Promoter Region Of Gene Research Articles

Identification and expression analysis of the HSP70 gene family under abiotic stresses in Litchi chinensis

HSP70 protein, as an important member of the heat shock protein (HSP) family, plays an important role in plant growth, development, and response to biotic and abiotic stresses. In order to explore the role of HSP70 gene family members in Litchi chinensis under low temperature, high temperature, drought, and salt stress, bioinformatics methods were used to identify the HSP70 gene family members within the entire L. chinensis genome. The expression of these genes under various abiotic stresses was then detected using quantitative real-time PCR (qRT-PCR). The results showed that the LcHSP70 gene family consisted of 18 members, which were unevenly distributed across ten L. chinensis chromosomes. The LcHSP70 protein contained 479-851 amino acids, with isoelectric points ranging from 5.07 to 6.95, and molecular weights from 52.44 kDa to 94.07 kDa. The predicted subcellular localization showed that LcHSP70 protein was present in the nucleus, cytoplasm, endoplasmic reticulum, mitochondria, and chloroplast. Phylogenetic analysis divided the LcHSP70 proteins into five subgroups, namely Ⅰ, Ⅱ, Ⅲ, Ⅳ, and Ⅵ. The promoter regions of the LcHSP70 genes contained various cis-acting elements related to plant growth, development, hormone response, and stress response. Moreover, the expression of LcHSP70 genes displayed distint tissue-specific expression level, categorized into universal expression and specific expression. From the selected 6 LcHSP70 genes (i.e., LcHSP70-1, LcHSP70-5, LcHSP70-10, LcHSP70-14, LcHSP70-16, and LcHSP70-18), their relative expression levels were assessed under different abiotic stresses using qRT-PCR. The results indicated that the gene family members exhibited diverse responses to low temperature, high temperature, drought, and salt stress, with significant variations in their expression levels across different time periods. These results provide a foundation for further exploration of the function of the LcHSP70 gene family.

Circadian rhythm response and its effect on photosynthetic characteristics of the Lhcb family genes in tea plant

BackgroundThe circadian clock, also known as the circadian rhythm, is responsible for predicting daily and seasonal changes in the environment, and adjusting various physiological and developmental processes to the appropriate times during plant growth and development. The circadian clock controls the expression of the Lhcb gene, which encodes the chlorophyll a/b binding protein. However, the roles of the Lhcb gene in tea plant remain unclear.ResultsIn this study, a total of 16 CsLhcb genes were identified based on the tea plant genome, which were distributed on 8 chromosomes of the tea plant. The promoter regions of CsLhcb genes have a variety of cis-acting elements including hormonal, abiotic stress responses and light response elements. The CsLhcb family genes are involved in the light response process in tea plant. The photosynthetic parameter of tea leaves showed rhythmic changes during the two photoperiod periods (48 h). Stomata are basically open during the day and closed at night. Real-time quantitative PCR results showed that most of the CsLhcb family genes were highly expressed during the day, but were less expressed at night.ConclusionsResults indicated that CsLhcb genes were involved in the circadian clock process of tea plant, it also provided potential references for further understanding of the function of CsLhcb gene family in tea plant.

Identification and expression profiling of SAPK gene family members in Dendrobium officinale

To investigate the potential roles of stress-activated protein kinase (SAPK) gene family members in Dendrobium officinale, we employed multiple bioinformatics methods to identify the members of this family. The physicochemical properties, chromosomal localization, phylogenetic relationship, gene structure, and cis-acting elements of each D. officinale SAPK (DoSAPK) member were analyzed. In addition, their expression profiles in different tissues and under the low-temperature or salt stress treatment were determined by real-time fluorescence quantitative PCR. The results showed that D. officinale carried eight DoSAPK family members, which belonged to three groups (groups Ⅰ, Ⅱ, and Ⅲ). These genes were located on seven chromosomes, and there were two pairs of genes with replication. The DoSAPK members within the same group had similar gene structures, conserved motifs, and secondary structures. The cis-acting elements in the promoter regions of DoSAPK genes included abundant hormone and stress response elements. DoSAPK family members presented tissue-specific expression in D. officinale. Furthermore, they were differentially expressed under the low-temperature or salt stress treatment, which suggested that they might be involved in the responses to low-temperature and salt stress. Intriguingly, DoSAPK1 might play a role in the abiotic stress responses. The results laid a foundation for in-depth study of the members and roles of the DoSAPK gene family.

CCT6A Regulates Cervical Cancer Cell Glycolysis and Proliferation under Hypoxic Conditions via the Telomerase Cajal Body Protein 1/Telomerase Reverse Tranase.

Cervical cancer is one of the most common female malignancies worldwide, a hypoxic microenvironment usually causes enhanced viability and glycolytic capacity of cervical cancer cells. The aim of this study was to investigate the association between chaperonin containing t-complex polypeptide 1 subunit 6A (CCT6A) and cell proliferation as well as hypoxic glycolysis. Bioinformatics analysis was conducted to explore the association between cervical cancer and its partner protein under hypoxic conditions, namely, CCT6A. Subsequently, the expression of CCT6A was silenced, and the effects of CCT6A silencing on cervical cancer cell proliferation, cell cycle, glycolysis-related proteins, and telomerase activity were examined. Bioinformatics analysis was performed to investigate the expression of CCT6A in cervical cancer under hypoxic conditions. The expression of CCT6A was silenced in cervical cancer cells, HeLa and SiHa, to study its effects on cell proliferation and hypoxic glycolysis. The localization of telomerase activity-related proteins, telomerase CTAB1 and TERT, was detected using immunofluorescence, and their interaction was assessed using immunoprecipitation. A cellular hypoxia model was established, and the products of the glycolysis reaction were detected. A nude mouse tumor model was constructed, and the changes in glycolysis-related proteins in tumor tissues were examined using Western blot, while Ki67 expression in tumor tissues was evaluated by immunohistochemistry. Telomerase activity was found to be enhanced in CCT6A-silenced cervical cancer cells, along with an increase in telomerase TCAB1 and TERT protein binding associated with telomerase activity. Additionally, the proportion of cells in the Gap 2/mitosis (G2/M) stage and the 5-ethynyl-2'-deoxyuridine (EdU) positivity rate were decreased in CCT6A-silenced cells, indicating a reduction in cell proliferation. The expression of cell cycle-related proteins, including cyclin E, CCNA2, and cyclin-dependent kinase 2 (CDK2), was suppressed. Furthermore, under a hypoxic environment, silencing CCT6A led to a significant reduction in cell viability and downregulation of glycolysis-related proteins, such as lactate dehydrogenase A (LDHA) and hexokinase 2 (HK2). Mechanistically, silencing CCT6A may reduce telomerase activity by inhibiting the TCAB1/TERT interaction. Additionally, TERT was found to activate the promoter region of the HK2 gene, and inhibition of TERT activity reduced the transcriptional level of HK2. The study primarily explored the involvement of CCT6A in cervical cancer, yet it did not account for the myriad of other elements potentially influencing cell proliferation and glycolysis. It is essential to recognize that cervical cancer's etiology is multifaceted, shaped by an array of genetic variations, environmental influences, and protein interactions. Silencing CCT6A could effectively attenuate the upregulation of cell proliferation and glycolytic function mediated by TCAB1/TERT in cervical cancer cells.

The phloem protein 2 (PP2) is positively regulated by ThNAC13 that enhances salt tolerance of Tamarix

NAC transcription factors play a significant role in regulating plant responses to biological processes and environmental stresses. Our previous study revealed 21 ThNAC proteins, and ThNAC13 is a nuclear protein that responds to saline stress in Tamarix hispida. To determine whether ThNAC13 regulates downstream genes, RNA sequencing (RNA-seq) and DNA affinity purification sequencing (DAP-seq) were used to identify the binding sites of ThNAC13 at the whole-genome level. In RNAi-silenced ThNAC13 transgenic Tamarix, a total of 4 downregulated differentially expressed genes (DEGs) and 114 peaks strongly associated with four different motifs were identified compared with those in empty vector transgenic Tamarix under salt stress. The c48149.graph_c0 gene contains the key domain of the phloem protein 2 (PP2) protein and is a candidate gene downstream of ThNAC13. Yeast one-hybrid (Y1H), chromatin immunoprecipitation (ChIP) and dual-luciferase reporter (LUC) assays were used to identify ThNAC13 as an upstream regulator of ThPP2. Transgenic Tamarix plants in which ThPP2 was transiently expressed or silenced via RNAi were used for functional gain and loss experiments. The ThPP2-overexpressing plants exhibited decreased hydrogen peroxide (H2O2), superoxide (O2•−), and electrolyte leakage (EL) and malondialdehyde (MDA) levels and increased superoxide dismutase (SOD), peroxidase (POD) and catalase (CAT) activity. In contrast, transient RNAi-mediated silencing of ThPP2 in Tamarix had the opposite effect. ThNAC13 binds to the promoter region of the ThPP2 gene and reduces the reactive oxygen species (ROS) accumulation and enhances antioxidant enzyme activity, positively regulating salt stress tolerance in Tamarix.

In-Depth Characterization of bZIP Genes in the Context of Endoplasmic Reticulum (ER) Stress in Brassica campestris ssp. chinensis.

Numerous studies have been conducted to investigate the genomic characterization of bZIP genes and their involvement in the cellular response to endoplasmic reticulum (ER) stress. These studies have provided valuable insights into the coordinated cellular response to ER stress, which is mediated by bZIP transcription factors (TFs). However, a comprehensive and systematic investigations regarding the role of bZIP genes and their involvement in ER stress response in pak choi is currently lacking in the existing literature. To address this knowledge gap, the current study was initiated to elucidate the genomic characteristics of bZIP genes, gain insight into their expression patterns during ER stress in pak choi, and investigate the protein-to-protein interaction of bZIP genes with the ER chaperone BiP. In total, 112 members of the BcbZIP genes were identified through a comprehensive genome-wide analysis. Based on an analysis of sequence similarity, gene structure, conserved domains, and responsive motifs, the identified BcbZIP genes were categorized into 10 distinct subfamilies through phylogenetic analysis. Chromosomal location and duplication events provided insight into their genomic context and evolutionary history. Divergence analysis estimated their evolutionary history with a predicted divergence time ranging from 0.73 to 80.71 million years ago (MYA). Promoter regions of the BcbZIP genes were discovered to exhibit a wide variety of cis-elements, including light, hormone, and stress-responsive elements. GO enrichment analysis further confirmed their roles in the ER unfolded protein response (UPR), while co-expression network analysis showed a strong relationship of BcbZIP genes with ER-stress-responsive genes. Moreover, gene expression profiles and protein-protein interaction with ER chaperone BiP further confirmed their roles and capacity to respond to ER stress in pak choi.

Association of polymorphism in the promotor area of the caprine BMPR1B gene with litter size and body measurement traits in Damani goats.

This study aimed to explore polymorphisms in the promoter region of the caprine BMPR1B (Bone morphogenetic protein receptor 1 beta) gene and its association with body measurement and litter size traits in Damani does. A total of 53 blood samples were collected to analyze the association between the BMPR1B gene polymorphism and 11 phenotypic traits in Damani female goats. The results revealed that three novel SNPs were identified in the promoter region of the caprine BMPR1B gene, including g.67 A > C (SNP1), g.170 G > A(SNP2), and g.501A > T (SNP3), among which the SNP1 and SNP2 were significantly (p < 0.05) associated with litter size and body measurement traits in Damani goats. In SNP1 the AC genotype could be used as a marker for litter size, and the CC genotype for body weight in Damani goats. In SNP2, the genotype GG was significantly (p < 0.05) associated with ear and head length. Therefore, we can conclude from the present study, that genetic variants AC and CC of the caprine BMPR1B gene could be used as genetic markers for economic traits through marker-assisted selection for the breed improvement program of the Damani goat.

Dissecting the role of mitogen-activated protein kinase Hog1 in yeast flocculation.

Living organisms are frequently exposed to multiple biotic and abiotic stress forms during their lifetime. Organisms cope with stress conditions by regulating their gene expression programs. In response to different environmental stress conditions, yeast cells activate different tolerance mechanisms, many of which share common signaling pathways. Flocculation is one of the key mechanisms underlying yeast survival under unfavorable environmental conditions, and the Tup1-Cyc8 corepressor complex is a major regulator of this process. Additionally, yeast cells can utilize different mitogen-activated protein kinase (MAPK) pathways to modulate gene expression during stress conditions. Here, we show that the high osmolarity glycerol (HOG) MAPK pathway is involved in the regulation of yeast flocculation. We observed that the HOG MAPK pathway was constitutively activated in flocculating cells, and found that the interaction between phosphorylated Hog1 and the FLO genes promoter region increased significantly upon sodium chloride exposure. We found that treatment of cells with cantharidin decreased Hog1 phosphorylation, causing a sharp reduction in the expression of FLO genes and the flocculation phenotype. Similarly, deletion of HOG1 in yeast cells reduced flocculation. Altogether, our results suggest a role for HOG MAPK signaling in the regulation of FLO genes and yeast flocculation.

The histone lysine acetyltransferase KAT2B inhibits cholangiocarcinoma growth: evidence for interaction with SP1 to regulate NF2-YAP signaling

BackgroundCholangiocarcinoma (CCA) is a highly malignant cancer of the biliary tract with poor prognosis. Further mechanistic insights into the molecular mechanisms of CCA are needed to develop more effective target therapy.MethodsThe expression of the histone lysine acetyltransferase KAT2B in human CCA was analyzed in human CCA tissues. CCA xenograft was developed by inoculation of human CCA cells with or without KAT2B overexpression into SCID mice. Western blotting, ChIP-qPCR, qRT-PCR, protein immunoprecipitation, GST pull-down and RNA-seq were performed to delineate KAT2B mechanisms of action in CCA.ResultsWe identified KAT2B as a frequently downregulated histone acetyltransferase in human CCA. Downregulation of KAT2B was significantly associated with CCA disease progression and poor prognosis of CCA patients. The reduction of KAT2B expression in human CCA was attributed to gene copy number loss. In experimental systems, we demonstrated that overexpression of KAT2B suppressed CCA cell proliferation and colony formation in vitro and inhibits CCA growth in mice. Mechanistically, forced overexpression of KAT2B enhanced the expression of the tumor suppressor gene NF2, which is independent of its histone acetyltransferase activity. We showed that KAT2B was recruited to the promoter region of the NF2 gene via interaction with the transcription factor SP1, which led to enhanced transcription of the NF2 gene. KAT2B-induced NF2 resulted in subsequent inhibition of YAP activity, as reflected by reduced nuclear accumulation of oncogenic YAP and inhibition of YAP downstream genes. Depletion of NF2 was able to reverse KAT2B-induced reduction of nuclear YAP and subvert KAT2B-induced inhibition of CCA cell growth.ConclusionsThis study provides the first evidence for an important tumor inhibitory effect of KAT2B in CCA through regulation of NF2-YAP signaling and suggests that this signaling cascade may be therapeutically targeted for CCA treatment.

Genome-wide identification and stress response analysis of BcaCPK gene family in amphidiploid Brassica carinata.

Calcium-dependent protein kinases (CPKs) are crucial for recognizing and transmitting Ca2+ signals in plant cells, playing a vital role in growth, development, and stress response. This study aimed to identify and detect the potential roles of the CPK gene family in the amphidiploid Brassica carinata (BBCC, 2n = 34) using bioinformatics methods. Based on the published genomic information of B. carinata, a total of 123 CPK genes were identified, comprising 70 CPK genes on the B subgenome and 53 on the C subgenome. To further investigate the homologous evolutionary relationship between B. carinata and other plants, the phylogenetic tree was constructed using CPKs in B. carinata and Arabidopsis thaliana. The phylogenetic analysis classified 123 family members into four subfamilies, where gene members within the same subfamily exhibited similar conserved motifs. Each BcaCPK member possesses a core protein kinase domain and four EF-hand domains. Most of the BcaCPK genes contain 5 to 8 introns, and these 123 BcaCPK genes are unevenly distributed across 17 chromosomes. Among these BcaCPK genes, 120 replicated gene pairs were found, whereas only 8 genes were tandem duplication, suggesting that dispersed duplication mainly drove the family amplification. The results of the Ka/Ks analysis indicated that the CPK gene family of B. carinata was primarily underwent purification selection in evolutionary selection. The promoter region of most BcaCPK genes contained various stress-related cis-acting elements. qRT-PCR analysis of 12 selected CPK genes conducted under cadmium and salt stress at various points revealed distinct expression patterns among different family members in response to different stresses. Specifically, the expression levels of BcaCPK2.B01a, BcaCPK16.B02b, and BcaCPK26.B02 were down-regulated under both stresses, whereas the expression levels of other members were significantly up-regulated under at least one stress. This study systematically identified the BcaCPK gene family in B. carinata, which contributes to a better understanding the CPK genes in this species. The findings also serve as a reference for analyzing stress responses, particularly in relation to cadmium and salt stress in B. carinata.

The transcription factor HMGB2 indirectly regulates APRIL expression and Gd-IgA1 production in patients with IgA nephropathy

Background IgA nephropathy (IgAN) is the most common primary glomerulonephritis worldwide. Proliferation-inducing ligand (APRIL) was identified as an important cause of glycosylation deficiency of IgA1 (Gd-IgA1), which can ‘trigger’ IgAN. Our previous study indicated that high migration group protein B2 (HMGB2) in peripheral blood mononuclear cells from patients with IgAN was associated with disease severity, but the underlying mechanism remains unclear. Materials and methods The location of HMGB2 was identified by immunofluorescence. qRT-PCR and Western blotting were used to measure HMGB2, HMGA1, and APRIL expression. Gd-IgA1 levels were detected by enzyme-linked immunosorbent assay (ELISA). In addition, we used DNA pull-down, protein profiling, and transcription factor prediction software to identify proteins bound to the promoter region of the APRIL gene. RNA interference and coimmunoprecipitation (Co-IP) were used to verify the relationships among HMGB2, high mobility group AT-hook protein 1 (HMGA1), and APRIL. Results HMGB2 expression was greater in IgAN patients than in HCs and was positively associated with APRIL expression in B cells. DNA pull-down and protein profiling revealed that HMGB2 and HMGA1 bound to the promoter region of the APRIL gene. The expression levels of HMGA1, APRIL, and Gd-IgA1 were downregulated after HMGB2 knockdown. Co-IP indicated that HMGB2 binds to HMGA1. The Gd-IgA1 concentration in the supernatant was reduced after HMGA1 knockdown. HMGA1 binding sites were predicted in the promoter region of the APRIL gene. Conclusion HMGB2 expression is greater in IgAN patients than in healthy controls; it promotes APRIL expression by interacting with HMGA1, thereby inducing Gd-IgA1 overexpression and leading to IgAN.

Abstract P43: Optimization of DNA Methyltransferase Inhibitors-based Combination to Target the Epigenome of Gastric Cancer

Abstract Aberrant DNA methylation is one of the critical epigenetic modifications that has become a widespread phenotype in solid tumours. Similar to haematological malignancies, many studies have shown that the DNA methylation landscape of solid tumours exhibits a pattern of either hypermethylation of the promoter regions of tumour-suppressive genes in advanced cancer or global hypomethylation in the early stages of carcinogenesis. Additionally, dysregulated DNA methylation also seems to remodel the tumour microenvironment and affect immune cells' response towards immune checkpoint inhibitors. In gastric cancer, comprehensive DNA methylation analyses have demonstrated that gastric tumours carry the CpG island methylation phenotype (CIMP), resulting in extended molecular-based subgroups such as the extremely high-methylation epigenotype (E-HME), HME, and low-methylation epigenotype (LME). Therefore, conventional histology grading and molecular subtyping are insufficient to address the multifaceted epigenome of gastric cancer. As such, current standard-of-care (SOC) therapy is not able to overcome cancer relapse and tumour metastasis. Clearly, this shows the therapeutic potential of using DNA hypomethylating drugs like DNA methyltransferase inhibitors (DNMTi) for solid tumours such as gastric cancer. In our study, we exploited the engineering-based Quadratic Phenotypic Optimization Platform (QPOP) to identify top-ranking DNMTi-based drug combinations across gastric cancer cell lines (GC-CL) and patient-derived organoids (GC-PDO). Based on QPOP analysis, we showed that a non-SOC drug combination, Docetaxel/5-Azacytidine is a frequently top-ranking. Besides, our present findings suggest that CIMP-high gastric cancer cells are more susceptible towards DNMT inhibitors through the specific reduction of DNMT1 levels. As we surveyed the effects of Docetaxel/5-Azacytidine in a CIMP-high GC-CL via Infinium methylation array, we observed robust gene methylation changes that are involved in non-canonical Wnt Signaling and EMT. On the other hand, proteomic analysis revealed a small subset of our panel of GC lines that are positive for the cancer-testis antigen, MageA4. Interestingly, the protein expression of MageA4 in some CIMP-high lines is restored upon treatment with DNMT inhibitors. This evinces that DNMTi can subject epigenetically silenced MageA4 gastric tumours to autologous T-cell receptor therapy. Taken altogether, we plan to further interrogate the mechanisms of DNMT inhibitors in sensitizing CIMP-high gastric tumours towards Taxanes by acting on the non-canonical Wnt pathway and EMT activators. Citation Format: Lissa Hooi, Vivien Koh, Dexter Kai Hao Thng, Jasmine Goh, Lim Jhin Jieh, Lee Rui Xue, Masturah Bte Mohd Abdul Rashid, Toh Tan Boon, Wei Peng Yong, Edward Kai-Hua Chow. Optimization of DNA Methyltransferase Inhibitors-based Combination to Target the Epigenome of Gastric Cancer [abstract]. In: Proceedings of Frontiers in Cancer Science; 2023 Nov 6-8; Singapore. Philadelphia (PA): AACR; Cancer Res 2024;84(8_Suppl):Abstract nr P43.

A network of transcription factors in complex with a regulating cell cycle cyclin orchestrates fungal oxidative stress responses

BackgroundResponse to oxidative stress is universal in almost all organisms and the mitochondrial membrane protein, BbOhmm, negatively affects oxidative stress responses and virulence in the insect fungal pathogen, Beauveria bassiana. Nothing further, however, is known concerning how BbOhmm and this phenomenon is regulated.ResultsThree oxidative stress response regulating Zn2Cys6 transcription factors (BbOsrR1, 2, and 3) were identified and verified via chromatin immunoprecipitation (ChIP)-qPCR analysis as binding to the BbOhmm promoter region, with BbOsrR2 showing the strongest binding. Targeted gene knockout of BbOsrR1 or BbOsrR3 led to decreased BbOhmm expression and consequently increased tolerances to free radical generating compounds (H2O2 and menadione), whereas the ΔBbOsrR2 strain showed increased BbOhmm expression with concomitant decreased tolerances to these compounds. RNA and ChIP sequencing analysis revealed that BbOsrR1 directly regulated a wide range of antioxidation and transcription-associated genes, negatively affecting the expression of the BbClp1 cyclin and BbOsrR2. BbClp1 was shown to localize to the cell nucleus and negatively mediate oxidative stress responses. BbOsrR2 and BbOsrR3 were shown to feed into the Fus3-MAPK pathway in addition to regulating antioxidation and detoxification genes. Binding motifs for the three transcription factors were found to partially overlap in the promoter region of BbOhmm and other target genes. Whereas BbOsrR1 appeared to function independently, co-immunoprecipitation revealed complex formation between BbClp1, BbOsrR2, and BbOsrR3, with BbClp1 partially regulating BbOsrR2 phosphorylation.ConclusionsThese findings reveal a regulatory network mediated by BbOsrR1 and the formation of a BbClp1-BbOsrR2-BbOsrR3 complex that orchestrates fungal oxidative stress responses.

Transcription factor DREF regulates expression of the microRNA gene bantam in Drosophila melanogaster.

The bantam gene encodes a vital microRNA and has a complex expression pattern in various tissues at different stages of Drosophila development. This microRNA is involved in the control of normal development of the ocular and wing imaginal discs, the central nervous system, and also in maintaining the undifferentiated state of stem cells in the ovaries of adult females. At the cellular level, bantam stimulates cell proliferation and prevents apoptosis. The bantam gene is a target of several conserved signaling cascades, in particular, Hippo. At the moment, at least ten proteins are known to directly regulate the expression of this gene in different tissues of Drosophila. In this study, we found that the bantam regulatory region contains motifs characteristic of binding sites for DREF, a transcription factor that regulates the expression of Hippo cascade genes. Using transgenic lines containing a full-length bantam lethality-rescuing deletion fragment and a fragment with a disrupted DREF binding site, we show that these motifs are functionally significant because their disruption at the bantam locus reduces expression levels in the larvae and ovaries of homozygous flies, which correlates with reduced vitality and fertility. The effect of DREF binding to the promoter region of the bantam gene on its expression level suggests an additional level of complexity in the regulation of expression of this microRNA. A decrease in the number of eggs laid and a shortening of the reproductive period in females when the DREF binding site in the regulatory region of the bantam gene is disrupted suggests that, through bantam, DREF is also involved in the regulation of Drosophila oogenesis.

The Transcription Factor Zfp335 Promotes Differentiation and Survival of Effector Th1 Cells by Directly Regulating Lmna Expression.

Ag-specific effector CD4+ T cells play a crucial role in defending against exogenous pathogens. However, the mechanisms governing the differentiation and function of IFN-γ-producing effector CD4+ Th1 cells in immune responses remain largely unknown. In this study, we elucidated the pivotal role of zinc finger protein 335 (Zfp335) in regulating effector Th1 cell differentiation and survival during acute bacterial infection. Mice with Zfp335 knockout in OT-II cells exhibited impaired Ag-specific CD4+ T cell expansion accompanied by a significant reduction in resistance to Listeria infection. Furthermore, Zfp335 deficiency restricted the effector CD4+ Th1 cell population and compromised their survival upon Listeria challenge. The expression of T-bet and IFN-γ was accordingly decreased in Zfp335-deficient Th1 cells. Mechanistically, Zfp335 directly bound to the promoter region of the Lmna gene and regulated its expression. Overexpression of Lmna was able to rescue the survival and function of Zfp335-deficient effector Th1 cells. Therefore, our study provides novel insights into the mechanisms governing effector Th1 cell differentiation and survival during acute infection.

The novel candidate gene Grb10 regulates the growth performance of Guizhou white goats

Growth factor receptor binding protein 10 (Grb10) regulates muscle growth but its function in goats is not well studied. This study examined the relationship between Grb10 gene polymorphism and growth traits in Guizhou white goats. Further, the effect of promoter polymorphism on Grb10 transcription was analyzed via a dual-luciferase assay. Quantitative reverse transcription-polymerase chain reaction analysis showed that the expression level of the Grb10 gene was the highest in the longissimus dorsi muscle (P < 0.01) and the lowest in the spleen. Moreover, sequencing results showed that the Grb10 gene had five single nucleotide polymorphisms. The association analysis showed that g.–246 C > T, g.72323 T > C, and g.178634 A > G loci were significantly associated with growth traits (P < 0.05). Linkage disequilibrium analysis of each locus showed that g.72323 T > C and g.72814 T > A loci had the strongest linkage (r2 = 0.482, D' = 0.73). Furthermore, according to an analysis conducted using TFDB4 software, it was predicted that a mutation in the promoter region of the g.–246 C > T locus resulted in the absence of four transcriptional regulatory factors. The results of dual-luciferase reporter assay further demonstrated that mutation of the g.–246 C > T locus reduced the transcriptional activity of the promoter region of the Grb10 gene. These findings suggest that Grb10 gene polymorphism could affect growth traits in goats.

Unveiling kiwifruit TCP genes: evolution, functions, and expression insights

ABSTRACT The TEOSINTE-BRANCHED1/CYCLOIDEA/PROLEFERATING-CELL-FACTORS (TCP) gene family is a plant-specific transcriptional factor family involved in leaf morphogenesis and senescence, lateral branching, hormone crosstalk, and stress responses. To date, a systematic study on the identification and characterization of the TCP gene family in kiwifruit has not been reported. Additionally, the function of kiwifruit TCPs in regulating kiwifruit responses to the ethylene treatment and bacterial canker disease pathogen (Pseudomonas syringae pv. actinidiae, Psa) has not been investigated. Here, we identified 40 and 26 TCP genes in Actinidia chinensis (Ac) and A. eriantha (Ae) genomes, respectively. The synteny analysis of AcTCPs illustrated that whole-genome duplication accounted for the expansion of the TCP family in Ac. Phylogenetic, conserved domain, and selection pressure analysis indicated that TCP family genes in Ac and Ae had undergone different evolutionary patterns after whole-genome duplication (WGD) events, causing differences in TCP gene number and distribution. Our results also suggested that protein structure and cis-element architecture in promoter regions of TCP genes have driven the function divergence of duplicated gene pairs. Three and four AcTCP genes significantly affected kiwifruit responses to the ethylene treatment and Psa invasion, respectively. Our results provided insight into general characters, evolutionary patterns, and functional diversity of kiwifruit TCPs.

Development of CRISPR/Cas9 systems to induce targeted mutations in the promoter region of the OsSRFP1 gene in rice

Rice is one of the most important staple food crops worldwide, especially in Asia. However, the climate change has been negatively affecting the arable land area, which leads to a severe reduction of the rice yield. In which, the salt stress is considered as one of the most critical abiotic stresses. The creation of rice varieties with salt tolerance is an urgent research direction. In this study, we successfully created two CRISPR/Cas9 vectors to introduce targeted mutations of two different positions in the promoter region of the OsSRFP1 , a gene involves in the Ubiquitination pathway and regulates important responses to multiple stresses. Cali from mature seeds of the Khang Dan 18 rice cultivar was used for Agrobacterium mediated transformation. Total 6 T0 rice lines showed CRISPR/Cas9 induced mutations in the targeted sites. Two of them were utilized for validating inheritance and segregation of induced mutations at the T1 generation. Importantly, homozygous mutant lines lacking the transgene were successfully identified at the T1 generation. These mutant lines are valuable materials for us to continue to assess gene expression, plant growth and development as well as salt tolerance in the further research work.

The Putative GATA Transcription Factor SbGATA22 as a Novel Regulator of Dhurrin Biosynthesis.

Cyanogenic glucosides are specialized metabolites produced by over 3000 species of higher plants from more than 130 families. The deployment of cyanogenic glucosides is influenced by biotic and abiotic factors in addition to being developmentally regulated, consistent with their roles in plant defense and stress mitigation. Despite their ubiquity, very little is known regarding the molecular mechanisms that regulate their biosynthesis. The biosynthetic pathway of dhurrin, the cyanogenic glucoside found in the important cereal crop sorghum (Sorghum bicolor (L.) Moench), was described over 20 years ago, and yet no direct regulator of the biosynthetic genes has been identified. To isolate regulatory proteins that bind to the promoter region of the key dhurrin biosynthetic gene of sorghum, SbCYP79A1, yeast one-hybrid screens were performed. A bait fragment containing 1204 base pairs of the SbCYP79A1 5' regulatory region was cloned upstream of a reporter gene and introduced into Saccharomyces cerevisiae. Subsequently, the yeast was transformed with library cDNA representing RNA from two different sorghum developmental stages. From these screens, we identified SbGATA22, an LLM domain B-GATA transcription factor that binds to the putative GATA transcription factor binding motifs in the SbCYP79A1 promoter region. Transient assays in Nicotiana benthamiana show that SbGATA22 localizes to the nucleus. The expression of SbGATA22, in comparison with SbCYP79A1 expression and dhurrin concentration, was analyzed over 14 days of sorghum development and in response to nitrogen application, as these conditions are known to affect dhurrin levels. Collectively, these findings suggest that SbGATA22 may act as a negative regulator of SbCYP79A1 expression and provide a preliminary insight into the molecular regulation of dhurrin biosynthesis in sorghum.

The Mediator Subunit OsMED16 Interacts with the WRKY Transcription Factor OsWRKY45 to Enhance Rice Resistance Against Magnaporthe oryzae.

Rice blast, caused by Magnaporthe oryzae (M. oryzae), is one of the most common and damaging diseases of rice that limits rice yield and quality. The mediator complex plays a vital role in promoting transcription by bridging specific transcription factors and RNA polymerase II. Here, we show that the rice mediator subunit OsMED16 is essential for full induction of the diterpenoid phytoalexin biosynthesis genes and resistance to the ascomycetous fungus M. oryzae. Mutants of Osmed16 show reduced expression of the DP biosynthesis genes and are markedly more susceptible to M. oryzae, while transgenicplants overexpressing OsMED16 increased the expression of the DP biosynthesis genes and significantly enhanced resistance to M. oryzae. Interestingly, OsMED16 is physically associated with the WRKY family transcription factor OsWRKY45, which interacts with the phytoalexin synthesis key regulator transcription factor OsWRKY62. Further, OsMED16-OsWRKY45-OsWRKY62 complex could bind to the promoter regions of phytoalexin synthesis-related genes and activate their gene expression. Our results show that OsMED16 may enhance rice tolerance to M. oryzae via directly manipulating phytoalexin de novo biosynthesis.