part responsible for fusion and estrogen signaling defects in T21 STBs. To determine if trisomic NRIP1 is sufficient to reduce estrogen signaling, we used Cre-recombinase to flip-in a doxycycline-inducible third copy of NRIP1 in a euploid iPS cell line (NRIP1^OE). We then BAP-differentiated NRIP1^OE alongside a GFP control (NRIP1^GFP) with 100ng/mL doxycycline and measured baseline fusion and STB marker expression. NRIP1^OE lines, like T21 lines, displayed significantly reduced fusion (p < 0.01) and expression of STB-related transcripts (ERVW-1 p< 0.01, CGB p< 0.001) compared to NRIP1^GFP. Finally, we measured estrogen-responsiveness of NRIP1^OE and NRIP1^GFP and found that NRIP1^OE lines also phenocopy diminished estrogen signaling that we observed in T21 lines when exposed to estradiol. In conclusion, our study provides strong evidence that NRIP1 contributes to physiological defects in T21 placentas and suggests that NRIP1 may serve as a potential target for therapeutic intervention. Our study also has broad implications for the precise modulation of estrogen signaling in trophoblast fate and endocrine function in all pregnancies. This study was funded by the John and Anna Sie Foundation as well as the NIH Fellowship 1F32HD116568-01. We also received substantial support for RNA-sequencing and iPS cell line reprogramming from the Linda Crnic Institute for Down Syndrome at the University of Colorado Anschutz Medical Campus.