Alternative splicing of helicase-like transcription factor (Hltf): Intron retention-dependent activation of immune tolerance at the feto-maternal interface

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.

The Role of Epigenetics in Placental Development and the Etiology of Preeclampsia

“…Table S1. High-throughput studies analyzing methylation profiles of different relevant tissues in the context of preeclampsia [42,43,47,54,55,56,57,58,59,60,61,62,63,64,65,66,67,68,69,70,71,72,73].…”

Helicase-like transcription factor (Hltf) gene-deletion promotes oxidative phosphorylation (OXPHOS) in colorectal tumors of AOM/DSS-treated mice

“…The development of global Hltf -deleted mice in collaboration with genOway (Lyon, France) was previously described [24–26]. Mice are fully congenic (N11) on the C57BL/6J genomic background.…”

“…Mice are fully congenic (N11) on the C57BL/6J genomic background. Global Hltf -deleted mice presented a neonatal lethal phenotype [24–26]. Hltf -deleted mice and their littermate controls (Hltf +/+) breathed freely at birth, and acquired a characteristic pink color suggesting normal lung and diaphragm function.…”

“…In this study, we combined our global Hltf -deleted mouse model [24–26] with AOM/DSS-treatment, to probe the effects of Hltf loss of expression on survival, colorectal tumor localization, multiplicity, morphology, and gene expression. To eliminate gender bias as well as genetic background effects [27], we used male mice congenic on the C57Bl/J6 background.…”

<p>Plausibility of trophoblastic-like regulation of cancer tissue</p>

“…Hence, (a) HLA-G antigens and PD-L1/L2 ligands expressed on these cells activate the matching inhibitory immune checkpoint receptors on the immune cells in situ, thus antagonizing the antigen-induced co-stimulation, (b) myeloid-derived suppressive cells (MDSC) 44,45 recruited by chemoattraction blunt the immune response, typically by inducing extra catabolism of tryptophan and arginine, and subsequent T cell depletion, (c) the complementmediated cytolysis is locally impeded by action of specific cell-surface anticomplement factors, and (d) the trophoblastic C-terminal truncated helicase-like transcription factor, an alternative splicing isoform, would lessen the cytotoxicity of natural killer cells. 46 Hence, the putative trophoblastic-like transdifferentiation of cancer cells and the related polyvalent mechanisms of local immune tolerance would explain why immune fighting against cancer has clearly a rather poor achievement. Fighting against infectious agents remains concurrently fully preserved through TLR activation on immune, stromal and trophoblastic or tumor cells (see the next section), and ensuing both release of chemokines and activation of the cytokine network.…”