Anthropoid primate–specific retroviral element THE1B controls expression of CRH in placenta and alters gestation length
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.