Fig 1.
THE1B is a candidate enhancer for placenta-specific regulation of CRH and other genes.
(A) Comparative genomic alignment of 16 primates and 3 nonprimate mammals (UCSC Genome Browser, hg38, accessed June 14, 2017). LTR element THE1B (red bar) is present in anthropoid primates (red lines of phylogenetic tree) and absent in prosimians and nonprimate mammals. (B) Alignment of PCR-amplified transcript (Sequenced Transcript) and reads from human placental transcriptome precapture (RNA-seq) and postcapture (Capture-seq) for THE1B-containing transcripts. Sequenced fusion transcript found in human and rhesus macaque joins THE1B to CRH exon 1. This transcript is not found in pre- or postcapture RNA-seq; however, THE1B is transcribed. Raw data can be found at GEO accession number GSE118289. (C) Heat map of coregulation of THE1B-associated genes in placenta relative to other tissues. THE1B-associated genes are less coregulated, and thus more divergent in expression, between placenta and other tissues when compared to a group of randomly selected genes. Numerical values and their corresponding colors represent the odds ratio of observing the effect of relative coexpression between tissue pairs, generated by nonparametric tests separately between each pair of tissues as described by Pavlicev and colleagues [33]. (D) Upper right, higher percentage of human placenta–enriched genes, compared with all genes, are associated with THE1B. (P = 0.015 by binomial test.) Lower left, the log2fold gene expression between human and mouse of THE1B-associated human placenta–enriched genes. Raw data can be found at GEO accessions GSE118289 and GSE43520 (human placenta), GSE43520 (mouse placenta), and GSE30611 (other human tissues). Numerical data can be found in S1 Data. CRH, corticotropin-releasing hormone; Endometr. strom., endometrial stromal cells; GEO, Gene Expression Omnibus; LTR, long terminal repeat; RNA-seq, RNA sequencing; THE1B, transposon-like human element 1B; UCSC, University of California, Santa Cruz.
Fig 2.
BAC transgenic mice exhibit placental CRH expression and delayed parturition, which are eliminated by THE1B deletion.
(A) Schematic of human BAC clone RP11-366K18 (gray) with CRH shown to scale (red). (B) Expression of human CRH measured by qPCR in adult tissues and placenta. (n = 2–4 per group, P < 0.0001 by two-way ANOVA with tissue type as source of variation. Error bars indicate the standard error of the mean.) (C) Gestation length of litters homozygous for Tg(BAC1), homozygous for Tg(BAC2), or wild-type control. BAC transgenic litters have a significantly longer gestation length than wild-type control litters. (n = 28 +/+, n = 9 Tg[BAC1]/Tg[BAC1], P < 0.0001 by one-way ANOVA with post hoc Tukey’s multiple comparisons test. n = 17 Tg[BAC2]/Tg[BAC2], P = 0.0006 compared to +/+ by one-way ANOVA with post hoc Tukey’s multiple comparisons test. Error bars indicate standard deviation.) (D) Gestation length of litters hemizygous for Tg(BAC1) or wild-type control. Litters receiving Tg(BAC1) only from the father are born significantly later than wild-type control litters. (n = 20 +/+, n = 17 Tg[BAC1]/+, P = 0.0003 by unpaired two-tailed t-test. Error bars indicate the standard deviation.) (E) Schematic of 5-kb region containing CRH (gray) and THE1B (red). Blue arrows represent PAM sequences targeted by CRISPR/Cas9. (F) Sequenced deletions of the indicated transgenic founder animals compared to the Tg(BAC1) parent line. Red text, THE1B element. Blue text, PAM sequences indicated in (A). (G) Placental expression of human CRH at E18.5 measured by qPCR. (n = 3 Tg[BAC1]/+, n = 6 Tg[CR1]/+, P = 0.0006 by one-way ANOVA with post hoc Tukey’s multiple comparisons test. n = 5 Tg[CR2]/+, P = 0.0035 compared to Tg[BAC1]/+ by one-way ANOVA with post hoc Tukey’s multiple comparisons test. Error bars indicate the standard error of the mean.) (H) Hypothalamic expression of human CRH measured by qPCR. (n = 3 Tg[BAC1]/+, n = 4 Tg[CR1]/+, P = 0.0033 by one-way ANOVA with post hoc Tukey’s multiple comparisons test. n = 4 Tg[CR2]/+, P = 0.0013 compared to Tg[CR1]/+ by one-way ANOVA with post hoc Tukey’s multiple comparisons test. Error bars indicate the standard error of the mean.) (I) Gestation length of litters homozygous for Tg(CR1), homozygous for Tg(CR2), or wild-type control. The gestation length of THE1B-deleted transgenic litters is not significantly different from wild-type control litters. (n = 39 +/+, n = 15 Tg[CR1]/Tg[CR1], n = 7 Tg[CR2]/Tg[CR2], P = 0.0568 by one-way ANOVA. Error bars indicate standard deviation.) Numerical data for B, C, D, G, H, and I can be found in S1 Data. BAC, bacterial artificial chromosome; CRISPR/Cas9, clustered regularly interspaced short palindromic repeat/CRISPR-associated 9; E18.5, embryonic day 18.5; HT, hypothalamus; ND, not detected; ns, not significant; PAM, protospacer adjacent motif; qPCR, quantitative PCR; skelM, skeletal muscle; THE1B, transposon-like human element 1B.
Fig 3.
THE1B 5′ insertion site creates novel binding site for transcription factor DLX3.
(A) Top left, sequence of 5′ insertion site of THE1B near CRH, predicted DLX3 binding site shown in red. Lower left, electrophoretic mobility shift assay demonstrating binding of DLX3-DDK to positive control (JRE probe) and the 5′ insertion site of THE1B (THE1B 5′ probe). The red arrow denotes the band formed by DLX3-DDK binding to labeled DNA probe; this binding is disrupted with the addition of anti-DDK but not isotype control mouse IgG. Top right, same sequence as left, predicted GATA2 binding site shown in red. Lower right, GATA2-DDK fails to bind to the 5′ insertion site of THE1B (THE1B 5′ probe) while binding positive control (TCR probe). Blue arrow denotes the supershifted band of anti-DDK, GATA2-DDK, and TCR probe. (B) DNA binding motif of transcription factor DLX3 [46]. This DLX3 binding site is formed by the insertion of THE1B (right of black bar) into the genome (left of black bar). (C) ChIP for DLX3 (black bars) and RNA polymerase II (white bar) with quantitative real-time PCR. DLX3 is significantly associated with the 5′ end of THE1B and positive control (JRE) in human term placenta. ChIP-qPCR data were normalized to the IgG control for each target and presented relative to negative control (JRE distal). (n = 3 for all, P < 0.0001 for THE1B and P = 0.0184 for JRE by one-way ANOVA with post hoc Dunnett’s multiple comparisons test relative to JRE distal. Error bars indicate the standard error of the mean.) (D) Immunohistochemical comparison of human CRH localization in junctional zone (“jz”) and labyrinth (“lab”) of Tg(BAC1)/+ and wild-type littermate control. Human CRH is predominantly localized to the labyrinth in Tg(BAC1) mouse placenta. Blue, nuclei with DAPI. Magenta pseudocolor, CRH (imaged on far red). Green, background fluorescence of RBCs. Scale bars, 100 μm. (E) Quantitative real-time PCR of Dlx3 and CRH in Tg(BAC1)/+ mouse placentas separated by dissection into junctional zone (“jz”) and labyrinth (“lab”). Dlx3 (black circle) is predominantly expressed in labyrinth, which is consistent with previous reports [47]. (n = 5 Tg(BAC1)/+, P = 0.0003 by paired two-tailed t-test. Box and whiskers indicate that all data points are displayed.) CRH (red triangle) is undetectable in junctional zone. Numerical data for C and E can be found in S1 Data. ChIP, chromatin immunoprecipitation; CRH, corticotropin-releasing hormone; DDK, aspartic acid–aspartic acid–lysine; DLX3, distal-less homeobox 3; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; IgG, immunoglobulin G; JRE, human glycoprotein hormone α-subunit junctional regulatory element; qPCR, quantitative PCR; RBC, red blood cell; TCR, T cell receptor; THE1B, transposon-like human element 1B.