Maternal postpartum depression (PPD) is a significant public health concern due to the severe negative impact on maternal and child health and well-being. In this study, we aimed to identify genes ...associated with PPD. To do this, we investigated genome-wide gene expression profiles of pregnant women during their third trimester of pregnancy and tested the association of gene expression with perinatal depressive symptoms. A total of 137 women from a cohort from the University of North Carolina, USA were assessed. The main phenotypes analysed were Edinburgh Postnatal Depression Scale (EPDS) scores at 2 months postpartum and PPD (binary yes/no) based on an EPDS cutoff of 10. Illumina NextSeq500/550 transcriptomic sequencing from whole blood was analysed using the edgeR package. We identified 71 genes significantly associated with postpartum depression scores at 2 months, after correction for multiple testing at 5% FDR. These included several interesting candidates including TNFRSF17, previously reported to be significantly upregulated in women with PPD and MMP8, a matrix metalloproteinase gene, associated with depression in a genome-wide association study. Functional annotation of differentially expressed genes revealed an enrichment of immune response-related biological processes. Additional analysis of genes associated with changes in depressive symptoms from recruitment to 2 months postpartum identified 66 genes significant at an FDR of 5%. Of these genes, 33 genes were also associated with depressive symptoms at 2 months postpartum. Comparing the results with previous studies, we observed that 15.4% of genes associated with PPD in this study overlapped with 700 core maternal genes that showed significant gene expression changes across multiple brain regions (P = 7.9e-05) and 29-53% of the genes were also associated with estradiol changes in a pharmacological model of depression (P values range = 1.2e-4-2.1e-14). In conclusion, we identified novel genes and validated genes previously associated with oestrogen sensitivity in PPD. These results point towards the role of an altered immune transcriptomic landscape as a vulnerability factor for PPD.
Breast cancer risk is strongly associated with an intergenic region on 11q13. We have previously shown that the strongest risk-associated SNPs fall within a distal enhancer that regulates CCND1. ...Here, we report that, in addition to regulating CCND1, this enhancer regulates two estrogen-regulated long noncoding RNAs, CUPID1 and CUPID2. We provide evidence that the risk-associated SNPs are associated with reduced chromatin looping between the enhancer and the CUPID1 and CUPID2 bidirectional promoter. We further show that CUPID1 and CUPID2 are predominantly expressed in hormone-receptor-positive breast tumors and play a role in modulating pathway choice for the repair of double-strand breaks. These data reveal a mechanism for the involvement of this region in breast cancer.
MicroRNAs are noncoding regulators of gene expression, which act by repressing protein translation and/or degrading mRNA. Many have been shown to drive tumorigenesis in cancer, but functional studies ...to understand their mode of action are typically limited to single-target genes. In this study, we use synthetic biotinylated miRNA to pull down endogenous targets of miR-182-5p. We identified more than 1000 genes as potential targets of miR-182-5p, most of which have a known function in pathways underlying tumor biology. Specifically, functional enrichment analysis identified components of both the DNA damage response pathway and cell cycle to be highly represented in this target cohort. Experimental validation confirmed that miR-182-5p-mediated disruption of the homologous recombination (HR) pathway is a consequence of its ability to target multiple components in that pathway. Although there is a strong enrichment for the cell cycle ontology, we do not see primary proliferative defects as a consequence of miR-182-5p overexpression. We highlight targets that could be responsible for miR-182-5p-mediated disruption of other biological processes attributed in the literature so far. Finally, we show that miR-182-5p is highly expressed in a panel of human breast cancer samples, highlighting its role as a potential oncomir in breast cancer.
Somatic mutation calling from next-generation sequencing data remains a challenge due to the difficulties of distinguishing true somatic events from artifacts arising from PCR, sequencing errors or ...mis-mapping. Tumor cellularity or purity, sub-clonality and copy number changes also confound the identification of true somatic events against a background of germline variants. We have developed a heuristic strategy and software (http://www.qcmg.org/bioinformatics/qsnp/) for somatic mutation calling in samples with low tumor content and we show the superior sensitivity and precision of our approach using a previously sequenced cell line, a series of tumor/normal admixtures, and 3,253 putative somatic SNVs verified on an orthogonal platform.
MicroRNAs (miRNAs) bind to mRNAs and target them for translational inhibition or transcriptional degradation. It is thought that most miRNA-mRNA interactions involve the seed region at the 5' end of ...the miRNA. The importance of seed sites is supported by experimental evidence, although there is growing interest in interactions mediated by the central region of the miRNA, termed centered sites. To investigate the prevalence of these interactions, we apply a biotin pull-down method to determine the direct targets of ten human miRNAs, including four isomiRs that share centered sites, but not seeds, with their canonical partner miRNAs.
We confirm that miRNAs and their isomiRs can interact with hundreds of mRNAs, and that imperfect centered sites are common mediators of miRNA-mRNA interactions. We experimentally demonstrate that these sites can repress mRNA activity, typically through translational repression, and are enriched in regions of the transcriptome bound by AGO. Finally, we show that the identification of imperfect centered sites is unlikely to be an artifact of our protocol caused by the biotinylation of the miRNA. However, the fact that there was a slight bias against seed sites in our protocol may have inflated the apparent prevalence of centered site-mediated interactions.
Our results suggest that centered site-mediated interactions are much more frequent than previously thought. This may explain the evolutionary conservation of the central region of miRNAs, and has significant implications for decoding miRNA-regulated genetic networks, and for predicting the functional effect of variants that do not alter protein sequence.
Nullbasic is a derivative of the HIV-1 transactivator of transcription (Tat) protein that strongly inhibits HIV-1 replication in lymphocytes. Here we show that lentiviral vectors that constitutively ...express a Nullbasic-ZsGreen1 (NB-ZSG1) fusion protein by the eEF1α promoter led to robust long-term inhibition of HIV-1 replication in Jurkat cells. Although Jurkat-NB-ZSG1 cells were infected by HIV-1, no virus production could be detected and addition of phorbol ester 12-myristate 13-acetate (PMA) and JQ1 had no effect, while suberanilohydroxamic acid (SAHA) modestly stimulated virus production but at levels 300-fold lower than those seen in HIV-1-infected Jurkat-ZSG1 cells. Virus replication was not recovered by coculture of HIV-1-infected Jurkat-NB-ZSG1 cells with uninfected Jurkat cells. Latently infected Jurkat latent 6.3 and ACH2 cells treated with latency-reversing agents produced measurable viral capsid (CA), but little or none was made when they expressed NB-ZSG1. When Jurkat cells chronically infected with HIV-1 were transduced with lentiviral virus-like particles conveying NB-ZSG1, a >3-log reduction in CA production was observed. Addition of PMA increased virus CA production but at levels 500-fold lower than those seen in nontransduced Jurkat cells. Transcriptome sequencing analysis confirmed that HIV-1 mRNA was strongly inhibited by NB-ZSG1 but indicated that full-length viral mRNA was made. Analysis of HIV-1-infected Jurkat cells expressing NB-ZSG1 by chromatin immunoprecipitation assays indicated that recruitment of RNA polymerase II (RNAPII) and histone 3 lysine 9 acetylation were inhibited. The reduction of HIV-1 promoter-associated RNAPII and epigenetic changes in viral nucleosomes indicate that Nullbasic can inhibit HIV-1 replication by enforcing viral silencing in cells.
HIV-1 infection is effectively controlled by antiviral therapy that inhibits virus replication and reduces measurable viral loads in patients below detectable levels. However, therapy interruption leads to viral rebound due to latently infected cells that serve as a source of continued viral infection. Interest in strategies leading to a functional cure of HIV infection by permanent viral suppression, which may be achievable, is growing. Here we show that a mutant form of the HIV-1 Tat protein, referred to as Nullbasic, can inhibit HIV-1 transcription in infected Jurkat T cell to undetectable levels. Analysis shows that Nullbasic alters the epigenetic state of the HIV-1 long terminal repeat promoter, inhibiting its association with RNA polymerase II. This study indicates that key cellular proteins and pathways targeted here can silence HIV-1 transcription. Further elucidation could lead to functional-cure strategies by suppression of HIV transcription, which may be achievable by a pharmacological method.
Background
Caveolae proteins play diverse roles in cancer development and progression. In prostate cancer, non‐caveolar caveolin‐1 (CAV1) promotes metastasis, while CAVIN1 attenuates CAV1‐induced ...metastasis. Here, we unveil a novel mechanism linking CAV1 to selective loading of exosomes with metastasis‐promoting microRNAs.
Results
We identify hnRNPK as a CAV1‐regulated microRNA binding protein. In the absence of CAVIN1, non‐caveolar CAV1 drives localisation of hnRPNK to multi‐vesicular bodies (MVBs), recruiting AsUGnA motif‐containing miRNAs and causing their release within exosomes. This process is dependent on the lipid environment of membranes as shown by cholesterol depletion using methyl‐β‐cyclodextrin or by treatment with n‐3 polyunsaturated fatty acids. Consistent with a role in bone metastasis, knockdown of hnRNPK in prostate cancer PC3 cells abolished the ability of PC3 extracellular vesicles (EV) to induce osteoclastogenesis, and biofluid EV hnRNPK is elevated in metastatic prostate and colorectal cancer.
Conclusions
Taken together, these results support a novel pan‐cancer mechanism for CAV1‐driven exosomal release of hnRNPK and associated miRNA in metastasis, which is modulated by the membrane lipid environment.
Highlights
1. Non‐caveolar caveolin‐1 drives non‐canonical localization of hnRNPK to multivesicular bodies (MVB) via membrane raft‐dependent mechanism.
2. MVB localized hnRNPK mediates EV loading of AsUGnA motif‐containing miRNA, which is required for EV‐induced in vitro osteoclastogenesiss.
3. Elevated EV hnRNPK in biofluids from metastatic cancer patients suggest a role as a biomarker.
The developing mouse kidney is currently the best-characterized model of organogenesis at a transcriptional level. Detailed spatial maps have been generated for gene expression profiling combined ...with systematic in situ screening. These studies, however, fall short of capturing the transcriptional complexity arising from each locus due to the limited scope of microarray-based technology, which is largely based on "gene-centric" models.
To address this, the polyadenylated RNA and microRNA transcriptomes of the 15.5 dpc mouse kidney were profiled using strand-specific RNA-sequencing (RNA-Seq) to a depth sufficient to complement spatial maps from pre-existing microarray datasets. The transcriptional complexity of RNAs arising from mouse RefSeq loci was catalogued; including 3568 alternatively spliced transcripts and 532 uncharacterized alternate 3' UTRs. Antisense expressions for 60% of RefSeq genes was also detected including uncharacterized non-coding transcripts overlapping kidney progenitor markers, Six2 and Sall1, and were validated by section in situ hybridization. Analysis of genes known to be involved in kidney development, particularly during mesenchymal-to-epithelial transition, showed an enrichment of non-coding antisense transcripts extended along protein-coding RNAs.
The resulting resource further refines the transcriptomic cartography of kidney organogenesis by integrating deep RNA sequencing data with locus-based information from previously published expression atlases. The added resolution of RNA-Seq has provided the basis for a transition from classical gene-centric models of kidney development towards more accurate and detailed "transcript-centric" representations, which highlights the extent of transcriptional complexity of genes that direct complex development events.
Recent RNA-sequencing studies have shown remarkable complexity in the mammalian transcriptome. The ultimate impact of this complexity on the predicted proteomic output is less well defined. We have ...undertaken strand-specific RNA sequencing of multiple cellular RNA fractions (>20 Gb) to uncover the transcriptional complexity of human embryonic stem cells (hESCs). We have shown that human embryonic stem (ES) cells display a high degree of transcriptional diversity, with more than half of active genes generating RNAs that differ from conventional gene models. We found evidence that more than 1000 genes express long 5' and/or extended 3'UTRs, which was confirmed by "virtual Northern" analysis. Exhaustive sequencing of the membrane-polysome and cytosolic/untranslated fractions of hESCs was used to identify RNAs encoding peptides destined for secretion and the extracellular space and to demonstrate preferential selection of transcription complexity for translation in vitro. The impact of this newly defined complexity on known gene-centric network models such as the Plurinet and the cell surface signaling machinery in human ES cells revealed a significant expansion of known transcript isoforms at play, many predicting possible alternative functions based on sequence alterations within key functional domains.
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