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Silencing in yeast: identification of clr4 targets.

Efficient handling of multiple reactions is a crucial prerequisite for productive RNA differential display (DD) analysis. To identify transcriptional targets of the histone H3 Lys9-specific methyltransferase Clr4, we applied a multiformat modification of DD to compare between clr4+ and clr4- transcriptomes of Schizosaccaromyces pombe. As a result, 14 differentially expressed bands were identified among 720 polymerase chain reaction (PCR) studied. The content of these bands was then analyzed by cloning, sequencing, and Northern analysis. In the final stage of verification, four Clr4 targets were isolated based on their expression in six Clr4 chromo and SET domain mutant strains. The step-by-step description of the multiformat DD provided below includes RNA purification, cDNA synthesis, 96-well PCR, electrophoretic separation of PCR products, isolation of DNA fragments from differentially expressed bands, and verification of candidate genes by Northern analysis.

Blotting, Northern↗

Transcriptional and histopathological profiling of skeletal muscle in Bla/J mice at the stage of dysferlinopathy manifestation.

Dysferlinopathy is a rare muscular dystrophy characterized by chronic muscle damage and ineffective regeneration. While late-stage morphological changes, such as fibroadipose replacement, are well described, the early molecular mechanisms driving muscle fiber loss and regenerative failure at the onset of the disease remain largely uncharacterized. To address this gap, we investigated the skeletal muscles of dysferlin-deficient Bla/J mice during the early manifestation stage (3 months of age). This exploratory study aimed to identify primary pathomorphogenetic events by correlating the transcriptomic profile of the tissue with its specific histopathological and ultrastructural alterations. We performed a comparative analysis of the m. gastrocnemius in 3-month-old Bla/J mice versus wild-type controls using RNA sequencing, RT-qPCR, histomorphometry and transmission electron microscopy. The results revealed atrophy and muscle fiber necrosis without the expected induction of Fbxo32 and Trim63 ubiquitin ligases, suggesting ubiquitin-proteasome system-independent muscle mass loss. Furthermore, the absence of Casp3, Bak1, and Bad induction, confirmed by the lack of active caspase-3, excluded apoptosis as the primary death mechanism. A differentiation block in satellite cells was confirmed by the lack of Myf5, Myod1, and Myog induction and a trend toward Tead4 suppression, pointing to an early failure of the reparative program. Exploratory RNA sequencing also identified a suppression of Prkn expression accompanied by LC3B-II-positive autophagosome accumulation. Immunohistochemical and immunofluorescent evaluation of the mitochondrial network (TOMM20) revealed abnormal accumulations and dense clumping, indicating impaired organelle clearance. Furthermore, ultrastructural analysis demonstrated internal organelle damage and the presence of myelin-like structures, consistent with a state of stalled mitophagy. Collectively, this exploratory study demonstrates that early muscle atrophy and myofiber necrosis in dysferlinopathy occur independently of canonical ubiquitin-proteasome and apoptotic pathways. Instead, the disease manifestation stage is structurally characterized by stalled mitochondrial clearance and a delayed regenerative response.

Animals↗

Non-EST based prediction of exon skipping and intron retention events using Pfam information.

Most of the known alternative splice events have been detected by the comparison of expressed sequence tags (ESTs) and cDNAs. However, not all splice events are represented in EST databases since ESTs have several biases. Therefore, non-EST based approaches are needed to extend our view of a transcriptome. Here, we describe a novel method for the ab initio prediction of alternative splice events that is solely based on the annotation of Pfam domains. Furthermore, we applied this approach in a genome-wide manner to all human RefSeq transcripts and predicted a total of 321 exon skipping and intron retention events. We show that this method is very reliable as 78% (250 of 321) of our predictions are confirmed by ESTs or cDNAs. Subsequent analyses of splice events within Pfam domains revealed a significant preference of alternative exon junctions to be located at the protein surface and to avoid secondary structure elements. Thus, splice events within Pfams are probable to alter the structure and function of a domain which makes them highly interesting for detailed biological investigation. As Pfam domains are annotated in many other species, our strategy to predict exon skipping and intron retention events might be important for species with a lower number of ESTs.

Algorithms↗

Transcriptome characterization elucidates signaling networks that control human ES cell growth and differentiation.

Human embryonic stem (hES) cells hold promise for generating an unlimited supply of cells for replacement therapies. To characterize hES cells at the molecular level, we obtained 148,453 expressed sequence tags (ESTs) from undifferentiated hES cells and three differentiated derivative subpopulations. Over 32,000 different transcripts expressed in hES cells were identified, of which more than 16,000 do not match closely any gene in the UniGene public database. Queries to this EST database revealed 532 significantly upregulated and 140 significantly downregulated genes in undifferentiated hES cells. These data highlight changes in the transcriptional network that occur when hES cells differentiate. Among the differentially regulated genes are several components of signaling pathways and transcriptional regulators that likely play key roles in hES cell growth and differentiation. The genomic data presented here may facilitate the derivation of clinically useful cell types from hES cells.

Antigens, CD↗

Breakpoint analysis of the pericentric inversion between chimpanzee chromosome 10 and the homologous chromosome 12 in humans.

During this study, we analysed the pericentric inversion that distinguishes human chromosome 12 (HSA12) from the homologous chimpanzee chromosome (PTR10). Two large chimpanzee-specific duplications of 86 and 23 kb were observed in the breakpoint regions, which most probably occurred associated with the inversion. The inversion break in PTR10p caused the disruption of the SLCO1B3 gene in exon 11. However, the 86-kb duplication includes the functional SLCO1B3 locus, which is thus retained in the chimpanzee, although inverted to PTR10q. The second duplication spans 23 kb and does not contain expressed sequences. Eleven genes map to a region of about 1 Mb around the breakpoints. Six of these eleven genes are not among the differentially expressed genes as determined previously by comparing the human and chimpanzee transcriptome of fibroblast cell lines, blood leukocytes, liver and brain samples. These findings imply that the inversion did not cause major expression differences of these genes. Comparative FISH analysis with BACs spanning the inversion breakpoints in PTR on metaphase chromosomes of gorilla (GGO) confirmed that the pericentric inversion of the chromosome 12 homologs in GGO and PTR have distinct breakpoints and that humans retain the ancestral arrangement. These findings coincide with the trend observed in hominoid karyotype evolution that humans have a karyotype close to an ancestral one, while African great apes present with more derived chromosome arrangements.

Animals↗

Insights into the immune transcriptome of the shrimp Litopenaeus vannamei: tissue-specific expression profiles and transcriptomic responses to immune challenge.

Infectious disease constitutes a major obstacle to the sustainability of shrimp aquaculture worldwide and a significant threat to natural populations of shrimp and other crustacea. The study of the shrimp immune system, including the response to viral infection, has been hampered by a relative lack of molecular genetic information and of tools suitable for high-throughput assessment of gene expression. In this report, the generation of a cDNA microarray encompassing 2,469 putative unigenes expressed in gills, circulating hemocytes, and hepatopancreas of Litopenaeus vannamei is described. The unigenes printed on the microarray were derived from the analyses of 7,021 expressed sequence tags obtained from standard cDNA libraries as well as from libraries generated by suppression subtractive hybridization, after challenging shrimp with a variety of immune stimuli. The general utility of the cDNA microarray was demonstrated by interrogating the array with labeled RNA from four different shrimp tissues (gills, hemocytes, hepatopancreas, and muscle) and by analyzing the transcriptomic response of shrimp to a lethal challenge with white spot syndrome virus. Our results indicate that white spot syndrome virus infection upregulates (in the hepatopancreas) genes encoding known and potential antimicrobial effectors, while some genes involved in protection from oxidative stress were found to be downregulated by the virus.

Animals↗

Comprehensive transcriptomics and proteomics analysis of neointima formation in human saphenous vein: implications for bypass graft disease.

Human saphenous veins (SVs) are widely used as grafts in coronary artery bypass (CABG) surgery but often fail due to neointima formation. Little is known, however, regarding the cellular, transcriptomic, and proteomic dynamics of neointima formation in human veins. Here, we performed transcriptomics and proteomics analysis in an ex vivo tissue culture model of neointima formation in human SVs procured for CABG surgery. Histological examination demonstrated significant elastin degradation and neointima formation (indicated by increased neointima area and neointima-to-media ratio) in SVs subjected to tissue culture. Analysis of data from 72 patients suggests that the progression of SV remodeling and neointima formation differs according to sex and body mass index, which is negatively associated with neointima formation in males only. RNA sequencing demonstrated upregulation of proinflammatory and proliferation-related genes during neointima formation and identified novel processes, including increased cellular stress and DNA damage responses, reflecting tissue trauma associated with vein harvesting. Proteomic analysis identified upregulated extracellular matrix-related and coagulation/thrombosis proteins and downregulated metabolic proteins. Spatial transcriptomics, used to infer regionally enriched gene expression, suggested dynamic alterations in fibroblast and vascular smooth muscle cell (VSMC) states during neointima formation. Specifically, we identified the emergence of HES1+ and matrix metalloproteinase 2- and 14-positive (MMP2+/MMP14+) expression in VSMCs and fibroblasts, respectively, during neointima formation. Furthermore, our data suggest that MIR647, identified through screening, maintains VSMC contractile gene expression. Our findings suggest dynamic transcriptomic and proteomic changes during neointima formation in human veins and provide useful mechanistic information for the pathogenesis of SV graft disease.NEW & NOTEWORTHY Using multiomics and spatial transcriptomics, we uncover dynamic molecular and cellular changes driving neointima proliferation in human saphenous veins, the most common conduit for bypass surgery. Our study highlights sex- and body mass index-associated differences, novel fibroblast and smooth muscle cell states, and a role for microRNA-647 in preserving vascular contractile phenotype. These findings provide new insight into the mechanisms of vein graft failure and may guide future strategies to improve coronary bypass outcomes.

Humans↗

A critical evaluation of differential display as a tool to identify genes involved in legume nodulation: looking back and looking forward.

Screening for differentially expressed genes is a straightforward approach to study the molecular basis of a biological system. In the last 10 years, differential screening technology has evolved rapidly and currently high-throughput tools for genome-wide transcript profiling, such as expressed sequence tags and microarray analysis, are becoming widely available. Here, an overview of this (r)evolution is given with emphasis on the differential display method, which for many years has been the preferred technique of scientists in diverse fields of research. Differential display has also been the method of choice for the identification of genes involved in the symbiotic interaction between Azorhizobium caulinodans and Sesbania rostrata. The advantages with respect to tissue specificity of this particular model system for legume nodulation and the results of a screening for early nodulation-related genes have been considered in the context of transcriptome analyses in other rhizobium-legume interactions.

Azorhizobium caulinodans↗

Nonuniform hybridization: a potential source of error in oligonucleotide-chip experiments with low amounts of starting material.

Low amounts of starting material are a significant limitation of gene-expression profiling of microprepared pathologic specimens. Linear RNA amplification has become the method of choice to overcome this problem. Thus, transcriptomal analyses by oligonucleotide-chips or cDNA microarrays are now feasible with labeled complementary RNA generated from total RNA samples in the lower nanogram range. However, in case of oligonucleotide-chips, it has been underestimated so far that individual complementary RNA molecules are shorter in length than and display a 3' bias in comparison to the sequence stretch represented by oligonucleotides on the chip. This can lead to incorrect interpretation of raw data. We have analyzed this problem testing ex vivo-microprepared endothelial cells with Affymetrix GeneChips U133A. Only a small subset of housekeeping genes showed adequate uniform hybridization. We developed a software tool for objective evaluation of oligonucleotide-chips based on automated analysis of as well as normalization to this subset of housekeeping genes. We analyzed the gene expression profile of microprepared lymphatic vascular endothelial cells. We show that optimized normalization prevented exclusion of angiopoietin-2, a lymphatic endothelial marker, from the lymphovascular transcriptome.

Endothelial Cells↗

A Multi-omics Exploration Revealing SLIT2 as a Prime Therapeutic Target for Peripheral Facial Paralysis: Integrating Single-Cell Transcriptomics and Plasma Proteome Data.

Peripheral facial paralysis (PFP) is a common neurological disorder characterized by facial-nerve dysfunction. Identifying therapeutic targets and understanding the molecular and cellular mechanisms underlying PFP are crucial for developing effective treatment strategies. This study combined Mendelian randomization (MR) analysis and single-cell RNA sequencing (scRNA-seq) to explore potential therapeutic candidates and their roles in PFP pathophysiology. The MR analysis included 1925 publicly available plasma protein cis-heritability instruments. Instrumental variables were selected for MR analysis to identify plasma proteins associated with PFP, followed by colocalization analysis to evaluate shared genetic variants between the identified proteins and PFP. After the initial identification of plasma proteins associated with Bell's palsy using MR analysis, a rat model of facial-nerve injury was established to further dissect underlying mechanisms at cellular and molecular levels. Using scRNA-seq technology, we delved deeply into cellular Heterogeneity and dynamic changes in gene expression in the facial-nerve nucleus tissues under both injured and control conditions, thereby achieving a systematic study ranging from macroscopic genetic associations to microscopic cellular functions. Finally, expression patterns were preliminarily validated by performing in vitro immunofluorescence analysis on the facial-nerve nucleus samples of SD rats. The MR analysis results identified 30 plasma proteins significantly associated with PFP, with nine target genes showing differential expression in the scRNA-seq data. Colocalization analysis demonstrated that slit guidance Ligand 2 (SLIT2), semaphorin 4D (SEMA4D), EGF containing fibulin extracellular matrix protein 1 (EFEMP1), and sprouty related EVH1 domain containing 2 (SPRED2) shared causal variants with PFP. SLIT2 was highly expressed in the microglia and inhibitory neurons in the experimental group, whereas SEMA4D showed elevated expression across multiple glial cell types in the same group. In contrast, EFEMP1 and SPRED2 showed distinct expression patterns in fibroblasts and oligodendrocytes. The role of SLIT2 has been previously well-documented in many central nervous system diseases. However, for the first time, this study detected SLIT2 alteration after facial-nerve injury. Altered intercellular signaling, particularly enhanced SLIT2-ROBO signaling between neurons and glial cells, was observed in the PFP group. Pseudotime analysis revealed dynamic SLIT2 expression during microglia and inhibitory neuron differentiation, mirroring changes in ROBO1 expression. Immunofluorescence analysis of rat facial-nerve nucleus samples verified that SLIT2 protein levels were significantly increased in the facial-nerve nuclei of injured samples. In conclusion, despite the fact that this study is primarily founded on animal models and despite notable differences existing between animals and humans in terms of the facial motor nucleus, this study successfully identified SLIT2 as potential therapeutic targets for PFP. The SLIT2-ROBO axis stands out as a particularly promising candidate. SLIT2 may play a role in modulating neuroimmune interactions and promoting nerve repair. These findings provide a foundation for future clinical studies and targeted interventions to enhance recovery from PFP. Future research should focus on human sample validation to enhance clinical translation.

Animals↗

Exploring (novel) gene expression during retinoid-induced maturation and cell death of acute promyelocytic leukemia.

During recent years, reports have shown that biological responses of acute promyelocytic leukemia (APL) cells to retinoids are more complex than initially envisioned. PML-RARalpha chimeric protein disturbs various biological processes such as cell proliferation, differentiation, and apoptosis. The distinct biological programs that regulate these processes stem from specific transcriptional activation of distinct (but overlapping) sets of genes. These programs are sometimes mutually exclusive and depend on whether the signals are delivered by RAR or RXR agonists. Furthermore, evidence that retinoid nuclear signaling by retinoid, on its own, is not enough to trigger these cellular responses is rapidly accumulating. Indeed, work with NB4 cells show that the fate of APL cells treated by retinoid depends on complex signaling cross-talk. Elucidation of the sequence of events and cascades of transcriptional regulation necessary for APL cell maturation will be an additional tool with which to further improve therapy by retinoids. In this task, the classical techniques used to analyze gene expression have proved time consuming, and their yield has been limited. Global analyses of the APL cell transcriptome are needed. We review the technical approaches currently available (differential display, complementary DNA microarrays), to identify novel genes involved in the determination of cell fate.

Apoptosis↗

Rhodopseudomonas palustris CGA009 has two functional ppsR genes, each of which encodes a repressor of photosynthesis gene expression.

The PpsR protein is a regulator of redox-dependent photosystem development in purple phototrophic bacteria. In contrast to most species, Rhodopseudomonas palustris contains two ppsR genes. We show that the inactivation of each of the R. palustris strain CGA009 ppsR genes results in an elevated level of formation of the photosystem under dark aerobic conditions. Absorption spectra of the two PpsR mutants revealed qualitative and quantitative differences in light-harvesting peak amplitude increases. A sequence difference in the helix-turn-helix DNA binding motif of PpsR2 (Arg 439 to Cys) between R. palustris strains CEA001 and CGA009 is shown to be a natural polymorphism that does not inactivate the repressor activity of the protein. To evaluate which photosynthesis genes are regulated by the two PpsR proteins, transcriptome profiles of the CGA009 and PpsR mutant strains were analyzed in microarray experiments. Transcription of most but not all photosystem genes was derepressed in the mutant strains to levels consistent with the in vivo absorption spectra, mathematical analyses of peak shapes and amplitudes, reaction center protein levels, and real-time PCR of selected mRNAs. Closely spaced PpsR binding motif repeats were identified 5' of genes that were derepressed in the transcriptome analysis of PpsR mutants. This work shows that both the PpsR1 and PpsR2 proteins from R. palustris strain CGA009 function as oxygen-responsive transcriptional repressors.

Aerobiosis↗

Abundant novel transcriptional units and unconventional gene pairs on human chromosome 22.

Novel transcriptional units (TUs) are EST-supported transcribed features not corresponding to known genes. Unconventional gene pairs (UGPs) are pairs of genes and/or TUs sharing exon-to-exon cis-antisense overlaps or putative bidirectional promoters. Computational TU and UGP discovery followed by manual curation was performed in the entire published 34.9-Mb human chromosome 22 euchromatic sequence. Novel TUs (n = 517) were as abundant as known genes (n = 492) and typically did not have nonprimate DNA and protein homologies. One hundred seventy-one (33%) of TUs, but only 13 (3%) of genes, both lacked nonprimate conservation and localized to gaps in the human-mouse BLASTZ alignment. Novel TUs were richer in exonic primate-specific interspersed repetitive elements (P = 0.001) and were more likely to rely on splice junctions provided by them, than were known genes: 19% of spliced TUs, versus 5% of spliced genes, had a splice site within a primate-specific repeat. Hence, novel TUs and known genes may represent different portions of the transcriptome. Two hundred nine (21%) of chromosome 22 transcripts participated in 77 cis-antisense and 42 promoter-sharing UGPs. Transcripts involved simultaneously in both UGP types were more common than was expected (P = 0.01). UGPs were nonrandomly distributed along the sequence: 89 (75%) clustered in distinct regions, the sum of which equaled 4.4 Mb (<13% of the chromosome). Eighty (67%) of the UGPs possessed significant locus structure differences between primates and rodents. Since some TUs may be functional noncoding transcripts and since the cis-regulatory potential of UGPs is well recognized, TUs and UGPs specific to the primate lineage may contribute to the genomic basis for primate-specific phenotypes.

Animals↗

Identifying secretomes in people, pufferfish and pigs.

The proteins processed by the secretory pathway (secretome) are critical players in the development of multi-cellular eukaryotic organisms but have yet to be comprehensively studied at the genomic level. In this study, we use the Target P algorithm to predict human (13-20% of proteins found in individual datasets) and Fugu (14%) secretomes based on analysis of their nearly complete proteomes. We combine internal processing with prediction software to automate secreted protein identification and overcome one of the major challenges associated with EST data: identification of the minority of clones that encode N-terminally-complete proteins. We discuss the use of these methods to predict secreted proteins in EST-based consensus sequence sets, and we validate these predictions using an assay for cell-free cotranslational translocation. Analysis of TIGR Porcine Gene Index 4.0 as a test dataset resulted in the identification of 352 N-terminally-complete, putative secreted proteins. In functional agreement with our predictions, 34 of 40 (85%) of these cDNAs were verified to be cotranslationally translocated in an in vitro translation system. The methods developed here are specifically designed to accept partial open reading frames and improve secreted protein predictions in eukaryotic transcriptomes, and are valuable for the analysis and annotation of eukaryotic EST databases.

Algorithms↗

Transcriptomic insights into exogenous fatty acid-enhanced halotolerance in Zygosaccharomyces rouxii.

BACKGROUND: High salinity restricts microbial growth during brine-based food fermentation. Although exogenous unsaturated fatty acids improve the salt tolerance of Zygosaccharomyces rouxii, the associated transcriptional mechanisms remain unclear. This study investigated the transcriptomic response of Z. rouxii CGMCC 3791 to palmitoleic acid (C16:1) under high salt conditions. RESULTS: Cells were cultured in yeast extract peptone dextrose (YPD) containing 120&#x2009;g&#x2009;L-1 NaCl, with or without 20&#x2009;&#x3bc;g&#x2009;mL-1 C16:1. They were analyzed by RNA sequencing. Principal component analysis clearly separated the two treatments. Using q&#x2009;<&#x2009;0.05 and |log2 fold change|&#x2009;>&#x2009;1, 23 differentially expressed genes were identified - three upregulated and 20 downregulated. INO1, MLS1, POX1, MEP2, and SOD5 were among the major responsive genes. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses indicated that peroxisome-associated functions, lipid metabolism, oxidative stress responses, nitrogen utilization, and mitogen-activated protein kinase (MAPK) signaling were the principal C16:1-responsive processes. CONCLUSION: Exogenous C16:1 elicited a focused transcriptional adjustment rather than broad transcriptome-wide reprogramming in salt-stressed Z. rouxii. The results indicated that peroxisome-linked lipid processes and redox regulation were candidate mechanisms underlying fatty-acid-associated halotolerance and provided targets for improving the robustness of high-salt food fermentation. &#xa9; 2026 Society of Chemical Industry.

Zygosaccharomyces rouxii↗

Diversity and evolution of conotoxins based on gene expression profiling of Conus litteratus.

Cone snails are attracting increasing scientific attention due to their unprecedented diversity of invaluable channel-targeted peptides. As arguably the largest and most successful evolutionary genus of invertebrates, Conus also may become the model system to study the evolution of multigene families and biodiversity. Here, a set of 897 expressed sequence tags (ESTs) derived from a Conus litteratus venom duct was analyzed to illuminate the diversity and evolution mechanism of conotoxins. Nearly half of these ESTs represent the coding sequences of conotoxins, which were grouped into 42 novel conotoxin cDNA sequences (seven superfamilies), with T-superfamily conotoxins being the dominant component. The gene expression profile of conotoxin revealed that transcripts are expressed with order-of-magnitude differences, sequence divergence within a superfamily increases from the N to the C terminus of the open reading frame, and even multiple scaffold-different mature peptides exist in a conotoxin gene superfamily. Most excitingly, we identified a novel conotoxin superfamily and three novel cysteine scaffolds. These results give an initial insight into the C. litteratus transcriptome that will contribute to a better understanding of conotoxin evolution and the study of the cone snail genome in the near future.

Amino Acid Sequence↗

Transcriptome analysis of Sinorhizobium meliloti during symbiosis.

BACKGROUND: Rhizobia induce the formation on specific legumes of new organs, the root nodules, as a result of an elaborated developmental program involving the two partners. In order to contribute to a more global view of the genetics underlying this plant-microbe symbiosis, we have mined the recently determined Sinorhizobium meliloti genome sequence for genes potentially relevant to symbiosis. We describe here the construction and use of dedicated nylon macroarrays to study simultaneously the expression of 200 of these genes in a variety of environmental conditions, pertinent to symbiosis. RESULTS: The expression of 214 S. meliloti genes was monitored under ten environmental conditions, including free-living aerobic and microaerobic conditions, addition of the plant symbiotic elicitor luteolin, and a variety of symbiotic conditions. Five new genes induced by luteolin have been identified as well as nine new genes induced in mature nitrogen-fixing bacteroids. A bacterial and a plant symbiotic mutant affected in nodule development have been found of particular interest to decipher gene expression at the intermediate stage of the symbiotic interaction. S. meliloti gene expression in the cultivated legume Medicago sativa (alfalfa) and the model plant M. truncatula were compared and a small number of differences was found. CONCLUSIONS: In addition to exploring conditions for a genome-wide transcriptome analysis of the model rhizobium S. meliloti, the present work has highlighted the differential expression of several classes of genes during symbiosis. These genes are related to invasion, oxidative stress protection, iron mobilization, and signaling, thus emphasizing possible common mechanisms between symbiosis and pathogenesis.

Cluster Analysis↗