Abstract
Hltf is regulated by intron retention, and global Hltf-deletion causes perinatal lethality from hypoglycemia. In heart, full-length Hltf is a transcriptional regulator of Hif-1α that controls transport systems. Thus, we tested the hypothesis that Hltf deletion from placenta caused or exacerbated neonatal hypoglycemia via Hif-1α regulation of nutrient transporters. RNA-seq data analyses identified significant changes in transcript expression and alternative splicing (AS) in E18.5 placentome. iPathwayGuide was used for gene ontology (GO) analysis of biological processes, molecular functions and cellular components. Elim pruning algorithm identified hierarchical relationships. The methylome was interrogated by Methyl-MiniSeq Epiquest analysis. GO analysis identified gene enrichment within biological processes. Protein expression was visualized with immunohistochemistry. Although two Hltf mRNA isoforms are quantifiable in most murine tissues, only the truncated Hltf isoform is expressed in placenta. The responsible intron retention event occurs in the absence of DNA methylation. iPathwayGuide analysis identified 157 target genes of 11,538 total genes with measured expression. These were obtained using a threshold of 0.05 for statistical significance (p-value) and a long fold change of expression with absolute value of at least 0.6. Hltf deletion altered transcription of trophoblast lineage-specific genes, and increased transcription of the Cxcr7 (p = 0.004) gene whose protein product is a co-receptor for human and simian immunodeficiency viruses. Concomitant increased Cxcr7 protein was identified with immunolabeling. Hltf deletion had no effect on transcription or site-specific methylation patterns of Hif-1α, the major glucose transporters, or System A amino acid transporters. There was no measureable evidence of uteroplacental dysfunction or fetal compromise. iPathGuide analysis revealed Hltf suppresses cytolysis (10/21 genes; p-value 1.900e-12; p-value correction: Elim pruning; GO:019835) including the perforin-granzyme pathway in uterine natural killer cells. Our findings 1) prove the truncated Hltf protein isoform is a transcription factor, 2) establish a functional link between AS of Hltf and immunosuppression at the feto-maternal interface, 3) correlate intron retention with the absence of DNA methylation, and 4) underscore the importance of differential splicing analysis to identify Hltf’s functional diversity.
Highlights
Pregnancy and malignancy share molecular mechanisms [1,2,3] in order to cope with environmental stress, alter the metabolomics of the microenvironment to promote growth and invasion, and achieve immune tolerance
All studies were conducted in accord with the NIH Guidelines for the Care and Use of Laboratory Animals, as reviewed and approved by the Animal Care and Use Committee at Texas Tech University Health Sciences Center (TTUHSC) [NIH Assurance of Compliance A3056-01; USDA Certification 74-R-0050, Customer 1481]
alternative splicing (AS) of Hltf in placenta is a model in which gene expression and cellular differentiation are mediated by functional Hltf protein from intron-retained transcripts that are not lost to nonsense-mediated decay (NMD)
Summary
Pregnancy and malignancy share molecular mechanisms [1,2,3] in order to cope with environmental stress, alter the metabolomics of the microenvironment to promote growth and invasion, and achieve immune tolerance. A second critical mechanism, which is an adaptive response to genotoxic stress, is ploidy [10]. Trophoblast stem cells of the placenta exit the mitotic cell cycle to become endoreplicating trophoblast giant cells [11]. These polyploid cells lack functional DNA damage response mechanisms [12, 13]. In the human pregnancy complication of preeclampsia, cytotrophoblasts display selective resistance to DNA damage, i.e. DNA damage is localized to the maternal side not the fetal side of the placenta [14]. Polyploidy is a hallmark of human cancers where endoreplication improves cellular adaptation [12] and drug resistance [13]
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