Promoter Fragment Research Articles

Ectopic MYBL2-Mediated Regulation of Androglobin Gene Expression.

Androglobin (ADGB) is a highly conserved and recently identified member of the globin superfamily. Although previous studies revealed a link to ciliogenesis and an involvement in murine spermatogenesis, its physiological function remains mostly unknown. Apart from FOXJ1-dependent regulation, the transcriptional landscape of the ADGB gene remains unexplored. We, therefore, aimed to obtain further insights into regulatory mechanisms governing ADGB expression. To this end, changes in ADGB promoter activity were examined using luciferase reporter gene assays in the presence of a set of more than 475 different exogenous transcription factors. MYBL2 and PITX2 resulted in the most pronounced increase in ADGB promoter-dependent luciferase activity. Subsequent truncation strategies of the ADGB promoter fragment narrowed down the potential MYBL2 and PITX2 binding sites within the proximal ADGB promoter. Furthermore, MYBL2 binding sites on the ADGB promoter were further validated via a guide RNA-mediated interference strategy using reporter assays. Chromatin immunoprecipitation (ChIP)-qPCR experiments illustrated enrichment of the endogenous ADGB promoter region upon MYBL2 and PITX2 overexpression. Consistently, ectopic MYBL2 expression induced endogenous ADGB mRNA levels. Collectively, our data indicate that ADGB is strongly regulated at the transcriptional level and might have functions beyond ciliogenesis.

Sex dimorphic response to osteocyte miR21 deletion in murine calvaria bone as determined by RNAseq analysis.

Low levels of microRNA (miR) 21 may explain the higher osteocyte apoptosis with Cx43-deficient and aged female mice. However, miR21 exerts a sex-divergent role in osteocytes, regulating bone mass and architecture through non-cell autonomous effects on osteoblasts and osteoclasts, via sex-specific regulation of osteocyte cytokine production. miR21 deficiency improves bone strength in females, and, to a higher extent, in male miR21-deficient mice. To understand the molecular basis for the effects of miR21 deletion, mRNA was isolated from miR21fl/fl (controls) or miR21-deficient (by deletion in cells expressing Cre recombinase under the control of the 8kb fragment of the DMP1 promoter: miR21ΔOt mice). miR21 was 50% lower in miR21ΔOt whole calvaria bone compared to control mice of the corresponding sex. RNAseq was performed in 4 samples/sex and genotype. There were 152 genes with <.05 P-value and >1 absolute log2 fold change in the male data analysis, and expression of most genes was higher in the miR21fl/fl group. Two of the genes, Actn3 and Myh4, had a false discovery rate < 0.1. Gene enrichment analysis of significant genes on both KEGG pathways and gene ontology (GO) gene sets shows that the significant genes were enriched in muscle contraction. Some muscle-related genes like Actn3 were included in multiple significant pathways. For females, only 65 genes had P-value <.05 and >1 absolute log2 fold change. Yet, no significant KEGG or GO pathways, including ≥5 significant genes, were seen, and no overlap of significant genes was found between male and female samples. Therefore, deletion of miR21 has a stronger effect on male transcriptome in calvaria, compared to females. Further, no enrichment of any pathway was detected in female samples. Thus, either there are no differences between 2 groups in female or the effect size is small, and a larger sample size is needed to uncover miR21-dependent differences.

Two IFNa3s mediate the regulation of IRF9 in the process of infection with Streptococcus iniae in yellowfin seabream, Acanthopagrus latus (Hottuyn, 1782)

IRF9 can play an antibacterial role by regulating the type I interferon (IFN) pathway. Streptococcus iniae can cause many deaths of yellowfin seabream, Acanthopagrus latus in pond farming. Nevertheless, the regulatory mechanism of type I IFN signalling by A. latus IRF9 (AlIRF9) against S. iniae remains elucidated. In our study, AlIRF9 has a total cDNA length of 3200 bp and contains a 1311 bp ORF encoding a presumed 436 amino acids (aa). The genomic DNA sequence of AlIRF9 has nine exons and eight introns, and AlIRF9 was expressed in various tissues, containing the stomach, spleen, brain, skin, and liver, among which the highest expression was in the spleen. Moreover, AlIRF9 transcriptions in the spleen, liver, kidney, and brain were increased by S. iniae infection. By overexpression of AlIRF9, AlIRF9 is shown as a whole-cell distribution, mainly concentrated in the nucleus. Moreover, the promoter fragments of −415 to +192 bp and −311 to +196 bp were regarded as core sequences from two AlIFNa3s. The point mutation analyses verified that AlIFNa3 and AlIFNa3-like transcriptions are dependent on both M3 sites with AlIRF9. In addition, AlIRF9 could greatly reduce two AlIFNa3s and interferon signalling factors expressions. These results showed that in A. latus, both AlIFNa3 and AlIFNa3-like can mediate the regulation of AlIRF9 in the process of infection with S. iniae.

Identification of transcription factor genes responsive to MeJA and characterization of a LaMYC2 transcription factor positively regulates lycorine biosynthesis in Lycoris aurea

Jasmonates (JAs) are among the main phytohormones, regulating plant growth and development, stress responses, and secondary metabolism. As the major regulator of the JA signaling pathway, MYC2 also plays an important role in plant secondary metabolite synthesis and accumulation. In this study, we performed a comparative transcriptome analysis of Lycoris aurea seedlings subjected to methyl jasmonate (MeJA) at different treatment times. A total of 31,193 differentially expressed genes (DEGs) were identified by RNA sequencing. Among them, 732 differentially expressed transcription factors (TFs) comprising 51 TF families were characterized. The most abundant TF family was WRKY proteins (80), followed by AP2/ERF-EFR (67), MYB (59), bHLH (52), and NAC protein (49) families. Subsequently, by calculating the Pearson's correlation coefficient (PCC) between the expression level of TF DEGs and the lycorine contents, 41 potential TF genes (|PCC| >0.8) involved in lycorine accumulation were identified, including 36 positive regulators and 5 negative regulators. Moreover, a MeJA-inducible MYC2 gene (namely LaMYC2) was cloned on the basis of transcriptome sequencing. Bioinformatic analyses revealed that LaMYC2 proteins contain the bHLH-MYC_N domain and bHLH-AtAIB_like motif. LaMYC2 protein is localized in the cell nucleus, and can partly rescue the MYC2 mutant in Arabidopsis thaliana. LaMYC2 protein could interact with most LaJAZs (especially LaJAZ3 and LaJAZ4) identified previously. Transient overexpression of LaMYC2 increased lycorine contents in L. aurea petals, which might be associated with the activation of the transcript levels of tyrosine decarboxylase (TYDC) and phenylalanine ammonia lyase (PAL) genes. By isolating the 887-bp-length promoter fragment upstream of the start codon (ATG) of LaTYDC, we found several different types of E-box motifs (CANNTG) in the promoter of LaTYDC. Further study demonstrated that LaMYC2 was indeed able to bind the E-box (CACATG) present in the LaTYDC promoter, verifying that the pathway genes involved in lycorine biosynthesis could be regulated by LaMYC2, and that LaMYC2 has positive roles in the regulation of lycorine biosynthesis. These findings demonstrate that LaMYC2 is a positive regulator of lycorine biosynthesis and may facilitate further functional research of the LaMYC2 gene, especially its potential regulatory roles in Amaryllidaceae alkaloid accumulation in L. aurea.

Plant-specific environmental and developmental signals regulate the mismatch repair protein MSH6 in Arabidopsis thaliana

The DNA mismatch repair (MMR) is a postreplicative system that guarantees genomic stability by correcting mispaired and unpaired nucleotides. In eukaryotic nuclei, MMR is initiated by the binding of heterodimeric MutS homologue (MSH) complexes to the DNA error or lesion. Among these proteins, MSH2-MSH6 is the most abundant heterodimer. Even though the MMR mechanism and proteins are highly conserved throughout evolution, physiological differences between species can lead to different regulatory features. Here, we investigated how light, sugar, and/or hormones modulate Arabidopsis thaliana MSH6 expression pattern. We first characterized the promoter region of MSH6. Phylogenetic shadowing revealed three highly conserved regions. These regions were analyzed by the generation of deletion constructs of the MSH6 full-length promoter fused to the β-glucuronidase (GUS) gene. Combined, our in silico and genetic analyses revealed that a 121-bp promoter fragment was necessary for MSH6 expression and contained potential cis-acting elements involved in light- and hormone-responsive gene expression. Accordingly, light exposure or sugar treatment of four-day old A. thaliana seedlings triggered an upregulation of MSH6 in shoot and root apical meristems. Appropriately, MSH6 was also induced by the stem cell inducer WUSCHEL. Further, the stimulatory effect of light was dependent on the presence of phyA. In addition, treatment of seedlings with auxin or cytokinin also caused an upregulation of MSH6 under darkness. Consistent with auxin signals, MSH6 expression was suppressed in the GATA23 RNAi line compared with the wild type. Our results provide evidence that endogenous factors and environmental signals controlling plant growth and development regulate the MSH6 protein in A. thaliana.

MiR-135b Aggravates Fusobacterium nucleatum-Induced Cisplatin Resistance in Colorectal Cancer by Targeting KLF13.

Cisplatin resistance is the main cause of colorectal cancer (CRC) treatment failure, and the cause has been reported to be related to Fusobacterium nucleatum (Fn) infection. In this study, we explored the role of Fn in regulating cisplatin resistance of CRC cells and its underlying mechanism involved. The mRNA and protein expressions were examined by qRT-PCR and western blot. Cell proliferation and cell apoptosis were assessed using CCK8 and flow cytometry assays, respectively. Dual-luciferase reporter gene assay was adopted to analyze the molecular interactions. Herein, our results revealed that Fn abundance and miR-135b expression were markedly elevated in CRC tissues, with a favorable association between the two. Moreover, Fn infection could increase miR-135b expression via a concentration-dependent manner, and it also enhanced cell proliferation but reduced apoptosis and cisplatin sensitivity by upregulating miR-135b. Moreover, KLF13 was proved as a downstream target of miR-135b, of which overexpression greatly diminished the promoting effect of miR-135b or Fn-mediated cisplatin resistance in CRC cells. In addition, it was observed that upstream 2.5kb fragment of miR-135b promoter could be interacted by β-catenin/TCF4 complex, which was proved as an effector signaling of Fn. LF3, a blocker of β-catenin/TCF4 complex, was confirmed to diminish the promoting role of Fn on miR-135b expression. Thus, it could be concluded that Fn activated miR-135b expression through TCF4/β-catenin complex, thereby inhibiting KLF13 expression and promoting cisplatin resistance in CRC.

Functional Analysis of the HbREF1 Promoter from Hevea brasiliensis and Its Response to Phytohormones

The rubber elongation factor (REF) is the most abundant protein in the latex of Hevea brasiliensis, which is closely related to natural rubber biosynthesis. In order to gain a deeper understanding of the transcriptional regulation mechanism of HbREF1, a 1758 bp genomic DNA fragment of the HbREF1 promoter was isolated. Promoter sequence analysis revealed several transcription factor binding sites in the HbREF1 promoter, such as bZIP, bHLH, EIL, AP2/ERF, MYB, and Trihelix. To assess the promoter activity, a series of HbREF1 promoter deletion derivatives were created and fused with firefly luciferase (LUC). The LUC image demonstrated that all of the HbREF1 promoters exhibited transcriptional activity. Furthermore, the assay revealed the presence of multiple regulatory elements within the promoter region that negatively regulate the transcriptional activity. Subsequent analysis of the transcriptional activity following treatment with phytohormones identified an ABA-responsive element located between −583 bp and −200 bp, an ET-responsive element between −718 bp and −583 bp, a JA-responsive element between −1758 bp and −1300 bp, and a SA-responsive element between −1300 bp and −718 bp. These results were largely consistent with the predictions of cis-acting elements. This study has established significant groundwork for future investigations into the regulatory mechanism of HbREF1.

Functional Characterization of CsF3Ha and Its Promoter in Response to Visible Light and Plant Growth Regulators in the Tea Plant.

Flavanone 3-hydroxylase (F3H) catalyzes trihydroxyflavanone formation into dihydroflavonols in the anthocyanin biosynthesis pathway, serving as precursors for anthocyanin synthesis. To investigate the CsF3Ha promoter's regulation in the 'Zijuan' tea plant, we cloned the CsF3Ha gene from this plant. It was up-regulated under various visible light conditions (blue, red, and ultraviolet (UV)) and using plant growth regulators (PGRs), including abscisic acid (ABA), gibberellic acid (GA3), salicylic acid (SA), ethephon, and methyl jasmonate (MeJA). The 1691 bp promoter sequence was cloned. The full-length promoter P1 (1691 bp) and its two deletion derivatives, P2 (890 bp) and P3 (467 bp), were fused with the β-glucuronidase (GUS) reporter gene, and were introduced into tobacco via Agrobacterium-mediated transformation. GUS staining, activity analysis, and relative expression showed that visible light and PGRs responded to promoter fragments. The anthocyanin content analysis revealed a significant increase due to visible light and PGRs. These findings suggest that diverse treatments indirectly enhance anthocyanin accumulation in 'Zijuan' tea plant leaves, establishing a foundation for further research on CsF3Ha promoter activity and its regulatory role in anthocyanin accumulation.

Alteration of the -35 and -10 sequences and deletion the upstream sequence of the -35 region of the promoter A1 of the phage T7 in dsDNA confirm the contribution of non-specific interactions with E. coli RNA polymerase to the transcription initiation process.

Transcription initiation is a multi-step process, in which the RNA polymerase holoenzyme binds to the specific promoter sequences to form a closed complex, which, through intermediate stages, isomerizes into an open complex capable of initiating the productive phase of transcription. The aim of this work was to determine the contribution of the -10 and -35 regions of the promoter, as well as the role of non-specific interactions, in the binding of RNA polymerase and the formation of an active initiation complex capable of transcription. Therefore, fragments of promoter DNA, derived from the strong promoter A1 of the phage T7, containing completely and partially altered elements -35 and -10, and devoid of an upstream region, were constructed using genetic engineering methods. Functional analyses of modified promoter fragments were carried out, checking their ability to form binary complexes with Escherichia coli RNA polymerase (RNAP) and the efficiency of converting binary complexes into triple complexes characteristic of the productive phase of transcription. The obtained results suggest that, in relation to the A1 promoter of the T7 phage, the most important role of the -35 region is carrying the open complex through the next phases of transcription initiation. The weakening of specific impacts within the region -35 is a reason for the defect associated with the transformation of the open complex, formed by a DNA fragment containing the completely altered -35 region, into elongation and the impairment of RNA synthesis. This leads to breaking contacts with the RNA polymerase holoenzyme, and destabilization and disintegration of the complex in the initial phase of productive transcription. This confirms the hypothesis of the so-called stressed intermediate state associated with the stage of transition from the open complex to the elongation complex. The experiments carried out in this work confirm also that the process of promoter localization and recognition, as well as the formation of binary complexes, is sequential in nature, and that the region located upstream of the -35 hexamer, and the hexamer itself, plays here an additive role.

PRRG4 regulates mitochondrial function and promotes migratory behaviors of breast cancer cells through the Src-STAT3-POLG axis

BackgroundBreast cancer is the leading cause of cancer death for women worldwide. Most of the breast cancer death are due to disease recurrence and metastasis. Increasingly accumulating evidence indicates that mitochondria play key roles in cancer progression and metastasis. Our recent study revealed that transmembrane protein PRRG4 promotes the metastasis of breast cancer. However, it is not clear whether PRRG4 can affect the migration and invasion of breast cancer cells through regulating mitochondria function.MethodsRNA-seq analyses were performed on breast cancer cells expressing control and PRRG4 shRNAs. Quantitative PCR analysis and measurements of mitochondrial ATP content and oxygen consumption were carried out to explore the roles of PRRG4 in regulating mitochondrial function. Luciferase reporter plasmids containing different lengths of promoter fragments were constructed. Luciferase activities in breast cancer cells transiently transfected with these reporter plasmids were analyzed to examine the effects of PRRG4 overexpression on promoter activity. Transwell assays were performed to determine the effects of PRRG4-regulated pathway on migratory behaviors of breast cancer cells.ResultsAnalysis of the RNA-seq data revealed that PRRG4 knockdown decreased the transcript levels of all the mitochondrial protein-encoding genes. Subsequently, studies with PRRG4 knockdown and overexpression showed that PRRG4 expression increased mitochondrial DNA (mtDNA) content. Mechanistically, PRRG4 via Src activated STAT3 in breast cancer cells. Activated STAT3 in turn promoted the transcription of mtDNA polymerase POLG through a STAT3 DNA binding site present in the POLG promoter region, and increased mtDNA content as well as mitochondrial ATP production and oxygen consumption. In addition, PRRG4-mediated activation of STAT3 also enhanced filopodia formation, migration, and invasion of breast cancer cells. Moreover, PRRG4 elevated migratory behaviors and mitochondrial function of breast cancer cells through POLG.ConclusionOur results indicate that PRRG4 via the Src-STAT3-POLG axis enhances mitochondrial function and promotes migratory behaviors of breast cancer cells.

CRISPRi-Mediated Treatment of Dominant Rhodopsin-Associated Retinitis Pigmentosa.

Rhodopsin (RHO) mutations such as Pro23His are the leading cause of dominantly inherited retinitis pigmentosa in North America. As with other dominant retinal dystrophies, these mutations lead to production of a toxic protein product, and treatment will require knockdown of the mutant allele. The purpose of this study was to develop a CRISPR-Cas9-mediated transcriptional repression strategy using catalytically inactive Staphylococcus aureus Cas9 (dCas9) fused to the Krüppel-associated box (KRAB) transcriptional repressor domain. Using a reporter construct carrying green fluorescent protein (GFP) cloned downstream of the RHO promoter fragment (nucleotides -1403 to +73), we demonstrate a ∼74-84% reduction in RHO promoter activity in RHOpCRISPRi-treated versus plasmid-only controls. After subretinal transduction of human retinal explants and transgenic Pro23His mutant pigs, significant knockdown of rhodopsin protein was achieved. Suppression of mutant transgene in vivo was associated with a reduction in endoplasmic reticulum (ER) stress and apoptosis markers and preservation of photoreceptor cell layer thickness.

Phytohormones as Regulators of Mitochondrial Gene Expression in Arabidopsis thaliana

The coordination of activities between nuclei and organelles in plant cells involves information exchange, in which phytohormones may play essential roles. Therefore, the dissection of the mechanisms of hormone-related integration between phytohormones and mitochondria is an important and challenging task. Here, we found that inputs from multiple hormones may cause changes in the transcript accumulation of mitochondrial-encoded genes and nuclear genes encoding mitochondrial (mt) proteins. In particular, treatments with exogenous hormones induced changes in the GUS expression in the reporter line possessing a 5′-deletion fragment of the RPOTmp promoter. These changes corresponded in part to the up- or downregulation of RPOTmp in wild-type plants, which affects the transcription of mt-encoded genes, implying that the promoter fragment of the RPOTmp gene is functionally involved in the responses to IAA (indole-3-acetic acid), ACC (1-aminocyclopropane-1-carboxylic acid), and ABA (abscisic acid). Hormone-dependent modulations in the expression of mt-encoded genes can also be mediated through mitochondrial transcription termination factors 15, 17, and 18 of the mTERF family and genes for tetratricopeptide repeat proteins that are coexpressed with mTERF genes, in addition to SWIB5 encoding a mitochondrial SWI/SNF (nucleosome remodeling) complex B protein. These genes specifically respond to hormone treatment, displaying both negative and positive regulation in a context-dependent manner. According to bioinformatic resources, their promoter region possesses putative cis-acting elements involved in responses to phytohormones. Alternatively, the hormone-related transcriptional activity of these genes may be modulated indirectly, which is especially relevant for brassinosteroids (BS). In general, the results of this study indicate that hormones are essential mediators that are able to cause alterations in the transcript accumulation of mt-related nuclear genes, which, in turn, trigger the expression of mt genes.

Molecular Cloning and Functional Characterization of the 5′ Regulatory Region of the SLC11A1 Gene from Yaks

The solute transport protein family 11 A1 (SLC11A1), also recognized as natural resistance-associated macrophage protein 1 (NRAMP1), represents a transmembrane protein encoded by the SLC11A1 gene. A variety of prior investigations have illuminated its involvement in conferring resistance or susceptibility to bacterial agents, positioning it as a promising candidate gene for breeding disease-resistant animals. Yaks (Bos grunniens), renowned inhabitants of the Qinghai-Tibet Plateau in China, stand as robust ruminants distinguished by their adaptability and formidable disease resistance. Notwithstanding these unique traits, there is scant literature on the SLC11A1 gene in the yak population. Our inquiry commences with the cloning of the 5′ regulatory region sequence of the Zhongdian yak SLC11A1 gene. We employ bioinformatics tools to identify transcription factor binding sites, delineating pivotal elements like enhancers and cis-acting elements. To ascertain the promoter activity of this region, we amplify four distinct promoter fragments within the 5′ regulatory region of the yak SLC11A1 gene. Subsequently, we design a luciferase reporter gene vector containing four site-specific deletion mutations and perform transient transfection experiments. Through these experiments, we measure and compare the activity of disparate gene fragments located within the 5′ regulatory region, revealing regions bearing promoter functionality and discerning key regulatory elements. Our findings validate the promoter functionality of the 5′ regulatory region, offering preliminary insights into the core and principal regulatory segments of this promoter. Notably, we identified single nucleotide polymorphisms (SNPs) that may be associated with important regulatory elements such as NF-1 and NF-1/L. This study provides a theoretical framework for in-depth research on the function and expression regulation mechanism of the yak SLC11A1 gene.

High-throughput screening system of citrus bacterial canker-associated transcription factors and its application to the regulation of citrus canker resistance

One of the main diseases that adversely impacts the global citrus industry is citrus bacterial canker (CBC), caused by the bacteria Xanthomonas citri subsp. citri (Xcc). Response to CBC is a complex process, with both protein-DNA as well as protein–protein interactions for the regulatory network. To detect such interactions in CBC resistant regulation, a citrus high-throughput screening system with 203 CBC-inducible transcription factors (TFs), were developed. Screening the upstream regulators of target by yeast-one hybrid (Y1H) methods was also performed. A regulatory module of CBC resistance was identified based on this system. One TF (CsDOF5.8) was explored due to its interactions with the 1-kb promoter fragment of CsPrx25, a resistant gene of CBC involved in reactive oxygen species (ROS) homeostasis regulation. Electrophoretic mobility shift assay (EMSA), dual-LUC assays, as well as transient overexpression of CsDOF5.8, further validated the interactions and transcriptional regulation. The CsDOF5.8–CsPrx25 promoter interaction revealed a complex pathway that governs the regulation of CBC resistance via H2O2 homeostasis. The high-throughput Y1H/Y2H screening system could be an efficient tool for studying regulatory pathways or network of CBC resistance regulation. In addition, it could highlight the potential of these candidate genes as targets for efforts to breed CBC-resistant citrus varieties.

Identifying cis-Acting Elements Associated with the High Activity and Endosperm Specificity of the Promoters of Genes Encoding Low-Molecular-Weight Glutenin Subunits in Common Wheat (Triticum aestivum).

Low-molecular-weight glutenin subunits (LMW-GSs) associated with bread-baking quality and flour nutrient quality accumulate in endosperms of common wheat and related species. However, the mechanism underlying the expression regulation of genes encoding LMW-GSs has not been fully elucidated. In this study, we identified LMW-D2 and LMW-D7, which are highly and weakly expressed, respectively, via the analysis of RNA-sequencing data of Chinese Spring wheat and wheat transgenic lines transformed with 5' deletion promoter fragments and GUS fusion constructs. The 605-bp fragment upstream of the LMW-D2 start codon could drive high levels of GUS expression in the endosperm. The truncated endosperm box located at the -300 site resulted in the loss of LMW-D2 promoter activity, and a single-nucleotide polymorphism on the GCN4 motif was closely related to the expression of LMW-GSs. TCT and TGACG motifs, as well as the others located on the 5' distal end, might also be involved in the transcription regulation of LMW-GSs. In transgenic lines, fusion proteins of LMW-GS and GUS were deposited into protein bodies. Our findings provide new insights into the mechanism underlying the transcription regulation of LMW-GSs and will contribute to the development of wheat endosperm as a bioreactor for the production of nutraceuticals, antibodies, vaccines, and medicinal proteins.

Targeting G3BP2 Sensitizes Acute Myeloid Leukemia to Venetoclax Treatment Via ELF1-MCL1 Axis

Venetoclax-based combination therapies have emerged as promising treatments for elderly acute myeloid leukemia (AML) patients. However, venetoclax resistance renders the treatment ineffective, mainly due to the upregulation of MCL1. Thus, new venetoclax-based combination therapy is under development to circumvent venetoclax resistance. The Ras-GTPase-activating protein SH3-domain binding protein 2 (G3BP2) is upgraded in several solid tumors. Targeting G3BP2 re-sensitized tumor cells to chemotherapy. To date, whether targeting G3BP2 sensitizes venetoclax treatment in AML and its role in leukemogenesis has not been defined. To this end, the primary AML cells and AML cells were collected and cultured with compound C108 (G3BP2 inhibitor) and venetoclax alone or both. The result showed that co-culture with compound C108 and venetoclax decreased the cell viability in primary AML and AML cells, indicating the potent synergy of G3BP2 inhibitor and venetoclax. The Quantitative real-time PCR (RT-qPCR) showed that G3BP2 was overexpressed in AML patients and was associated with poor prognosis. Knockdown (KD) of G3BP2 decreased proliferation and induced apoptosis in AML cells. In addition, G3BP2 inhibition impeded the tumorigenicity in the subcutaneous AML xenograft tumor model. These data demonstrated that G3BP2 sensitized AML to venetoclax treatment and is critical in AML development. The RNA-seq analysis was performed to investigate the role of G3BP2 in venetoclax resistance and leukemogenesis. The results showed that differentially expressed genes (DEG) were enriched in apoptotic processes when G3BP2 were downregulated. The immunoblotting results indicated that G3BP2 knockdown decreased the expression of MCL1 and BCL2, and vice versa. Moreover, we found that the transcription factor ELF1 was positively correlated with G3BP2 by the immunoblotting and RNA-seq analysis. The result was further validated by the Pearson correlation analysis based on the online database. The expression of ELF1 was decreased when G3BP2 were downregulated. Thus, we concluded that G3BP2 is an upstream gene of ELF1 and MCL1. To further investigate the relationship among G3BP2, ELF1, and MCL1, the RNA binding protein immunoprecipitation assay (RIP) and mRNA stability analysis was performed. The results suggested that G3BP2 interacted with the ELF1 mRNA to enhance its stability. We employed bioinformatics analysis via the JASPAR database and RPISeq tools and found ELF1 may facilitate the transcription of MCL1, which was validated in the following study. The expression of MCL1 was restored in the G3BP2 KD cells when ELF1 was overexpressed. We subsequently performed CHIP-qPCR, dual-luciferase reporter gene assays with truncated MCL1 promoter fragments. A 576bp of the binding region upstream of MCL1 promoter was defined, indicating that ELF1 regulated MCL1 expression by modulating its transcription activity. Taken together, our data suggested that targeting G3BP2 decreases MCL1 expression by disturbing ELF1 mRNA stability, thus increasing the sensitivity to venetoclax treatment. We finally established the intra-venous tumorigenesis xenograft model to verify the role of G3BP2 downregulation in venetoclax-treated leukemia mice. The NCG immunodeficient mice were transplanted with U937 cells and administrated with venetoclax daily from 7-21 days post transplantation (Figure 1A). The data showed that G3BP2 KD in combination with venetoclax reduced clinical scores and prolonged the overall survival of AML mice (combination vs. venetoclax: p&amp;lt;0.01; combination vs. G3BP2 KD: p&amp;lt;0.05, Figure 1B). The reduced leukemic burden was observed when targeting G3BP2 in combination with venetoclax. Meanwhile, we verified the decreased expression of ELF1 and MCL1 in mice targeting G3BP2 in combination with venetoclax by immunofluorescence (IF) and flow cytometry analysis. These data supported the synergic effect of G3BP2 inhibition and venetoclax treatment in AML. Our findings emphasize the critical role of G3BP2 in conferring resistance to venetoclax in AML by stabilizing ELF1 mRNA to regulate MCL1 expression. Targeting G3BP2 represents an appealing therapeutic strategy for patients with venetoclax-resistant AML.

Promoter cloning and activities analysis of JmLFY, a key gene for flowering in Juglans mandshurica.

Juglans mandshurica (Manchurian walnut) is a precious timber and woody grain and oil species in Northeast China. The heterodichogamous characteristic phenomenon resulted in the non-synchronous flowering and development of male and female flowers, which limited the mating and the yield and quality of fruits. LFY is a core gene in the flowering regulatory networks, which has been cloned in J. mandshurica, and the function has also been verified preliminarily. In this study, the JmLFY promoter sequence with different lengths of 5'-deletion (pLFY1-pLFY6) were cloned and conducted bioinformatics analysis, the promoter activities were analyzed by detecting their driving activity to GUS gene in the tobacco plants that transformed with different promoter sequence stably or transiently. After that, the interaction between JmSOC1 and JmLFY gene promoter was also analyzed via yeast single-hybrid. The results showed that the promoter sequence contains core cis-acting elements essential for eukaryotic promoters, hormone response elements, defense- and stress-responsive elements, flowering-related elements, etc. Transgenic tobacco plants with pLFY1 were obtained by Agrobacterium infection using the pCAMBIA1301 expression vector, and the GUS gene driven by the JmLFY promoter was detected to express in the leaf, stem, flower, and root of the transformed tobacco plant, which indicated that the obtained JmLFY promoter had driving activity. GUS histochemical staining and enzyme activity detection showed that promoter fragments with different lengths had promoter activity and could respond to the induction of long photoperiod, low temperature, salicylic acid (SA), IAA, GA3, and methyl jasmonate (MeJA). The core regulatory region of JmLFY gene promoter in J. mandshurica was between -657 bp and -1,904 bp. Point-to-point validation of yeast single-hybrid confirmed the interaction between JmSOC1 and JmLFY gene promoter, which indicated that JmLFY gene is the downstream target of JmSOC1. These results reveal relevant factors affecting JmLFY gene expression and clarify the molecular mechanism of JmLFY gene regulation in the flower developmental partially, which will provide a theoretical basis for regulating the flowering time by regulating JmLFY gene expression in J. mandshurica.

Association of the Arabidopsis oleoyl Δ12-desaturase FAD2 with pre-cis-Golgi stacks at endoplasmic reticulum-Golgi-exit sites.

The unsaturation of phospholipids influences the function of membranes. In Arabidopsis thaliana, the oleoyl Δ12-desaturase FAD2 converts oleic (18:1Δ9 ) to linoleic acid (18:2Δ9,12 ) and influences phospholipid unsaturation in different cellular membranes. Despite its importance, the precise localization of Arabidopsis FAD2 has not been unambiguously described. As FAD2 is thought to modify phospholipid-associated fatty acids at the endoplasmic reticulum (ER), from where unsaturates are distributed to other cellular sites, we hypothesized that FAD2 locates to ER subdomains enabling trafficking of lipid intermediates through the secretory pathway. Fluorescent FAD2 fusions used to test this hypothesis were first assessed for functionality by heterologous expression in yeast (Saccharomyces cerevisiae), and in planta by Arabidopsis fad2 mutant rescue upon ectopic expression from an intrinsic FAD2 promoter fragment. Light sheet fluorescence, laser scanning confocal or spinning disc microscopy of roots, leaves, or mesophyll protoplasts showed the functional fluorescence-tagged FAD2 variants in flattened donut-shaped structures of ~0.5-1 μm diameter, in a pattern not resembling mere ER association. High-resolution imaging of coexpressed organellar markers showed fluorescence-tagged FAD2 in a ring-shaped pattern surrounding ER-proximal Golgi particles, colocalizing with pre-cis-Golgi markers. This localization required the unusual C-terminal retention signal of FAD2, and deletion or substitutions in this protein region resulted in relaxed distribution and diffuse association with the ER. The distinct association of FAD2 with pre-cis-Golgi stacks in Arabidopsis root and leaf tissue is consistent with a contribution of FAD2 to membrane lipid homeostasis through the secretory pathway, as verified by an increased plasma membrane liquid phase order in the fad2 mutant.

B-252 In Vitro Expression Analysis of Variants in the Upstream Region of Genes Related to Familial Hypercholesterolemia

Abstract Background Familial hypercholesterolemia (FH) is commonly described as an autosomal dominant disorder caused mainly by variants in genes LDLR, APOB, and PCSK9, resulting in severe hypercholesterolemia. However, the substantial number of individuals who are clinically diagnosed but negative for known FH-causing variants indicates that relevant variants outside of frequently analyzed regions might exist. Considering the increasing number of promoter variants reported as likely to cause FH and the relevance of in vitro expression assays in the pathogenicity evaluation of these variants, this work aimed to examine the functionality of variants LDLR rs36218923-T and APOB rs934197-T, identified in a Brazilian FH cohort. Methods Upstream fragments of LDLR (-861 bp to +85 bp) and APOB (-1460 bp to +127 bp) were PCR amplified from genomic DNA of two FH patients carrying LDLR rs36218923-T and APOB rs934197-T in heterozygosis and homozygosis, respectively. After linearization and PGK promoter removal of the pGL4.53 vector using Kpn I and Hind III restriction enzymes, fragments were cloned upstream of the Firefly Luciferase (luc2) coding region using the NEBuilder HiFi DNA Assembly Cloning Kit. Site-directed mutagenesis was also performed using partially overlapping primers with 3´-overhangs and Platinum SuperFi II DNA Polymerase to produce different constructs, including one containing LDLR rs879254375-G, which is a known FH-causing variant. All constructs were confirmed by Sanger sequencing. pGL4.53 constructs containing either wild type or variant alleles and pNL1.1.TK (control of transfection variation, expressing NanoLuc Luciferase) were co-transfected into HepG2 cells. Wild type constructs were considered positive controls, while promoterless pGL4.53 was considered negative control. Luciferase assays were carried out 48 h after transfection using the Nano-Glo Dual-Luciferase Reporter Assay System. After normalization of luminescence units of each assay (Firefly/Nanoluc), luciferase activity was determined as the mean of four transfections with the assay performed in triplicate. Results The construct carrying LDLR rs36218923-T variant caused a significant mean reduction of luciferase activity (75,4% ± 2,9% SEM) over the promoterless construct relative to LDLR wild type construct. However, the construct harboring the LDLR rs879254375-G variant caused a more substantial reduction of luciferase activity relative to the wild type construct (91,2% ± 0,9% SEM). This reduction was consistent with shown by the previous study that reported LDLR rs879254375-G as a functional variant, which corroborates the methods adopted in this study. APOB rs934197-T was previously reported as functional based on a CAT assay using constructs of two tandemly arranged 30 bp fragments of APOB promoter, but in our study, the construct carrying the entire APOB promoter sequence and harboring rs934197-T variant did not cause a significant difference in mean luciferase activity compared to APOB wild type construct (P = 0.564). It is important to mention that our APOB upstream constructs had the additional variants rs617314-G, rs1560357-T, rs1625764-A, and rs1800481-G, which are considered benign because they occur at high frequency in the general population (0.82-1). Conclusion Our results suggest that LDLR rs36218923-T is likely to contribute to FH phenotype and emphasize the importance of performing in vitro expression assays in determining relevant upstream variants for FH molecular characterization.

Functional characterization of the MSP-C6 promoter as a potential tool for mesocarp-preferential expression of transgenes

Modification of lipid composition in the mesocarp tissue of oil palm involves genetic manipulation of multiple genes. More than one mesocarp-preferential promoter is necessary for the expression of individual transgenes in the same plant to obviate transcriptional gene silencing. This study aimed to identify genes that are preferentially expressed in the mesocarp tissue and characterize selected candidate mesocarp-preferential promoters. Ten transcripts that were preferentially expressed in the mesocarp tissue were identified from the analysis of 82 transcriptome datasets of 12 different oil palm tissues. The expression of two candidate genes, MSP-C1 and MSP-C6, was verified to be preferentially expressed in the mesocarp tissues and shown to have a low expression level in non-mesocarp tissues by reverse transcription quantitative real-time PCR (RT-qPCR). MSP-C6 promoter fragments of different lengths were transformed into tomato plants for further characterization. Both unripe and ripe fruits of transgenic tomato plants transformed with a construct harboring the MSP-C6-F1 (2014 bp) promoter were shown to have high beta-glucuronidase (GUS) activities. The findings of this study suggest the potential applications of the MSP-C6 promoter as a molecular tool for genetic engineering of novel traits in fruit crops.