New opportunities to overcome T cell dysfunction: the role of transcription factors and how to target them.

Immune checkpoint blockade (ICB) therapies, which block inhibitory receptors on T cells, can be efficacious in reinvigorating dysfunctional T cell responses. However, most cancers do not respond to these therapies and even in those that respond, tumors can acquire resistance. New strategies are needed to rescue and recruit T cell responses across patient populations and disease states. In this review, we define mechanisms of T cell dysfunction, focusing on key transcription factor (TF) networks. We discuss the complex and sometimes contradictory roles of core TFs in both T cell function and dysfunction. Finally, we review strategies to target TFs using small molecule modulators, which represent a challenging but highly promising opportunity to tune the T cell response toward sustained immunity.

Dedifferentiation-like reprogramming of degenerative nucleus pulposus cells into notochordal-like cells by defined factors.

The extensive degeneration of functional somatic cells and the depletion of endogenous stem/progenitor populations present significant challenges to tissue regeneration in degenerative diseases. Currently, a cellular reprogramming approach enabling directly generating corresponding progenitor populations from degenerative somatic cells remains elusive. The present study focused on intervertebral disc degeneration (IVDD) and identified a three-factor combination (OCT4, FOXA2, TBXT [OFT]) that could induce the dedifferentiation-like reprogramming of degenerative nucleus pulposus cells (dNPCs) toward induced notochordal-like cells (iNCs). Single-cell transcriptomics dissected the transitions of cell identity during reprogramming. Further, OCT4 was found to directly interact with bromodomain PHD-finger transcription factor to remodel the chromatin during the early phases, which was crucial for initiating this dedifferentiation-like reprogramming. In rat models, intradiscal injection of adeno-associated virus carrying OFT generated iNCs from in situ dNPCs and reversed IVDD. These results collectively present a proof-of-concept for dedifferentiation-like reprogramming of degenerated somatic cells into corresponding progenitors through the development of a factor-based strategy, providing a promising approach for regeneration in degenerative disc diseases.

The ScAPD1-like gene from the desert moss Syntrichia caninervis enhances resistance to Verticillium dahliae via phenylpropanoid gene regulation.

Soloist is a member of a distinct and small subfamily within the AP2/ERF transcriptional factor family that play important roles in plant biotic and abiotic stress responses. There are limited studies of Soloist genes and their functions are poorly understood. We characterized the abiotic and biotic stress tolerance function of the ScSoloist gene (designated as ScAPD1-like) from the desert moss Syntrichia caninervis. ScAPD1-like responded to multiple abiotic, biotic stresses and plant hormone treatments. ScAPD1-like protein located to the nucleus and bound to several DNA elements. Overexpression of ScAPD1-like in Arabidopsis did not alter abiotic stress resistance or inhibit Pseudomonas syringae pv. tomato (Pst) DC3000 infection. However, overexpression of ScAPD1-like significantly increased the resistance of transgenic Arabidopsis and S. caninervis to Verticillium dahliae infection, decreased reactive oxygen species accumulation and improved reactive oxygen species scavenging activity. ScAPD1-like overexpression plants altered the abundance of transcripts for lignin synthesis and promoted lignin accumulation in Arabidopsis. ScAPD1-like directly bind to RAV1, AC elements, and TATA-box in the promoters of AtPAL1 and AtC4H genes, respectively, in vitro. Chromatin immunoprecipitation-quantitative polymerase chain reaction assays demonstrated ScAPD1-like directly bound to PAL and C4H genes promoters in Arabidopsis and their homologs in S. caninervis. In S. caninervis, ScAPD1-like overexpression and RNAi directly regulated the abundance of ScPAL and ScC4H transcripts and modified the metabolites of phenylpropanoid pathway. We provide insight into the function of Soloist in plant defense mechanisms that likely occurs through activation of the phenylpropanoid biosynthesis pathway. ScAPD1-like is a promising candidate gene for breeding strategies to improve resistance to Verticillium wilt.

Genomic and transcriptomic studies on flavonoid biosynthesis in Lagerstroemia indica.

BACKGROUND: Lagerstroemia indica is a widely cultivated ornamental woody shrub/tree of the family Lythraceae that is used as a traditional medicinal plant in East Asia and Egypt. However, unlike other ornamental woody plants, its genome is not well-investigated, which hindered the discovery of the key genes that regulate important traits and the synthesis of bioactive compounds. RESULTS: In this study, the genomic sequences of L. indica were determined using several next-generation sequencing technologies. Altogether, 324.01 Mb sequences were assembled and 98.21% (318.21 Mb) of them were placed in 24 pseudo-chromosomes. The heterozygosity, repeated sequences, and GC residues occupied 1.65%, 29.17%, and 38.64% of the genome, respectively. In addition, 28,811 protein-coding gene models, 327 miRNAs, 552 tRNAs, 214 rRNAs, and 607 snRNAs were identified. The intra- and interspecies synteny and Ks analysis revealed that L. indica exhibits a hexaploidy. The co-expression profiles of the genes involved in the phenylpropanoid (PA) and flavonoid/anthocyanin (ABGs) pathways with the R2R3 MYB genes (137 members) showed that ten R2R3 MYB genes positively regulate flavonoid/anthocyanin biosynthesis. The colors of flowers with white, purple (PB), and deep purplish pink (DPB) petals were found to be determined by the levels of delphinidin-based (Dp) derivatives. However, the substrate specificities of LiDFR and LiOMT probably resulted in the different compositions of flavonoid/anthocyanin. In L. indica, two LiTTG1s (LiTTG1-1 and LiTTG1-2) were found to be the homologs of AtTTG1 (WD40). LiTTG1-1 was found to repress anthocyanin biosynthesis using the tobacco transient transfection assay. CONCLUSIONS: This study showed that the ancestor L. indica experienced genome triplication approximately 38.5 million years ago and that LiTTG1-1 represses anthocyanin biosynthesis. Furthermore, several genes such as LiDFR, LiOMTs, and R2R3 LiMYBs are related to anthocyanin biosynthesis. Further studies are required to clarify the mechanisms and alleles responsible for flower color development.

Single-cell transcriptome landscape elucidates the cellular and developmental responses to tomato chlorosis virus infection in tomato leaf.

Plant viral diseases compromise the growth and yield of the crop globally, and they tend to be more serious under extreme temperatures and drought climate changes. Currently, regulatory dynamics during plant development and in response to virus infection at the plant cell level remain largely unknown. In this study, single-cell RNA sequencing on 23 226 individual cells from healthy and tomato chlorosis virus-infected leaves was established. The specific expression and epigenetic landscape of each cell type during the viral infection stage were depicted. Notably, the mesophyll cells showed a rapid function transition in virus-infected leaves, which is consistent with the pathological changes such as thinner leaves and decreased chloroplast lamella in virus-infected samples. Interestingly, the F-box protein SKIP2 was identified to play a pivotal role in chlorophyll maintenance during virus infection in tomato plants. Knockout of the SlSKIP2 showed a greener leaf state before and after virus infection. Moreover, we further demonstrated that SlSKIP2 was located in the cytomembrane and nucleus and directly regulated by ERF4. In conclusion, with detailed insights into the plant responses to viral infections at the cellular level, our study provides a genetic framework and gene reference in plant-virus interaction and breeding in the future research.

Functional characterization of large yellow croaker (Larimichthys crocea) Peroxiredoxin IV (PrxIV) gene promoter.

Peroxiredoxin IV (PrxIV), which possesses an N-terminal signal peptide, is the only secretable protein in Prx family. PrxIV can protect cells against reactive oxygen species (ROS) and act as a DAMP to promote infection-independent immune response. However, the characterization and regulation of promoters of PrxIV genes are rarely reported. In this study, a 1511-bp 5'-flanking sequence of large yellow croaker (Larimichthys crocea) PrxIV (LcPrxIV) was cloned and characterized. DNA truncation combined with luciferase activity assay revealed that a fragment of -781/+20 contained in the plasmid LcPrxIV-P3 exhibited the highest promoter activity. It could initiate the luciferase expression up to 44.6-fold when compared to control plasmid pGL3-Basic. TFSEARCH analysis revealed many recognizing sequences of transcriptional factors exist within this 1511-bp sequence, including Foxo and CREB. Altogether, four putative binding sites located in three recognizing sequences of CREB were identified. Notably, co-transfection of LcPrxIV-P3 with LcCREB led to a significant 2.48-fold increase of the LcPrxIV-P3 promoter activity (P<0.01). Furthermore, the mutation at putative binding sites A, B, and all four sites of CREB in the LcPrxIV-P3 caused the significant decrease of activation on LcPrxIV-P3 promoter activity, suggesting these two sites may be the main binding sites of CREB in LcPrxIV promoter. In addition, the oxidative stress caused by hydrogen peroxide, rather than immune stimuli such as Poly (I: C), LPS, LTA, or PGN could lead to the elevation of LcPrxIV-P3 promoter activity. When the concentration of hydrogen peroxide reached 500 µM, the promoter activity of LcPrxIV-P3 could be up-regulated to 1.47-fold, which was extremely significantly different from the control (P<0.001). These results help to elucidate the regulatory mechanisms of LcPrxIV gene expression, and the role of LcPrxIV in protecting cells against oxidative stress or in oxidoreduction-dependent signal transduction.

Aspergillus oryzae PrtR alters transcription of individual peptidase genes in response to the growth environment.

Aspergillus oryzae PrtR is an ortholog of the transcription factor PrtT, which positively regulates the transcription of extracellular peptidase genes in Aspergillus niger and Aspergillus fumigatus. To identify the genes under the control of PrtR and elucidate its regulatory mechanism in A. oryzae, prtR gene disruption mutants were generated. The control strain clearly showed a halo on media containing skim milk as the nitrogen source, whereas the ΔprtR strain formed a smaller halo. Measurement of acid peptidase activity revealed that approximately 84% of acidic endopeptidase and 86% of carboxypeptidase activities are positively regulated by PrtR. As the transcription of the prtR gene varied depending on culture conditions, especially with or without a protein substrate, it was considered that its transcription would be regulated in response to a nitrogen source. In addition, contrary to previous expectations, PrtR was found to act both in promoting and repressing the transcription of extracellular peptidase genes. The mode of regulation varied from gene to gene. Some genes were regulated in the same manner in both liquid and solid cultures, whereas others were regulated in different ways depending on the culture conditions. Furthermore, PrtR has been suggested to regulate the transcription of peptidase genes that are closely associated with other transcription factors. KEY POINTS: • Almost all peptidase genes in Aspergillus oryzae are positively regulated by PrtR • However, several genes are regulated negatively by PrtR • PrtR optimizes transcription of peptidase genes in response to culture conditions.

Mechanistic study of transcription factor Sox18 during heart development.

Heart development is a delicate and complex process regulated by coordination of various signaling pathways. In this study, we investigated the role of sox18 in heart development by modulating Wnt/ß-Catenin signaling pathways. Our spatiotemporal expression analysis revealed that sox18 is mainly expressed in the heart, branchial arch, pharyngeal arch, spinal cord, and intersegmental vessels at the tailbud stage of Xenopus tropicalis embryo. Overexpression of sox18 in the X. tropicalis embryos causes heart edema, while loss-of-function of sox18 can change the signal of developmental heart marker gata4 at different stages, suggesting that sox18 plays an essential role in the development of the heart. Knockdown of SOX18 in human umbilical vein endothelial cells suggests a link between Sox18 and ß-CATENIN, a key regulator of the Wnt signaling pathway. Sox18 negatively regulates islet1 and tbx3, the downstream factors of Wnt/ß-Catenin signaling, during the linear heart tube formation and the heart looping stage. Taken together, our findings highlight the crucial role of Sox18 in the development of the heart via inhibiting Wnt/ß-Catenin signaling.

An alfalfa MYB-like transcriptional factor MsMYBH positively regulates alfalfa seedling drought resistance and undergoes MsWAV3-mediated degradation.

Drought is a major threat to alfalfa (Medicago sativa L.) production. The discovery of important alfalfa genes regulating drought response will facilitate breeding for drought-resistant alfalfa cultivars. Here, we report a genome-wide association study of drought resistance in alfalfa. We identified and functionally characterized an MYB-like transcription factor gene (MsMYBH), which increases the drought resistance in alfalfa. Compared with the wild-types, the biomass and forage quality were enhanced in MsMYBH overexpressed plants. Combined RNA-seq, proteomics and chromatin immunoprecipitation analysis showed that MsMYBH can directly bind to the promoters of MsMCP1, MsMCP2, MsPRX1A and MsCARCAB to improve their expression. The outcomes of such interactions include better water balance, high photosynthetic efficiency and scavenge excess H2O2 in response to drought. Furthermore, an E3 ubiquitin ligase (MsWAV3) was found to induce MsMYBH degradation under long-term drought, via the 26S proteasome pathway. Furthermore, variable-number tandem repeats in MsMYBH promoter were characterized among a collection of germplasms, and the variation is associated with promoter activity. Collectively, our findings shed light on the functions of MsMYBH and provide a pivotal gene that could be leveraged for breeding drought-resistant alfalfa. This discovery also offers new insights into the mechanisms of drought resistance in alfalfa.

FNDC5/irisin facilitates muscle-adipose-bone connectivity through ubiquitination-dependent activation of runt-related transcriptional factors RUNX1/2.

In the past decade, the cleavage protein irisin derived from fibronectin type III domain-containing protein 5 (FNDC5) in exercise-stimulated skeletal muscle has increasingly become a biomarker associated with metabolic syndrome and osteoporosis in humans. However, it is unclear how this protein facilitates muscle-adipose-bone connectivity in metabolic and skeletal homeostasis. In this study, we unexpectedly observed that the FNDC5 gene can be markedly activated during the differentiation of brown adipocytes but not white adipocytes, and that FNDC5 is specifically expressed in mouse brown adipose tissues (BATs). But unlike it in the skeletal muscles, the expression of FNDC5/irisin in BAT is promoted by cold exposure rather than exercise in mice. Analysis of promoter activity and chromatin immunoprecipitation further showed that peroxisome proliferator-activated receptor γ coactivator-1α and thyroid hormone receptors cooperate on the FNDC5 gene promoter to induce its transcription. We found that FNDC5/irisin stimulates the runt-related transcriptional factors RUNX1/2 via a focal adhesion kinase-dependent pathway in both bone and subcutaneous white adipose tissues. Mechanistically, focal adhesion kinase is stimulated by FNDC5/irisin and then facilitates E3 ubiquitin-protein ligase WW domain-containing protein 2 to ubiquitinate and subsequently activate RUNX1/2, culminating in the activation of osteoblast-related or thermogenesis-related genes. Interestingly, the PR domain containing protein 16 that is crucial for subcutaneous white adipose "browning" and skeletal development was found to form a complex with RUNX1/2 in a WW domain-containing protein 2-dependent manner. These findings elucidate a signaling mechanism by which FNDC5/irisin supports the muscle-adipose-bone connectivity, especially BAT-bone connectivity.

Integrated microRNA and whole-transcriptome sequencing reveals the involvement of small and long non-coding RNAs in the fiber growth of ramie plant.

BACKGROUND: MicroRNAs (miRNAs) and long non-coding RNAs (lncRNAs) are the two main types of non-coding RNAs that play crucial roles in plant growth and development. However, their specific roles in the fiber growth of ramie plant (Boehmeria nivea L. Gaud) remain largely unknown. METHODS: In this study, we performed miRNA and whole-transcriptome sequencing of two stem bark sections exhibiting different fiber growth stages to determine the expression profiles of miRNAs, lncRNAs, and protein-encoding genes. RESULTS: Among the identified 378 miRNAs and 6,839 lncRNAs, 88 miRNAs and 1,288 lncRNAs exhibited differential expression. Bioinformatics analysis revealed that 29 and 228 differentially expressed protein-encoding genes were targeted by differentially expressed miRNAs and lncRNAs, respectively, constituting eight putative competing endogenous RNA networks. lncR00022274 exhibited downregulated expression in barks with growing fibers. It also had an antisense overlap with the MYB gene, BntWG10016451, whose overexpression drastically increased the xylem fiber number and secondary wall thickness of fibers in the stems of transgenic Arabidopsis, suggesting the potential association of lncR00022274-BntWG10016451 expression with fiber growth. CONCLUSIONS: These findings provide insights into the roles of ncRNAs in the regulation of fiber growth in ramie, which can be used for the biotechnological improvement of its fiber yield and quality in the future.

Insights into ZmWAKL in maize kernel development: genome-wide investigation and GA-mediated transcription.

BACKGROUND: The functional roles of the Wall Associated Kinase (WAK) and Wall Associated Kinase Like (WAKL) families in cellular expansion and developmental processes have been well-established. However, the molecular regulation of these kinases in maize development is limited due to the absence of comprehensive genome-wide studies. RESULTS: Through an in-depth analysis, we identified 58 maize WAKL genes, and classified them into three distinct phylogenetic clusters. Moreover, structural prediction analysis showed functional conservation among WAKLs across maize. Promoter analysis uncovered the existence of cis-acting elements associated with the transcriptional regulation of ZmWAKL genes by Gibberellic acid (GA). To further elucidate the role of WAKL genes in maize kernels, we focused on three highly expressed genes, viz ZmWAKL38, ZmWAKL42 and ZmWAKL52. Co-expression analyses revealed that their expression patterns exhibited a remarkable correlation with GA-responsive transcription factors (TF) TF5, TF6, and TF8, which displayed preferential expression in kernels. RT-qPCR analysis validated the upregulation of ZmWAKL38, ZmWAKL42, ZmWAKL52, TF5, TF6, and TF8 following GA treatment. Additionally, ZmWAKL52 showed significant increase of transcription in the present of TF8, with ZmWAKL52 localizing in both the plasma membrane and cell wall. TF5 positively regulated ZmWAKL38, while TF6 positively regulated ZmWAKL42. CONCLUSIONS: Collectively, these findings provide novel insights into the characterization and regulatory mechanisms of specific ZmWAKL genes involved in maize kernel development, offering prospects for their utilization in maize breeding programs.

Structural insights into the transcriptional regulator NalC, a key component of the MexAB-OprM efflux pump system, from Pseudomonas aeruginosa.

Pseudomonas aeruginosa is an opportunistic pathogen with significant public health implications due to its multi-drug resistance (MDR). Among the mechanisms that mediate MDR, the NalC protein, a member of the TetR family of transcriptional regulators, modulates the mexAB-oprM operon, thus facilitating the efflux pump system. The resistance-nodulation-division (RND) family of multidrug efflux pumps plays a crucial role in expelling a broad spectrum of antimicrobial compounds, serving as a key adaptive mechanism. Structural analyses revealed that NalC adopts a modular architecture consisting of distinct domains involved in ligand recognition and transcriptional regulation. The N-terminal domain of NalC contains a DNA-binding helix-turn-helix motif, which interacts with specific DNA sequences in the PA3720-armR operon region. This interaction initiates the transcriptional activation of the efflux pump system. On the other hand, the C-terminal domain of NalC exhibits a highly dynamic structure and is implicated in ligand sensing and signal transduction. Our findings suggest potential binding sites for small molecules that could act as allosteric modulators, thereby providing new avenues for the development of therapeutic strategies targeting MDR Pseudomonas aeruginosa.

A dramatic decline in fruit citrate induced by mutagenesis of a NAC transcription factor, AcNAC1.

Citrate is a common primary metabolite which often characterizes fruit flavour. The key regulators of citrate accumulation in fruit and vegetables are poorly understood. We systematically analysed the dynamic profiles of organic acid components during the development of kiwifruit (Actinidia spp.). Citrate continuously accumulated so that it became the predominate contributor to total acidity at harvest. Based on a co-expression network analysis using different kiwifruit cultivars, an Al-ACTIVATED MALATE TRANSPORTER gene (AcALMT1) was identified as a candidate responsible for citrate accumulation. Electrophysiological assays using expression of this gene in Xenopus oocytes revealed that AcALMT1 functions as a citrate transporter. Additionally, transient overexpression of AcALMT1 in kiwifruit significantly increased citrate content, while tissues showing higher AcALMT1 expression accumulated more citrate. The expression of AcALMT1 was highly correlated with 17 transcription factor candidates. However, dual-luciferase and EMSA assays indicated that only the NAC transcription factor, AcNAC1, activated AcALMT1 expression via direct binding to its promoter. Targeted CRISPR-Cas9-induced mutagenesis of AcNAC1 in kiwifruit resulted in dramatic declines in citrate levels while malate and quinate levels were not substantially affected. Our findings show that transcriptional regulation of a major citrate transporter, by a NAC transcription factor, is responsible for citrate accumulation in kiwifruit, which has broad implications for other fruits and vegetables.

DNA damage-inducible transcript 3 deficiency promotes bone resorption in murine periodontitis models.

BACKGROUND AND OBJECTIVE: Periodontitis is a multifactorial inflammatory disease that leads to the destruction of supporting structures of the teeth. DNA damage-inducible transcript 3 (DDIT3) plays crucial roles in cell survival and differentiation. DDIT3 regulates bone mass and osteoclastogenesis in femur. However, the role of DDIT3 in periodontitis has not been elucidated. This research aimed to explore the role and mechanisms of DDIT3 in periodontitis. METHODS: DDIT3 gene knockout (KO) mice were generated using a CRISPR/Cas9 system. Experimental periodontitis models were established to explore the role of DDIT3 in periodontitis. The expression of DDIT3 in periodontal tissues was detected by quantitative real-time polymerase chain reaction (qRT-PCR) and immunohistochemistry (IHC). The alveolar bone phenotypes were observed by micro-CT and stereomicroscopy. The inflammation levels and osteoclast activity were examined by histological staining, immunostaining, and qRT-PCR. Bone marrow-derived macrophages (BMMs) were isolated to confirm the effects of DDIT3 on osteoclast formation and function in vitro. RESULTS: The increased expression of DDIT3 in murine inflamed periodontal tissues was detected. DDIT3 knockout aggravated alveolar bone loss and enhanced expression levels of inflammatory cytokines in murine periodontitis models. Increased osteoclast formation and higher expression levels of osteoclast-specific markers were observed in the inflamed periodontal tissues of KO mice. In vitro, DDIT3 deficiency promoted the formation of tartrate-resistant acid phosphatase (TRAP)-positive multinucleated osteoclasts and the bone resorption activity of mature osteoclasts. CONCLUSIONS: Our results demonstrate that DDIT3 deletion aggravated alveolar bone loss in experimental periodontitis through enhanced inflammatory reactions and osteoclastogenesis. The anti-inflammation and the inhibition of bone loss by DDIT3 in murine periodontitis provides a potential novel therapeutic strategy for periodontitis.

GadR4 mediates the acid resistance and pathogenicity of Listeria monocytogenes 10403S by negatively regulating the gadT2/gadD2 cluster.

Listeria monocytogenes is an important foodborne pathogen that can survive under acidic conditions. The glutamate decarboxylase (GAD) system is one of the acid resistance systems of L. monocytogenes. It usually comprises two glutamate transporters (GadT1/T2) and three glutamate decarboxylases (GadD1/D2/D3). Among them, gadT2/gadD2 contributes most significantly to the acid resistance of L. monocytogenes. However, the regulation mechanisms of gadT2/gadD2 still remain unclear. The results of this study indicated that gadT2/gadD2 deletion significantly decreases the survival rate of L. monocytogenes under different acidic conditions, including brain and heart infusion (BHI) broth, with a pH of 2.5, 2% citric acid, 2% acetic acid and 2% lactic acid. Further, gadT2/gadD2 cluster was expressed in the representative strains in response to alkaline stress rather than acid stress. To explore the regulation of gadT2/gadD2, we knocked out the five transcriptional factors belonging to the Rgg family in L. monocytogenes 10403S. We found that the deletion of gadR4, which exhibits the highest homology with the gadR of Lactococcus lactis, resulted in a significant increase in the survival rate of L. monocytogenes upon acid stress. Western blot analysis showed that gadR4 deletion significantly increased the gadD2 expression of L. monocytogenes under alkaline and neutral conditions. Furthermore, the gfp reporter gene showed that gadR4 deletion significantly increased the expression of the gadT2/gadD2 cluster. Adhesion and invasion assays indicated that gadR4 deletion significantly increased the rates of adhesion and invasion of L. monocytogenes to epithelial Caco-2 cells. Virulence assays showed that gadR4 knockout significantly improved the colonization ability of L. monocytogenes in the livers and spleens of the infected mice. Taken together, our results showed that GadR4, a transcription factor belonging to the Rgg family, negatively regulates the gadT2/gadD2 cluster, thus, reducing the acid stress tolerance and pathogenicity of L. monocytogens 10403S. Our results provide a better understanding of the regulation of the GAD system of L. monocytogenes and a novel approach to potentially prevent and control listeriosis.

PLAGL2 promotes the stemness and is upregulated by transcription factor E2F1 in human lung cancer.

This study mainly focuses on revealing the role of PLAGL2 in lung cancer stemness. In vitro and in vivo experiments were performed to evaluate the effects of PLAGL2 on lung cancer cell stemness. Mechanistic analysis using luciferase reporter and ChIP assays were implemented to reveal the underlying mechanisms. The transcriptional factor E2F1 transcriptionally activated PLAGL2 expression via directly binding to PLAGL2 promoter in lung cancer cells. Moreover, PLAGL2 promoted the stemness of lung cancer cells dependent on E2F1-mediated transcriptional activation. This study provides a potential target for lung cancer progression.

p63 immunoexpression in hair follicles of normal and alopecia X-affected skin of Pomeranian dogs.

BACKGROUND: Alopecia X in Pomeranians is caused by a hair cycle deregulation, associated with downregulation of key regulatory genes of the Wnt and Shh pathways, and stem-cell markers. However, the pathogenesis remains unclear. p63 is an important transcription factor correlated with the aforementioned hair cycle modulating genes. HYPOTHESIS/OBJECTIVES: The aim of this study was to highlight possible changes of p63 immunohistochemical expression within the hair follicles in canine alopecia X compared with normal skin. ANIMALS: Skin biopsies from 19 alopecia X-affected and six control Pomeranians were analysed. MATERIALS AND METHODS: Serial histological sections of skin biopsies harbouring anagen, telogen and kenogen hair follicles were immunohistochemically evaluated for differences in p63 expression in the affected and control samples. RESULTS: Dogs with alopecia X had a significantly decreased immunoexpression of p63 in telogen and kenogen hair follicles. CONCLUSIONS AND CLINICAL RELEVANCE: The decrease of p63 immunoexpression observed in canine alopecia X suggests an involvement of p63 in hair cycle.

Chemical inhibitors of the conserved bacterial transcriptional regulator DksA1 suppressed quorum sensing-mediated virulence of Pseudomonas aeruginosa.

Pseudomonas aeruginosa is a Gram-negative opportunistic pathogen whose virulence is dependent on quorum sensing (QS). DksA1, an RNA polymerase-binding transcriptional regulator, plays a role in determining a number of phenotypes, including QS-mediated virulence. We therefore envisioned that DksA1 inhibitors may help to control P. aeruginosa infection. Here, we screened a library of 6970 chemical compounds and identified two compounds (henceforth termed Dkstatins) that specifically suppressed DksA1 activity. Treatment with these two compounds also substantially decreased the production of elastase and pyocyanin, dominant virulence determinants of P. aeruginosa, and protected murine hosts from lethal infection from a prototype strain of P. aeruginosa, PAO1. The Dkstatins also suppressed production of homoserine lactone (HSL)-based autoinducers that activate P. aeruginosa QS. The level of 3-oxo-C12-HSL produced by Dkstatin-treated wildtype PAO1 closely resembled that of the ΔdksA1 mutant. RNA-Seq analysis showed that transcription levels of QS- and virulence-associated genes were markedly reduced in Dkstatin-treated PAO1 cells, indicating that Dkstatin-mediated suppression occurs at the transcriptional level. Importantly, Dkstatins increased the antibiotic susceptibilities of PAO1, particularly to protein synthesis inhibitors, such as tobramycin and tetracycline. Co-immunoprecipitation assays demonstrated that these Dkstatins interfered with DksA1 binding to the ß subunit of RNA polymerase, pointing to a potential mechanism of action. Collectively, our results illustrate that inhibition of P. aeruginosa QS may be achieved via DksA1 inhibitors and that Dkstatins may serve as potential lead compounds to control infection.

Id2 and Id4 are not the major negative regulators of oligodendrocyte differentiation during early central nervous system development.

Myelin sheathes ensure the rapid conduction of neural impulse and provide nutritional support for neurons. Myelin sheathes are formed by differentiated oligodendrocytes (OLs) in the central nervous system. During OL development, the differentiation of oligodendrocyte progenitor cells (OPCs) into mature OLs is controlled by both positive differentiation factors (drivers) and negative regulatory factors (brakes). Previous studies have suggested Id2 and Id4 as the key negative factors for OL differentiation. However, these conclusions were mainly based on in vitro studies and the reported OL phenotype in Id4 mutants appear to be mild. In this study, we systematically investigated the in vivo function of Id2 and Id4 genes in OL differentiation in their genetic mutants and in embryonic chicken spinal cord. Our results showed that disruption of Id4 has no effect on OL differentiation and maturation, whereas Id2 mutants and Id2/Id4 compound mutants display a mild and transient precocity of OL differentiation. In agreement with these loss-of-function studies, Id2, but not Id4, is weakly expressed in OPCs. Despite their minor roles in OL differentiation, forced expression of Id2 and Id4 in embryonic chicken spinal cords strongly inhibit the differentiation of OPCs. Taken together, our detailed functional and expressional studies strongly suggest that Id2 and Id4 are not the major in vivo repressors of OPC differentiation during animal development, shedding new light on the molecular regulation of early OL development.