Unique Transcripts Research Articles

TGFβ and Hippo Signaling Pathways Coordinate to Promote Acinar to Ductal Metaplasia in Human Pancreas.

Acinar-to-ductal metaplasia (ADM) serves as a precursor event in the development of pancreatic ductal adenocarcinoma (PDAC) upon constitutive environmental and genetical stress. While the role of ADM in PDAC progression has been established, the molecular mechanisms underlying human ADM remain elusive. We previously demonstrated the induction of ADM in human acinar cells through the transforming growth factor beta (TGFβ) signaling pathway. We aim to investigate the interaction between TGFβ and Hippo pathways in mediating ADM. RNA-sequencing was conducted on sorted normal primary human acinar, ductal, and AD (acinar cells that have undergone ADM) cells. ATAC-seq analysis was utilized to reveal the chromatin accessibility in these three cell types. ChIP-Seq of YAP1, SMAD4, and H3K27ac was performed to identify the gene targets of YAP1 and SMAD4. The role of YAP1/TAZ in ADM-driven cell proliferation, as well as in oncogenic KRAS driven proliferation, was assessed using sphere formation assay. AD cells have a unique transcription profile, with upregulated genes in open chromatin states in acinar cells. YAP1 and SMAD4 co-occupy the loci of ADM-related genes, including PROM1, HES1, and MMP7, co-regulating biological functions such as cell adhesion, cell migration, and inflammation. Overexpression of YAP1/TAZ promoted acinar cell proliferation but still required the TGFβ pathway. YAP1/TAZ were also crucial for TGFβ-induced sphere formation and were necessary for KRAS-induced proliferation. Our study reveals the intricate transition between acinar and AD states in human pancreatic tissues. It unveils the complex interaction between the Hippo and TGF-β pathways during ADM, highlighting the pivotal role of YAP1/TAZ and SMAD4 in PDAC initiation.

Recent insights into the functions and mechanisms of antisense RNA: emerging applications in cancer therapy and precision medicine.

The antisense RNA molecule is a unique DNA transcript consisting of 19-23 nucleotides, characterized by its complementary nature to mRNA. These antisense RNAs play a crucial role in regulating gene expression at various stages, including replication, transcription, and translation. Additionally, artificial antisense RNAs have demonstrated their ability to effectively modulate gene expression in host cells. Consequently, there has been a substantial increase in research dedicated to investigating the roles of antisense RNAs. These molecules have been found to be influential in various cellular processes, such as X-chromosome inactivation and imprinted silencing in healthy cells. However, it is important to recognize that in cancer cells; aberrantly expressed antisense RNAs can trigger the epigenetic silencing of tumor suppressor genes. Moreover, the presence of deletion-induced aberrant antisense RNAs can lead to the development of diseases through epigenetic silencing. One area of drug development worth mentioning is antisense oligonucleotides (ASOs), and a prime example of an oncogenic trans-acting long noncoding RNA (lncRNA) is HOTAIR (HOX transcript antisense RNA). NATs (noncoding antisense transcripts) are dysregulated in many cancers, and researchers are just beginning to unravel their roles as crucial regulators of cancer's hallmarks, as well as their potential for cancer therapy. In this review, we summarize the emerging roles and mechanisms of antisense RNA and explore their application in cancer therapy.

Generation of DNAzyme in Bacterial Cells by a Bacterial Retron System.

DNAzymes are catalytically active single-stranded DNAs in which DNAzyme 10-23 (Dz 10-23) consists of a catalytic core and a substrate-binding arm that reduces gene expression through sequence-specific mRNA cleavage. However, the in vivo application of Dz 10-23 depends on exogenous delivery, which leads to its inability to be synthesized and stabilized in vivo, thus limiting its application. As a unique reverse transcription system, the bacterial retron system can synthesize single-stranded DNA in vivo using ncRNA msr/msd as a template. The objective of this work is to reduce target gene expression using Dz 10-23 generated in vivo by the retron system. In this regard, we successfully generated Dz 10-23 by cloning the Dz 10-23 coding sequence into the retron msd gene and tested its ability to reduce specific gene expression by examining the mRNA levels of cfp encoding cyan fluorescence protein and other functional genes such as mreB and ftsZ. We found that Dz had different repressive effects when targeting different mRNA regions, and in general, the repressive effect was stronger when targeting downstream of mRNAs. Our results also suggested that the reduction effect was due to cleavage of the substrate mRNA by Dz 10-23 rather than the antisense effect of the substrate-binding arm. Therefore, this study not only provided a retron-based method for the intracellular generation of Dz 10-23 but also demonstrated that Dz 10-23 could reduce gene expression by cleaving target mRNAs in cells. We believe that this new strategy would have great potential in the regulation of gene expression.

Enhanced bovine genome annotation through integration of transcriptomics and epi-transcriptomics datasets facilitates genomic biology.

The accurate identification of the functional elements in the bovine genome is a fundamental requirement for high-quality analysis of data informing both genome biology and genomic selection. Functional annotation of the bovine genome was performed to identify a more complete catalog of transcript isoforms across bovine tissues. A total of 160,820 unique transcripts (50% protein coding) representing 34,882 unique genes (60% protein coding) were identified across tissues. Among them, 118,563 transcripts (73% of the total) were structurally validated by independent datasets (PacBio isoform sequencing data, Oxford Nanopore Technologies sequencing data, de novo assembled transcripts from RNA sequencing data) and comparison with Ensembl and NCBI gene sets. In addition, all transcripts were supported by extensive data from different technologies such as whole transcriptome termini site sequencing, RNA Annotation and Mapping of Promoters for the Analysis of Gene Expression, chromatin immunoprecipitation sequencing, and assay for transposase-accessible chromatin using sequencing. A large proportion of identified transcripts (69%) were unannotated, of which 86% were produced by annotated genes and 14% by unannotated genes. A median of two 5' untranslated regions were expressed per gene. Around 50% of protein-coding genes in each tissue were bifunctional and transcribed both coding and noncoding isoforms. Furthermore, we identified 3,744 genes that functioned as noncoding genes in fetal tissues but as protein-coding genes in adult tissues. Our new bovine genome annotation extended more than 11,000 annotated gene borders compared to Ensembl or NCBI annotations. The resulting bovine transcriptome was integrated with publicly available quantitative trait loci data to study tissue-tissue interconnection involved in different traits and construct the first bovine trait similarity network. These validated results show significant improvement over current bovine genome annotations.

Microfluidic-assisted single-cell RNA sequencing facilitates the development of neutralizing monoclonal antibodies against SARS-CoV-2.

As a class of antibodies that specifically bind to a virus and block its entry, neutralizing monoclonal antibodies (neutralizing mAbs) have been recognized as a top choice for combating COVID-19 due to their high specificity and efficacy in treating serious infections. Although conventional approaches for neutralizing mAb development have been optimized for decades, there is an urgent need for workflows with higher efficiency due to time-sensitive concerns, including the high mutation rate of SARS-CoV-2. One promising approach is the identification of neutralizing mAb candidates via single-cell RNA sequencing (RNA-seq), as each B cell has a unique transcript sequence corresponding to its secreted antibody. The state-of-the-art high-throughput single-cell sequencing technologies, which have been greatly facilitated by advances in microfluidics, have greatly accelerated the process of neutralizing mAb development. Here, we provide an overview of the general procedures for high-throughput single-cell RNA-seq enabled by breakthroughs in droplet microfluidics, introduce revolutionary approaches that combine single-cell RNA-seq to facilitate the development of neutralizing mAbs against SARS-CoV-2, and outline future steps that need to be taken to further improve development strategies for effective treatments against infectious diseases.

The RNA-binding KH-domain in the unique transcription factor of the malaria parasite is responsible for its transcriptional regulatory activity.

To date, only a small number of regulatory transcription factors have been predicted from the genome of Plasmodium and Apicomplexan parasites. We previously identified a nuclear factor named Prx regulatory element-binding protein (PREBP) from Plasmodium falciparum. PREBP had been suggested to bind to the cis-element in the promoter of an antioxidant pf1-cys-prx gene, thereby promoting the expression of downstream genes. PREBP has 4 putative K homology (KH) domains, which are known to bind RNA and single-stranded DNA. In this study, to understand the detailed action of PREBP in parasite cells, we first observed that in living parasite cells, PREBP was localized in the nucleus in the trophozoite and schizont stages, in which the expression of the target pf1-cys-prx was enhanced. The interaction of PREBP and the cis-element of pf1-cys-prx in the parasite cells was also confirmed. Further, the activities of PREBP deletion mutants were analyzed, and regions with repeated KH domains in PREBP seemed to be responsible for the recognition of the cis-element. These results led us to hypothesize that Plasmodium and other Apicomplexan parasites might have a transcription factor family with KH domains. Bioinformatic analysis revealed a putative ortholog group including PREBP and several Plasmodium and Apicomplexan factors with KH domains. One of the P. falciparum-derived factors, which were included in the putative ortholog group, was found to be localized at the nucleus in the trophozoite stage, indicating that it might be a novel transcription factor. The discovery of PREBP and putative transcription factors with KH domains suggested that multi-functional proteins with KH domains possibly evolved in the Apicomplexan organisms. They might play key roles in transcriptional regulatory processes that are essential for living organisms and may even represent unique drug targets for malaria therapy.

Comparison of Single Cell Transcriptome Sequencing Methods: Of Mice and Men.

Single cell RNAseq has been a big leap in many areas of biology. Rather than investigating gene expression on a whole organism level, this technology enables scientists to get a detailed look at rare single cells or within their cell population of interest. The field is growing, and many new methods appear each year. We compared methods utilized in our core facility: Smart-seq3, PlexWell, FLASH-seq, VASA-seq, SORT-seq, 10X, Evercode, and HIVE. We characterized the equipment requirements for each method. We evaluated the performances of these methods based on detected features, transcriptome diversity, mitochondrial RNA abundance and multiplets, among others and benchmarked them against bulk RNA sequencing. Here, we show that bulk transcriptome detects more unique transcripts than any single cell method. While most methods are comparable in many regards, FLASH-seq and VASA-seq yielded the best metrics, e.g., in number of features. If no equipment for automation is available or many cells are desired, then HIVE or 10X yield good results. In general, more recently developed methods perform better. This also leads to the conclusion that older methods should be phased out, and that the development of single cell RNAseq methods is still progressing considerably.

MicroRNA-mRNA Analysis Reveals Tissue-Specific Regulation of microRNA in Mangrove Clam (Geloina erosa).

Geloina erosa is an important benthic animal in the mangrove, serving as an indicator organism for coastal environmental pollution. This study aimed to investigate the tissue-specific expression of miRNAs and their regulatory roles in predicted targets in G. erosa. Through miRNA sequencing and co-expression network analysis, we extensively studied the miRNA expression in three tissues: gills, hepatopancreas, and muscle. The results revealed a total of 1412 miRNAs, comprising 1047 known miRNAs, and 365 newly predicted miRNAs. These miRNAs exhibited distinct tissue-specific expression patterns. In the miRNA target gene prediction, a total of 7404 potential predicted targets were identified, representing approximately 33% of all unique transcripts associated with miRNAs. Further co-expression network analysis revealed nine modules, each showing a positive correlation with specific tissues (gills, hepatopancreas, or muscle). The blue module showed a significant correlation with gills (r = 0.83, p-value = 0.006), the black module was significantly related to the hepatopancreas (r = 0.78, p-value = 0.01), and the purple module was significantly correlated with muscle (r = 0.83, p-value = 0.006). Within these modules, related miRNAs tended to cluster together, while their correlations with other modules were relatively weak. Functional enrichment analysis was performed on miRNAs and their predicted targets in each tissue. In the gills, miRNAs primarily regulate immune-related genes, substance transport, and cytoskeletal organization. In the hepatopancreas, miRNAs suppressed genes involved in shell formation and played a role in cellular motor activity and metabolism. In muscle, miRNAs participate in metabolism and photoreceptive processes, as well as immune regulation. In summary, this study provides valuable insights into the tissue-specific regulation of miRNAs in G. erosa, highlighting their potential roles in immune response, metabolism, and environmental adaptation. These findings offer important clues for understanding the molecular mechanisms and biological processes in G. erosa, laying the foundation for further validation and elucidation of these regulatory relationships.

Identification of promoter targets by Aureochrome 1a in the diatom Phaeodactylum tricornutum.

Aureochromes (AUREOs) are unique blue light receptors and transcription factors found only in stramenopile algae. While each of the four AUREOs identified in the diatom Phaeodactylum tricornutum may have a specific function, PtAUREO1a has been shown to have a strong impact on overall gene regulation, when light changes from red to blue light conditions. Despite its significance, the molecular mechanism of PtAUREO1a is largely unexplored. To comprehend the overall process of gene regulation by PtAUREO1a, we conducted a series of in vitro and in vivo experiments, including pull-down assays, yeast one-hybrid experiments, and phenotypical characterization using recombinant PtAUREOs and diatom mutant lines expressing a modified PtAureo1a gene. We describe the distinct light absorption properties of four PtAUREOs and the formation of all combinations of their potential dimers. We demonstrate the capability of PtAUREO1a and 1b to activate the genes, diatom-specific cyclin 2, PtAureo1a, and PtAureo1c under both light and dark conditions. Using mutant lines expressing a modified PtAUREO1a protein with a considerably reduced light absorption, we found novel evidence that PtAUREO1a regulates the expression of PtLHCF15, which is essential for red light acclimation. Based on current knowledge, we present a working model of PtAUREO1a gene regulation properties.

TRNA-Cys gene clusters exhibit high variability in Arabidopsis thaliana

Although most of the genes encoding tRNAs in plants are dispersed throughout the genome, a fraction of them form tRNA gene clusters. In Arabidopsis thaliana, the smallest of tRNA clusters on chromosome 5 consists of four tRNA-Cys-GCA genes placed within repeating units of 0.4 kbp. A systematic analysis of the genomic sequences of syntenic regions from various ecotypes of A. thaliana showed that the general structure of the cluster, consisting of a tRNA-Cys pseudogene followed by repeating units containing tRNA-Cys genes, is well conserved. However, there is significant heterogeneity in the number of repeating units between different ecotypes. A unique feature of this cluster is the presence of putative transposable elements (Helitron). In addition, two further tRNA-Cys gene mini-clusters (gene pairs) in A. thaliana were identified. RNA-seq-based evaluation of expression of tRNA-Cys-GCA genes showed a positive signal for 11 out of 13 unique transcripts. An analysis of the conservation of the tRNA-Cys clusters from A. thaliana with the corresponding regions from four other Arabidopsis species suggests a sequence of events that led to the divergence of these regions.

Carbohydrates and carbohydrate degradation gene abundance and transcription in Atlantic waters of the Arctic

Carbohydrates are chemically and structurally diverse, represent a substantial fraction of marine organic matter and are key substrates for heterotrophic microbes. Studies on carbohydrate utilisation by marine microbes have been centred on phytoplankton blooms in temperate regions, while far less is known from high-latitude waters and during later seasonal stages. Here, we combine glycan microarrays and analytical chromatography with metagenomics and metatranscriptomics to show the spatial heterogeneity in glycan distribution and potential carbohydrate utilisation by microbes in Atlantic waters of the Arctic. The composition and abundance of monomers and glycan structures in POM varied with location and depth. Complex fucose-containing sulfated polysaccharides, known to accumulate in the ocean, were consistently detected, while the more labile β-1,3-glucan exhibited a patchy distribution. Through ‘omics analysis, we identify variations in the abundance and transcription of carbohydrate degradation-related genes across samples at the community and population level. The populations contributing the most to transcription were taxonomically related to those known as primary responders and key carbohydrate degraders in temperate ecosystems, such as NS4 Marine Group and Formosa. The unique transcription profiles for these populations suggest distinct substrate utilisation potentials, with predicted glycan targets corresponding to those structurally identified in POM from the same sampling sites. By combining cutting-edge technologies and protocols, we provide insights into the carbohydrate component of the carbon cycle in the Arctic during late summer and present a high-quality dataset that will be of great value for future comparative analyses.

Biological, prognostic, and therapeutic impact of the epigenome in CLL

Chronic lymphocytic leukemia (CLL) is characterized by widespread alterations in the genetic and epigenetic landscapes which seem to underlie the variable clinical manifestations observed in patients. Over the last decade, epigenomic studies have described the whole-genome maps of DNA methylation and chromatin features of CLL and normal B cells, identifying distinct epigenetic mechanisms operating in tumoral cells. DNA methylation analyses have identified that the CLL methylome contains imprints of the cell of origin, as well as of the proliferative history of the tumor cells, with both being strong independent prognostic predictors. Moreover, single-cell analysis revealed a higher degree of DNA methylation noise in CLL cells, which associates with transcriptional plasticity and disease aggressiveness. Integrative analysis of chromatin has uncovered chromatin signatures, as well as regulatory regions specifically active in each CLL subtype or in Richter transformed samples. Unique transcription factor (TF) binding motifs are overrepresented on those regions, suggesting that altered TF networks operate from disease initiation to progression as nongenetic factors mediating the oncogenic transcriptional profiles. Multiomics analysis has identified that response to treatment is modulated by an epigenetic imprint, and that treatments affect chromatin through the activity of particular set of TFs. Additionally, the epigenome is an axis of therapeutic vulnerability in CLL, as it can be targeted by inhibitors of histone modifying enzymes, that have shown promising preclinical results. Altogether, this review aims at summarizing the major findings derived from published literature to distill how altered epigenomic mechanisms contribute to CLL origin, evolution, clinical behavior, and response to treatment.

Investigating the Role of APOE Genotype in Human Microglia

AbstractBackgroundAlthough the pivotal role of apolipoprotein E () in AD has been recognized for three decades, its function in different brain cells is far from understood. Knockout studies of the APOE gene may shed light of the function of ApoE. The aim of the present study was to unravel its function in induced pluripotent stem cell (iPSC) derived microglia.MethodWe studied two human iPSC‐derived microglia cell cultures, one derived from a healthy control (Bioni010‐C – endogenous genotype apolipoprotein E34 (APOE34)) and one from an AD patient (UKBi011‐A – endogenous genotype APOE44) and their edited isogenic cell‐lines (for UKBi011‐A, APOE22 and APOE33 and for Bioni010‐C, hemizygous due to gene editing, APOE2‐ and APOE3‐ (32971461, 29753274)). Additionally, APOE KO lines were generated from both backgrounds. We performed whole genome transcriptomic (IlluminaTruSequencing) and proteomic (TMT‐labelled Nano‐LC peptidomic MS) analysis (n = six per genotype). For transcriptomic data, log2‐normalized intensities were analysed with (in R); there were 13115 unique transcripts. For proteomics analysis, after global normalization, the data were locally normalized (by protein), the limma package was used to calculate FDR‐corrected p‐values (1% cut off); there were 3975 (Bioni010 samples) and 4349 (UKBi011 samples) proteins. In the quality control analysis for the proteomics, we excluded one outlier of the six per line based on principle component/Euclidean distance analysis. We extracted pairwise comparisons to APOE KO in transcriptomic (|logFC|>0.5, FDR <0.05) and proteomic (|logFC|>0 and FDR <0.05) data.ResultTwenty transcripts (including APOE) were concordantly differentially expressed across all comparisons between APOE‐expressing and KO. These are broadly involved in processes related to mitochondrial function/organization, apoptosis, transcriptional regulation, immune functions, extracellular matrix, and energy metabolism among others. Seven proteins were concordantly differentially expressed across all pairwise comparisons with APOE KO, involved in extracellular matrix, immune signalling, energy metabolism, and cytoskeletal structure.ConclusionOur study finds APOE knockout leads to a number of changes related to immune, mitochondrial, and extracellular matrix genes, suggesting that APOE in human iPSC‐derived microglia cells influences immune and mitochondrial functions.

Dissecting embryonic and extraembryonic lineage crosstalk with stem cell co-culture

Embryogenesis necessitates harmonious coordination between embryonic and extraembryonic tissues. Although stem cells of both embryonic and extraembryonic origins have been generated, they are grown in different culture conditions. In this study, utilizing a unified culture condition that activates the FGF, TGF-β, and WNT pathways, we have successfully derived embryonic stem cells (FTW-ESCs), extraembryonic endoderm stem cells (FTW-XENs), and trophoblast stem cells (FTW-TSCs) from the three foundational tissues of mouse and cynomolgus monkey (Macaca fascicularis) blastocysts. This approach facilitates the co-culture of embryonic and extraembryonic stem cells, revealing a growth inhibition effect exerted by extraembryonic endoderm cells on pluripotent cells, partially through extracellular matrix signaling. Additionally, our cross-species analysis identified both shared and unique transcription factors and pathways regulating FTW-XENs. The embryonic and extraembryonic stem cell co-culture strategy offers promising avenues for developing more faithful embryo models and devising more developmentally pertinent differentiation protocols.

Differential Isoforms Signature of Splicing Factor Mutant Myeloid Neoplasia

Exclusivity of splicing factor (SF) mutations is a near-dogma in myeloid neoplasia (MN). Such exclusivity is often reflected in morphopathologic features as in the case of SF3B1 mutations associated with ringed sideroblasts. Consequences of SF mutations are hard to reconcile given the central role of RNA splicing in normal physiology and the multitude of alternative splicing changes occurring upon mutations. Historically, distinct gene isoforms play key roles in hematopoiesis and might be tumor associated. We performed global transcript level expression analysis to identify SF specific differentially expressed isoforms associated with isoform disequilibrium. Such analysis prompted to the distinction between common and exclusive isoforms possibly discerning novel mechanistic insights in the pathogenesis of SF mutant MN. Of particular importance is the discovery of novel isoforms not expressed in normal condition. Total RNA isolated from 100 whole bone marrows of MN patients with six SF mutations ( DDX41, PRPF8, SF3B1, SRSF2, U2AF1, ZRSR2) and healthy subjects was subjected to RNASeq library prep (Kapa RNAHyperPrep Kit with rRNA depletion). Transcript abundance was generated using spliced alignment to the genome by STAR followed by Salmon-basedquantification. We then applied bootstrapping to capture variation in abundance estimates since transcript-level quantification is amenable to variation, in contrast with gene level, due to the difficulty of unique mapping of short read sequences. Differential isoform testing was done using edgeR accounting for overdispersion due to isoform uncertainty. Mutant samples were selected with VAF >15% for better estimation of isoforms vs clonality. Differential SF isoform usage was defined in comparison to healthy controls. In total, 1681 unique transcripts in 1418 unique genes (positively/negatively dysregulated) at a threshold of .05 P-value and absolute logfold change of 2.0 were selected. Among patients with SF mutations, patients carrying sole U2AF1 had the highest number of distinct isoforms (n=189), which accounted for about 20% of isoforms in the entire cohort of U2AF1 mutant patients. DDX41 mutations had the second highest (n=161), while ZRSR2 had the lowest number of distinct isoforms(n=48). We categorized the features in groups I1-I6 according to the number of SF sharing isoforms. I1 indicates the group of patients carrying isoforms which where distinct per each SF ( Fig1). U2AF1I1 included genes previously reported to be associated with U2AF1 ( e.g., GNAS) and genes restricted to neutrophil degranulation. DDX41I1 was enriched in innate immune response gene isoforms including HLA-DQA1 and SAMHD1 and known genes predisposing to myeloid and blood disorders ( e.g. CSF1R, RPL15). SRSF2 isoform signature was mainly characterized by upregulation of INF-gamma and beta, IFN-induced OAS1 and TLR response pathways ( e.g., IRAK2). Specific isoforms were shared between SFs. SF3B1 shared 35 isoforms with PRPF8 and 8 with SRSF2, including Stathmin1, a gene expressed in high proliferative MN phenotypes. Different isoforms in the same gene were also associated with different groups. For instance, DDX5 isoform-219 producing a 65-residues transcript was shared between PRPF8 and SF3B1 (I2) while isoform-234 producing 614-residues transcript was shared between PRPF8, DDX41, and ZRSR2 mutants (I3). Beside the unique isoform signature per each SF, we imputed any differential isoform changes in myeloid genes often associated with SF mutations. Isoforms for the TET triad were analyzed and resulted in a 110 residues transcript (TET2-208) significantly downregulated (FDR < .05) in all SF, except SRSF2. We then focused on the discovery of exclusive isoforms. In total, 115 unique transcripts (lncRNAs, 20; protein coding, 95) were expressed in all SF mutant patients but not expressed in healthy controls. GDF15 isoform-201 encoding a 308-residues transcript was detected in SF3B1, DDX41, and ZRSR2 mutants. LncRNAs have gained attention in regulating gene expression in MN. We found 9 novel lncRNA transcripts in DDX41, SF3B1, PRPF8, U2AF1 and ZRSR2 mutants, with one being common in all of them (ENSG00000273445 chromosome 2p11.2). In sum, our study focusing solely on transcript level analysis describes the first isoform landscape of SF mutant MN and launches the idea that isoform analysis might shed light on the diversity and uniqueness of SF mutations.

Single Nuclei Multiomics Analysis of Transcriptional and Chromatin Accessibility of Tumor Cells Uncovers Molecular Signatures and Regulatory Elements of Malignant Clonal Evolution in Multiple Myeloma

Category Multiple Myeloma and Plasma Cell Dyscrasias: Basic and Translational Introduction Multiple myeloma (MM) is a hematological cancer resulting from abnormal proliferation of plasma cells (PC) in bone marrow. The progression of the disease from Monoclonal Gammopathy of Undetermined Significance (MGUS), Smoldering Multiple Myeloma (SMM), to MM involves multiple drivers and dynamic interactions of premalignant/malignant cells. In the advancement of single-cell technologies, the same-cell readouts of nucleus RNA sequencing (snRNA-seq) and Assay for Transposase-Accessible Chromatin sequencing (snATAC-seq) provide a less biased measurement of gene expressions and chromatin accessible regions. Profiling and analyzing both omics data at different stages of MM may reveal essential molecular signatures and regulatory elements for microenvironmental modification and evolution. These insights could benefit MM patients as novel targets for early therapeutic intervention at different stages of progression. Methods Bone marrow aspirates were collected from 33 individuals: Healthy donors (n=3), patients with MGUS (n=2), SMM (n=5), MM (n=21), and Plasma Cell Leukemia (PCL; n=2). Viable mononuclear cells underwent CD138+ sorting and nuclei isolation prior to library preparation. Libraries were prepared with the Chromium Multiome kit (10X Genomics) and sequenced on NovaSeq S4 flow cells at UAMS. The clonal V(D)J arrangements of each sample and expression level from HD samples were used to distinguish malignant (MM) from normal plasma cells (PC). The MM data from snATAC-seq and snRNA-seq were integrated using Signac with the weighted nearest neighbor methods from Seurat. All inter- and intra-tumor clusters were evaluated for markers of high-risk disease, differential gene expression, differentially accessible regions, and transcription factor activities. Results We performed snRNA-seq and snATAC-seq sequencing to generate multi-stage, molecular classified-specific chromatin accessibility and transcriptional profiles of plasma cell neoplasms comprising 439,134 cells across 33 samples. The malignant plasma cells for each sample were extracted by the unique clonotypic signature of the immunoglobulin gene rearrangement. Of these, we detected the highest clonality in MGUS samples, suggesting clonal competition at the premalignant stage. Based on our primary microarray data, we detected the overexpression of molecular signatures for most cells in each sample, confirming the intratumor cell heterogeneity. Furthermore, we used the ratio of 70-gene signature of aggressive disease and proliferation signatures to investigate the clonal composition of MM cells corresponding to the risk of relapse. Surprisingly, the proportion of high-risk cells increased respectively from low to high-risk samples. The expansion of high-risk clonality might exhibit competition among malignant cell subpopulations regarding microenvironmental adaptation. The multiomics integration generated less distinct clusters between MGUS and SMM, indicating the gradual complex alterations of subclonal gene expression and chromatin accessibility in malignant transformation. Interestingly, most accessible regions were found in intronic/distal intergenic regions implying the long-range gene regulation from cis-regulatory elements. Based on the positive/negative correlation of binding motifs within differentially accessible regions, we identified unique enriched transcription factors for each disease stage, suggesting the mechanism patterns in the evolutionary process of malignant cells. Conclusion In the largest multiomics study to date of CD138+ cells, we demonstrate the malignant clonal evolution using gene expression and chromatin accessibility profiling from patients with early-stage disease to highly aggressive form. Throughout the time of disease progression, we found an increase in the proportion of malignant cells with high-risk signatures. Also, we uncover unique signatures of chromatin accessibility for each molecular classification that can further refine our understanding of functional heterogeneity in multiple myeloma. Collectively, our joint single nuclei profiling reveals additional insights into the malignant clonal evolution and investigates novel regulatory elements involved in disease progression.

Genome-Wide Identification and Expression Analysis of the Walnut C-Repeat Binding Factor Gene Family under Low-Temperature Stress

The walnut (Juglans regia) is a nut with a high nutritional value and has been recognized throughout the world as an economically important woody plant. However, the walnut is vulnerable to abiotic stresses, especially low-temperature freezes, which can severely impede their growth and development, resulting in substantial financial losses. The CBF (C-repeat binding factor) gene, a unique plant transcription factor classified within the AP2/ERF (ethylene response factor) family, plays a vital role in the process of plants coping with abiotic stress, particularly low temperatures. This study utilized bioinformatics techniques to identify eight JrCBF genes within the walnut genome, distributed across six chromosomes. The upstream promoter sequences of these genes are rich in cis-regulatory elements related to hormonal responses and non-biotic stresses. Transcriptome data and qRT-PCR analysis revealed that JrCBF1 and JrCBF2 were significantly upregulated under low temperatures. JrCBF1 and JrCBF2 also responded positively to high-temperature stress. Under drought stress, JrCBF5 and JrCBF8 had a significant difference in their expression relative to other genes. To gain further insights into their functionality, subcellular localization experiments were conducted on JrCBF1 and JrCBF2, confirming their nuclear localization. These results provide valuable insights into the specific functions of CBF gene in enhancing the resistance of walnut to abiotic stress.

Prevalently carried HLA allele associates with inflammation and poor prognosis in HF patients via co-stimulatory, adhesion and MHC-II enhanceosome regulation

Abstract Background Heart failure with reduced ejection fraction (HFrEF) is associated with impaired inflammation resolution, adverse remodeling, restricted function, and poor prognosis. Furthermore, immune cells play crucial roles in pathological remodeling in cardiovascular diseases. Thus identifying subsets of patients with unique inflammatory profiles may aid in better risk stratification and precision therapies for chronic HFrEF patients with altered immune profiles. Methods and Results A pilot study using scRNA-seq of circulating immune cells (n=16) showed strong T cell activation in HFrEF patients, which was validated via flow cytometry in HFrEF patients and healthy controls (n=180 patients) with a shift into the memory/effector phenotype (p<0.0001). Thus, HFrEF patients may have unique MHC signatures promoting aberrant T cell activation, leading to chronic inflammation and pathology. We investigated antigen-presenting cells (APCs) that activate CD4+ T cells via MHC-II and costimulatory molecules. Indeed, we found MHC-II and costimulatory molecule signatures associated with HFrEF and T cell activation, such as HLA-DRB5 (DRB5) and ICAM-1, indicating both T cell activation potential and a pro-inflammatory status. To study whether HLA-DRB5 selectively promotes immune dysregulation, circulating monocytes from healthy DRB5 carriers and non-carriers were investigated using scRNA-seq showing enhanced antigen presentation, co-stimulatory and inflammatory molecule expression (e.g. HLA-DRA, ICAM-2, JAK2). ScRNA-seq confirmed HLA-DRB5 regulation of MHC-II, inflammatory and adhesion (e.g. ITGAM and ITGB1) properties by silencing HLA-DRB5 in THP1 cells. In silico analysis of monocyte epigenome data predicted unique transcription factors (e.g. ERG) controlling of HLA-DRB5 via regulatory elements suggesting a role in selective MHC-II molecule activation. To gain insights as to whether HLA carrier status might associate with prognosis, we performed HLA sequencing on age and gender matched HFrEF patients (n=93). During follow up of this cohort we identified HLA-DRB5*0101, which belongs to the DR2-supertype, as a predictor for death (p<0.05). We hypothesized whether HLA-DRB5 expression could impact T cell infiltration into the heart. Thus, we stimulated naïve T cells with pre-treated THP1-derived DCs (stress v. no stress cardiac organoid medium) and found HLA-DRB5 silencing reduced the infiltration of T cells into unstressed cardiac organoids compared to control group. Conclusions This is the first study to provide mechanistic insights for how HLA-DRB5 regulates innate and adaptive immunity in the context of heart failure, while also showing corresponding changes in T cell activation, infiltration capacity and prognosis in HFrEF patients. These data provide novel insights for risk stratification of heart failure patients and novel immunological interventions which may improve prognosis in HFrEF patients.

Plasmodial Transcription Factors and Chromatin Modifiers as Drug Targets

The complex life cycle of the malaria parasite Plasmodium requires the parasite to adequately adapt to different conditions. For this reason, Plasmodium strictly controls its gene expression, and given its evolutionary distance from the human host, the involved factors may figure as attractive potential drug targets. In recent years, several unique transcription factors and chromatin modifiers have been identified and partially characterized in Plasmodium falciparum and in the murine species P. yoelii and P. berghei. This review unites data from studies focusing on drug development against enigmatic plant-like AP2-transcription factors and chromatin modifiers, such as histone acetyl transferases and deacetylases and histone methyltransferases and demethylases. Considering the reported success of inhibition of both factors, these may be included as targets to effectively combat the parasite by perturbing its control of gene expression.

Proinflammatory islet antigen reactive CD4 T cells are linked with response to alefacept in type 1 diabetes.

Variation in the preservation of β cell function in clinical trials in type 1 diabetes (T1D) has emphasized the need to define biomarkers to predict treatment response. The T1DAL trial targeted T cells with alefacept (LFA-3-Ig) and demonstrated C-peptide preservation in ~30% of new onset T1D subjects. We analyzed islet antigen reactive (IAR) CD4 T cells in PBMC samples collected prior to treatment from alefacept- and placebo-treated subjects using flow cytometry and single cell RNA-sequencing. IAR CD4 T cells at baseline had heterogenous phenotypes. Transcript profiles formed phenotypic clusters of cells along a trajectory based on increasing maturation and activation, and T cell receptor (TCR) chains showed clonal expansion. Notably, the frequency of IAR CD4 T cells with a memory phenotype and a unique transcript profile (Cluster 3) were inversely correlated with C-peptide preservation in alefacept-, but not placebo-treated subjects. Cluster 3 cells had a proinflammatory phenotype characterized by expression of the transcription factor BHLHE40 and the cytokines GM-CSF and TNF-α, and shared TCR chains with effector memory-like clusters. Our results suggest IAR CD4 T cells as a potential baseline biomarker of response to therapies targeting the CD2 pathway and warrant investigation for other T cell-related therapies.