PLoS One. 2018 Jul 5;13(7):e0200211. doi: 10.1371/journal.pone.0200211.

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

Kaur G1, Helmer RA1, Smith LA2, Martinez-Zaguilan R3, Dufour JM1, Chilton BS1.
1 Department of Cell Biology & Biochemistry, Texas Tech University Health Sciences Center, Lubbock, Texas, United States of America.
2 Department of Pathology, Texas Tech University Health Sciences Center, Lubbock, Texas, United States of America.
3 Department of Cell Physiology & Molecular Biophysics, Texas Tech University Health Sciences Center, Lubbock, Texas, United States of America.

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.

PMID: 29975766

 

Supplement:

Helicase-like transcription factor (HLTF/Hltf) is an alternatively spliced tumor suppressor in humans and mice.  In mice, full-length, isoform 1 (exons 1-25) encodes a protein with DNA damage repair capabilities, and isoform 2 (exons 1-21 with exon 21 extended via a partial intron retention event) encodes a C-terminal truncation mutant lacking the DNA repair domain.  Similar to some other tumor suppressors, Hltf has important functions in development. Hltf-deletion causes brain [1] and cardiac [2] defects and perinatal lethality. Whole genome sequencing showed Hltf regulates the G2/M transition of the cell cycle in heart and brain. This shared phenotype is consistent with Hltf’s role in DNA damage repair, and was attributed to the protein encoded by the full-length isoform based its ratio to truncated transcripts in brain (5:1) and heart (26:1). Because there is often a functional link between placental insufficiency and cardiac defects, the placental transcriptome was evaluated for Hltf-deletion effects.

 

The placenta is a transient structure that maintains the most intimate affiliation between two genetically dissimilar organisms: the mother and her conceptus. The exclusive expression of the Hltf C-terminal truncation mutant that is incapable of DNA damage repair is evidence of functional selection pressure for alternative splicing in trophoblast giant cells that have large polyploidy nuclei resulting from selective endoreduplication [3] concomitant with the accumulation of DNA damage [4]. As shown in Figure 1, Hltf-deletion from placental cells that function in the calibrated response to an invasive threat modulates the expression of 26/627 genes; p = 7.800e-4; P-value correction, Elim pruning. Chief among these is the perforin-granzyme pathway in uterine NK cells that near term has a putative role in extracellular matrix remodeling. In Hltf-deleted placenta this pathway is transcriptionally upregulated coincident with increased Mmp12 (LogFC 0.740, p = 0.026) expression and a loss of structural integrity of the extracellular matrix (Figure 2). The extracellular matrix mitigates the migratory behavior of trophoblast (implantation) and cancer (metastasis) cells [5, 6]. Thus Hltf has a role in immune and extracellular matrix homeostasis.

 

 

Figure 1. Immune Response. Differentially expressed genes that are annotated to immune response are ranked based on the significance of their measured (log fold-change) logFC. The box and the whisker plot on the left summarizes the distribution of all the differentially increased (red) and decreased (blue) genes that are annotated to this GO:0006955 term.

 

 

Figure 2. Hltf immunolabeling and picrosirius red connective tissue stain. Truncated Hltf protein was robustly expressed (Sigma-Aldrich HPA 015284 to human HLTF aa 164-300) in trophoblast giant cells (TGCs) of control placenta (A), and completely absent in TGCs of Hltf-deleted placenta (B). Minus antibody controls for wild type (C) and Hltf-deleted (D) placenta shows negligible nonspecific immunostaining. Picrosirius red staining shows a normal collagen pattern in the extracellular matrix of control placenta (E) compared with a disrupted pattern in the ECM of Hltf-deleted placenta (F). 40x magnification

 

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