Putative Transcription Factor Research Articles

Meta-Analysis of Antioxidant Mutants Reveals Common Alarm Signals for Shaping Abiotic Stress-Induced Transcriptome in Plants.

Aims: Reactive oxygen species (ROS) are key regulators of plant growth, development, and stress tolerance. Stress-induced changes in ROS levels trigger multilevel signaling. However, the precise mechanisms by which ROS signals are translated into changes in gene expression remain poorly defined. Focusing on six key antioxidant enzymes, we performed a meta-analysis of transcriptome data available in public databases to analyze ROS-mediated control of nuclear gene expression. Results: An information-guided pipeline was developed, which identified 19 putative transcription factors (TFs), as components in a "common alarm signal cascade" pathway following perception of changes in ROS levels. Crucially, 30%-35% of the abiotic stress transcriptome signatures had binding sites for common alarm signal-transcription factors (CAS-TFs) in their promoter regions. Furthermore, Phloem Early Dof 2 (PEAR2), DNA binding with one finger 5.8 (DOF5.8), and Obf-Binding Protein 3 (OBP3) were identified as top-ranked TFs on the basis of a cumulative DAPseq (DNA-affinity purification sequencing) score on the promoters of selected genes regulating core pathways of salt, drought, heat, and cold stress tolerance. Innovation: This study identifies a set of CAS-TFs that may play a major role in shaping the transcriptome of abiotic stress-induced ROS signaling. Ranking analysis identified PEAR2, DOF5.8, and OBP3 as the top-ranked CAS-TFs that regulated known markers of abiotic stress tolerance. Conclusion: The current findings suggest a major role of ROS in the abiotic stress signaling and also identify a set of TFs that take part in the signaling. Taken together, these findings suggested that the common alarm signal cascade underpins broad-range tolerance against multistress conditions. The identification of associated ROS-responsive CAS-TFs may provide novel targets for crop improvement.

Neuronal transcription of autism gene PTCHD1 is regulated by a conserved downstream enhancer sequence

Patched domain-containing 1 (PTCHD1) is a well-established susceptibility gene for autism spectrum disorder (ASD) and intellectual disability (ID). Previous studies have suggested that alterations in the dosage of PTCHD1 may contribute to the etiology of both ASD and ID. However, there has not yet been a thorough investigation regarding mechanisms that regulate PTCHD1 expression. We sought to characterize the Ptchd1 promoter in a mouse neuronal model, as well as to identify and validate cis regulatory elements. We defined specific regions of the Ptchd1 promoter essential for robust expression in P19-induced neurons. Evolutionarily-conserved putative transcription factor binding sites within these regions were subsequently identified. Using a pairwise comparison of chromatin accessibility between mouse forebrain and liver tissues, a candidate regulatory region, ~ 9.1 kbp downstream of the Ptchd1 stop codon was defined. This region harbours two ENCODE-predicted enhancer cis-regulatory elements. Further, using DNase footprint analysis, a putative YY1-binding motif was also identified. Genomic deletion of the entire 8 kbp downstream open chromatin region attenuated Ptchd1 transcription by over 60% in our neuronal model, corroborating its predicted regulatory function. This study provides mechanistic insights related to the expression of PTCHD1, and provides important context to interpret genetic and genomic variation at this locus which may influence neurodevelopment.

In Vivo Reprogramming of Adult Liver Sinusoidal Vascular Endothelial Cells into a Hematopoietic Stem and Progenitor Cell Niche

Hematopoietic stem and progenitor cells (HSPCs) reside in specialized microenvironments known as the HSPC niche. Vascular endothelial cells (ECs) are one of the most abundant and significant components of the niche, providing known supportive factors such as E-selectin, CXCL12, and SCF. During marrow stress, extramedullary hematopoiesis can occur in the liver or spleen, but the process is inefficient. The sinusoidal vessels of the liver share many similarities with marrow sinusoids. Here, we aim to identify the transcription factor (TF) code that specifies the vascular EC fate in HSPC marrow niche by conducting differential gene expression analysis with the non-niche supportive ECs of the liver. We performed scRNAseq analysis on FACS-isolated ECs from adult zebrafish kidney marrow and liver. We found that these ECs express conserved sinusoidal genes, such as sele, flt4 and gpr182. However, marrow ECs have significantly higher expression of HSPC niche supportive genes, including mrc1a, lyve1b and cxcl12a. We further identified putative TFs that are uniquely upregulated in the marrow ECs, namely tfec, mafbb, foxp4, irf8 and hoxb8a. The expression of these candidate TFs was conserved in the mammalian bone marrow ECs, as shown by relevant mouse bone marrow scRNA-seq and human bone marrow RNA-seq analyses. To determine whether these candidate TFs can functionally specify a HSPC niche EC fate, we selectively overexpressed them using a zebrafish liver sinusoidal EC-specific enhancer in vivo. Upon the overexpression of tfec and mafbb together, we found adult zebrafish liver sinusoidal ECs significantly upregulated key genes known for HSPC niche supportive functions in both embryonic and adult zebrafish HSPC niches, including mrc1a (log2FC=4.9, p<0.005) and lyve1b (log2FC=3.6, p<0.005) and dab2 (log2FC=5.0, p<0.005). Overexpression of TFEC and MAFB in human iPSC-derived ECs showed upregulation of HPSC niche supportive genes that we previously identified from zebrafish in vivo studies, including LYVE1 (log2FC=5.6, p<0.005) and DAB2 (log2FC=1.1, p<0.005) . Notably, previously published HSPC niche genes, including CXCL12(log2FC=5.1 p< 0.005) , PTN (log2FC=2.6 p<0.005) and JAG1 (log2FC=1.7 p<0.005), are also upregulated upon TFEC and MAFB overexpression. Furthermore, gene ontology analysis revealed an enrichment of genes related to cell adhesion (p<0.005), migration (p<0.005), and extravasation pathways (p=0.014). A colony-forming unit (CFU) assay of CD34+CD45+ HSPCs co-cultured with TFEC and MAFB overexpressing hiPSC-derived ECs had significantly greater numbers of CFUs compared to those co-cultured with hiPSC-derived ECs without the TF overexpression: CFU-GEMM (23.3±2.4 vs. 10.0±2.1, p=0.004), CFU-G (89.3±13.4 vs. 46.0±4.2, p=0.027), and CFU-E (42.3±3.3 vs. 26.7±0.9, p=0.021). Our findings revealed a TF code that specifies the HSPC niche supportive fate of vascular ECs and distinguishes vascular ECs in the hematopoietic niche vs. non-niche organs. These results may translate into novel therapies targeting the sinusoidal vascular niches to better stimulate and support human HSPCs.

A novel transcriptional repressor specifically regulates xylanase gene 1 in Trichoderma reesei

BackgroundThe well-known industrial fungus Trichoderma reesei has an excellent capability of secreting a large amount of cellulases and xylanases. The induced expression of cellulase and xylanase genes is tightly controlled at the transcriptional level. However, compared to the intensive studies on the intricate regulatory mechanism of cellulase genes, efforts to understand how xylanase genes are regulated are relatively limited, which impedes the further improvement of xylanase production by T. reesei via rational strain engineering.ResultsTo identify transcription factors involved in regulating xylanase gene expression in T. reesei, yeast one-hybrid screen was performed based on the promoters of two major extracellular xylanase genes xyn1 and xyn2. A putative transcription factor named XTR1 showing significant binding capability to the xyn1 promoter but not that of xyn2, was successfully isolated. Deletion of xtr1 significantly increased the transcriptional level of xyn1, but only exerted a minor promoting effect on that of xyn2. The xylanase activity was increased by ~ 50% with XTR1 elimination but the cellulase activity was hardly affected. Subcellular localization analysis of XTR1 fused to a green fluorescence protein demonstrated that XTR1 is a nuclear protein. Further analyses revealed the precise binding site of XTR1 and nucleotides critical for the binding within the xyn1 promoter. Moreover, competitive EMSAs indicated that XTR1 competes with the essential transactivator XYR1 for binding to the xyn1 promoter.ConclusionsXTR1 represents a new transcriptional repressor specific for controlling xylanase gene expression. Isolation and functional characterization of this new factor not only contribute to further understanding the stringent regulatory network of xylanase genes, but also provide important clues for boosting xylanase biosynthesis in T. reesei.

Jaw size variation is associated with a novel craniofacial function for galanin receptor 2 in an adaptive radiation of pupfishes.

Understanding the genetic basis of novel adaptations in new species is a fundamental question in biology. Here we demonstrate a new role for galr2 in vertebrate craniofacial development using an adaptive radiation of trophic specialist pupfishes endemic to San Salvador Island, Bahamas. We confirmed the loss of a putative Sry transcription factor binding site upstream of galr2 in scale-eating pupfish and found significant spatial differences in galr2 expression among pupfish species in Meckel's cartilage using in situ hybridization chain reaction (HCR). We then experimentally demonstrated a novel role for Galr2 in craniofacial development by exposing embryos to Garl2-inhibiting drugs. Galr2-inhibition reduced Meckel's cartilage length and increased chondrocyte density in both trophic specialists but not in the generalist genetic background. We propose a mechanism for jaw elongation in scale-eaters based on the reduced expression of galr2 due to the loss of a putative Sry binding site. Fewer Galr2 receptors in the scale-eater Meckel's cartilage may result in their enlarged jaw lengths as adults by limiting opportunities for a circulating Galr2 agonist to bind to these receptors during development. Our findings illustrate the growing utility of linking candidate adaptive SNPs in non-model systems with highly divergent phenotypes to novel vertebrate gene functions.

<i>In silico</i> Analysis of <i>Dreb2a</i> Gene among Diploid <i>Oryza</i> Species Reveal Gene Retrogression in Cultivated Species

The dehydration-responsive element-binding (DREB) genes are types of plant-specific transcription factors that bind to DRE/CRT elements in response to abiotic stresses. It belongs to the AP2 superfamily and contains highly conserved APETELA2 (AP2) domain. In this study, comparative analysis of DREB2A gene from diploid Oryza species was performed using BLASTN approach. The OglDREB2A sequence (KU159742.1) was used as query against 11 diploid Oryza species in the Gramene database for identification of DREB2A orthologs. Molecular characterization, subcellular localization, conserved motifs and promoter analysis were also performed using bioinformatics tools. Molecular characterization of DREB2A protein in the diploid Oryza showed an AP2 conserved domain and nuclear localized protein which is similar to putative transcription factor involved in stress responses. Motif analysis using MEME suites identified 7 significant motifs across all the diploid Oryza species with motif 2, 3, 4 and 5 as the common motifs with ~50 amino acids length. Analysis of 1kb upstream of TSS of DREB2A of the 11 diploid Oryza species revealed many CAREs associated with stress (DRE, LTRE, ERE, G-box, TCC-motif and Sp1) and hormonal regulations (ABREs, MeJA, GARE, and GC-motif) with ABRE as the common element in all the diploid species. Additionally, wild species has more number of cis elements than the cultivated species suggesting a possible role in genome evolution. Overall, the 11 DREB2A orthologs are conserved in terms of sequence homology and protein structure but cultivated species has less number of transcripts than their progenitors suggesting gene retrogression in cultivated Oryza species.

Panicle transcriptome of high-yield mutant indica rice reveals physiological mechanisms and novel candidate regulatory genes for yield under reproductive stage drought stress

BackgroundReproductive stage drought stress (RDS) is a major global threat to rice production. Due to climate change, water scarcity is becoming an increasingly common phenomenon in major rice-growing areas worldwide. Understanding RDS mechanisms will allow candidate gene identification to generate novel rice genotypes tolerant to RDS.ResultsTo generate novel rice genotypes that can sustain yield under RDS, we performed gamma-irradiation mediated mutation breeding in the drought stress susceptible mega rice variety, MTU1010. One of the mutant MM11 (MTU1010 derived mutant11) shows consistently increased performance in yield-related traits under field conditions consecutively for four generations. In addition, compared to MTU1010, the yield of MM11 is sustained in prolonged drought imposed during the reproductive stage under field and in pot culture conditions. A comparative emerged panicle transcriptome analysis of the MTU1010 and MM11 suggested metabolic adjustment, enhanced photosynthetic ability, and hormone interplay in regulating yield under drought responses during emerged panicle development. Regulatory network analysis revealed few putative significant transcription factor (TF)-target interactions involved in integrated signalling between panicle development, yield and drought stress.ConclusionsA gamma-irradiate rice mutant MM11 was identified by mutation breeding, and it showed higher potential to sustain yield under reproductive stage drought stress in field and pot culture conditions. Further, a comparative panicle transcriptome revealed significant biological processes and molecular regulators involved in emerged panicle development, yield and drought stress integration. The study extends our understanding of the physiological mechanisms and candidate genes involved in sustaining yield under drought stress.

Regulation of fungal raw-starch-degrading enzyme production depends on transcription factor phosphorylation and recruitment of the Mediator complex

Filamentous fungus can produce raw-starch-degrading enzyme (RSDE) that efficiently degrades raw starch below starch gelatinization temperature. Employment of RSDE in starch processing can save energy. A key putative transcription factor PoxRsrA (production of raw-starch-degrading enzyme regulation in Penicilliumoxalicum) was identified to regulate RSDE production in P. oxalicum; however, its regulatory mechanism remains unclear. Here we show that PoxRsrA1434–1730 was the transcriptional activation domain, with essential residues, D1508, W1509 and M1510. SANT (SWI3, ADA2, N-CoR and TFIIIB)-like domain 1 (SANT1) bound to DNA at the sequence 5′-RHCDDGGD-3′ in the promoter regions of genes encoding major amylases, with an essential residue, R866. SANT2 interacted with a putative 3-hydroxyisobutyryl-CoA hydrolase, which suppressed phosphorylation at tyrosines Y1127 and Y1170 of PoxRsrA901–1360, thereby inhibiting RSDE biosynthesis. PoxRsrA1135–1439 regulated mycelial sporulation by interacting with Mediator subunit Med6, whereas PoxRsrA1440–1794 regulated RSDE biosynthesis by binding to Med31. Overexpression of PoxRsrA increased sporulation and RSDE production. These findings provide insights into the regulatory mechanisms of fungal RSDE biosynthesis.

Single-cell transcriptome analysis dissects lncRNA-associated gene networks in Arabidopsis

The plant genome produces an extremely large collection of long noncoding RNAs (lncRNAs) that are generally expressed in a context-specific manner and have pivotal roles in regulation of diverse biological processes. Here, we mapped the transcriptional heterogeneity of lncRNAs and their associated gene regulatory networks at single-cell resolution. We generated a comprehensive cell atlas at the whole-organism level by integrative analysis of 28 published single-cell RNA sequencing (scRNA-seq) datasets from juvenile Arabidopsis seedlings. We then provided an in-depth analysis of cell-type-related lncRNA signatures that show expression patterns consistent with canonical protein-coding gene markers. We further demonstrated that the cell-type-specific expression of lncRNAs largely explains their tissue specificity. In addition, we predicted gene regulatory networks on the basis of motif enrichment and co-expression analysis of lncRNAs and mRNAs, and we identified putative transcription factors orchestrating cell-type-specific expression of lncRNAs. The analysis results are available at the single-cell-based plant lncRNA atlas database (scPLAD; https://biobigdata.nju.edu.cn/scPLAD/). Overall, this work demonstrates the power of integrative single-cell data analysis applied to plant lncRNA biology and provides fundamental insights into lncRNA expression specificity and associated gene regulation.

A pathogen-induced putative NAC transcription factor mediates leaf rust resistance in barley

Leaf rust, caused by Puccinia hordei, is one of the most widespread and damaging foliar diseases affecting barley. The barley leaf rust resistance locus Rph7 has been shown to have unusually high sequence and haplotype divergence. In this study, we isolate the Rph7 gene using a fine mapping and RNA-Seq approach that is confirmed by mutational analysis and transgenic complementation. Rph7 is a pathogen-induced, non-canonical resistance gene encoding a protein that is distinct from other known plant disease resistance proteins in the Triticeae. Structural analysis using an AlphaFold2 protein model suggests that Rph7 encodes a putative NAC transcription factor with a zinc-finger BED domain with structural similarity to the N-terminal DNA-binding domain of the NAC transcription factor (ANAC019) from Arabidopsis. A global gene expression analysis suggests Rph7 mediates the activation and strength of the basal defence response. The isolation of Rph7 highlights the diversification of resistance mechanisms available for engineering disease control in crops.

Epigenetic reprogramming around IFN1 and IFNy2 promoters in rainbow trout cells inoculated with infectious pancreatic necrosis virus (IPNV).

Infectious pancreatic necrosis virus (IPNV) has proven to effectively evade the host antiviral responses. This study clarifies whether the modulation of the antiviral immune response exerted by IPNV involves epigenetic mechanisms. An in-silico characterization of the rainbow trout IFN1 and IFNγ2 promoters was performed, identifying the islands or sequences rich in CpG dinucleotides and the putative transcription factor binding sites (TBS) for both gene promoters. RTS11 cells (rainbow trout monocyte/macrophage) were infected with IPNV, and the course of viral infection was followed up to 48h post infection (hpi). Infected cells showed increased IFN1 and IFNγ2 transcriptional expression at 6 and 24 hpi, respectively. IPNV infection caused increases and decreases in global IFNγ2 promoter methylation at 6 and 24 hpi, respectively. The CpG dinucleotides at positions -392 and+38 of this promoter were the most sensitive to methylation changes. The IFN1 promoter remained fully unmethylated during the course of the infection, similar to the control. The changes in the methylation pattern observed for the IFNγ2 promoter were coincident with the changes in DNA methyltransferase (DNMT) expression levels, increasing at 6 hpi and decreasing below basal level at 24 hpi. Similarly, the H4 histones associated with the IFN1 and IFNγ2 promoters were hyperacetylated at 6 hpi, subsequently decreasing their acetylation below basal levels at 24 hpi, in both promoters. Coincidentally with the above, overexpression of histone acetyltransferase (HAT) was observed at 6 hpi and of histone deacetylase (HDAC) at 24 hpi, with return to baseline of HAT. These results suggest that IPNV would epigenetically modulate the expression of IFN1 by changing acetylation levels of the histones H4 associated with its promoter. Also, the modulation of the expression of IFNy2 would be by switching methylation/demethylation levels of its promoter, in addition to changes in acetylation levels of histones H4 associated with this promoter. This study is the first to demonstrate the effect of epigenetic reprogramming after IPNV infection in salmonid cells, demonstrating that promoter methylation/demethylation level and changes in the histone code associated with promoters may play a role in the modulation of the immune response induced by the virus.

Integrated metabolome, full-length sequencing, and transcriptome analyses unveil the molecular mechanisms of color formation of the canary yellow and red bracts of Bougainvillea × buttiana 'Chitra'.

Bougainvillea is a typical tropical flower of great ornamental value due to its colorful bracts. The molecular mechanism behind color formation is not well-understood. Therefore, this research conducted metabolome analysis, transcriptome analysis, and multi-flux full-length sequencing in two color bracts of Bougainvillea × buttiana 'Chitra' to investigate the significantly different metabolites (SDMs) and differentially expressed genes (DEGs). Overall, 261 SDMs, including 62 flavonoids and 26 alkaloids, were detected, and flavonols and betalains were significantly differentially accumulated among the two bracts. Furthermore, the complete-length transcriptome of Bougainvillea × buttiana was also developed, which contained 512 493 non-redundant isoforms. Among them, 341 210 (66.58%) displayed multiple annotations in the KOG, GO, NR, KEGG, Pfam, Swissprot, and NT databases. RNA-seq findings revealed that 3610 DEGs were identified between two bracts. Co-expression analysis demonstrated that the DEGs and SDMs involved in flavonol metabolism (such as CHS, CHI, F3H, FLS, CYP75B1, kaempferol, and quercetin) and betacyanin metabolism (DODA, betanidin, and betacyanins) were the main contributors for the canary yellow and red bract formation, respectively. Further investigation revealed that several putative transcription factors (TFs) might interact with the promoters of the genes mentioned above. The expression profiles of the putative TFs displayed that they may positively and negatively regulate the structural genes' expression profiles. The data revealed a potential regulatory network between important genes, putative TFs, and metabolites in the flavonol and betacyanin biosynthesis of Bougainvillea × buttiana 'Chitra' bracts. These findings will serve as a rich genetic resource for future studies that could create new color bracts.

Conservation of putative transcription factor binding sites of co-expressed Plasmopara halstedii genes in two Phytophthora species

Oomycetes, a large group of fungus-like organisms, include some destructive plant pathogens causing enormous economic damage. Phylogenetically, oomycetes belong to the kingdom Straminipila and have diverse lifestyles, including saprotrophs and both general and specialized pathogens of various eukaryotic supergroups. A rapid increase in genomic studies and next-generation sequencing technologies have led to significant progress in understanding oomycete lifestyles. However, their genetics, including transcriptional regulation, have been studied to a much lesser extent. Here, we provide a cross-species analysis of oomycete promoter for providing a first step towards elucidating gene regulation networks related to pathogenicity and life cycle stages. The clustered DNA sequences of Plasmopara halstedii transcriptome time-series expression level dataset from a preliminary study have been used as a core reference for cross-species comparisons. Using a computational pipeline, 46 potential transcription factor binding site (TFBS) motifs in 25 clusters with functionally conserved downstream genes of downy mildew and two Phytophthora species, regardless of the gene expression levels of Phytophthora transcriptomes, were found. This can now be followed up by knock-out experiments in oomycete species amenable for genetic modification.

Sugar composition and transcriptome analysis in developing ‘Fengtang’ plum (Prunus salicina Lindl.) reveal candidate genes regulating sugar accumulation

Sweetness is an important attribute of fruit quality, which directly affects consumers' preference for fresh fruit and is mostly determined by carbohydrate composition. ‘Fengtang’ plum (Prunus salicina Lindl.) is recognized for its high soluble sugar content, but the sugar composition and the molecular mechanisms underlying sugar overproduction are not fully understood. In this work, the sugar components were analyzed using gas chromatography-mass spectrometry combined with transcription profiles from RNA-sequencing and Quantitative Real-time PCR during fruit development. The target metabolic group showed that sucrose was the dominant sugar component in mature fruit, followed by glucose, fructose, and sorbitol. Based on the transcriptome data and qRT-PCR validation, we identified 12 key structural genes that significantly responded to corresponding component accumulation: sucrose synthase (PsSUS4), sucrose phosphate synthase (PsSPS2), neutral invertase (PsNINV1/3/4), phosphoglucomutase (PsPGM1), UTP-glucose-1-phosphate uridylyl transferase (PsUGP1/2), hexose kinase (PsHXK1/3), sugar transport protein (PsSTP1), and Sugars Will Eventually be Exported Transporter (PsSWEET4). In which PsSUS4 and PsSPS2, whose encoding proteins immediately catalyze sucrose synthesis, were selected to be silenced using the virus-induced gene silencing technology. Silencing of PsSUS4 or PsSPS2 resulted in decreased sucrose content by 27.6% and 8%, respectively, compared with the control, verifying their important roles in sucrose accumulation. Subsequently, sugar metabolism networks in this high-sugar plum were constructed with 12 key structural genes, 72 putative transcription factors, and 4 major sugar components. These results might facilitate a better understanding of the molecular mechanisms of sugar accumulation in ‘Fengtang’ plum and provide a framework for future fruit quality improvement.

Abstract P1144: Single-cell RNA-Sequencing Of The Murine Cardiac Conduction System Reveals Transcriptional Regulators Associated With Conduction Development And Disease

Rhythmic beating of the heart is driven by a small subset of heart cells, altogether known as the cardiac conduction system (CCS). The CCS consists of unique components including the sinoatrial node (SAN), the atrioventricular node (AVN), the bundle of His, bundle branches and Purkinje fiber (PF) system. Due to obstacles inherent in studying the CCS, including small cell numbers, large cell-type heterogeneity and complex anatomy, our understanding of the transcriptional networks driving CCS development remains limited. Here, we harnessed single-cell RNA-sequencing (scRNAseq) and bioinformatic analyses to uncover putative DNA-binding transcription factors (TFs) as well as transcriptional co-regulators enriched within the entire developing murine CCS. Comparison of multiple independent prior datasets, spanning all major components of the CCS, revealed previously known TFs necessary for conduction development including, Islet 1 ( Isl1 ; avg log fold change 0.34, p= 1E-192) in the SAN; T-box transcription factor 5 (Tbx5 ; avg log fold change 0.54, p= 2.1E-110) in the AVN; and ETS variant transcription factor 1 ( Etv1; avg log fold change 0.89, p= 6.7E-105) in the PFs, among others. Notably, an additional 16 novel DNA-binding transcription factors and 40 transcription co-regulators were enriched within specific CCS components at statistical significance. To further evaluate the transcriptional landscape of individual subcomponents within the SAN (compact versus transitional cells), we also performed scRNAseq on FACS-isolated SAN cells from embryonic day 16.5 Hcn4 -GFP transgenic mice revealing differential expression of a host of TFs, both established and novel. Finally, our murine findings were then cross-referenced to over 15 human GWAS studies related to cardiac rhythm abnormalities, revealing an association of a subset of these murine CCS-enriched transcription factors and cardiac arrhythmias in humans. In conclusion, this study provides a comprehensive gene expression atlas of transcription factors and transcriptional co-regulators within individual CCS components at single-cell resolution, laying the groundwork for future studies into the transcriptional regulation of cardiac conduction development and disease.

Engineering transcriptional regulation of pentose metabolism in Rhodosporidiumtoruloides for improved conversion of xylose to bioproducts

Efficient conversion of pentose sugars remains a significant barrier to the replacement of petroleum-derived chemicals with plant biomass-derived bioproducts. While the oleaginous yeast Rhodosporidiumtoruloides (also known as Rhodotorulatoruloides) has a relatively robust native metabolism of pentose sugars compared to other wild yeasts, faster assimilation of those sugars will be required for industrial utilization of pentoses. To increase the rate of pentose assimilation in R.toruloides, we leveraged previously reported high-throughput fitness data to identify potential regulators of pentose catabolism. Two genes were selected for further investigation, a putative transcription factor (RTO4_12978, Pnt1) and a homolog of a glucose transceptor involved in carbon catabolite repression (RTO4_11990). Overexpression of Pnt1 increased the specific growth rate approximately twofold early in cultures on xylose and increased the maximum specific growth by 18% while decreasing accumulation of arabitol and xylitol in fast-growing cultures. Improved growth dynamics on xylose translated to a 120% increase in the overall rate of xylose conversion to fatty alcohols in batch culture. Proteomic analysis confirmed that Pnt1 is a major regulator of pentose catabolism in R.toruloides. Deletion of RTO4_11990 increased the growth rate on xylose, but did not relieve carbon catabolite repression in the presence of glucose. Carbon catabolite repression signaling networks remain poorly characterized in R.toruloides and likely comprise a different set of proteins than those mainly characterized in ascomycete fungi.

Molecular characterization, transcriptional regulation of sea perch Moloney leukemia virus 10 and its antiviral function against VHSV.

Moloney leukemia virus 10 (MOV10) is a conserved RNA helicase and has multiple biological functions in mammals, but its role remains poorly understood in bony fish. Here, we cloned a MOV10 homolog from sea perch (Lateolabrax japonicus), which contained 23 exons and 22 introns, with an open reading frame of 3000 bp encoding 1000 amino acids. Tissue distribution analysis showed that MOV10 was high expressed in blood of sea perch. Promoter analysis revealed several putative multiple transcription factors binding sites, including upstream transcription factor 1, GATA-box, transcription initiation factor IIB, activator protein 1 and two interferon (IFN) stimulated response elements. Further analysis found that IFNc, IFNh, and IFNγ could not only activate IFN regulatory factor (IRF) 1 expression which in turn led to the induction of MOV10, but also prompted the expression of IRF10 to hinder excessive MOV10 expression. Moreover, IRF2 also suppressed MOV10 expression that was initiated by IRF1. Viral hemorrhagic septicemia virus (VHSV) infection upregulated MOV10 expression in vivo and in vitro, which in turn, enhanced IFNh expression and exhibited strong antiviral activity against VHSV proliferation. This study provides a basis to investigate the immune escape of VHSV by affecting the biological function of transcription factors in the signaling pathways associated with antiviral molecules.

Heat shock response in Sulfolobus acidocaldarius and first implications for cross-stress adaptation

Sulfolobus acidocaldarius, a thermoacidophilic crenarchaeon, frequently encounters temperature fluctuations, oxidative stress, and nutrient limitations in its environment. Here, we employed a high-throughput transcriptomic analysis to examine how the gene expression of S. acidocaldarius changes when exposed to high temperatures (92 °C). The data obtained was subsequently validated using quantitative reverse transcription-PCR (qRT-PCR) analysis. Our particular focus was on genes that are involved in the heat shock response, type-II Toxin-Antitoxin systems, and putative transcription factors. To investigate how S. acidocaldarius adapts to multiple stressors, we assessed the expression of these selected genes under oxidative and nutrient stresses using qRT-PCR analysis. The results demonstrated that the gene thβ encoding the β subunit of the thermosome, as well as hsp14 and hsp20, play crucial roles in the majority of stress conditions. Furthermore, we observed overexpression of at least eight different TA pairs belonging to the type II TA systems under all stress conditions. Additionally, four common transcription factors: FadR, TFEβ, CRISPR loci binding protein, and HTH family protein were consistently overexpressed across all stress conditions, indicating their significant role in managing stress. Overall, this work provides the first insight into molecular players involved in the cross-stress adaptation of S. acidocaldarius.

Identification and Expressional Analysis of Putative PRDI-BF1 and RIZ Homology Domain-Containing Transcription Factors in Mulinia lateralis.

Mollusca represents one of the ancient bilaterian groups with high morphological diversity, while the formation mechanisms of the precursors of all germ cells, primordial germ cells (PGCs), have not yet been clarified in mollusks. PRDI-BF1 and RIZ homology domain-containing proteins (PRDMs) are a group of transcriptional repressors, and PRDM1 (also known as BLIMP1) and PRDM14 have been reported to be essential for the formation of PGCs. In the present study, we performed a genome-wide retrieval in Mulinia lateralis and identified 11 putative PRDMs, all of which possessed an N-terminal PR domain. Expressional profiles revealed that all these prdm genes showed specifically high expression levels in the given stages, implying that all PRDMs played important roles during early development stages. Specifically, Ml-prdm1 was highly expressed at the gastrula stage, the key period when PGCs arise, and was specifically localized in the cytoplasm of two or three cells of blastula, gastrula, or trochophore larvae, matching the typical characteristics of PGCs. These results suggested that Ml-prdm1-positive cells may be PGCs and that Ml-prdm1 could be a candidate marker for tracing the formation of PGCs in M. lateralis. In addition, the expression profiles of Ml-prdm14 hinted that it may not be associated with PGCs of M. lateralis. The present study provides insights into the evolution of the PRDM family in mollusks and offers a better understanding of the formation of PGCs in mollusks.

Poster 116: Integrated scRNA-seq and spatial transcriptomics analysis uncovers distinct cellular populations and transcriptomes in human hip synovium between patients with femoroacetabular impingement and osteoarthritis.

Objectives: Osteoarthritis (OA) is a common degenerative joint disease marked by joint dysfunction and chronic pain. Previous studies have outlined a variety of complex factors associated with cartilage degradation, inflammation, and pathological changes in knee synovium. However, hip and knee OA have been established as immunologically distinct types of joint diseases due to unique cytokine profiles of synovial fluid, and data regarding hip OA synovium is scarce1. Evidence suggests that primary hip OA may result from cam-type femoroacetabular impingement (FAI). Thus, cam-type FAI has been considered a unique early-phase model of hip OA, allowing scientists to identify regulators implicated in hip OA development. Integrated single-cell RNA sequencing (scRNA-seq) and spatial transcriptomics (Spatial-seq) provides a powerful means to better understand unique genetic markers underlying signaling pathways and cellular interactions that may be involved in disease progression (i.e., FAI to hip OA). In this study, we hypothesize that FAI and hip OA patients have distinct transcriptomic profiles and signaling pathways in their synovial tissues. We aim to identify the underlying molecular mechanisms by using scRNA-seq, Spatial-seq, and bioinformatic analyses. Methods: De-identified human hip synovial tissues were harvested from patients diagnosed with cam- type FAI or with OA (and accompanying cam morphology) according to approved IRB protocols at the University of Rochester (UR). Tissue samples were digested for scRNA-seq and sectioned for Spatial-seq separately and submitted to UR Genomics Research Center (10X Genomics). Distinct cell populations and differentially expressed genes (DEGs) were identified using bioinformatic tools including Seurat2, Monocle3, RcisTarget4, and CellChat5 R packages. Functional analysis for identified cell subsets was annotated by Gene Ontology term6. Differentiation trajectories of non-hematopoietic cells and cell-cell crosstalk were also evaluated. Results: Unsupervised clustering of FAI and OA hip synovial cells identified four major conserved groups: NK cells, endothelial cells, myeloid cells, and non-hematopoietic cells. Sub-clustering of integrated FAI and OA non-hematopoietic cells further yielded 5 distinct cell types including lining, sublining and transitional fibroblast-like synoviocytes (FLS) as well as endothelial cells and pericytes (Fig. 1A). In comparison to FAI synovium, OA synovium exhibited 4.5-, 5- and 8.5-fold increase in IL11+/CD82+ sublining FLS (cluster 5), pericytes (cluster 7) and SPP1+/KDR+ capillary endothelial cells (cluster 9), respectively (Fig. 1B). To further investigate the origin of these populations, we performed pseudotime analysis and revealed that pericytes may differentiate from C34+/C3+/CXCL14+ sublining perivascular FLS (cluster 6) (Fig. 2A)6. To determine the putative transcription factors (TFs) regulating the differentiation process between these two cell populations, we identified that KLF4, SOX5 and CREB3L2 (CAMP Responsive Element Binding Protein 3 Like 2) are potential key regulators using DNA-binding motif analysis. We also observed an opposite trend in expression of KLF4 and CREB3L2, with the highest expression levels of KLF4 in sublining perivascular FLS (Fig. 2B). Importantly, by integrating scRNA-seq and Spatial-seq datasets, we were able to map spatial location of identified cell clusters in the hip joint synovium, allowing us to further interrogate cell-cell crosstalk. For example, we observed that pericytes are in close proximity to SPP1+/KDR+ capillary endothelial cells (cluster 9) and identified that VISFATIN- (ITGA5+ITGB1) and SPP1-CD44 are critical signaling pathways between these two cell populations in OA synovium (Fig. 3B). Conclusions: OA is the most prevalent degenerative joint disorder and is a primary cause of worldwide disability without any available disease modifying therapies. We believe that our findings will reveal key genetic markers related to hip OA progression and facilitate the development of targeted drug deliveries for future therapeutic applications. Our scRNA-seq analysis revealed unique transcriptomic patterns in the synovial cells between FAI and hip OA patients. Integration of non-hematopoietic cells revealed a significant increase in the number of pericytes and endothelial cells present in OA vs. FAI synovium. This is supported by the evidence of increased vasculature in OA, which in turn may promote enhanced infiltration of myeloid cells, cytokines and chemokines into the synovium, leading to an advanced joint inflammation and disease progression7. Furthermore, we identified that both pericytes and capillary endothelial cells can express visfatin (an adipokine) and may modulate synovial inflammation in OA via integrin-mediated signaling pathways8. This observation aligns with previous studies that highlight a significant contribution of endothelial cell-pericyte interactions to OA pathogenesis8. In addition, our finding of decreased expression of KLF4 in pericytes is consistent with previous reports showing that KLF4 inactivation enhances the expression of pericyte markers9. Our next step is to integrate our scRNA- seq and Spatial-seq datasets of non-hematopoietic cells with myeloid cells to further elucidate how altered cell-cell crosstalk and signaling pathways in synovium may lead to hip OA progression. Indeed, we have analyzed myeloid cells separately, and observed elevated accumulation of “hybrid” macrophages expressing pro- and anti-inflammatory markers (IL10+/IL1 +/IL1 +) in the hip OA compared to FAI synovium.