Single-cell multiplex chromatin and RNA interactions in ageing human brain.

Dynamically organized chromatin complexes often involve multiplex chromatin interactions and sometimes chromatin-associated RNA1-3. Chromatin complex compositions change during cellular differentiation and ageing, and are expected to be highly heterogeneous among terminally differentiated single cells4-7. Here we introduce the multinucleic acid interaction mapping in single cells (MUSIC) technique for concurrent profiling of multiplex chromatin interactions, gene expression and RNA-chromatin associations within individual nuclei. When applied to 14 human frontal cortex samples from older donors, MUSIC delineated diverse cortical cell types and states. We observed that nuclei exhibiting fewer short-range chromatin interactions were correlated with both an 'older' transcriptomic signature and Alzheimer's disease pathology. Furthermore, the cell type exhibiting chromatin contacts between cis expression quantitative trait loci and a promoter tends to be that in which these cis expression quantitative trait loci specifically affect the expression of their target gene. In addition, female cortical cells exhibit highly heterogeneous interactions between XIST non-coding RNA and chromosome X, along with diverse spatial organizations of the X chromosomes. MUSIC presents a potent tool for exploration of chromatin architecture and transcription at cellular resolution in complex tissues.

Prenatal diagnosis of hereditary diffuse gastric cancer: a case report.

BACKGROUND: Hereditary diffuse gastric cancer(HDGC) is a kind of malignant gastric cancer that is difficult to find in the early stage. However, this late onset and incomplete penetrance hereditary cancer, and its prenatal diagnosis have rarely been reported previously. CASE PRESENTATION: A 26-year-old woman was referred to genetic counseling for an ultrasonography of fetal choroid plexus cyst at 17 weeks of gestation. The ultrasonographic evaluation showed bilateral choroid plexus cysts(CPC) in the lateral ventricles, and the women showed a family history of gastric cancer and breast cancer. Trio copy number sequencing identified a pathogenic CDH1 deletion in the fetus and unaffected mother. The CDH1 deletion was found in three of the five family members tested, segregation among affected family members. The couple finally decided to terminate the pregnancy after genetic counseling by hospital geneticists due to the uncertainty of the occurrence of HDGC in the future. CONCLUSIONS: In prenatal diagnosis, a family history of cancer should be widely concerned, and prenatal diagnosis of hereditary tumors requires extensive cooperation between the prenatal diagnosis structure and the pathology department.

The Effect of High CDC6 Levels on Predicting Poor Prognosis in Colorectal Cancer.

INTRODUCTION: We investigated the function of cell division cycle 6 (CDC6) on the prognosis in colorectal carcinoma (CRC). METHODS: CDC6 protein expression levels in 121 patients with colorectal cancer and adjacent normal mucosa were detected by immunohistochemistry. RESULTS: Compared to adjacent normal tissues, CDC6 mRNA level was overexpressed in CRC tissues. Moreover, CDC6 protein levels were expressed up to 93.39% (113/121) in CRC tissues in the cell nucleus or cytoplasm. However, there were only 5.79% (7/121) in normal mucosal tissues with nuclear expression. CDC6 expression was significantly correlated with TNM stage and tumor metastasis. The 5-year survival rate was lower in the high CDC6 expression group than the low group. After silencing of CDC6 expression in SW620 cells, cell proliferation was slowed, the tumor clones were decreased, and the cell cycle was arrested in G1 phase. In multivariate analysis, increased CDC6 protein expression levels in colon cancer tissues were associated with cancer metastasis, TNM stage, and patient survival time. CONCLUSION: CDC6 is highly expressed in CRC, and downregulation of CDC6 can slow the growth of CRC cells in vitro. It is also an independent predictor for poor prognosis and may be a useful biomarker for targeted therapy and prognostic evaluation.

ZFX acts as a transcriptional activator in multiple types of human tumors by binding downstream of transcription start sites at the majority of CpG island promoters.

High expression of the transcription factor ZFX is correlated with proliferation, tumorigenesis, and patient survival in multiple types of human cancers. However, the mechanism by which ZFX influences transcriptional regulation has not been determined. We performed ChIP-seq in four cancer cell lines (representing kidney, colon, prostate, and breast cancers) to identify ZFX binding sites throughout the human genome. We identified ~9,000 ZFX binding sites and found that the majority of the sites are in CpG island promoters. Moreover, genes with promoters bound by ZFX are expressed at higher levels than genes with promoters not bound by ZFX. To determine if ZFX contributes to regulation of the promoters to which it is bound, we performed RNA-seq analysis after knockdown of ZFX by siRNA in prostate and breast cancer cells. Many genes with promoters bound by ZFX were downregulated upon ZFX knockdown, supporting the hypothesis that ZFX acts as a transcriptional activator. Surprisingly, ZFX binds at +240 bp downstream of the TSS of the responsive promoters. Using Nucleosome Occupancy and Methylome Sequencing (NOMe-seq), we show that ZFX binds between the open chromatin region at the TSS and the first downstream nucleosome, suggesting that ZFX may play a critical role in promoter architecture. We have also shown that a closely related zinc finger protein ZNF711 has a similar binding pattern at CpG island promoters, but ZNF711 may play a subordinate role to ZFX. This functional characterization of ZFX provides important new insights into transcription, chromatin structure, and the regulation of the cancer transcriptome.

Single-cell multiplex chromatin and RNA interactions in aging human brain.

The dynamically organized chromatin complexes often involve multiplex chromatin interactions and sometimes chromatin-associated RNA (caRNA) 1-3. Chromatin complex compositions change during cellular differentiation and aging, and are expected to be highly heterogeneous among terminally differentiated single cells 4-7. Here we introduce the Multi-Nucleic Acid Interaction Mapping in Single Cell (MUSIC) technique for concurrent profiling of multiplex chromatin interactions, gene expression, and RNA-chromatin associations within individual nuclei. Applied to 14 human frontal cortex samples from elderly donors, MUSIC delineates diverse cortical cell types and states. We observed the nuclei exhibiting fewer short-range chromatin interactions are correlated with an "older" transcriptomic signature and with Alzheimer's pathology. Furthermore, the cell type exhibiting chromatin contacts between cis expression quantitative trait loci (cis eQTLs) and a promoter tends to be the cell type where these cis eQTLs specifically affect their target gene's expression. Additionally, the female cortical cells exhibit highly heterogeneous interactions between the XIST non-coding RNA and Chromosome X, along with diverse spatial organizations of the X chromosomes. MUSIC presents a potent tool for exploring chromatin architecture and transcription at cellular resolution in complex tissues.

Genome-wide analysis of the interplay between chromatin-associated RNA and 3D genome organization in human cells.

The interphase genome is dynamically organized in the nucleus and decorated with chromatin-associated RNA (caRNA). It remains unclear whether the genome architecture modulates the spatial distribution of caRNA and vice versa. Here, we generate a resource of genome-wide RNA-DNA and DNA-DNA contact maps in human cells. These maps reveal the chromosomal domains demarcated by locally transcribed RNA, hereafter termed RNA-defined chromosomal domains. Further, the spreading of caRNA is constrained by the boundaries of topologically associating domains (TADs), demonstrating the role of the 3D genome structure in modulating the spatial distribution of RNA. Conversely, stopping transcription or acute depletion of RNA induces thousands of chromatin loops genome-wide. Activation or suppression of the transcription of specific genes suppresses or creates chromatin loops straddling these genes. Deletion of a specific caRNA-producing genomic sequence promotes chromatin loops that straddle the interchromosomal target sequences of this caRNA. These data suggest a feedback loop where the 3D genome modulates the spatial distribution of RNA, which in turn affects the dynamic 3D genome organization.

FOXC1 Binds Enhancers and Promotes Cisplatin Resistance in Bladder Cancer.

Chemotherapy resistance is traditionally attributed to DNA mutations that confer a survival advantage under drug selection pressure. However, in bladder cancer and other malignancies, we and others have previously reported that cancer cells can convert spontaneously to an aggressive drug-resistant phenotype without prior drug selection or mutational events. In the current work, we explored possible epigenetic mechanisms behind this phenotypic plasticity. Using Hoechst dye exclusion and flow cytometry, we isolated the aggressive drug-resistant cells and analyzed their chromatin accessibility at regulatory elements. Compared to the rest of the cancer cell population, the aggressive drug-resistant cells exhibited enhancer accessibility changes. In particular, we found that differentially accessible enhancers were enriched for the FOXC1 transcription factor motif, and that FOXC1 was the most significantly overexpressed gene in aggressive drug-resistant cells. ChIP-seq analysis revealed that differentially accessible enhancers in aggressive drug-resistant cells had a higher FOXC1 binding, which regulated the expression of adjacent cancer-relevant genes like ABCB1 and ID3. Accordingly, cisplatin treatment of bladder cancer cells led to an increased FOXC1 expression, which mediated cell survival and conversion to a drug-resistant phenotype. Collectively, these findings suggest that FOXC1 contributes to phenotypic plasticity by binding enhancers and promoting a mutation-independent shift towards cisplatin resistance in bladder cancer.

MicroRNA­142­3p suppresses cell proliferation, invasion and epithelial­to­mesenchymal transition via RAC1­ERK1/2 signaling in colorectal cancer.

Aberrant expression of microRNAs (miRNAs/miRs) is associated with the initiation and progression of colorectal cancer (CRC), but how they regulate colorectal tumorigenesis is still unknown. The present study was designed to investigate the expression profile of miRNAs in human CRC tissues, and to reveal the molecular mechanism of miRNA­142­3p in suppressing colon cancer cell proliferation. The expression of miRNA was examined using an Exiqon miRNA array. Bioinformatics was used to predict the target genes of differentially expressed miRNAs and to analyze their biological function in CRC. The effect of miR­142­3p in colon cancer cells was evaluated in vitro using cell proliferation, colony formation and Transwell assays. Dual­luciferase reporter gene assays were performed to investigate the association between miR­142­3p and Rac family small GTPase 1 (RAC1). The effect of miR­142­3p regulation on colon cancer proliferation was assessed through western blotting and quantitative polymerase chain reaction analysis. Compared with their expression in adjacent non­cancer mucosal tissues, 76 miRNAs were upregulated and 102 miRNAs were downregulated in CRC. One of the most significantly and differentially regulated miRNAs was miR­142­3p, which was downregulated in 81.0% (51/63) of primary CRC tissues. After transfection of miR­142­3p mimics into colon cancer cells, proliferation and colony formation were decreased, and migration and invasion were markedly suppressed. RAC1 was a possible target of miR­142­3p, which was confirmed by dual­luciferase reporter assay. Transfection of miR­142­3p mimics decreased the levels of RAC1 and suppressed epithelial­to­mesenchymal transition in colon cancer cells. The phosphorylation of extraceullar signal­regulated kinase (ERK) was decreased significantly by the inhibition of RAC1 or transfection of miR­142­3p mimics in colon cancer cells. In conclusion, aberrant miRNAs are implicated in CRC. Decreased expression of miR­142­3p may be associated with CRC tumorigenesis via Rac1­ERK signaling.

Functional Mapping of AGO-Associated Zika Virus-Derived Small Interfering RNAs in Neural Stem Cells.

Viral interfering RNA (viRNA) has been identified from several viral genomes via directly deep RNA sequencing of the virus-infected cells, including zika virus (ZIKV). Once produced by endoribonuclease Dicer, viRNAs are loaded onto the Argonaute (AGO) family proteins of the RNA-induced silencing complexes (RISCs) to pair with their RNA targets and initiate the cleavage of target genes. However, the identities of functional ZIKV viRNAs and their viral RNA targets remain largely unknown. Our recent study has shown that ZIKV capsid protein interacted with Dicer and antagonized its endoribonuclease activity, which requires its histidine residue at the 41st amino acid. Accordingly, the engineered ZIKV-H41R loss-of-function (LOF) mutant virus no longer suppresses Dicer enzymatic activity nor inhibits miRNA biogenesis in NSCs. By combining AGO-associated RNA sequencing, deep sequencing analysis in ZIKV-infected human neural stem cells (NSCs), and miRanda target scanning, we defined 29 ZIKV derived viRNA profiles in NSCs, and established a complex interaction network between the viRNAs and their viral targets. More importantly, we found that viRNA production from the ZIKV mRNA is dependent on Dicer function and is a limiting factor for ZIKV virulence in NSCs. As a result, much higher levels of viRNAs generated from the ZIKV-H41R virus-infected NSCs. Therefore, our mapping of viRNAs to their RNA targets paves a way to further investigate how viRNAs play the role in anti-viral mechanisms, and perhaps other unknown biological functions.

Genome-wide analysis of HOXC4 and HOXC6 regulated genes and binding sites in prostate cancer cells.

Aberrant expression of HOXC6 and HOXC4 is commonly detected in prostate cancer. The high expression of these transcription factors is associated with aggressive prostate cancer and can predict cancer recurrence after treatment. Thus, HOXC4 and HOXC6 are clinically relevant biomarkers of aggressive prostate cancer. However, the molecular mechanisms by which these HOXC genes contribute to prostate cancer is not yet understood. To begin to address the role of HOXC4 and HOXC6 in prostate cancer, we performed RNA-seq analyses before and after siRNA-mediated knockdown of HOXC4 and/or HOXC6 and also performed ChIP-seq to identify genomic binding sites for both of these transcription factors. Our studies demonstrate that HOXC4 and HOXC6 co-localize with HOXB13, FOXA1 and AR, three transcription factors previously shown to contribute to the development of prostate cancer. We suggest that the aberrantly upregulated HOXC4 and HOXC6 proteins may compete with HOXB13 for binding sites, thus altering the prostate transcriptome. This competition model may be applicable to many different human cancers that display increased expression of a HOX transcription factor.

The Zika Virus Capsid Disrupts Corticogenesis by Suppressing Dicer Activity and miRNA Biogenesis.

Zika virus (ZIKV) causes microcephaly and disrupts neurogenesis. Dicer-mediated miRNA biogenesis is required for embryonic brain development and has been suggested to be disrupted upon ZIKV infection. Here we mapped the ZIKV-host interactome in neural stem cells (NSCs) and found that Dicer is specifically targeted by the capsid from ZIKV, but not other flaviviruses, to facilitate ZIKV infection. We identified a capsid mutant (H41R) that loses this interaction and does not suppress Dicer activity. Consistently, ZIKV-H41R is less virulent and does not inhibit neurogenesis in vitro or corticogenesis in utero. Epidemic ZIKV strains contain capsid mutations that increase Dicer binding affinity and enhance pathogenicity. ZIKV-infected NSCs show global dampening of miRNA production, including key miRNAs linked to neurogenesis, which is not observed after ZIKV-H41R infection. Together these findings show that capsid-dependent suppression of Dicer is a major determinant of ZIKV immune evasion and pathogenesis and may underlie ZIKV-related microcephaly.

The Enigmatic HOX Genes: Can We Crack Their Code?

Homeobox genes (HOX) are a large family of transcription factors that direct the formation of many body structures during early embryonic development. There are 39 genes in the subgroup of homeobox genes that constitute the human HOX gene family. Correct embryonic development of flies and vertebrates is, in part, mediated by the unique and highly regulated expression pattern of the HOX genes. Disruptions in these fine-tuned regulatory mechanisms can lead to developmental problems and to human diseases such as cancer. Unfortunately, the molecular mechanisms of action of the HOX family of transcription factors are severely under-studied, likely due to idiosyncratic details of their structure, expression, and function. We suggest that a concerted and collaborative effort to identify interacting protein partners, produce genome-wide binding profiles, and develop HOX network inhibitors in a variety of human cell types will lead to a deeper understanding of human development and disease. Within, we review the technological challenges and possible approaches needed to achieve this goal.

TRIM9-Mediated Resolution of Neuroinflammation Confers Neuroprotection upon Ischemic Stroke in Mice.

Excessive and unresolved neuroinflammation is a key component of the pathological cascade in brain injuries such as ischemic stroke. Here, we report that TRIM9, a brain-specific tripartite motif (TRIM) protein, was highly expressed in the peri-infarct areas shortly after ischemic insults in mice, but expression was decreased in aged mice, which are known to have increased neuroinflammation after stroke. Mechanistically, TRIM9 sequestered ß-transducin repeat-containing protein (ß-TrCP) from the Skp-Cullin-F-box ubiquitin ligase complex, blocking IκBα degradation and thereby dampening nuclear factor κB (NF-κB)-dependent proinflammatory mediator production and immune cell infiltration to limit neuroinflammation. Consequently, Trim9-deficient mice were highly vulnerable to ischemia, manifesting uncontrolled neuroinflammation and exacerbated neuropathological outcomes. Systemic administration of a recombinant TRIM9 adeno-associated virus that drove brain-wide TRIM9 expression effectively resolved neuroinflammation and alleviated neuronal death, especially in aged mice. These findings reveal that TRIM9 is essential for resolving NF-κB-dependent neuroinflammation to promote recovery and repair after brain injury and may represent an attractive therapeutic target.

A Prostate Cancer Risk Element Functions as a Repressive Loop that Regulates HOXA13.

Prostate cancer (PCa) is the leading cancer among men in the United States, with genetic factors contributing to ∼42% of the susceptibility to PCa. We analyzed a PCa risk region located at 7p15.2 to gain insight into the mechanisms by which this noncoding region may affect gene regulation and contribute to PCa risk. We performed Hi-C analysis and demonstrated that this region has long-range interactions with the HOXA locus, located ∼873 kb away. Using the CRISPR/Cas9 system, we deleted a 4-kb region encompassing several PCa risk-associated SNPs and performed RNA-seq to investigate transcriptomic changes in prostate cells lacking the regulatory element. Our results suggest that the risk element affects the expression of HOXA13 and HOTTIP, but not other genes in the HOXA locus, via a repressive loop. Forced expression of HOXA13 was performed to gain further insight into the mechanisms by which this risk element affects PCa risk.

Zika Virus NS4A and NS4B Proteins Deregulate Akt-mTOR Signaling in Human Fetal Neural Stem Cells to Inhibit Neurogenesis and Induce Autophagy.

The current widespread outbreak of Zika virus (ZIKV) infection has been linked to severe clinical birth defects, particularly microcephaly, warranting urgent study of the molecular mechanisms underlying ZIKV pathogenesis. Akt-mTOR signaling is one of the key cellular pathways essential for brain development and autophagy regulation. Here, we show that ZIKV infection of human fetal neural stem cells (fNSCs) causes inhibition of the Akt-mTOR pathway, leading to defective neurogenesis and aberrant activation of autophagy. By screening the three structural proteins and seven nonstructural proteins present in ZIKV, we found that two, NS4A and NS4B, cooperatively suppress the Akt-mTOR pathway and lead to cellular dysregulation. Corresponding proteins from the closely related dengue virus do not have the same effect on neurogenesis. Thus, our study highlights ZIKV NS4A and NS4B as candidate determinants of viral pathogenesis and identifies a mechanism of action for their effects, suggesting potential targets for anti-ZIKV therapeutic intervention.

Identification of two severe fever with thrombocytopenia syndrome virus strains originating from reassortment.

Recently, a novel bunyavirus, severe fever with thrombocytopenia syndrome virus (SFTSV), was isolated in central China. The virus can cause multi-clinical symptoms: severe fever, thrombocytopenia, leukocytopenia, with a mortality rate of ~10%. Several studies show that SFTSV could undergo rapid evolution via gene mutation and homologous recombination. However, as an important evolutionary force for segmented-genome viruses, reassortment has not been reported in SFTSV. In this study, we identified two SFTSV strains of which the S segment has different origin from M and L, suggesting that reassortment might be potential force driving rapid change of SFTSV. This result might shed new light on the evolutionary behavior of the novel virus.