RNA profiling of laser microdissected human trophoblast subtypes at mid-gestation reveals a role for cannabinoid signaling in invasion.

Human placental architecture is complex. Its surface epithelium, specialized for transport, forms by fusion of cytotrophoblast progenitors into multinucleated syncytiotrophoblasts. Near the uterine surface, these progenitors assume a different fate, becoming cancer-like cells that invade its lining and blood vessels. The latter process physically connects the placenta to the mother and shunts uterine blood to the syncytiotrophoblasts. Isolation of trophoblast subtypes is technically challenging. Upon removal, syncytiotrophoblasts disintegrate and invasive cytotrophoblasts are admixed with uterine cells. We used laser capture to circumvent these obstacles. This enabled isolation of syncytiotrophoblasts and two subpopulations of invasive cytotrophoblasts from cell columns and the endovascular compartment of spiral arteries. Transcriptional profiling revealed numerous genes, the placental or trophoblast expression of which was not known, including neurotensin and C4ORF36. Using mass spectrometry, discovery of differentially expressed mRNAs was extended to the protein level. We also found that invasive cytotrophoblasts expressed cannabinoid receptor 1. Unexpectedly, screening agonists and antagonists showed that signals from this receptor promote invasion. Together, these results revealed previously unseen gene expression patterns that translate to the protein level. Our data also suggested that endogenous and exogenous cannabinoids can affect human placental development.

Placental transcriptomes in the common aneuploidies reveal critical regions on the trisomic chromosomes and genome-wide effects.

OBJECTIVE: Chromosomal aberrations are frequently associated with birth defects and pregnancy losses. Trisomy 13, Trisomy 18 and Trisomy 21 are the most common, clinically relevant fetal aneusomies. This study used a transcriptomics approach to identify the molecular signatures at the maternal-fetal interface in each aneuploidy. METHODS: We profiled placental gene expression (13-22 weeks) in T13 (n = 4), T18 (n = 4) and T21 (n = 8), and in euploid pregnancies (n = 4). RESULTS: We found differentially expressed transcripts (≥2-fold) in T21 (n = 160), T18 (n = 80) and T13 (n = 125). The majority were upregulated and most of the misexpressed genes were not located on the relevant trisomic chromosome, suggesting genome-wide dysregulation. A smaller number of the differentially expressed transcripts were encoded on the trisomic chromosome, suggesting gene dosage. In T21, <10% of the genes were transcribed from the Down syndrome critical region (21q21-22), which contributes to the clinical phenotype. In T13, 15% of the upregulated genes were on the affected chromosome (13q11-14), and in T18, the percentage increased to 24% (18q11-22 region). CONCLUSION: The trisomic placental (and possibly fetal) phenotypes are driven by the combined effects of genome-wide phenomena and increased gene dosage from the trisomic chromosome. © 2016 John Wiley & Sons, Ltd.

Titin-truncating variants in hiPSC cardiomyocytes induce pathogenic proteinopathy and sarcomere defects with preserved core contractile machinery.

Titin-truncating variants (TTNtv) are the single largest genetic cause of dilated cardiomyopathy (DCM). In this study we modeled disease phenotypes of A-band TTNtv-induced DCM in human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) using genome editing and tissue engineering technologies. Transcriptomic, cellular, and micro-tissue studies revealed that A-band TTNtv hiPSC-CMs exhibit pathogenic proteinopathy, sarcomere defects, aberrant Na+ channel activities, and contractile dysfunction. These phenotypes establish a dual mechanism of poison peptide effect and haploinsufficiency that collectively contribute to DCM pathogenesis. However, TTNtv cellular defects did not interfere with the function of the core contractile machinery, the actin-myosin-troponin-Ca2+ complex, and preserved the therapeutic mechanism of sarcomere modulators. Treatment of TTNtv cardiac micro-tissues with investigational sarcomere modulators augmented contractility and resulted in sustained transcriptomic changes that promote reversal of DCM disease signatures. Together, our findings elucidate the underlying pathogenic mechanisms of A-band TTNtv-induced DCM and demonstrate the validity of sarcomere modulators as potential therapeutics.

Pooled Screens Identify GPR108 and TM9SF2 as Host Cell Factors Critical for AAV Transduction.

Adeno-associated virus (AAV) has been used extensively as a vector for gene therapy. Despite its widespread use, the mechanisms by which AAV enters the cell and is trafficked to the nucleus are poorly understood. In this study, we performed two pooled, genome-wide screens to identify positive and negative factors modulating AAV2 transduction. Genome-wide libraries directed against all human genes with four designs per gene or eight designs per gene were transduced into U-2 OS cells. These pools were transduced with AAV2 encoding EGFP and sorted based on the intensity of EGFP expression. Analysis of enriched and depleted barcodes in the sorted samples identified several genes that putatively decreased AAV2 transduction. A subset of screen hits was validated in flow cytometry and imaging studies. In addition to KIAA0319L (AAVR), we confirmed the role of two genes, GPR108 and TM9SF2, in mediating viral transduction in eight different AAV serotypes. Interestingly, GPR108 displayed serotype selectivity and was not required for AAV5 transduction. Follow-up studies suggested that GPR108 localized primarily to the Golgi, where it may interact with AAV and play a critical role in mediating virus escape or trafficking. Cumulatively, these results expand our understanding of the process of AAV transduction in different cell types and serotypes.

On the Function of Trans-Splicing: No Evidence for Widespread Proteome Diversification in Trypanosomes.

A long-standing mystery of genomic/transcriptomic structure involves spliced leader trans-splicing (SLTS), in which short RNA "tags" transcribed from a distinct genomic locus is added near the 5' end of RNA transcripts by the spliceosome. SLTS has been observed in diverse eukaryotes in a phylogenetic pattern implying recurrent independent evolution. This striking convergence suggests important functions for SLTS, however no general novel function is known. Recent findings of frequent alternative SLTS (ALT-TS) suggest that ALT-TS could impart widespread functionality. Here, we tested the hypothesis that ALT-TS diversifies proteomes by comparing splicing patterns in orthologous genes between two deeply diverged trypanosome parasites. We also tested proteome diversification functions of ALT-TS by utilizing ribosome profiling sequence data. Finally, we investigated ALT-TS as a mechanism to regulate the expression of unproductive transcripts. Although our results indicate the functional importance of some cases of trans-splicing, we find no evidence for the hypothesis that proteome diversification is a general function of trans-splicing.