EP300 facilitates human trophoblast stem cell differentiation.

Early placenta development involves cytotrophoblast differentiation into extravillous trophoblast (EVT) and syncytiotrophoblast (STB). Defective trophoblast development and function may result in severe pregnancy complications, including fetal growth restriction and pre-eclampsia. The incidence of these complications is increased in pregnancies of fetuses affected by Rubinstein-Taybi syndrome, a developmental disorder predominantly caused by heterozygous mutations in CREB-binding protein (CREBBP) or E1A-binding protein p300 (EP300). Although the acetyltransferases CREBBP and EP300 are paralogs with many overlapping functions, the increased incidence of pregnancy complications is specific for EP300 mutations. We hypothesized that these complications have their origin in early placentation and that EP300 is involved in that process. Therefore, we investigated the role of EP300 and CREBBP in trophoblast differentiation, using human trophoblast stem cells (TSCs) and trophoblast organoids. We found that pharmacological CREBBP/EP300 inhibition blocks differentiation of TSCs into both EVT and STB lineages, and results in an expansion of TSC-like cells under differentiation-inducing conditions. Specific targeting by RNA interference or CRISPR/Cas9-mediated mutagenesis demonstrated that knockdown of EP300 but not CREBBP, inhibits trophoblast differentiation, consistent with the complications seen in Rubinstein-Taybi syndrome pregnancies. By transcriptome sequencing, we identified transforming growth factor alpha (TGFA, encoding TGF-α) as being strongly upregulated upon EP300 knockdown. Moreover, supplementing differentiation medium with TGF-α, which is a ligand for the epidermal growth factor receptor (EGFR), likewise affected trophoblast differentiation and resulted in increased TSC-like cell proliferation. These findings suggest that EP300 facilitates trophoblast differentiation by interfering with at least EGFR signaling, pointing towards a crucial role for EP300 in early human placentation.

TBX2, a Novel Regulator of Labour.

Background and Objectives: Therapeutic interventions targeting molecular factors involved in the transition from uterine quiescence to overt labour are not substantially reducing the rate of spontaneous preterm labour. The identification of novel rational therapeutic targets are essential to prevent the most common cause of neonatal mortality. Based on our previous work showing that Tbx2 (T-Box transcription factor 2) is a putative upstream regulator preceding progesterone withdrawal in mouse myometrium, we now investigate the role of TBX2 in human myometrium. Materials and Methods: RNA microarray analysis of (A) preterm human myometrium samples and (B) myometrial cells overexpressing TBX2 in vitro, combined with subsequent analysis of the two publicly available datasets of (C) Chan et al. and (D) Sharp et al. The effect of TBX2 overexpression on cytokines/chemokines secreted to the myometrium cell culture medium were determined by Luminex assay. Results: Analysis shows that overexpression of TBX2 in myometrial cells results in downregulation of TNFα- and interferon signalling. This downregulation is consistent with the decreased expression of cytokines and chemokines of which a subset has been previously associated with the inflammatory pathways relevant for human labour. In contrast, CXCL5 (C-X-C motif chemokine ligand 5), CCL21 and IL-6 (Interleukin 6), previously reported in relation to parturition, do not seem to be under TBX2 control. The combined bioinformatical analysis of the four mRNA datasets identifies a subset of upstream regulators common to both preterm and term labour under control of TBX2. Surprisingly, TBX2 mRNA levels are increased in preterm contractile myometrium. Conclusions: We identified a subset of upstream regulators common to both preterm and term labour that are activated in labour and repressed by TBX2. The increased TBX2 mRNA expression in myometrium collected during a preterm caesarean section while in spontaneous preterm labour compared to tissue harvested during iatrogenic preterm delivery does not fit the bioinformatical model. We can only explain this by speculating that the in vivo activity of TBX2 in human myometrium depends not only on the TBX2 expression levels but also on levels of the accessory proteins necessary for TBX2 activity.

Total bile acids in the maternal and fetal compartment in relation to placental ABCG2 expression in preeclamptic pregnancies complicated by HELLP syndrome.

OBJECTIVE: To investigate total bile acid (TBA) levels in maternal (MB) and umbilical cord blood (UCB) in normotensive, preeclamptic (PE), and PE pregnancies complicated by hemolysis elevated liver enzymes and low platelets (HELLP) syndrome in the context of ABCG2 placental gene expression levels, a recently reported placental bile acid transporter. METHODS: TBA levels were determined in 83 paired MB and UCB samples of normotensive, PE and PE/HELLP pregnancies and in 22 paired arterial and venous UCB samples from uncomplicated term pregnancies. ABCG2 gene expression was measured in 104 human placentas by reverse transcriptase quantitative polymerase chain reaction. RESULTS: Overall, TBA levels in MB are higher compared to levels in UCB (p<0.0001), but this comparison looses statistical significance for the 11 PE/HELLP cases. TBA levels in maternal blood are increased in PE/HELLP compared to PE pregnancies (p=0.016). TBA levels in arterial and venous UCB from 22 normotensive pregnancies are not statistically different. ABCG2 expression is reduced in pregnancies where preeclampsia is further complicated by HELLP syndrome. ABCG2 expression in human placenta is not correlated with TBA levels in either the maternal or fetal compartment. CONCLUSION: Increased maternal TBA levels in PE/HELLP pregnancies indicate a relation between bile acids in the maternal circulation and HELLP syndrome. As overall TBA levels in maternal blood are increased compared to UCB, we conclude that the placenta partly protects the fetus from increased maternal TBA levels. This consistent difference in TBA levels between the maternal and fetal compartment is unrelated to the placental expression of ABCG2.

Expression of placental FLT1 transcript variants relates to both gestational hypertensive disease and fetal growth.

The recent discovery of additional alternative spliced FLT1 transcripts encoding novel soluble (s)FLT1 protein isoforms complicates both the predictive value and functional implications of sFLT1 in preeclampsia. We investigated FLT1 expression levels and splicing patterns in placentas of normotensive and preeclamptic women, and established the tissue specificity of all FLT1 transcript variants. mRNA levels of sFLT1 splice variants were determined by real-time polymerase chain reaction in 21 normal human tissues and placental biopsies from 91 normotensive and 55 preeclamptic women. Cellular localization of placental FLT1 expression was established by RNA in situ hybridization. Of all tissues investigated, placenta has by far the highest FLT1 mRNA expression level, mainly localized in the syncytiotrophoblast layer. More than 80% of placental transcripts correspond to sFLT1_v2. Compared with normotensive placenta, preeclamptic placenta has ≈3-fold higher expression of all FLT1 transcript variants (P<0.001), with a slight shift in favor of sFLT1_v1. Although to a lesser degree, transcript levels are also increased in placenta from normotensive women that deliver a small for gestational age neonate. We conclude that sFLT isoform-specific assays could potentially improve the accuracy of current sFLT1 assays for the prediction of preeclampsia. However, placental FLT1 transcript levels are increased not only in preeclampsia but also in normotensive pregnancy with a small for gestational age fetus. This may indicate a common pathway involved in the development of both conditions but complicates the use of circulating sFLT1 protein levels for the prediction or diagnosis of preeclampsia alone.

Initial characterization of C16orf89, a novel thyroid-specific gene.

BACKGROUND: Thyroid hormone is prerequisite for proper fetal and postnatal neurodevelopment, growth, and metabolism. Although much progress has been made in the characterization of genes implicated in thyroid development and function, the majority of genes involved in this process are still unknown. We have previously applied serial analysis of gene expression (SAGE) to identify novel genes preferentially expressed in the thyroid, and this has resulted in the characterization of DUOX2 and IYD (also known as DEHAL1), two genes encoding essential enzymes in the production of thyroid hormone. In the current study we characterize the gene C16orf89, which is linked to another thyroid-specific SAGE tag CCAGCTGCCT. METHODS: We establish tissue-specific expression of C16orf89 using novel tissue-specific SAGE libraries and quantitative polymerase chain reaction. In addition, we characterize the C16orf89 gene and protein, and analyze its mRNA expression in response to thyrotropin and during mouse development. RESULTS: C16orf89 is predominantly expressed in human thyroid tissue with a specificity intermediate between thyroid transcription factors and proteins involved in thyroid hormone synthesis. C16orf89 shows the same expression pattern as Nkx2-1 (thyroid transcription factor 1) from embryonic day (E) 17.5 onward in the developing mouse thyroid and lung. The developmental timing of C16orf89 mRNA expression is similar to that of the iodide transporter Slc5a5 (also known as Nis). Both transcripts are detected from E17.5 in the developing thyroid. This is clearly later than the onset of Tg mRNA expression (from E14.5), while Nkx2-1 and Iyd mRNA can already be detected in the E12.5 thyroid. In in vitro cell culture C16orf89 expression is stimulated by thyrotropin. The major splice variant encodes a 361 amino acid protein that is well conserved between mammals, contains an N-terminal signal peptide, is secreted in a glycosylated form, and does not contain any known functional domain. CONCLUSIONS: We present a novel gene highly expressed in thyroid that encodes a currently enigmatic protein.

Genes differentially expressed in thyroid carcinoma identified by comparison of SAGE expression profiles.

To identify transcripts that distinguish malignant from benign thyroid disease serial analysis of gene expression (SAGE) profiles of papillary thyroid carcinoma and of normal thyroid are compared. Of the 21,000 tags analyzed, 204 tags are differentially expressed with statistical significance in the tumor. Thyroid tumor specificity of these transcripts is determined in silico using the tissue preferential expression (TPE) algorithm. TPE values demonstrate that 42 tags of the 204 are thyroid tumor specific. BC013035, a cDNA encoding a novel protein, is up-regulated from 0 to 24 tags in the thyroid tumor SAGE library. In a tissue panel of 30 thyroid tumors and 12 controls, it has an expression pattern similar to thyroid peroxidase, indicating possible involvement of BC013035 in thyroid differentiation. A tag coding for extracellular matrix protein 1 (ECM1) is absent in the normal thyroid SAGE library and present 55 times in the tumor. ECM1, a protein recently associated with angiogenesis and expressed in metastatic breast carcinoma, is up-regulated in 50% of all thyroid carcinoma and absent in normal controls and follicular adenoma. In conclusion, SAGE analysis and subsequent determination of TPE values facilitates the rapid distinction of genes specifically expressed in cancer tissues.