Integrated RNA-seq Analysis Research Articles

Investigating the Role of Inflammatory Response in Polycystic Ovary Syndrome Using Integrated RNA-Seq Analysis.

An important factor in the pathogenesis of polycystic ovary syndrome (PCOS) is chronic low-grade inflammation. However, the exact pathophysiology of PCOS is currently unknown, which makes clinical diagnosis and the development of effective treatments more difficult. We aimed to investigate the role of the inflammatory response in initiating and progressing PCOS. 13 control granulosa cell samples and 15 granulosa cell samples from patients with PCOS were obtained from the GSE102293, GSE34526, and GSE5850 datasets. The gene set variation analysis (GSVA) method was used to calculate the inflammatory response score. Subsequently, the genes associated with inflammation in the hub were identified using differential expression analysis and weighted gene co-expression network analysis (WGCNA). The findings were confirmed by analysis of independent datasets and examination of clinical samples by qRT-PCR analysis. A consensus cluster analysis was conducted to categorize the PCOS samples into subtypes related to inflammation. Functional enrichment and analysis of immune cell infiltration were conducted to explore the potential mechanisms involved. Additionally, the CMap database was utilized to predict potential drugs, and the results were confirmed through molecular docking. During the training cohort analysis, we identified five distinct genes (TGFBR2, ICAM3, WIPF1, SLC11A1, and NCF2) that could serve as potential diagnostic markers for PCOS. The expression levels of these genes were confirmed through validation in both the test set and clinical samples. In training cohort, two distinct inflammatory patterns (C1 and C2) were identified, and the C2 subtype exhibited activated immune- and inflammation-related pathways. Esmolol was shown to have potential as a drug to treat PCOS and it showed good results for molecular binding at TGFBR2, ICAM3, WIPF1, SLC11A1, and NCF2 proteins. Five diagnostic biomarkers and two inflammation-related molecular types associated with PCOS were identified, and esmolol was a potential drug for PCOS treatment. Our findings provided new diagnostic markers and potential small-molecule drugs for PCOS diagnosis and prevention.

Dissection of mRNA ac4C acetylation modifications in AC and Nr fruits: insights into the regulation of fruit ripening by ethylene

N4-acetylcytidine (ac4C) modification of mRNA has been shown to be present in plant RNAs, but its regulatory function in plant remains largely unexplored. In this study, we investigated the differentially expressed mRNAs, lncRNAs and acetylation modifications of mRNAs in tomato fruits from both genotypes. By comparing wild-type (AC) tomato and the ethylene receptor-mutant (Nr) tomato from mature green (MG) to six days after the breaker (Br6) stage, we identified differences in numerous key genes related to fruit ripening and observed the corresponding lncRNAs positively regulated the target genes expression. At the post-transcriptional level, the acetylation level decreased and increased in AC and Nr tomatoes from MG to Br6 stage, respectively. The integrated analysis of RNA-seq and ac4C-seq data revealed the potential positive role of acetylation modification in regulating gene expression. Furthermore, we found differential acetylation modifications of certain transcripts (ACO, ETR, ERF, PG, CesA, β-Gal, GAD, AMY, and SUS) in AC and Nr fruits which may explain the differences in ethylene production, fruit texture, and flavor during their ripening processes. The present study provides new insights into the molecular mechanisms by which acetylation modification differentially regulates the ripening process of wild-type and mutant tomato fruits deficient in ethylene signaling.Graphical

Integrated transcriptome and small RNA sequencing revealing miRNA-mediated regulatory network of bicolour pattern formation in Pericallis hybrida ray florets

Flower spots of plants are of evolutionary importance and ornamental value, but research on regulatory mechanisms is still limited. Cineraria (Pericallis hybrida) is an important ornamental plant with various bicolour cultivars. In this study, we performed transcriptome and small RNA sequencing to investigate the regulatory network of bicolour pattern formation in P. hybrida ray florets. Through transcriptome sequencing, we mainly focused on differentially expressed genes (DEGs) involved in flavonoid biosynthesis. Gene expression analysis showed that PhLAR (Unigene0092951), PhMYB8 (Unigene0068595) and PhbHLH137 (Unigene0091507) were up-regulated in the colourless region. Additionally, we identified 234 known miRNAs and 280 novel miRNAs by sRNA sequencing, and 49 differentially expressed miRNAs (DEmiRs) were screened. Integrated analysis of RNA-seq and sRNA-seq data revealed that miR172-AP2 and miR156-SPL modules were crucial in regulating bicolour pattern formation. When PhAP2 was transiently silenced in cineraria capitulum by virus-induced gene silencing (VIGS), the colourless region of ray florets increased and the coloured region changed from carmine to pink. Meanwhile, PhCHS2, PhDFR3 and PhANS were down-regulated, while PhLAR was up-regulated significantly. It was hypothesized that PhAP2 functions as an inhibitor of bicolour pattern appearance and could influence flavonoid biosynthesis. Overall, our study revealed a possible regulatory network of miRNA-mediated bicolour pattern appearance in P. hybrida, which could enrich the post-transcriptional regulatory mechanisms of flower spots formation in ornamental plants.

The XPO1-FOXC1-HOX Functional Axis Opens New Therapeutic Avenues to Treat DEK-NUP214 AML Patients

Introduction: t(6;9)(p23;q34.1)/DEK-NUP214 patients represent a discrete group of younger AML patients recognised as a separate disease entity in the World Health Organization classification of myeloid neoplasms. They typically display a dismal prognosis, higher relapse rate and a striking co-occurrence with FLT3-ITD mutations in > 70% of cases. DEK is a nuclear factor that has been attributed multifunctional roles, including gene regulation, while its fusion partner, NUP214, plays a pivotal role in nuclear export by interacting with transport receptors such as XPO1. However, the precise mechanism by which DEK-NUP214 drives leukaemia remains unclear. Aims: Application of a multi-omics profiling to understand the oncogenic mechanisms underlying DEK-NUP214 fusion protein and identify potential therapeutical vulnerabilities. Methods: A comprehensive multi-omics screening (whole genome sequencing, targeted sequencing, transcriptomics, (phosphor)proteomics, mass cytometry (CyTOF) and in vitro drug screening) was conducted on 57 untreated primary AML samples from cytogenetically poor-risk AML, including four cases with t(6;9) translocation (complex karyotype (n=20), -7/del(7) (n=15), KMT2A rearrangements (not including t(9;11)) (n=12), t(6;9) (n=4) and other poor-risk karyotypes (inv(3), -5/del(5), t(3;12)/+8 or -17/del(17)) (n=6)). The t(6;9)/DEK-NUP214 cell line FKH-1, was utilised as a model for in vitro experiments and compared with other AML cell lines (OCI-AML3, KASUMI-1, THP-1, P31-FUJ, MV4-11 and K562). In vitro drug treatments were conducted in the cell lines and patient samples using Cell-Titer&Glo, and transcriptional changes were evaluated using qPCR and RNA-seq. To examine the function of selected target genes of DEK-NUP214, we used lentiviral shRNA system followed by cell cycle, apoptosis, differentiation, and cell proliferation assays. Results: An integrative RNA-seq analysis of our cohort and a separate series of 691 AML cases that included three additional t(6;9) cases (Leucegene, https://leucegene.ca/) revealed 128 genes significantly upregulated and 74 downregulated in t(6;9) patients compared to other AML samples. The overexpressed genes included some known leukaemia mediators (FOXC1, HOXA and HOXB genes), the genes previously reported in t(6;9) patients (EYA3, SESN1 and PRDM2) and novel genes with roles in haematopoiesis or cancer cell survival that showed the most significant differences (NFIX and GGT5). The significant overexpression of these genes was also confirmed in the t(6;9) FKH-1 cell line. In vitro drug screening of 527 licenced or investigational drugs allowed us to identify the compounds that were most effective and selective for t(6;9) patients in comparison with other AML cytogenetic groups. The two XPO1 inhibitors (Selinexor and Eltanexor) included in our drug panel were within the four top-ranked compounds. Of note, qPCR and RNA-seq analyses in the cell lines and primary patient samples showed that inhibition of XPO1 resulted in significant downregulation of the expression of FOXC1, NFIX, EYA3, HOXA and HOXB genes, highlighting a functional axis linking these target genes, XPO1 and the fusion. In line with this finding, silencing of FOXC1 in the FKH-1 cells led to an increase in apoptosis, lower cell proliferation and reduced clonogenic capacity, accompanied by a concomitant downregulation of HOXA and HOXB genes. Collectively, these findings support a key role of FOXC1 in DEK-NUP214-driven AML. Conclusion: This study offers valuable insights into the molecular and pathological mechanisms underlying t(6;9)-AML, revealing a functional interaction between DEK-NUP214, XPO1 and FOXC1, and providing evidence to consider XPO1 inhibition as a potential new avenue to treat these patients.

Diagnostic Utility of Comprehensive RNA-Seq Analysis in Adult B-ALL

Introduction Adult B-cell acute lymphoblastic leukemia (B-ALL) comprises 75% of adult ALL cases. The classification and risk stratification of patients have conventionally relied on cytogenetics analysis. However, cytogenetics has limitations due to its reliance on metaphase cells, which can mitigate classification accuracy. In recent years, transcriptome sequencing (RNA-seq) of large pediatrics ALL cohorts has expanded the number of recurrent gene fusions recognized as disease defining events and informed risk stratification [Brady et al. 2022, Mullighan et al. 2017]. This study aims to evaluate the diagnostic utility of RNA-seq for clinical subtype classification in adult B-ALL in a large representative cohort. Methods The study cohort comprised 338 adult patients (25-65 years) with newly diagnosed ALL (UKALL14, ISRCTN66541317, NCT01085617). Paired-end RNA-seq data at 60 Million reads per sample were generated. Analysis focused on the detection of disease-defining gene fusions and gene expression signatures. Three RNA fusion callers (FusionCatcher, FuSeq, and STAR-Fusion) and three expression-based classifiers (ALLSorts, ALLspice, and ALLCatchR) were used for consensus fusion calling and expression-based classification, respectively [Nicorici et al. 2014, Vu et al. 2018, Haas et al. 2019, Schmidt et al., Mäkinen et al., Bader et al. 2022]. Integration of classification results from expression-based classifiers and the consensus gene fusions provides 4 levels of RNA-derived subtype information, denoted as “RNA evidence” in this study. These RNA-seq based classification results were compared with clinical cytogenetics findings. Results Out of 338 samples in this cohort, cytogenetics unequivocally classified 73% (246/338) of the samples. By integrating RNA expression-based classification with fusion subtypes, we confidently classified 87% (295/338) of the cohort with at least 2 levels of RNA evidence. In total, consensus gene fusions were identified in 74% (249/338), while expression-based classifiers assigned a confident subtype to 82% (319/338). Subsequently, we compared cytogenetics with RNA-seq derived classification and found a high concordance of 98% (240/246) between the two approaches. Only 6 (<2%) samples with cytogenetics subtypes were not classified by RNA-seq, attributed to specific subtype characteristics (near-haploid) or lower RNA purity/quality. 27% (92/338) of subjects were not classified by cytogenetics (31 missing/failed and 61 indeterminate cytogenetics). Of the 31 samples with failed/missing cytogenetics, RNA-seq confidently assigned a class in 74% (23/31). For the remaining 61 cases with indeterminate cytogenetic (complex/other), RNA-seq resolved classification in 74% (45/61). Taken together, this improved the classification for 20% (68/338) of the entire cohort that was previously unclassified through cytogenetics. Notably, RNA-seq-exclusive findings were enriched in subtypes like DUX4, PAX5alt, and ZNF384, which are challenging for cytogenetics to discern. Importantly, integration of RNA-seq with cytogenetics led to a confident subtype classification for 93% of the cohort (314/338), a significant improvement over the initial 73% based on cytogenetics alone. Conclusions In conclusion, our study highlights the relevance of integrated RNA-seq analysis for reliable and comprehensive subtype classification in adult B-ALL. The high concordance between cytogenetics and RNA-derived subtypes underscores their complementary roles, providing valuable insights into disease mechanisms and guiding personalized treatment strategies.

Identification of GUCA2A and COL3A1 as prognostic biomarkers in colorectal cancer by integrating analysis of RNA-Seq data and qRT-PCR validation

By 2030, it is anticipated that there will be 2.2 million new instances of colorectal cancer worldwide, along with 1.1 million yearly deaths. Therefore, it is critical to develop novel biomarkers that could help in CRC early detection. We performed an integrated analysis of four RNA-Seq data sets and TCGA datasets in this study to find novel biomarkers for diagnostic, prediction, and as potential therapeutic for this malignancy, as well as to determine the molecular mechanisms of CRC carcinogenesis. Four RNA-Seq datasets of colorectal cancer were downloaded from the Sequence Read Archive (SRA) database. The metaSeq package was used to integrate differentially expressed genes (DEGs). The protein–protein interaction (PPI) network of the DEGs was constructed using the string platform, and hub genes were identified using the cytoscape software. The gene ontology and KEGG pathway enrichment analysis were performed using enrichR package. Gene diagnostic sensitivity and its association to clinicopathological characteristics were demonstrated by statistical approaches. By using qRT-PCR, GUCA2A and COL3A1 were examined in colon cancer and rectal cancer. We identified 5037 differentially expressed genes, including (4752 upregulated, 285 downregulated) across the studies between CRC and normal tissues. Gene ontology and KEGG pathway analyses showed that the highest proportion of up-regulated DEGs was involved in RNA binding and RNA transport. Integral component of plasma membrane and mineral absorption pathways were identified as containing down-regulated DEGs. Similar expression patterns for GUCA2A and COL3A1 were seen in qRT-PCR and integrated RNA-Seq analysis. Additionally, this study demonstrated that GUCA2A and COL3A1 may play a significant role in the development of CRC.

Loss of ZBED6 Protects Against Sepsis-Induced Muscle Atrophy by Upregulating DOCK3-Mediated RAC1/PI3K/AKT Signaling Pathway in Pigs.

Sepsis-induced muscle atrophy often increases morbidity and mortality in intensive care unit (ICU) patients, yet neither therapeutic target nor optimal animal model is available for this disease. Here, by modifying the surgical strategy of cecal ligation and puncture (CLP), a novel sepsis pig model is created that for the first time recapitulates the whole course of sepsis in humans. With this model and sepsis patients, increased levels of the transcription factor zinc finger BED-type containing 6 (ZBED6) in skeletal muscle are shown. Protection against sepsis-induced muscle wasting in ZBED6-deficient pigs is further demonstrated. Mechanistically, integrated analysis of RNA-seq and ChIP-seq reveals dedicator of cytokinesis 3 (DOCK3) as the direct target of ZBED6. In septic ZBED6-deficient pigs, DOCK3 expression is increased in skeletal muscle and myocytes, activating the RAC1/PI3K/AKT pathway and protecting against sepsis-induced muscle wasting. Conversely, opposite gene expression patterns and exacerbated muscle wasting are observed in septic ZBED6-overexpressing myotubes. Notably, sepsis patients show increased ZBED6 expression along with reduced DOCK3 and downregulated RAC1/PI3K/AKT pathway. These findings suggest that ZBED6 is a potential therapeutic target for sepsis-induced muscle atrophy, and the established sepsis pig model is a valuable tool for understanding sepsis pathogenesis and developing its therapeutics.

Integrated RNA-Seq Analysis for Identification of Differentially Expressed Gene in Medullo-Blastoma

Integrated RNA-Seq Analysis for Identification of Differentially Expressed Gene in Medullo-Blastoma

Abstract 2440: HOTAIR functionality and regulation in ovarian cancer stem cells

Abstract Ovarian cancer (OC) is the fifth leading cause of cancer-related death among American women. Persistence of OC stem cells (OCSCs) is believed to contribute to resistance to platinum-based chemotherapy and disease relapse. We have previously shown that epigenetic changes in OCSCs play a role in post-therapy OCSC persistence and demonstrated the potential to target OCSC using epigenetic therapies. HOXC transcript antisense RNA (HOTAIR) has been shown to be associated with chemoresistance and overexpressed in high-grade serous OC (HGSOC). HOTAIR interacts with Polycomb Repressive Complex 2 (PRC2) and plays a key role in chromatin remodeling. Because HOTAIR is a known epigenetic regulator of differentiation and developmental genes in OC, we hypothesized that HOTAIR regulates OCSCs and epigenetic targeting HOTAIR will prevent tumor relapse. To produce loss-of-function phenotypes of HOTAIR and investigate the function of this gene, we utilized the paired CRISPR guide RNA design to delete the functional sites of HOTAIR in a panel high grade serous OC cell lines (Kuramochi, OVCAR3) without affecting nearby protein-coding gene. Knockout of HOTAIR re-sensitized OC cells to platinum treatment and significantly decreased (P<0.001) the OCSC population and stemness-related phenotypes, including inhibiting stemness-associated gene expression ALDH1A1, Notch3, Sox9, and PROM1 and in vitro spheroid forming ability under low attachment conditions. Integrated analysis of RNA-seq and ATAC-seq on control and HOTAIR knockout cells revealed HOTAIR altered global chromatin dynamics to change downstream gene expression, particularly, the NF-kB pathway. In xenograft assays using HGSOC cells, combining a HOTAIR inhibitor with an EZH2 inhibitor and chemotherapy significantly decreased in vivo tumor formation and increased mouse survival. In summary, depletion of HOTAIR functionally decreased OCSC phenotypes and malignant potential and reprogrammed HGSOC to a less stem-like phenotype. There is an urgent need to develop new therapeutic strategies to target OCSCs and overcome chemoresistant OC, and the results of this study suggest that targeting HOTAIR in combination with epigenetic therapies may represent a novel therapeutic strategy to prevent tumor relapse in OC. Citation Format: Weini Wang, Yanchi Zhou, Ji Wang, Ali Ozes, Heather M. O’Hagan, Anirban Mitra, Kenneth P. Nephew. HOTAIR functionality and regulation in ovarian cancer stem cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2440.

Integrated analysis of RNA-seq in hepatocellular carcinoma reveals competing endogenous RNA network composed of circRNA, lncRNA, and mRNA.

Circular RNAs (circRNAs) and long non-coding RNAs (lncRNAs) have been hypothesized to have important roles in the etiology of hepatocellular carcinoma (HCC). However, the synergistic effect of circRNA and lncRNA in the pathogenesis of HCC has rarely been studied. In this study, the Gene Expression Omnibus database was used to get the expression profiles of circRNAs, micro RNAs (miRNAs), lncRNAs, and messenger RNAs (mRNAs) in HCC tissues and normal tissues. The accession numbers for this database are GSE101728, GSE155949, and GSE108724. We found 291 differentially overexpressed lncRNAs and 541 differentially overexpressed mRNA in GSE101728, 30 differentially overexpressed circRNA in GSE155949, and 48 significantly downregulated miRNA in GSE198724. Meanwhile, based on Pearson correlation test, we established lncRNA-mRNA networks. We constructed lncRNA/circRNA-miRNA pairs through Starbase database prediction and identified the common miRNAs. The intersection of co-predicted miRNAs and the 48 significantly low expression miRNAs in GSE198724 were included in the following study. miRDB, Targetscan, miRwalk, and lncRNA-related mRNA jointly determined the miRNA-mRNA portion of the circRNA/lncRNA-miRNA-mRNA co-expression network. And, among 55 differentially expressed mRNA in circRNA/lncRNA-miRNA-mRNA network, CPEB3, EFNB3, FATA4, growth hormone receptor, GSTZ1, KLF8, MFAP4, PAIP2B, PHACTR3, PITPNM3, RPS6KA6, RSPO3, SLITRK6, SMOC1, STEAP4, SYT1, TMEM132E, TSPAN11, and ZFPM2 were intimately related to the prognosis of HCC patients in Kaplan-Meier plotter analysis (P < .05). We have discovered that the prognosis-related lncRNAs/circRNAs-miRNA-mRNA network plays a significant role in the pathogenesis of HCC. These findings may offer fresh perspectives for further research into the pathogenesis of HCC and the search for novel treatments for HCC.

Identification of Hub Genes Associated with Breast Cancer Using Integrated Gene Expression Data with Protein-Protein Interaction Network

Breast cancer (BC) is the most incident cancer type among women. BC is also ranked as the second leading cause of death among all cancer types. Therefore, early detection and prediction of BC are significant for prognosis and in determining the suitable targeted therapy. Early detection using morphological features poses a significant challenge for physicians. It is therefore important to develop computational techniques to help determine informative genes, and hence help diagnose cancer in its early stages. Eight common hub genes were identified using three methods: the maximal clique centrality (MCC), the maximum neighborhood component (MCN), and the node degree. The hub genes obtained were CDK1, KIF11, CCNA2, TOP2A, ASPM, AURKB, CCNB2, and CENPE. Enrichment analysis revealed that the differentially expressed genes (DEGs) influenced multiple pathways. The most significant identified pathways were focal adhesion, ECM-receptor interaction, melanoma, and prostate cancer pathways. Additionally, survival analysis using Kaplan–Meier was conducted, and the results showed that the obtained eight hub genes are promising candidate genes to serve as prognostic and diagnostic biomarkers for BC. Furthermore, a correlation study between the clinicopathological factors in BC and the eight hub genes was performed. The results showed that all eight hub genes are associated with the clinicopathological variables of BC. Using an integrated analysis of RNASeq and microarray data, a protein-protein interaction (PPI) network was developed. Eight hub genes were identified in this study, and they were validated using previous studies. Additionally, Kaplan-Meier was used to verify the prognostic value of the obtained hub genes.

Defining cell-specific physical basis of gene expression and signatures of functional chromatin 3D interactions: integrated analysis of single-cell 3D chromatin models, RNA/ATAC-seq, and epigenetic data.

Three-dimensional structures of chromatin play important roles in gene regulation. Hi-C measurement provided a wealth of information on chromatin interactions. Emerging evidence suggests functional chromatin interactions are related to gene expression. However, gene promoters and their regulatory elements can be separated by 103-106 bp apart. Therefore, examining 3D chromatin conformations and identifying functionally long-range interactions are important for deciphering the structure-function relationship of 3D genome. Our recent work of CHROMATIX deep sampling algorithms can identify non-random chromatin interactions (5-7% of total Hi-C). These can be used to generate large ensembles of 3D single-cell chromatin conformations, accurately reproducing both single-cell capture and imaging studies, as well as population Hi-C measurements. In this study, we examine predicted ensembles of single-cell 3D chromatin conformations of loci among leukemia K562 cells, lymphoblastoid GM12878 cells, and lung fibroblast IMR90 cells. We identified small subsets of non-random chromatin interactions from Hi-C data based on large random ensembles of 3D chromatin chains folded in confined nuclear volume. These non-random interactions were used to generate ensembles of 5.0×104 single-cell 3D conformations for individual loci. We further quantified the heterogeneity of 3D chromatin conformations by clustering, with each spatial cluster and its proportion defined. In addition, we carried out an integrated analysis of RNA-seq, ATAC-seq, and epigenetic data of these cell lines. Results show that cell-specific functional chromatin interactions provide 3D basis for differential gene expression. There are well-defined signatures of 3D subpopulations of chromatin conformations involving distinctive patterns of epigenetic marks, and these signatures are strongly associated with gene differential expression.

The Deubiquitinase USP39 Promotes Esophageal Squamous Cell Carcinoma Malignancy as a Splicing Factor.

Esophageal squamous cell carcinoma (ESCC) is an aggressive epithelial malignancy and the underlying molecular mechanisms remain elusive. Here, we identify that the ubiquitin-specific protease 39 (USP39) drives cell growth and chemoresistance by functional screening in ESCC, and that high expression of USP39 correlates with shorter overall survival and progression-free survival. Mechanistically, we provide evidence for the role of USP39 in alternative splicing regulation. USP39 interacts with several spliceosome components. Integrated analysis of RNA-seq and RIP-seq reveals that USP39 regulates the alternative splicing events. Taken together, our results indicate that USP39 functions as an oncogenic splicing factor and acts as a potential therapeutic target for ESCC.

Epigenetic regulation of seed-specific gene expression by DNA methylation valleys in castor bean

BackgroundUnderstanding the processes governing angiosperm seed growth and development is essential both for fundamental plant biology and for agronomic purposes. Master regulators of angiosperm seed development are expressed in a seed-specific manner. However, it is unclear how this seed specificity of transcription is established. In some vertebrates, DNA methylation valleys (DMVs) are highly conserved and strongly associated with key developmental genes, but comparable studies in plants are limited to Arabidopsis and soybean. Castor bean (Ricinus communis) is a valuable model system for the study of seed biology in dicots and source of economically important castor oil. Unlike other dicots such as Arabidopsis and soybean, castor bean seeds have a relatively large and persistent endosperm throughout seed development, representing substantial structural differences in mature seeds. Here, we performed an integrated analysis of RNA-seq, whole-genome bisulfite sequencing, and ChIP-seq for various histone marks in the castor bean.ResultsWe present a gene expression atlas covering 16 representative tissues and identified 1162 seed-specific genes in castor bean (Ricinus communis), a valuable model for the study of seed biology in dicots. Upon whole-genome DNA methylation analyses, we detected 32,567 DMVs across five tissues, covering ~33% of the castor bean genome. These DMVs are highly hypomethylated during development and conserved across plant species. We found that DMVs have the potential to activate transcription, especially that of tissue-specific genes. Focusing on seed development, we found that many key developmental regulators of seed/endosperm development, including AGL61, AGL62, LEC1, LEC2, ABI3, and WRI1, were located within DMVs. ChIP-seq for five histone modifications in leaves and seeds clearly showed that the vast majority of histone modification peaks were enriched within DMVs, and their remodeling within DMVs has a critical role in the regulation of seed-specific gene expression. Importantly, further experiment analysis revealed that distal DMVs may act as cis-regulatory elements, like enhancers, to activate downstream gene expression.ConclusionsOur results point to the importance of DMVs and special distal DMVs behaving like enhancers, in the regulation of seed-specific genes, via the reprogramming of histone modifications within DMVs. Furthermore, these results provide a comprehensive understanding of the epigenetic regulator roles in seed development in castor bean and other important crops.

Integrative RNA-seq and ATAC-seq analyses of phosphodiesterase 6 mutation-induced retinitis pigmentosa.

Inhibition of poly-ADP-ribose polymerase 1 (PARP1) could relieve phosphodiesterase 6 mutation-induced retinitis pigmentosa (RP). However, the mechanism related to PARP1 overexpression in the RP has not been clarified. We attempted to explore the potential mechanism related to PARP1 regulating RP. ATAC-seq and RNA-seq were performed for retina tissues of C3H and rd1 mice. The differentially expressed genes (DEGs) were identified, followed by the construction of PARP1-DEG co-expression and protein-protein interaction (PPI) networks. Gene ontology-biological process and pathway enrichment of DEGs were performed by clusterProfiler software. The overlapped genes that might play regulatory roles in PARP1 expression were mined by integrated analysis of RNA-seq and ATAC-seq data. A total of 1061 DEGs were identified between C3H and rd1 group. Co-expression network was constructed with 313 PARP1-gene co-expression pairs. The down-regulated DEGs were closely related to visual perception and light stimulus-related biological process, while the up-regulated DEGs were significantly enriched in phototransduction and PPAR signaling pathway. PPI network was constructed with 202 nodes and 375 edges, which was clustered into 3 modules. Module 1 genes were closely related to detection of light stimulus, visual perception related biological process and phototransduction pathway (involved with Gnat1/Guca1b/Gnat2/Sag/Pde6g). By integrated analysis of the RNA-seq and ATAC-seq, the overlapped up-regulated genes were Asxl3 and Nyap2, while the down-regulated genes were Tmem136 and Susd3. Gnat1 may play a key role in RP development by interacting with PARP1. Susd3 may play a regulatory role in PARP1 expression and affect RP formation.

Identification and Validation of Constructing the Prognostic Model With Four DNA Methylation-Driven Genes in Pancreatic Cancer

Background: Pancreatic cancer (PC) is a highly aggressive gastrointestinal tumor and has a poor prognosis. Evaluating the prognosis validly is urgent for PC patients. In this study, we utilized the RNA-sequencing (RNA-seq) profiles and DNA methylation expression data comprehensively to develop and validate a prognostic signature in patients with PC. Methods: The integrated analysis of RNA-seq, DNA methylation expression profiles, and relevant clinical information was performed to select four DNA methylation-driven genes. Then, a prognostic signature was established by the univariate, multivariate Cox, and least absolute shrinkage and selection operator (LASSO) regression analyses in The Cancer Genome Atlas (TCGA) dataset. GSE62452 cohort was utilized for external validation. Finally, a nomogram model was set up and evaluated by calibration curves. Results: Nine DNA methylation-driven genes that were related to overall survival (OS) were identified. After multivariate Cox and LASSO regression analyses, four of these genes (RIC3, MBOAT2, SEZ6L, and OAS2) were selected to establish the predictive signature. The PC patients were stratified into two groups according to the median risk score, of which the low-risk group displayed a prominently favorable OS compared with the high-risk group, whether in the training (p < 0.001) or validation (p < 0.01) cohort. Then, the univariate and multivariate Cox regression analyses showed that age, grade, risk score, and the number of positive lymph nodes were significantly associated with OS in PC patients. Therefore, we used these clinical variables to construct a nomogram; and its performance in predicting the 1-, 2-, and 3-year OS of patients with PC was assessed via calibration curves. Conclusion: A prognostic risk score signature was built with the four alternative DNA methylation-driven genes. Furthermore, in combination with the risk score, age, grade, and the number of positive lymph nodes, a nomogram was established for conveniently predicting the individualized prognosis of PC patients.

Integrative RNA-Seq analysis of Capsicum annuum L.-Phytophthora capsici L. pathosystem reveals molecular cross-talk and activation of host defence response.

The online version contains supplementary material available at 10.1007/s12298-021-01122-y.

Abstract P305: Patient-specific Ipsc-derived Cardiomyocytes Reveal Abnormal Regulation Of Fgf16 In A Familial Atrial Septal Defect

Aims: Congenital heart disease (CHD) frequently occurs in newborns due to abnormal formation of the heart or major blood vessels. Mutations in the GATA4 gene, which encodes GATA binding protein 4, are responsible for atrial septal defect (ASD), a common CHD. This study aims to gain insights into the molecular mechanisms of CHD using human induced pluripotent stem cells (iPSCs) from a family cohort with ASD. Methods and Results: Patient-specific iPSCs possess the same genetic information as the donor and can differentiate into various cell types from all three germ layers in vitro , thus presenting a promising approach for disease modeling and molecular mechanism research. Here, we generated a patient-specific iPSC line (iPSC-G4 T280M ) from a family cohort carrying a hereditary ASD mutation in GATA4 gene (T280M), as well as a human embryonic stem cell line (ESC-G4 T280M ) carrying the isogenic T280M mutation using the CRISPR/Cas9 genome editing method. The GATA4-mutant iPSCs and ESCs were then differentiated into cardiomyocytes (CMs) to model GATA4 mutation-associated ASD. We observed an obvious defect in cell proliferation in cardiomyocytes derived from both GATA4 T280M -mutant iPSCs (iPSC-G4 T280M -CMs) and ESCs (ESC-G4 T280M -CMs), while the impaired proliferation ability of iPSC-G4 T280M -CMs could be restored by gene correction. Integrated analysis of RNA-Seq and ChIP-Seq data indicated that FGF16 is a direct target of wild-type GATA4. However, the T280M mutation obstructed GATA4 occupancy at the FGF16 promoter region, leading to impaired activation of FGF16 transcription. Overexpression of FGF16 in GATA4-mutant cardiomyocytes rescued the cell proliferation defect. The direct relationship between GATA4 T280M and ASD was demonstrated in a human iPSC model for the first time. Conclusions: In summary, our study revealed the molecular mechanism of the GATA4 T280M mutation in ASD. Understanding the roles of the GATA4-FGF16 axis in iPSC-CMs will shed light on heart development and provide novel insights for the treatment of ASD and other CHD disorders.

Abstract MP42: Macrophage Chromatin Remodeling Complex Subunit Baf60a In Atherosclerosis

Objectives: Atherosclerosis is a primary medical concern due to the increasing prevalence and lack of effective treatment that reverses the disease progression. While multiple genetic mutations/variations have been associated with atherosclerotic risk, the development of atherosclerosis in response to risk factors is also contributed by factors at the epigenetic and chromatin level. The SWI/SNF chromatin remodeling complex alters DNA accessibility and chromatin structure to modulate gene expression in response to external and internal cellular environments, potentially serving as an underexplored compartment of CVD and atherosclerosis development. Approach and Results: In an effort to identify key chromatin remodeling compartments involved in atherosclerotic foam cell formation, primary bone marrow-derived macrophages (BMDMs) were exposed to a pro-atherogenic environment, specifically to oxidized Low-density lipoprotein (oxLDL). We observed drastically and time-dependent decrease in the protein abundance of BAF60a, a transcription factor interacting subunit of the SWI/SNF chromatin remodeling complex. Macrophage-specific Baf60a deletion in ApoE deficient mice showed over 30% increase in aortic plaque formation after 16-weeks of western diet feeding (N=12, p=0.0097). Integrated analysis of RNA-seq and ATAC-seq regulatory network in oxLDL treated BAF60a deficient BMDMs identified a shift of chromatin accessibility, transcription factor footprint, and a reshape in gene expression patterns that regulate extracellular matrix organization, focal adhesion, integrin signaling, inflammation, and lead to foam cell formation which ultimately contribute to the atherosclerotic plaque build-up. Conclusions: Our data demonstrated the role of macrophage BAF60a in atherogenesis and suggested a potential therapeutic target to treat atherosclerosis, presumably by compensating for the loss of Baf60a.

Patient-specific iPSC-derived cardiomyocytes reveal abnormal regulation of FGF16 in a familial atrial septal defect.

Congenital heart disease (CHD) frequently occurs in newborns due to abnormal formation of the heart or major blood vessels. Mutations in the GATA4 gene, which encodes GATA binding protein 4, are responsible for atrial septal defect (ASD), a common CHD. This study aims to gain insights into the molecular mechanisms of CHD using human-induced pluripotent stem cells (iPSCs) from a family cohort with ASD. Patient-specific iPSCs possess the same genetic information as the donor and can differentiate into various cell types from all three germ layers in vitro, thus presenting a promising approach for disease modelling and molecular mechanism research. Here, we generated a patient-specific iPSC line (iPSC-G4T280M) from a family cohort carrying a hereditary ASD mutation in GATA4 gene (T280M), as well as a human embryonic stem cell line (ESC-G4T280M) carrying the isogenic T280M mutation using the CRISPR/Cas9 genome editing method. The GATA4-mutant iPSCs and ESCs were then differentiated into cardiomyocytes (CMs) to model GATA4 mutation-associated ASD. We observed an obvious defect in cell proliferation in cardiomyocytes derived from both GATA4T280M-mutant iPSCs (iPSC-G4T280M-CMs) and ESCs (ESC-G4T280M-CMs), while the impaired proliferation ability of iPSC-G4T280M-CMs could be restored by gene correction. Integrated analysis of RNA-Seq and ChIP-Seq data indicated that FGF16 is a direct target of wild-type GATA4. However, the T280M mutation obstructed GATA4 occupancy at the FGF16 promoter region, leading to impaired activation of FGF16 transcription. Overexpression of FGF16 in GATA4-mutant cardiomyocytes rescued the cell proliferation defect. The direct relationship between GATA4T280M and ASD was demonstrated in a human iPSC model for the first time. In summary, our study revealed the molecular mechanism of the GATA4T280M mutation in ASD. Understanding the roles of the GATA4-FGF16 axis in iPSC-CMs will shed light on heart development and provide novel insights for the treatment of ASD and other CHD disorders.