Negative Transcriptional Regulator Research Articles

Differential regulation of MYC expression by PKHD1/Pkhd1 in human and mouse kidneys: phenotypic implications for recessive polycystic kidney disease.

Autosomal recessive polycystic kidney disease (ARPKD; MIM#263200) is a severe, hereditary, hepato-renal fibrocystic disorder that leads to early childhood morbidity and mortality. Typical forms of ARPKD are caused by pathogenic variants in the PKHD1 gene, which encodes the fibrocystin/polyductin (FPC) protein. MYC overexpression has been proposed as a driver of renal cystogenesis, but little is known about MYC expression in recessive PKD. In the current study, we provide the first evidence that MYC is overexpressed in kidneys from ARPKD patients and confirm that MYC is upregulated in cystic kidneys from cpk mutant mice. In contrast, renal MYC expression levels were not altered in several Pkhd1 mutant mice that lack a significant cystic kidney phenotype. We leveraged previous observations that the carboxy-terminus of mouse FPC (FPC-CTD) is proteolytically cleaved through Notch-like processing, translocates to the nucleus, and binds to double stranded DNA, to examine whether the FPC-CTD plays a role in regulating MYC/Myc transcription. Using immunofluorescence, reporter gene assays, and ChIP, we demonstrate that both human and mouse FPC-CTD can localize to the nucleus, bind to the MYC/Myc P1 promoter, and activate MYC/Myc expression. Interestingly, we observed species-specific differences in FPC-CTD intracellular trafficking. Furthermore, our informatic analyses revealed limited sequence identity of FPC-CTD across vertebrate phyla and database queries identified temporal differences in PKHD1/Pkhd1 and CYS1/Cys1 expression patterns in mouse and human kidneys. Given that cystin, the Cys1 gene product, is a negative regulator of Myc transcription, these temporal differences in gene expression could contribute to the relative renoprotection from cystogenesis in Pkhd1-deficient mice. Taken together, our findings provide new mechanistic insights into differential mFPC-CTD and hFPC-CTD regulation of MYC expression in renal epithelial cells, which may illuminate the basis for the phenotypic disparities between human patients with PKHD1 pathogenic variants and Pkhd1-mutant mice.

Study on identification of a three-microRNA panel in serum for diagnosing neonatal early onset sepsis

Background Comparing with other diseases, early onset sepsis (EOS) is a global health concern in neonatal period for its high morbidity and mortality rates. In recent years, many studies have contributed to the figure out the expression patterns of circulating micro-RNAs (miRNAs) in different diseases and progressions, which could function as diagnostic biomarkers for EOS. The purpose of this study was to analyze the expression patterns of selected miRNAs and evaluate their diagnostic value for early detection and treatment. Methods This was a prospective cross-sectional study conducted from 1 July 2021 to 30 June 2022. We collected surplus peripheral blood and demographic statistics of septic neonates and non-infected neonates during the first 24 h after delivery and obtained 11 candidate miRNAs by literature screening. First, we extracted the candidate miRNAs from the serum of selected neonates and analyzed their expression levels, and then the receiver operating characteristic (ROC) curve was used to select the differentially expressed miRNAs. We analyzed their sensitivity and specificity and obtained the best diagnostic panel. Finally, with the help of differentially expressed miRNAs, we performed gene ontology (GO) enrichment and protein–protein interaction (PPI) analyses by their target genes. Results In patients with EOS, three miRNAs (mir-223-3p, mir-15a-5p, and mir-17-5p) in serum were significantly downregulated, and mir-146a-5p, mir-1-3p, and mir-16-5p were upregulated. The diagnostic value of these miRNAs (miR-15a-5p, AUC = 0.67; miR-223-3p, AUC = 0.72; miR-16-5p, AUC = 0.68; miR‐17‐5p, AUC = 0.70; miR-1-3p, AUC = 0.69; miR‐146a‐5p, AUC = 0.72) was moderate, and the diagnostic panel constructed by miR-15a-5p, miR-223-3p, and miR-16-5p possessed a comparatively higher diagnostic value (AUC = 0.85, sensitivity: 74.6%, specificity: 86%), indicating that their combined application may be a promising biomarker for clinical diagnosis of EOS. According to GO enrichment analysis, most proteins encoded by target genes were located in the cytosol as for cellular component (CC), for molecular function (MF), most proteins acted as regulators in protein binding, and for biological process (BP). Most genes function in positive or negative regulation of transcription from RNA polymerase II promoter, and the top 10 hub genes were CDKN1A, YAP1, CCNE1, CCND1, CKK6, ERBB4, CHEK1, DICER1, VEGFA, and APP by rank degree after PPI construction. Conclusions The three-miRNA panels (miR-15a-5p, miR-223-3p, and miR-16-5p) may be a novel noninvasive biological marker for EOS screening.

Genetic variations in G-quadruplex forming sequences affect the transcription of human disease-related genes.

Guanine-rich DNA strands can fold into non-canonical four-stranded secondary structures named G-quadruplexes (G4s). G4s folded in proximal promoter regions (PPR) are associated either with positive or negative transcriptional regulation. Given that single nucleotide variants (SNVs) affecting G4 folding (G4-Vars) may alter gene transcription, and that SNVs are associated with the human diseases' onset, we undertook a novel comprehensive study of the G4-Vars genome-wide (G4-variome) to find disease-associated G4-Vars located into PPRs. We developed a bioinformatics strategy to find disease-related SNVs located into PPRs simultaneously overlapping with putative G4-forming sequences (PQSs). We studied five G4-Vars disturbing in vitro the folding and stability of the G4s located into PPRs, which had been formerly associated with sporadic Alzheimer's disease (GRIN2B), a severe familiar coagulopathy (F7), atopic dermatitis (CSF2), myocardial infarction (SIRT1)and deafness (LHFPL5). Results obtained in cultured cells for these five G4-Vars suggest that the changes in the G4s affect the transcription, potentially contributing to the development of the mentioned diseases. Collectively, data reinforce the general idea that G4-Vars may impact on the different susceptibilities to human genetic diseases' onset, and could be novel targets for diagnosis and drug design in precision medicine.

Identification of SYT10 as a potential prognostic biomarker in esophageal cancer by comprehensive analysis of a mRNA-pseudogene/lncRNA-miRNA ceRNA network

BackgroundEsophageal carcinoma (ESCA) is often diagnosed at the advanced stages, has a poor survival rate and overall is one of the deadliest cancers world-wide. Recent studies have elaborated the significance of non-coding RNAs such as pseudogenes, long non-coding RNAs (lncRNAs) and microRNAs (miRNAs) in cancer progression. In this study, we constructed a four-component competing endogenous RNA (ceRNA) network in ECSA and suggested an RNA with prognostic potential. Materials and methodsExpression profiles of mRNAs, pseudogenes, lncRNAs and miRNAs were collected from The Cancer Genome Atlas (TCGA) database. A ceRNA network was then constructed based on the differentially-expressed RNAs (DERs). KEGG (Kyoto Encyclopedia of Genes and Genomes) and GO (Gene Ontology) functional analysis and PPI (Protein-Protein Interaction) network analysis was carried out on differentially-expressed (DE) mRNAs of the ceRNA network. Survival analysis was carried out on a selection of RNAs with the highest degree centrality ranks to discover potential prognostic biomarkers. ResultsA four-component ceRNA network with 529 nodes and 729 edges was constructed. The most significant GO biological process terms included positive regulation of transcription from RNA polymerase II promoter, negative regulation of transcription from RNA polymerase II promoter and positive regulation of transcription, DNA-templated. The analysis of KEGG pathways showed that DE mRNAs were significantly enriched in pathways such as pathways in cancer, PI3K-Akt signaling pathways and MicroRNAs in cancer. Among the RNAs that were found to be associated with survival, Synaptotagmin 10 (SYT10) had the highest hazard ratio and thus, proved to be a potential prognostic biomarker for ESCA. ConclusionOur study presented a four-component ceRNA network for ESCA, and identified RNA candidates that were associated with survival of ECSA. Further experimental evaluations and precise validation studies are needed for their clinical significance and roles in the progression of ESCA.

Vitamin D3 mitigates autoimmune inflammation caused by activation of myeloid dendritic cells in SLE.

Systemic lupus erythematosus (SLE) is an autoimmune disease in which defective T cells, immune complex deposition and other immune system alterations contribute to pathological changes of multiple organ systems. The vitamin D metabolite c is a critical immunomodulator playing pivotal roles in the immune system. Epidemiological evidence indicates that vitamin D deficiency is correlated with the severity of SLE. Our aim is to investigate the effects of 1,25(OH)2D3 (VitD3) on the activation of myeloid dendritic cells (mDCs) by autologous DNA-containing immune complex (DNA-ICs), and the effects of VitD3 on immune system balance during SLE. We purified DNA-ICs from the serum of SLE patients and isolated mDCs from normal subjects. In vitro studies showed that DNA-ICs were internalized and consumed by mDCs. VitD3 blocked the effects of DNA-ICs on RelB, IL-10 and TNF-α in mDCs. Further analysis indicated that DNA-ICs stimulated histone acetylation in the RelB promoter region, which was inhibited by VitD3. Knockdown of the histone deacetylase 3 gene (HDAC3) blocked these VitD3-mediated effects. Co-culture of mDCs and CD4+ T cells showed that VitD3 inhibited multiple processes mediated by DNA-ICs, including proliferation, downregulation of IL-10, TGF-β and upregulation of TNF-α. Moreover, VitD3 could also reverse the effects of DNA-IC-induced imbalance of CD4+ CD127- Foxp3+ T cells and CD4+ IL17+ T cells. Taken together, our results indicated that autologous DNA-ICs stimulate the activation of mDCs in the pathogenesis of SLE, and VitD3 inhibits this stimulatory effects of DNA-ICs by negative transcriptional regulation of RelB gene and maintaining the Treg/Th17 immune cell balance. These results suggest that vitamin D may have therapeutic value for the treatment of SLE.

A dual function for the chromatin organizer Special A-T rich Binding Protein 1 in B-lineage cells.

SATB1 (Special A-T rich Binding protein 1) is a cell type-specific factor that regulates the genetic network in developing T cells and neurons. In T cells, SATB1 is required for lineage commitment, VDJ recombination, development and maturation. Considering that its expression varies during B-cell differentiation, the involvement of SATB1 needs to be clarified in this lineage. Using a KO mouse model in which SATB1 was deleted from the pro-B-cell stage, we examined the consequences of SATB1 deletion in naive and activated B-cell subsets. Our model indicates first, unlike its essential function in T cells, that SATB1 is dispensable for B-cell development and the establishment of a broad IgH repertoire. Second, we show that SATB1 exhibits an ambivalent function in mature B cells, acting sequentially as a positive and negative regulator of Ig gene transcription in naive and activated cells, respectively. Third, our study indicates that the negative regulatory function of SATB1 in B cells extends to the germinal center response, in which this factor limits somatic hypermutation of Ig genes.

Complex origin, evolution, and diversification of non-canonically organized OVATE-OFP and OVATE-Like OFP gene pair across Embryophyta

Ovate Family Proteins (OFP) is a plant-specific gene family of negative transcriptional regulators. Till-date, a handful of in-silico studies have provided glimpses into family size, expansion patterns, and genic features across all major plant lineages. A major lacuna exists in understanding origin of organisation complexity of members such as those arranged in a head-to-head manner which may lead to transcriptional co-regulation via a common bi-directional promoter. To address this gap, we investigated the origin, organization and evolution of two head-to-head arranged gene pairs of homologs of AtOFP2-AtOFP17, and, AtOFP4-AtOFP20 across Archaeplastida. The ancestral forms of AtOFP2, AtOFP4, AtOFP17, and AtOFP20 are likely to have evolved in last common ancestors of Embryophyta (land plants) given their complete absence in Rhodophyta and Chlorophyta. The OFP gene family originated and expanded in Bryophyta, including protein variants with complete (OVATE-OFP) or partial (OVATE-Like OFP) OVATE domain; with head-to-head organization present only in Spermatophyta (gymnosperms and angiosperms). Ancestral State Reconstruction revealed the origin of head-to-head organized gene pair in gymnosperms, with both genes being OVATE-OFP (homologs of AtOFP2/4). Phylogenetic reconstruction and copy number analysis suggests the presence of a single copy of the head-to-head arranged pair of OFP2/4 (OVATE)-OFP17/20 (OVATE-Like) in all angiosperms except Brassicaceae, and a duplication event in last common ancestor of core Brassicaceae approximately 32–54 MYA leading to origin of AtOFP2-AtOFP17 and AtOFP4-AtOFP20 as paralogs. Synteny analysis of genomic regions harbouring homologs of AtOFP2-AtOFP17, AtOFP4-AtOFP20 and AtOFP2/4-AtOFP17/20 across angiosperms suggested ancestral nature of AtOFP2-AtOFP17 gene pair. The present study thus establishes the orthology and evolutionary history of two non-canonically organised gene pairs with variation in their OVATE domain. The non-canonical organisation, atleast in Brassicaceae, has the potential of generating complex transcriptional regulation mediated via a common bi-directional promoter. The study thus lays down a framework to understand evolution of gene and protein structure, transcriptional regulation and function across a phylogenetic lineage through comparative analyses.

Farnesoid X receptor represses human sulfotransferase 1A3 expression through direct binding to the promoter.

Human sulfotransferases 1A3 (SULT1A3) has received particular interest, due to their functions of catalyzing the sulfonation of numerous phenolic substrates, including bioactive endogenous molecules and therapeutic agents. However, the regulation of SULT1A3 expression and the underlying mechanism remain unclear. Here, we aimed to investigate the regulation effects of bile acid-activated farnesoid X receptor (FXR) on SULT1A3 expression, and to shed light on the mechanism thereof. Our results demonstrated that FXR agonists (CDCA and GW4064) significantly inhibit the expression of SULT1A3 at mRNA and protein levels. In addition, overexpression of FXR led to decrease in SULT1A3 expression and knockdown of FXR significantly induced the expression of SULT1A3 in protein and mRNA levels, confirming that FXR expression manifestly showed negative regulatory effect on basal SULT1A3 expression. Furthermore, a combination of luciferase reporter gene and CHIP assays showed that FXR repressed SULT1A3 transcription through direct binding to the region at base pair positions -664 to -654. In conclusion, this study for the first time confirmed FXR was a negative transcriptional regulator of human SULT1A3 enzyme.

Genome-wide characterization of NmrA-like proteins and the regulatory function of soybean GmNmrA6 in response to salt and oxidative stresses

NmrA (nitrogen metabolic regulation A)-like proteins are a group of negative transcriptional regulators involved in nitrogen metabolism. However, their biological functions in response to abiotic stress in oil crops still remain largely unknown. In this work, 42 putative NmrA-like protein encoding genes (GmNmrAs) were obtained from soybean genome. Bioinformatic analyses indicated that they were unevenly anchored on 13 chromosomes, highly conserved in gene structure and motif composition, and have gone through 27 segmental duplication events. Gene expression analyses showed that all GmNmrAs were ubiquitously expressed with overlapped but differential expression patterns. Biological function analysis in transgenic soybean exhibited that overexpression of GmNmrA6 promoted the growth and enhanced the tolerance of salt and oxidative stress in transgenic plants. Further ion content and antioxidant enzyme activity analysis revealed that transgenic soybean accumulated less sodium, hydrogen peroxide and malondialdehyde in the leaves, accompanied with an increased catalase activity and up-regulated the expression level of stress-related marker gene. The observations in this study revealed the evolutionary relationships and structure of GmNmrAs in soybean and the crucial function of GmNmrA6 in plant growth and the tolerance of abiotic stresses.

Estrogens receptors, nuclear coactivator 1 and ligand-dependent corepressor expression are altered early during induced ovarian follicular persistence in dairy cattle

Failure of ovulation can lead to follicular persistence, one of the main components of the pathogenesis of cystic ovarian disease (COD) in dairy cattle. Follicular persistence causes the permanence of a functional follicular structure in the ovary, which alters the cyclicity of the female and causes infertility. The aim of the present study was to evaluate the expression of estrogen receptors (ESR) 1 and 2, and the coregulatory proteins NCOA1, NRIP1 and LCOR by immunohistochemistry, in antral and preovulatory/persistent follicles in a model of follicular persistence induced by low levels of progesterone, to detect incipient changes during COD development, on the expected day of ovulation (P0) and after 5 (P5), 10 (P10) and 15 (P15) days of follicular persistence. Twenty-five Holstein cows were used, which were distributed in 5 groups: control group (n = 5), group P0 (n = 5), group P5 (n = 5), group P10 (n = 5), group P15 (n = 5). ESR1 expression was lower in antral follicles of the P5 (theca), P10 and P15 (theca and granulosa) groups relative to the control group (p < 0.05), and also lower in granulosa cells of persistent follicles of the P5, P10 and P15 groups than in dominant follicles of the control group (p < 0.05), without differences in theca cells. ESR2 expression showed no differences between groups. The ESR1:ESR2 balance favored ESR2 expression along the development of persistent follicles, as from 5 days of persistence (p < 0.05). NCOA1 expression was higher in granulosa cells of both antral and persistent follicles from the P0 group relative to the P5 and P10 groups, but showed no differences with the control and P15 groups (p < 0.05). Theca cells of antral and persistent follicles showed higher expression in the P0 and P15 groups in relation to the control, P5 and P10 groups (p < 0.05). No differences were detected for NRIP1 in antral, dominant and persistent follicles between groups. LCOR expression showed a decrease in granulosa cells of antral follicles from all persistence groups relative to the control group (p < 0.05). In theca cells, antral follicles of the P10 group showed lower LCOR expression than the control group (p < 0.05). LCOR expression was similar for dominant and persistent follicles. Considering that the ESR1:ESR2 balance favored ESR2 expression along the development of persistent follicles, as well as the decreased LCOR and NCOA1 expression, we may assume that, at the early stages of persistence, there is a negative regulation of ESR transcription. This coincides with the effects of estrogens through ESR on proliferation and apoptosis among other processes that favor follicular persistence. The results obtained provide relevant information in the knowledge of local events during the development of follicular persistence that could explain the failures in the reversion of the disease through hormonal treatments and the high recurrence rates reported for COD. In addition, it contributes to the study and identification of possible therapeutic targets, for the design of new treatments.

Systematic profiling of subtelomeric silencing factors in budding yeast.

Subtelomeric gene silencing is the negative transcriptional regulation of genes located close to telomeres. This phenomenon occurs in a variety of eukaryotes with salient physiological implications, such as cell adherence, virulence, immune-system escape, and aging. The process has been widely studied in the budding yeast Saccharomyces cerevisiae, where genes involved in this process have been identified mostly on a gene-by-gene basis. Here, we introduce a quantitative approach to study gene silencing, that couples the classical URA3 reporter with GFP monitoring, amenable to high-throughput flow cytometry analysis. This dual silencing reporter was integrated into several subtelomeric loci in the genome, where it showed a gradual range of silencing effects. By crossing strains with this dual reporter at the COS12 and YFR057W subtelomeric query loci with gene-deletion mutants, we carried out a large-scale forward screen for potential silencing factors. The approach was replicable and allowed accurate detection of expression changes. Results of our comprehensive screen suggest that the main players influencing subtelomeric silencing were previously known, but additional potential factors underlying chromatin conformation are involved. We validate and report the novel silencing factor LGE1, a protein with unknown molecular function required for histone H2B ubiquitination. Our strategy can be readily combined with other reporters and gene perturbation collections, making it a versatile tool to study gene silencing at a genome-wide scale.

Dyrk1a Phosphorylation of α-Synuclein Mediating Apoptosis of Dopaminergic Neurons in Parkinson's Disease.

To investigate the role of aberrant Dyrk1a expression in phosphorylation modification at the α-synuclein serine 129 (Ser129) site to analyze its molecular mechanism in mediating apoptosis of PD. The protein level of P-α-synuclein (Ser129), α-synuclein, Bcl-2, Bax, active caspase 3, GSK3β, PI3K, AKT, and cyclinD1 were detected. The mRNA transcript levels of Dyrk1a and DAT and protein levels of IL-1β, IL-6, COX-2, and TNF-α were detected. P-α-synuclein (Ser129), α-synuclein, Bax, active caspase 3, GSK3β, and cyclinD1 expressions were decreased in Dyrk1a-AAV-ShRNA (P < 0.05), and Bcl-2, AKT, and PI3K expressions were increased (P < 0.05). Increased TH protein expression was shown in Dyrk1a-AAV-ShRNA (P < 0.05). Dyrk1a mRNA was decreased in the Dyrk1a-AAV-ShRNA group (P < 0.05), and DAT mRNA was increased (P < 0.05). IL-1β, IL-6, COX-2, and TNF-α protein levels were decreased in Dyrk1al-AAV-Sh-RNA (P < 0.05). Transcriptome sequencing showed that Fam220a, which was expected to activate STAT family protein binding activity and participate in the negative regulation of transcription through RNA polymerase II and protein dephosphorylation showed differentially upregulated expression. The untargeted metabolome showed that the major compounds in the Dyrk1a-AAV-ShRNA group were hormones and transmission mediators and the most metabolism-related pathways. Fam220a showed differentially upregulated expression, and differentially expressed genes were enriched for the neuroactive ligand-receptor interaction, vascular smooth muscle contraction, and melanogenesis-related pathways. Abnormal Dyrk1a expression can affect α-synuclein phosphorylation modifications, and dyrk1a knockdown activates the PI3K/AKT pathway and reduces dopaminergic neuron apoptosis. It provides a theoretical basis for the group to further investigate the molecular mechanism.

PnMYB4 negatively modulates saponin biosynthesis in Panax notoginseng through interplay with PnMYB1.

Saponins are the main triterpenoid ingredients from Panax notoginseng, a well-known Chinese medicine, and are important sources for producing drugs to prevent and treat cerebrovascular and cardiovascular diseases. However, the transcriptional regulatory network of saponin biosynthesis in P. notoginseng is largely unknown. In the present study we demonstrated that one R2R3-MYB transcription factor, designated PnMYB4, acts as a repressor of saponin accumulation. Suppression of PnMYB4 in P. notoginseng calli significantly increased the saponin content and the expression level of saponin biosynthetic genes. PnMYB4 directly bound to the promoters of key saponin biosynthetic genes, including PnSS, PnSE, and PnDS, to repress saponin accumulation. PnMYB4 and the activator PnMYB1 could interacted with PnbHLH, which is a positive regulator of saponin biosynthesis, to modulate the biosynthesis of saponin. PnMYB4 competed with PnMYB1 for binding to PnbHLH, repressing activation of the promoters of saponin structural genes induced by the PnMYB1-PnbHLH complex. Our study reveals that a complex regulatory module of saponin biosynthesis is associated with positive and negative MYB transcriptional regulators and provides a theoretical basis for improving the content of saponins and efficacy of P. notoginseng.

LncRNA miR663AHG represses the development of colon cancer in a miR663a-dependent manner

The MIR663AHG gene encodes both miR663AHG and miR663a. While miR663a contributes to the defense of host cells against inflammation and inhibits colon cancer development, the biological function of lncRNA miR663AHG has not been previously reported. In this study, the subcellular localization of lncRNA miR663AHG was determined by RNA-FISH. miR663AHG and miR663a were measured by qRT-PCR. The effects of miR663AHG on the growth and metastasis of colon cancer cells were investigated in vitro and in vivo. CRISPR/Cas9, RNA pulldown, and other biological assays were used to explore the underlying mechanism of miR663AHG. We found that miR663AHG was mainly distributed in the nucleus of Caco2 and HCT116 cells and the cytoplasm of SW480 cells. The expression level of miR663AHG was positively correlated with the level of miR663a (r = 0.179, P = 0.015) and significantly downregulated in colon cancer tissues relative to paired normal tissues from 119 patients (P < 0.008). Colon cancers with low miR663AHG expression were associated with advanced pTNM stage (P = 0.021), lymph metastasis (P = 0.041), and shorter overall survival (hazard ratio = 2.026; P = 0.021). Experimentally, miR663AHG inhibited colon cancer cell proliferation, migration, and invasion. The growth of xenografts from RKO cells overexpressing miR663AHG was slower than that of xenografts from vector control cells in BALB/c nude mice (P = 0.007). Interestingly, either RNA-interfering or resveratrol-inducing expression changes of miR663AHG or miR663a can trigger negative feedback regulation of transcription of the MIR663AHG gene. Mechanistically, miR663AHG could bind to miR663a and its precursor pre-miR663a, and prevent the degradation of miR663a target mRNAs. Disruption of the negative feedback by knockout of the MIR663AHG promoter, exon-1, and pri-miR663A-coding sequence entirely blocked these effects of miR663AHG, which was restored in cells transfected with miR663a expression vector in rescue experiment. In conclusion, miR663AHG functions as a tumor suppressor that inhibits the development of colon cancer through its cis-binding to miR663a/pre-miR663a. The cross talk between miR663AHG and miR663a expression may play dominant roles in maintaining the functions of miR663AHG in colon cancer development.

Using lung-on-a-chip to characterize HIF-1α-dependent inflammatory responses during acute Streptococcus pneumoniaeinfection

Abstract Current models used to study Streptococcus pneumoniae (SP) infections do not account for complex organ microenvironments or species-specific signaling pathways. To address this, we created a vascularized 3D lung-on-a-chip (LoC) that better recapitulates the physiologic lung, including cellular components, immune cell recruitment, blood flow, and oxygen tension. Hypoxia inducible factor 1-alpha (HIF-1α) is a transcription factor known to activate inflammatory pathways during infectious diseases, but its role in acute SP infection remains unclear. LoC was used to validate HIF-1α upregulation and characterize its role in immune cell recruitment. Whole human blood with fluorescently-labeled immune cells were perfused in LoC +/− BAY 87-2243 (HIF-1α chemical inhibitor). HIF1α-dependent inflammatory mediators involved in SP infection were identified by scRNA-seq and confirmed at the protein level by Western blot and ELISA. Preliminary studies of LoC stimulated with SP demonstrated HIF-1α early target gene activation. LoC showed increased CD15 +-neutrophil recruitment from perfused whole blood in microvessels to tissue and airway during SP infection, partially mediated by HIF-1α. IL-17C was identified as the top differentially expressed gene activated by SP and 2D protein analysis showed increased IL-17C levels during HIF-1α knockdown, suggesting that HIF-1α is a negative regulator of IL-17C transcription. These preliminary findings suggest that HIF-1α mediates immune cell recruitment during acute SP infection and support the use of LoC as a pre-clinical human model that can identify key mediators during pneumonia.

RNA-DNA interactomes of three prokaryotes uncovered by proximity ligation

Proximity ligation approaches, which are widely used to study the spatial organization of the genome, also make it possible to reveal patterns of RNA-DNA interactions. Here, we use RedC, an RNA-DNA proximity ligation approach, to assess the distribution of major RNA types along the genomes of E. coli, B. subtilis, and thermophilic archaeon T. adornatum. We find that (i) messenger RNAs preferentially interact with their cognate genes and the genes located downstream in the same operon, which is consistent with polycistronic transcription; (ii) ribosomal RNAs preferentially interact with active protein-coding genes in both bacteria and archaea, indicating co-transcriptional translation; and (iii) 6S noncoding RNA, a negative regulator of bacterial transcription, is depleted from active genes in E. coli and B. subtilis. We conclude that the RedC data provide a rich resource for studying both transcription dynamics and the function of noncoding RNAs in microbial organisms.

Poly(ADP-ribose)-binding protein RCD1 is a plant PARylation reader regulated by Photoregulatory Protein Kinases

Poly(ADP-ribosyl)ation (PARylation) is a reversible post-translational protein modification that has profound regulatory functions in metabolism, development and immunity, and is conserved throughout the eukaryotic lineage. Contrary to metazoa, many components and mechanistic details of PARylation have remained unidentified in plants. Here we present the transcriptional co-regulator RADICAL-INDUCED CELL DEATH1 (RCD1) as a plant PAR-reader. RCD1 is a multidomain protein with intrinsically disordered regions (IDRs) separating its domains. We have reported earlier that RCD1 regulates plant development and stress-tolerance by interacting with numerous transcription factors (TFs) through its C-terminal RST domain. This study suggests that the N-terminal WWE and PARP-like domains, as well as the connecting IDR play an important regulatory role for RCD1 function. We show that RCD1 binds PAR in vitro via its WWE domain and that PAR-binding determines RCD1 localization to nuclear bodies (NBs) in vivo. Additionally, we found that RCD1 function and stability is controlled by Photoregulatory Protein Kinases (PPKs). PPKs localize with RCD1 in NBs and phosphorylate RCD1 at multiple sites affecting its stability. This work proposes a mechanism for negative transcriptional regulation in plants, in which RCD1 localizes to NBs, binds TFs with its RST domain and is degraded after phosphorylation by PPKs.

SCAP contributes to embryonic angiogenesis by negatively regulating KISS-1 expression in mice

Sterol regulatory element-binding protein (SREBP) cleavage-activating protein (SCAP) is indispensable in organ development because it maintains intracellular cholesterol homeostasis. The vessel is not widely conceived of as a cholesterol-sensitive tissue, so the specific role of SCAP in angiogenesis has not been paid attention to. As an important component of the vascular mesoderm, vascular smooth muscle cells (VSMCs) are widely involved in each step of angiogenesis. Here, we report for the first time that VSMC-specific ablation of SCAP inhibits VSMC proliferation and migration, interacting with endothelial cells (ECs), and finally causes defective embryonic angiogenesis in mice. Mechanistically, we demonstrated that SCAP ablation in VSMCs leads to the upregulation of KISS-1 protein, consequently resulting in suppressed activation of the MAPK/ERK signaling pathway and downregulation of matrix metalloproteinase 9 (MMP9) and vascular endothelial-derived growth factor (VEGF) expression to prevent angiogenesis. Importantly, we found that SCAP promotes the cleavage and nuclear translocation of SREBP2, which acts as a negative transcription regulator, regulating KISS-1 expression. Our findings suggest that SCAP contributes to embryonic angiogenesis by negatively regulating KISS-1 expression in mice and provide a new point of view for therapeutic targets of vascular development.

Abstract 2605: LOXL3 knock out affects pathways which involve cytoskeleton regulation, proliferation and apoptosis in glioblastoma cells

Abstract Lysyl oxidase-like 3 (LOXL3), belonging to the lysyl oxidase family of copper-dependent amino oxidase, is responsible for the crosslinking of collagen and elastin. In tumors, the presence of LOXL3 has been associated with genomic stability, cell proliferation, and metastasis. In silico analysis has shown that glioblastoma was among the tumors with the highest LOXL3 expression levels. Moreover, LOXL3 expression increased with malignancy grade amongst diffusely infiltrative astrocytomas (from grade 2 to 4). In the present work, CRISPR-Cas9 gene editing was used to knock out LOXL3 in the human glioma U87MG cell line to evaluate the role of LOXL3 in glioblastoma. Analyses of protein expression level and genomic mutation mediated by CRISPR-Cas9 by two different sgRNA (denominated sgRNA1 and 2) were performed and the knock out efficiency and genome editing were confirmed. Transcriptome analysis of these cells revealed a downregulation of genes that coded for proteins involved in microtubule organization, cell division, and vesicle trafficking. On the other hand, upregulated genes when LOXL3 was knocked out in U87MG cells were involved in actin cytoskeleton, negative regulation of gene transcription, including chromatin remodeling, and cell-cell adhesion. Functional in vitro assays showed that LOXL3 knocked out cells presented decreased cell proliferation due to longer cell cycles with increased G1 and G2/M checkpoints compared to control cells. Additionally, LOXL3 knocked out U87MG cells presented a higher percentage of cells in the early phase of apoptosis with an increment after temozolomide treatment. Our results suggest an important role of LOXL3 in glioblastoma possibly mediated by cytoskeleton regulation, affecting cell proliferation, cell cycle, and cell death. These results reinforced the importance of LOXL3 in tumors, especially in glioblastoma, highlighting it as a potential therapeutic target. Citation Format: Talita S. Laurentino, Roseli S. Soares, Antonio M. Lerario, Suely K. Marie, Sueli Mieko Oba-Shinjo. LOXL3 knock out affects pathways which involve cytoskeleton regulation, proliferation and apoptosis in glioblastoma cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2605.

Construction and analysis of protein–protein interaction networks based on nuclear proteomics data of the desiccation-tolerant Xerophyta schlechteri leaves subjected to dehydration stress

ABSTRACT In order to understand the mechanism of desiccation tolerance in Xerophyta schlechteri, we carried out an in silico study to identify hub proteins and functional modules in the nuclear proteome of the leaves. Protein–protein interaction networks were constructed and analyzed from proteome data obtained from Abdalla and Rafudeen. We constructed networks in Cytoscape using the GeneMania software and analyzed them using a Network Analyzer. Functional enrichment analysis of key proteins in the respective networks was done using GeneMania network enrichment analysis, and GO (Gene Ontology) terms were summarized using REViGO. Also, community analysis of differentially expressed proteins was conducted using the Cytoscape Apps, GeneMania and ClusterMaker. Functional modules associated with the communities were identified using an online tool, ShinyGO. We identified HSP 70–2 as the super-hub protein among the up-regulated proteins. On the other hand, 40S ribosomal protein S2–3 (a protein added by GeneMANIA) was identified as a super-hub protein associated with the down-regulated proteins. For up-regulated proteins, the enriched biological process terms were those associated with chromatin organization and negative regulation of transcription. In the down-regulated protein-set, terms associated with protein synthesis were significantly enriched. Community analysis identified three functional modules that can be categorized as chromatin organization, anti-oxidant activity and metabolic processes.