Exploring the transcriptomic profile of human monkeypox virus via CAGE and native RNA sequencing approaches.

In this study, we employed short- and long-read sequencing technologies to delineate the transcriptional architecture of the human monkeypox virus and to identify key regulatory elements that govern its gene expression. Specifically, we conducted a transcriptomic analysis to annotate the transcription start sites (TSSs) and transcription end sites (TESs) of the virus by utilizing Cap Analysis of gene expression sequencing on the Illumina platform and direct RNA sequencing on the Oxford Nanopore technology device. Our investigations uncovered significant complexity in the use of alternative TSSs and TESs in viral genes. In this research, we also detected the promoter elements and poly(A) signals associated with the viral genes. Additionally, we identified novel genes in both the left and right variable regions of the viral genome.IMPORTANCEGenerally, gaining insight into how the transcription of a virus is regulated offers insights into the key mechanisms that control its life cycle. The recent outbreak of the human monkeypox virus has underscored the necessity of understanding the basic biology of its causative agent. Our results are pivotal for constructing a comprehensive transcriptomic atlas of the human monkeypox virus, providing valuable resources for future studies.

Predicting active enhancers with DNA methylation and histone modification.

BACKGROUND: Enhancers play a crucial role in gene regulation, and some active enhancers produce noncoding RNAs known as enhancer RNAs (eRNAs) bi-directionally. The most commonly used method for detecting eRNAs is CAGE-seq, but the instability of eRNAs in vivo leads to data noise in sequencing results. Unfortunately, there is currently a lack of research focused on the noise inherent in CAGE-seq data, and few approaches have been developed for predicting eRNAs. Bridging this gap and developing widely applicable eRNA prediction models is of utmost importance. RESULTS: In this study, we proposed a method to reduce false positives in the identification of eRNAs by adjusting the statistical distribution of expression levels. We also developed eRNA prediction models using joint gene expressions, DNA methylation, and histone modification. These models achieved impressive performance with an AUC value of approximately 0.95 for intra-cell prediction and 0.9 for cross-cell prediction. CONCLUSIONS: Our method effectively attenuates the noise generated by stochastic RNA production, resulting in more accurate detection of eRNAs. Furthermore, our eRNA prediction model exhibited significant accuracy in both intra-cell and cross-cell validation, highlighting its robustness and potential application in various cellular contexts.

Improving the annotation of the cattle genome by annotating transcription start sites in a diverse set of tissues and populations using Cap Analysis Gene Expression sequencing.

Understanding the genomic control of tissue-specific gene expression and regulation can help to inform the application of genomic technologies in farm animal breeding programs. The fine mapping of promoters [transcription start sites (TSS)] and enhancers (divergent amplifying segments of the genome local to TSS) in different populations of cattle across a wide diversity of tissues provides information to locate and understand the genomic drivers of breed- and tissue-specific characteristics. To this aim, we used Cap Analysis Gene Expression (CAGE) sequencing, of 24 different tissues from 3 populations of cattle, to define TSS and their coexpressed short-range enhancers (<1 kb) in the ARS-UCD1.2_Btau5.0.1Y reference genome (1000bulls run9) and analyzed tissue and population specificity of expressed promoters. We identified 51,295 TSS and 2,328 TSS-Enhancer regions shared across the 3 populations (dairy, beef-dairy cross, and Canadian Kinsella composite cattle from 2 individuals, 1 of each sex, per population). Cross-species comparative analysis of CAGE data from 7 other species, including sheep, revealed a set of TSS and TSS-Enhancers that were specific to cattle. The CAGE data set will be combined with other transcriptomic information for the same tissues to create a new high-resolution map of transcript diversity across tissues and populations in cattle for the BovReg project. Here we provide the CAGE data set and annotation tracks for TSS and TSS-Enhancers in the cattle genome. This new annotation information will improve our understanding of the drivers of gene expression and regulation in cattle and help to inform the application of genomic technologies in breeding programs.

African Swine Fever Virus and Host Response: Transcriptome Profiling of the Georgia 2007/1 Strain and Porcine Macrophages.

African swine fever virus (ASFV) has a major global economic impact. With a case fatality in domestic pigs approaching 100%, it currently presents the largest threat to animal farming. Although genomic differences between attenuated and highly virulent ASFV strains have been identified, the molecular determinants for virulence at the level of gene expression have remained opaque. Here, we characterize the transcriptome of ASFV genotype II Georgia 2007/1 (GRG) during infection of the physiologically relevant host cells, porcine macrophages. In this study, we applied cap analysis gene expression sequencing (CAGE-seq) to map th0e 5' ends of viral mRNAs at 5 and 16 h postinfection. A bioinformatics analysis of the sequence context surrounding the transcription start sites (TSSs) enabled us to characterize the global early and late promoter landscape of GRG. We compared transcriptome maps of the GRG isolate and the lab-attenuated BA71V strain that highlighted GRG virulence-specific transcripts belonging to multigene families, including two predicted MGF 100 genes, I7L and I8L. In parallel, we monitored transcriptome changes in the infected host macrophage cells. Of the 9,384 macrophage genes studied, transcripts for 652 host genes were differentially regulated between 5 and 16 h postinfection compared with only 25 between uninfected cells and 5 h postinfection. NF-κB activated genes and lysosome components such as S100 were upregulated, and chemokines such as CCL24, CXCL2, CXCL5, and CXCL8 were downregulated. IMPORTANCE African swine fever virus (ASFV) causes hemorrhagic fever in domestic pigs, with case fatality rates approaching 100% and no approved vaccines or antivirals. The highly virulent ASFV Georgia 2007/1 strain (GRG) was the first isolated when ASFV spread from Africa to the Caucasus region in 2007, then spreading through Eastern Europe and, more recently, across Asia. We used an RNA-based next-generation sequencing technique called CAGE-seq to map the starts of viral genes across the GRG DNA genome. This has allowed us to investigate which viral genes are expressed during early or late stages of infection and how this is controlled, comparing their expression to the nonvirulent ASFV-BA71V strain to identify key genes that play a role in virulence. In parallel, we investigated how host cells respond to infection, which revealed how the ASFV suppresses components of the host immune response to ultimately win the arms race against its porcine host.

The choice of negative control antisense oligonucleotides dramatically impacts downstream analysis depending on the cellular background.

BACKGROUND: The lymphatic and the blood vasculature are closely related systems that collaborate to ensure the organism's physiological function. Despite their common developmental origin, they present distinct functional fates in adulthood that rely on robust lineage-specific regulatory programs. The recent technological boost in sequencing approaches unveiled long noncoding RNAs (lncRNAs) as prominent regulatory players of various gene expression levels in a cell-type-specific manner. RESULTS: To investigate the potential roles of lncRNAs in vascular biology, we performed antisense oligonucleotide (ASO) knockdowns of lncRNA candidates specifically expressed either in human lymphatic or blood vascular endothelial cells (LECs or BECs) followed by Cap Analysis of Gene Expression (CAGE-Seq). Here, we describe the quality control steps adopted in our analysis pipeline before determining the knockdown effects of three ASOs per lncRNA target on the LEC or BEC transcriptomes. In this regard, we especially observed that the choice of negative control ASOs can dramatically impact the conclusions drawn from the analysis depending on the cellular background. CONCLUSION: In conclusion, the comparison of negative control ASO effects on the targeted cell type transcriptomes highlights the essential need to select a proper control set of multiple negative control ASO based on the investigated cell types.

Distinct roles of nucleosome sliding and histone modifications in controlling the fidelity of transcription initiation.

Regulation of gene expression starts from the transcription initiation. Regulated transcription initiation is critical for generating correct transcripts with proper abundance. The impact of epigenetic control, such as histone modifications and chromatin remodelling, on gene regulation has been extensively investigated, but their specific role in regulating transcription initiation is far from well understood. Here we aimed to better understand the roles of genes involved in histone H3 methylations and chromatin remodelling on the regulation of transcription initiation at a genome-scale using the budding yeast as a study system. We obtained and compared maps of transcription start site (TSS) at single-nucleotide resolution by nAnT-iCAGE for a strain with depletion of MINC (Mot1-Ino80C-Nc2) by Mot1p and Ino80p anchor-away (Mot1&Ino80AA) and a strain with loss of histone methylation (set1Δset2Δdot1Δ) to their wild-type controls. Our study showed that the depletion of MINC stimulated transcription initiation from many new sites flanking the dominant TSS of genes, while the loss of histone methylation generates more TSSs in the coding region. Moreover, the depletion of MINC led to less confined boundaries of TSS clusters (TCs) and resulted in broader core promoters, and such patterns are not present in the ssdΔ mutant. Our data also exhibits that the MINC has distinctive impacts on TATA-containing and TATA-less promoters. In conclusion, our study shows that MINC is required for accurate identification of bona fide TSSs, particularly in TATA-containing promoters, and histone methylation contributes to the repression of transcription initiation in coding regions.

CAGE-seq analysis of osteoblast derived from cleidocranial dysplasia human induced pluripotent stem cells.

Non-coding RNAs (ncRNAs) comprise a major portion of transcripts and serve an essential role in biological processes. Although the importance of major transcriptomes in osteogenesis has been extensively studied, the function of ncRNAs in human osteogenesis remains unclear. Previously, we developed hiPSCs from patients with cleidocranial dysplasia (CCD) caused by runt-related transcription factor 2 (RUNX2) haploinsufficiency. To gain insight into ncRNAs in osteogenesis, we surveyed differential ncRNA expression profiling and promoter differences of RUNX2 using patient-specific iPSCs and cap analysis gene expression (CAGE) technology to define the promoter landscape. Revertant iPSCs (Rev1 iPSCs) edited by CRISPR/Cas9 system to harbor mutation-corrected RUNX2 exhibited increased proximal promoter expression of RUNX2, while CCD iPSCs did not. We identified 2271 ncRNA genes with altered expression levels before and after differentiation, 31 of which showed at least 20-fold higher expression in Rev1 iPSCs. Bioinformatic analysis also categorized AC007392.3, LINC00379, RP11-122D10.1, and RP11-90J7.2 as enhancer regulatory regions, and HOXA-AS2, MIR219-2, and RP11-834C11.3 as dyadic regulatory regions of these ncRNAs. In addition, two miRNAs, termed MIR199A2 and MIR152, were found to have high enrichment of osteogenic-related terms. Upon further examination of the role of MIR152 on osteoblast differentiation, we found that MIR152 knockdown induced upregulation of ALP and COL1A1 in Saos-2 cells. Thus, ncRNAs were found to regulate the osteogenic differentiation potentials of hiPSCs that are used for bone regeneration and repair owing to their differentiation potentials. These data allow understanding ncRNA profiles of hiPSCs during osteogenesis.

Analysis of the wnt1 regulatory chromosomal landscape.

One of the earliest patterning events in the vertebrate neural plate is the specification of mes/r1, the territory comprising the prospective mesencephalon and the first hindbrain rhombomere. Within mes/r1, an interface of gene expression defines the midbrain-hindbrain boundary (MHB), a lineage restriction that separates the mesencephalon and rhombencephalon. wnt1 is critical to mes/r1 development and functions within the MHB as a component of the MHB gene regulatory network (GRN). Despite its importance to these critical and early steps of vertebrate neurogenesis, little is known about the factors responsible for wnt1 transcriptional regulation. In the zebrafish, wnt1 and its neighboring paralog, wnt10b, are expressed in largely overlapping patterns, suggesting co-regulation. To understand wnt1 and wnt10b transcriptional control, we used a comparative genomics approach to identify relevant enhancers. We show that the wnt1-wnt10b locus contains multiple cis-regulatory elements that likely interact to generate the wnt1 and wnt10b expression patterns. Two of 11 conserved enhancers tested show activity restricted to the midbrain and MHB, an activity that is conserved in the distantly related spotted gar orthologous elements. Three non-conserved elements also play a likely role in wnt1 regulation. The identified enhancers display dynamic modes of chromatin accessibility, suggesting controlled deployment during embryogenesis. Our results suggest that the control of wnt1 and wnt10b expression is under complex regulation involving the interaction of multiple enhancers.