Placental transcriptome co-expression analysis reveals conserved regulatory programs across gestation
© The Author(s) 2017
Received: 3 March 2016
Accepted: 7 December 2016
Published: 3 January 2017
Mammalian development in utero is absolutely dependent on proper placental development, which is ultimately regulated by the placental genome. The regulation of the placental genome can be directly studied by exploring the underlying organisation of the placental transcriptome through a systematic analysis of gene-wise co-expression relationships.
In this study, we performed a comprehensive analysis of human placental co-expression using RNA sequencing and intergrated multiple transcriptome datasets spanning human gestation. We identified modules of co-expressed genes that are preserved across human gestation, and also identifed modules conserved in the mouse indicating conserved molecular networks involved in placental development and gene expression patterns more specific to late gestation. Analysis of co-expressed gene flanking sequences indicated that conserved co-expression modules in the placenta are regulated by a core set of transcription factors, including ZNF423 and EBF1. Additionally, we identified a gene co-expression module enriched for genes implicated in the pregnancy pathology preeclampsia. By using an independnet transcriptome dataset, we show that these co-expressed genes are differentially expressed in preeclampsia.
This study represents a comprehensive characterisation of placental co-expression and provides insight into potential transcriptional regulators that govern conserved molecular programs fundamental to placental development.
The placenta is the first human tissue to start developing once the embryo implants into to the mother’s uterus shortly after conception. At implantation, placental trophoblast cells begin to invade into the lining of the uterus, where they colonise and transform the mother’s spiral arteries and the extra-embryonic tissue placental tissue establishes its own network of blood vessels. Together these processes facilitate the exchange of all nutrients, gases and waste throughout pregnancy. Normal placental function is dependent on appropriate growth and development of its structural components, which are underpinned by the fine-tuned regulation of gene expression. Consequently, alterations to placental gene regulation are thought to be a major contributor to pregnancy pathologies. Several studies aimed at elucidating the molecular basis of placental development have utilised high-throughput gene expression technologies, such as RNA sequencing (RNA-Seq) and microarrays, and show that the placenta undergoes global shifts in gene expression across human gestation [1–4]. They also show that placentas from pre-eclamptic pregnancies feature a distinct expression signature [5–9], and that some of these expression differences arise approximately six months before the condition manifests . Recently, two placental transcriptome studies employing RNA-Seq have described the breadth of gene expression in the human placenta and show that the placenta exhibits unique patterns of exon splicing and greater than four-fold enrichment for >800 genes compared to other human tissues [11, 12].
A common feature in previous studies on placental gene regulation is that expression data are typically summarised at the gene level for between-group comparisons, widely known as differential expression. With differential expression, the greatest significance is attributed to individual genes where the differences between groups reach an appropriate significance threshold. Although differential expression analyses have unquestionable utility, the inherent natural organisation of the transcriptome remains largely unexplored. Conversely, co-expression analyses that consider the gene-wise relationships in gene expression data have cast new light on previously unappreciated patterns of transcriptional organisation with regards to processes and functions such as lipid metabolism , cancer , human brain development and neuropathology [15–17], and embryonic development . Gene co-expression analyses identify groups of genes where expression levels are highly correlated across samples. By leveraging the inter-individual expression variability between biological samples, a co-expression analysis can enable the identification of higher-order relationships among genes. Further post hoc characterisation of these relationships can then provide insight into the biological processes arising from the underlying transcriptional program. Therefore, to gain a new perspective on placental genome regulation across human gestation and between human and mouse, we performed a comprehensive analysis of placental gene co-expression.
To explore patterns of gene co-expression in the healthy human term placenta, we performed single-strand 100-base paired-end total RNA-Seq for 16 samples, obtaining a total of 1.32 billion paired reads with and average of 83 million reads per library. The mapping rate was 94.6 ±16.6% with an average of 26.2 ±8.8 million uniquely mapped pairs per library overlapping annotated genes (Additional file 1: Figure 1a). By summarising the RNA-Seq reads by counting the number of overlaps with hg19 genes (see “Methods”), we detected 15,861 genes (including both coding and non-coding RNAs) above the threshold of >1 read count per million, which we show is an accurate threshold of detection based on quantification of spiked synthetic RNAs (Additional file 1: Figure 1b and c). The normalised gene expression values were also highly correlated (Additional file 1: Figure 2), with a Pearson’s correlation coefficient for each pair being 0.97 ±0.01.
Constructing a weighted human placental co-expression gene network
Each module was then summarised by calculating the module eigengene for each sample, which is the first principal component of gene expression values for the module. Therefore, the eigengene represents a weighted average of gene expression. For each gene, we then define its membership in each module as the absolute correlation between the gene’s expression and the module’s eigengene, and represent this correlation as kME . Genes are assigned to modules if they have an absolute kME>0.7. Note that by quantifying membership through correlation, module membership for each gene is no longer binary and allows genes to be members of more than one module (Additional file 1: Figure 3), thus connecting modules in a network.
Co-expression module characteristics
No. of genes
Variance explained by eigengene
Top ten hub genes (kME>0.9)
ZNF845, ZNF808, GPR160, GIN1, ATP5J, ZNF567, ANAPC10, C8orf59, MRPS36, RBM7
EPHA10, ARIH2OS, TUBD1, FLJ42102, KIAA0101, RPL13AP20, CD96, PDE6A, GGT8P, SLC35F1
NOTCH3, PLXND1, PALM, CSPG4, ARHGEF17, DCHS1, MARK4, KIRREL, LTBP4, AXL
HMMR, CASC5, DEPDC1, CDK1, KIF15, CCNA2, AIM1, TTK, ESCO2, EXO1
ATP2A1, C11orf35, P2RY2, CCDC33, ASIC3, KIFC2, IL17REL, CLIC3, MTVR2, RBBP8NL
HN1, ASAP3, SLC12A8, ASPHD2, B3GNT7, IL17RE, PRG2, NOG, IL2RB, PIPOX
SNORD114-29, CDH11, FAM198B, SNORD114-7, SNORD114-10, FKBP7, SNORD114-14, C4orf32, SNORD114-26, SNORD113-2
SBF1, ULK1, STRA6, DOT1L, BCAR1, TMEM184A, B3GNT8, SLC25A22, C19orf71, INTS1
SELL, S100A12, LRRK2, CYTIP, MNDA, ACSL1, FPR2, TGFA, LOC100505806, TMEM71
MTHFS, TTTY15, RPS4Y1, TXLNG2P, TTTY10, KDM5D, UTY, EIF1AY, ZFY, PRKY
PGAP3, GPR137, PRR5, ARTN, C10orf10, C7orf43, ALDH4A1, EFS, RELL2, ADIRF
PVRL4, ARHGEF4, NDRG1, INHBA, SYDE1, INHA, MIR210HG, C8orf58, SIGLEC6, PDZD7
FAM195B, FBXL15, BRAT1, AKAP2, SCAND1, EME2, CCDC85B, C19orf60, PGLS, TSR3
As our dataset featured equal number of samples from male and female fetuses, we expected that at least one co-expression module would be correlated with fetal sex status and would serve as a positive control. To test this, we performed a chromosomal enrichment test which identified module M10 to be significantly enriched for Y chromosome genes (Fisher exact test, Bonferroni p=2.9×10−12, OR=29.4, Additional file 1: Figure 4). Accordingly, M10 eigengene expression was also significantly higher for male samples (t-test, p =3.5×10−5, CI=0.27−0.57, Additional file 1: Figure 5).
As placental gene expression has previously been shown to be influenced by method of delivery and the onset of labor , we tested for an association of delivery method (operative vaginal, unassisted vaginal and cesarean section) and found no significant associations for any co-expression module (ANOVA tests with Bonferroni correction, all p>0.05). We further tested for eigengene correlations with birthweight and gestational age at delivery and found that M3 eigengene expression was moderately correlated with birthweight (Pearson’s r=0.53, Student asymptotic p=0.035, Additional file 1: Figure 6), however this correlation failed to remain significant after Bonferroni correction.
Co-expression modules are reproducible
Key co-expression modules are preserved across human gestation and conserved in the mouse
Given that the human placenta undergoes significant growth and remodeling throughout the nine months of gestation , we reasoned that if particular co-expression modules were involved in core placental functions, then these modules would be reproducible using gene expression data from earlier gestational time points. To test this hypothesis, we obtained microarray gene expression data from placental tissue collected during the first (GSE28551)  and second trimesters (GSE5999) . Although these datasets contain expression data for substantially fewer genes after filtering and annotation (57.6% and 63.9% of detectable genes in the RNA-Seq dataset, respectively), the module preservation statistics indicate that a majority of modules are nevertheless preserved across gestation at a low to moderate level of significance (Fig. 3). In particular, M4 shows moderate preservation Z summary >5 across all gestational time points, indicating a conserved pattern of gene regulation throughout human gestation. In contrast, the M2 module is highly preserved in the third trimester datasets Z summary >10 with little to no evidence of preservation during the first or second trimesters, suggesting M2 genes constitute a molecular program more specific to third trimester placental functions.
Preserved modules feature a core set of transcription factor motifs
Modules of co-expressed genes are implicated in pregnancy complications
Discussion and conclusions
By conducting this comprehensive co-expression network analysis of the human placental transcriptome, we reveal previously unappreciated aspects of transcriptional organisation at the fetal-maternal interface. This analysis entailed the integration of multiple gene expression datasets and curated databases, which enabled the testing of specific hypotheses regarding placental genome regulation.
Our results demonstrate that a large proportion of the placental transcriptome is organised into distinct modules of co-expressed genes, some of which are preserved across gestation, and conserved between human and mouse. The reproducibility of these networks, constructed from independent datasets and different platforms (RNA-Seq and microarrays) suggest a fundamental modular organisation of the placental transcriptome. Moreover, our cross-species analysis demonstrates that certain aspects of human placental transcriptional organisation are highly preserved in the mouse, indicating the evolutionary conservation of molecular processes which drive mammalian placental development.
When comparing the de novo human and mouse networks, five genes were identified as M3/m3 intramodular hub genes (kME>0.9) in both species (ARHGEF17, DOCK6, MAP3K9 OSBPL7, and PRR12), demonstrating a high degree of inter-species module reproducibility. These hub genes are centrally located within the M3 module and may be critical components of the network. Of particular interest, DOCK6 mutations in humans are associated with extreme placental angiopathy and a severely abnormal placental phenotype , while DOCK6 expression is reported to be down-regulated in placentas from growth-restricted fetuses . Similarly, OSBPL7, an oxysterol-binding protein, is also reported to be differentially expressed in placentas from preeclamptic pregnancies . For genes that do not have any previously reported placental phenotype association, these could be potential novel candidates for involvement in placental development. Given the size of the M3 co-expression module, it is reasonable to expect that these genes would be involved in multiple cellular processes. The results of the gene ontology analysis do indicate that M3 genes are involved in processes such as cell adhesion, cardiovascular system development, growth-factor binding and extracellular matrix structre. Together, there results suggests that the M3 co-expression network may be involved in multiple levels of placental development and regulation.
Investigation of the TFs that potentially regulate co-expression revealed that the most preserved modules are predicted to be regulated by a core set of transcription factors, including the M3 genes EBF1 and ZNF423, which potentially target a high proportion of genes in the most highly preserved modules. Although the effects of ZNF423 and EBF1 on placental gene regulation remain largely unexplored, ZNF423 appears to be a multi-functional transcription factor associated with B cell regulation, retinoic acid signalling, notch signalling, DNA damage response pathways, adipogenesis and cancer . Furthermore, homozygous mutation in the homologous gene in mice (Zfp423) results in smaller ataxic pups who die shortly after birth . This indicates a critical role for ZNF423 in development. EBF1 can act as both a transcriptional activator and repressor and has known roles in tumour suppression . When EBF1 binds DNA directly as a dimer, it can activate transcription via p300-CBP co-activation . In other contexts, the same DNA binding dimer in conjunction with ZNF423 can recruit the nucleosome remodelling and deacetylase (NuRD) complex, triggering EBF1-mediated transcriptional repression . The observation that EBF1 and ZNF423 are co-expressed in the placenta and members of the M3 module, and their widespread targeting potential across modules of co-expressed genes indicates that these TFs are candidate key regulators of transcription in the placenta.
The identification of M12 being enriched for genes implicated in PE demonstrates the utility of a co-expression analysis for identifying genes that may respond to the pathology, or may indeed underlie its aetiology. This guilt-by-association approach, clustered genes implicated in PE (M12) in a completely unsupervised manner, suggesting expression differences in these genes are driven by a set of common factors. The observation that several M12 hub genes are up-regulated in PE, and show highly correlated patterns of expression, implies that expression of other genes within this module is likely driven by the same underlying factors, together indicating that these genes are implicated in placental development. Moreover, the M12 network is preserved in the first trimester (Fig. 3), the period where the pathogenesis of PE is considered to have its origins . Furthermore, these patterns of co-expression do not appear to be conserved in the mouse. Although human and mouse placental development have many similarities, it is also important to note that mice do not develop preeclampsia. Together, these findings indicate further investigation of the involvement of M12 genes and their upstream regulators in human placental development may be a valuable way of generating new hypotheses regarding the placental origins of PE.
Concordant with our results, several M12 hub genes such as NDRG1, INHA, INHBA were central to both protein-protein interaction networks  and co-expression networks  implicated in PE in previous studies. Of particular interest, the intramodular M12 hub gene PVRL4, which is up-regulated in PE , is expressed more highly in the placenta compared to other human tissues . PVRL4 is a potent mediator of epithelial cell colony formation  and is also highly expressed in ovarian cancer tissue . Furthermore, cleaved PVRL4 is elevated in the serum of patients with ovarian cancer and is correlated with PVRL4 expression , suggesting that maternal serum PVRL4 may hold potential as a biomarker of PE. Together, these results suggest a potential role for M12 genes in the pathogenesis of PE.
One limitation of our study is the number of samples we have used to construct our co-expression networks, and the expression levels of some hub genes are relatively low. However, we are confident that our expression measurements are reasonably accurate at these levels as we emperically determined a threshold of detection using spike-in RNAs (Additional file 1: Figure 1). Furthermore, we have bolstered our analysis by incorporating multiple independent datasets to validate our results assess the preservation of co-expression networks. Secondly, as different placental biopsies can feature differing contributions of maternal versus fetal cells between different gestational ages and sampling methodologies, there are inherent limitations in comparing data between studies. This may be one underlying factor in driving the differences we observe between our dataset and the third trimeser validation dataset. We also recognise that the second trimester gene expression data (GSE5999) were from basal plate tissue collected from pre-term birth deliveries so they may not be directly comparable to the villous tissue data collected from uncomplicated pregnancies. Additionally, the mouse placental tissue we have re-analyzed (SRA062227) was collected at approximately mid gestation (E11.5) therefore the comparison with the late gestation human tissue should be interpreted with some caution. However, given the rarity of some of samples used in our analysis, we are of the opinion that the comparisons made still have value.
Several new questions arise from this comprehensive co-expression network analysis. Firstly, are patterns of co-expression altered in placental pathologies? Our analysis of independent expression datasets from PE placentas provide compelling preliminary evidence that M12 genes are up-regulated in PE, which warrants further investigation into the regulators of M12 genes. Secondly, what genetic and environmental factors influence co-expression? A comprehensive assessment of genotypes and environmental factors such as maternal diet has the potential to reveal drivers of placental expression variation. Thirdly, does silencing of hub genes shift module co-expression and influence placental cell phenotype and behavior? Functional studies aimed toward elucidating the biological function of co-expression modules may yield new insights into how placental development is regulated.
In summary, we show that a weighted gene co-expression network analysis can provide novel insights into the functional organisation of the placental transcriptome. To the best of our knowledge, the networks described herein have not been described previously, and emphasise that these findings could not be revealed through conventional gene-level summaries or differential expression experiments. In typical differential expression analyses, emphasis is placed on genes where the expression differences reach an appropriate level of significance. Although such experiments have contributed significantly to our understanding of placental genome regulation, the significance of each gene is typically determined in isolation, subsequently failing to connect genes in a manner that reflects the functional organisation of the transcriptome. By connecting genes in a manner that reflects underlying genome regulatory programs, we have exposed previously unappreciated aspects of the placental transcriptional landscape and provide a framework for multiple avenues of post hoc inquiry.
Ethics and consent
Ethics approval was granted by the Central Northern Adelaide Health Service Ethics of Human Research Committee (Approval #2005082) and the University of Adelaide Human Research Ethics Committee (H-137-2006). Written, informed consent was obtained from all patients.
Third trimester placenta samples were collected from primiparous women with singleleton pregnancies classified as being uncomplicated by using the criteria described in reference . Placenta samples were collected and dissected within one hour post-delivery at the Lyell McEwin Health Service, South Australia in accordance with our ethical approval (see ethics statement). Placental villous tissue was obtained by first taking a full-thickness sections and then removing the membranes and basal plate tissue before dissecting villous tissue from the middle of the section. No tissue or sample pooling was performed at any step. Samples of villous tissue were then incubated in RNAlater solution (Invitrogen) at 4 degrees celsius for 24 hours before being stored at -80 degrees celsius. Full sample details are listed in Additional file 1: Table 1.
RNA was extracted from 16 placental samples using TRIzol following the manufacturer’s protocol. All samples were spiked with 96 External RNA Controls Consortium (ERCC) ExFold RNA transcripts. Ribosomal RNAs were depleted from samples using Ribo-Zero Gold and sequencing libraries were prepared using Illumina®;TruSeq®;Stranded Total RNA Sample Preparation kits. Sequencing was performed on the Illumina Hi-Seq 2500 using a 100bp paired-end protocol at the Australian Cancer Genomics Facility in Adelaide.
Sequence adapters were trimmed using AdapterRemoval with options –trimns, –minlength 20. Trimmed RNA-Seq reads were aligned to known UCSC hg19 genes and the hg19 genome using Bowtie 2 v2.1.0 and TopHat v2.0.9 with options –library-type=fr-firststrand –mate-inner-dist -20 –mate-std-dev 180. UCSC hg19 reference genome and transcriptome was obtained through Illumina iGenomes (support.illumina.com/sequencing/sequencing_software/igenome.html).
Sequence data processing
Aligned RNA-Seq reads were summarised using the summarizeOverlaps algorithm with the UCSC known genes hg19 GTF file using the the options overlapMode=“Union”, ignoreStrand=FALSE, singleEnd=FALSE, fragments=TRUE  to generate a table of unique read counts per gene for each sample (this summarized data is available through NCBI GEO, GSE77085). Only genes >1FPKM were retained (15,861 genes) and count data were transformed and quantile-normalised using the Voom method  to produce a numeric matrix of normalised expression values on the log2 scale. All samples were processed in the same way, with all sequencing libraries created in the same batch and sequenced together. However, we nevertheless checked systematic differences between samples (Additional file 1: Figures 9 and 10) and found no evidence of batch effects or systematic shifts in gene expression.
To construct the network of co-expressed genes, we selected the most variable upper third of genes in the placental RNA-Seq dataset using the Weighted Gene Co-expression Network Analysis methods implemented in the WGCNA R package . Briefly, gene expression values were used to construct a signed co-expression network by computing a Pearson’s correlation matrix, which is then used to compute an adjacency matrix by raising the correlation matrix to a power. We chose a power of eight, which was determined by plotting scale-free fit and mean connectivity as a function of power (Additional file 1: Figure 11) using the scale-free topology criteria outlined in . By raising the absolute value of the correlation to a power, the construction of co-expression networks emphasises high correlations at the expense of low correlations . The interconnectedness (topological overlap) of each gene pair was calculated using the adjacency matrix, which was then used as input for average linkage hierarchical clustering.
Gene modules were then defined as branches of the resulting clustering tree, with the branches cut into defined modules using the dynamic tree-cut algorithm . Gene modules were then summarised by calculating module eigengenes, which are defined as the first principal components of the module expression profiles. As module eigengenes capture the maximum amount of variation of gene expression within a module, the eigengene is considered a representative value (or weighted average) of module gene expression . For each module, the gene membership value (kME) is defined as the correlation between the standardised gene expression values for each gene and the module eigengene for each sample . We assigned genes to modules if they had a high module membership defined as kME>0.7, and genes with a value below this threshold were assigned to the M0 (grey) module. Note that using this method allows genes to be members of more than one module.
To evaluate the preservation of human third trimester placenta gene modules in independent placenta gene expression datasets, we used the WGCNA modulePreservation function to generate module preservation statistics . These methods test whether the density and connectivity patterns of gene modules defined in our reference dataset are preserved in independent datasets. We used the Z summary statistic to summarise the evidence for significant module preservation compared to a random sample of all network genes reiterated over 100 permutations per dataset. We adopted the thresholds suggested by Langfelder et al , who indicate Z summary<2 implies no evidence for module preservation, 2<Z summary<10 implies weak to moderate preservation, and Z summary>10 implies strong evidence for module preservation.
RNA-seq validation dataset
We used the raw RNA-Seq reads from 20 human third trimester placenta samples as previously described in a separate analysis of the human placental transcriptome . In this current study, RNA-Seq reads were aligned to the human reference genome and UCSC known genes (hg19) using Tophat 2 with the options –library-type=fr-unstranded –segment-length=18. For the mouse expression data, we obtained RNA-Seq fastq files for 23 samples from the NCBI short read archive (SRA062227). Reads were aligned to mm10 genome and UCSC known genes using Tophat2 with the options –library-type=fr-unstranded –read-mismatches 3 –read-edit-dist 3. Alignment bam files were summarised to obtain the number of unique read counts per gene using the summarizeOverlaps function in the genomicAlignments R package  with the options ignore.strand=TRUE, paired=FALSE, mode=“union” followed by log2 counts per million transformation and quantile normalisation. To enable the comparison of human and mouse datasets, mouse gene identifiers were converted to orthologous human gene identifiers using Ensembl Biomart and the biomaRt R package. Only mouse genes with one-to-one orthologues in the human dataset were included and mouse genes with no corresponding human gene were removed from the analysis.
Microarray validation datasets
For second trimester placenta, Affymetrix CEL files for 27 samples (GSE5999) were pre-processed, background subtracted and normalised using the robust multi-average (RMA) algorithm . Pre-processed and normalised data from 16 first trimester placenta samples (GSE28551) and third trimester preeclampsia samples (GSE44711) were downloaded directly from NCBI GEO. Only probes that mapped uniquely to human genes using the bioconductor package biomaRt were retained. In cases where multiple probes mapped to the same gene, we selected the probe with the highest mean expression. Differential expression testing of GSE44711 was performed using linear models (lmFit and eBayes functions) and a rotation gene set test (mroast function) in the limma R package .
Gene lists for each module were tested for enrichment of gene ontology (GO) terms using Fisher exact tests to compute p-values for statistical over-representation of GO terms using the GOstats bioconductor package  with all the detectable genes (15,861) in our placental gene expression dataset used as the background set.
Transcription factor motif enrichment
The genes within each co-expression gene module were analysed for enrichment of transcription factor (TF)-binding sites (TFBS) against a background gene set of all detectable genes in the placenta dataset (15,861) using the oPOSSUM program and the JASPAR vertebrate core profiles [52, 53]. For each gene, we searched for TFBS motifs in the conserved regions of the 10kb upstream/downstream sequences using a conservation cut-off of 0.4, a matrix score threshold of 85% and a minimum specificity of 8-bits. The highly enriched TFBSs were identified by ranking TFs using results from Fisher tests and Z-score rankings.
SB was supported by an Australian Postgraduate Award from the Australian Government Department for Education and Training, and PhD scholarships from the Channel 7 Children’s research foundation and Healthy Development Adelaide, and the National Health and Medical Research Council (NHMRC). CTR is supported by an NHMRC Senior Research Fellowship (APP1020749). Funding for this research was provided by NHMRC through project grant APP1059120.
Availability of data and materials
The dataset supporting the conclusions of this article is available in the NCBI GEO repository under accession GSE77085 [https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE77085].
SB, TB-M, SJB and CTR conceived the project and designed the components. S.B carried out all bioinformatics and statistical analyses. SB, TB-M, SJB, CS, VC, GAD and CTR discussed the interpretation of the data. KS and CTR contributed RNA-Seq data. SB, TB-M and CTR prepared the manuscript. All authors reviewed and approved the manuscript.
The authors declare that they have no competing interests.
Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver(http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.
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