Members of the abscisic acid (ABA)-responsive element (ABRE) binding factor (ABF) and ABA-responsive element binding protein (AREB) families play essential roles in the regulation of ABA signaling pathway activity and shape the ability of plants to adapt to a range of stressful environmental conditions. To date, however, systematic genome-wide analyses focused on the ABF/AREB gene family in wheat are lacking. Here, we identified 35 ABF/AREB genes in the wheat genome, designated TaABF1-TaABF35 according to their chromosomal distribution. These genes were further classified, based on their phylogenetic relationships, into three groups (A-C), with the TaABF genes in a given group exhibiting similar motifs and similar numbers of introns/exons. Cis-element analyses of the promoter regions upstream of these TaABFs revealed large numbers of ABREs, with the other predominant elements that were identified differing across these three groups. Patterns of TaABF gene expansion were primarily characterized by allopolyploidization and fragment duplication, with purifying selection having played a significant role in the evolution of this gene family. Further expression profiling indicated that the majority of the TaABF genes from groups A and B were highly expressed in various tissues and upregulated following abiotic stress exposure such as drought, low temperature, low nitrogen, etc., while some of the TaABF genes in group C were specifically expressed in grain tissues. Regulatory network analyses revealed that four of the group A TaABFs (TaABF2, TaABF7, TaABF13, and TaABF19) were centrally located in protein-protein interaction networks, with 13 of these TaABF genes being regulated by 11 known miRNAs, which play important roles in abiotic stress resistance such as drought and salt stress. The two primary upstream transcription factor types found to regulate TaABF gene expression were BBR/BPC and ERF, which have previously been reported to be important in the context of plant abiotic stress responses. Together, these results offer insight into the role that the ABF/AREB genes play in the responses of wheat to abiotic stressors, providing a robust foundation for future functional studies of these genes.

BACKGROUND: Endothelial-to-Mesenchymal Transformation (EndMT) plays key roles in endothelial dysfunction during the pathological progression of atherosclerosis; however, its detailed mechanism remains unclear. Herein, we explored the biological function and mechanisms of upstream stimulating factor 1 (USF1) in EndMT during atherosclerosis.
METHODS: The in vivo and in vitro atherosclerotic models were established in high fat diet-fed ApoE-/- mice and ox-LDL-exposed human umbilical vein endothelial cells (HUVECs). The plaque formation, collagen and lipid deposition, and morphological changes in the aortic tissues were evaluated by hematoxylin and eosin (HE), Masson, Oil red O and Verhoeff-Van Gieson (EVG) staining, respectively. EndMT was determined by expression levels of EndMT-related proteins. Target molecule expression was detected by RT-qPCR and Western blotting. The release of pro-inflammatory cytokines was measured by ELISA. Migration of HUVECs was detected by transwell and scratch assays. Molecular mechanism was investigated by dual-luciferase reporter assay, ChIP, and Co-IP assays.
RESULTS: USF1 was up-regulated in atherosclerosis patients. USF1 knockdown inhibited EndMT by up-regulating CD31 and VE-Cadherin, while down-regulating α-SMA and vimentin, thereby repressing inflammation, and migration in ox-LDL-exposed HUVECs. In addition, USF1 transcriptionally activated ubiquitin-specific protease 14 (USP14), which promoted de-ubiquitination and up-regulation of NLR Family CARD Domain Containing 5 (NLRC5) and subsequent Smad2/3 pathway activation. The inhibitory effect of sh-USF1 or sh-USP14 on EndMT was partly reversed by USP14 or NLRC5 overexpression. Finally, USF1 knockdown delayed atherosclerosis progression via inhibiting EndMT in mice.
CONCLUSIONS: Our findings indicate the contribution of the USF1/USP14/NLRC5 axis to atherosclerosis development via promoting EndMT, which provide effective therapeutic targets.

Roots are the main organ for water uptake and the earliest part of a plant\'s response to drought, making them of great importance to our understanding of the root system\'s response to drought. However, little is known about the underlying molecular mechanisms that control root responses to drought stress. Here, we identified and functionally characterized the AP2/ERF family transcription factor (TF) PtrABR1 and the upstream target gene zinc-finger protein TF PtrYY1, which respond to drought stress by promoting the growth and development of lateral roots in Populus trichocarpa. A root-specific induction of PtrABR1 under drought stress was explored. The overexpression of PtrABR1 (PtrABR1-OE) promoted root growth and development, thereby increasing tolerance to drought stress. In addition, PtrYY1 is directly bound to the promoter of PtrABR1 under drought stress, and the overexpression of PtrYY1 (PtrYY1-OE) promoted lateral root growth and development and increased tolerance to drought stress. An RNA-seq analysis of PtrABR1-OE with wild-type (WT) poplar identified PtrGH3.6 and PtrPP2C44, which share the same pattern of expression changes as PtrABR1. A qRT-PCR and cis-element analysis further suggested that PtrGH3.6 and PtrPP2C44 may act as potential downstream targets of PtrABR1 genes in the root response pathway to drought stress. In conclusion, these results reveal a novel drought regulatory pathway in which PtrABR1 regulates the network through the upstream target gene PtrYY1 and the potential downstream target genes PtrGH3.6 and PtrPP2C44, thereby promoting root growth and development and improving tolerance to drought stress.

Cervical cancer (CC) represents a major global health concern, characterized by chemoresistance and immune evasion mechanisms. Circular RNAs (circRNAs), which play a crucial role in cancer pathogenesis, particularly in the case of CC, have gained significant attention. The primary objective of this study was to investigate the functional significance of circRNAs in chemoresistant CC. A significant upregulation of circPRDM4 expression in chemoresistant CC cells. To investigate the functional consequences, we conducted circPRDM4 knockdown experiments, which resulted in the effective blockade of immune escape mechanisms employed by chemoresistant CC cells. Furthermore, circPRDM4 knockdown demonstrated a significant suppression of tumorigenesis in CC cells, highlighting its contribution to the oncogenic potential of CC. Investigating the regulatory mechanisms involved, we found that the transcriptional factor upstream stimulatory factor 1 (USF1) acts as an inducer of circPRDM4 expression. Remarkably, USF1 was found to effectively modulate CC cell immune escape via its interaction with circPRDM4. Moreover, our results revealed that USF1 is intricately involved in CC cell tumorigenesis through the regulation of circPRDM4. Collectively, our study elucidates the significant roles of circPRDM4 and its upstream regulator USF1 in chemoresistant CC cells. These findings underscore the importance of circRNAs in CC pathogenesis and provide valuable insights into the mechanisms underlying immune escape and tumorigenesis.

Syntaxin-6 (STX6), a protein of the syntaxin family, is located in the trans-Golgi network and is involved in a variety of intracellular membrane transport events. STX6 is overexpressed in different human malignant tumors. However, little is known about its exact function and molecular mechanism in hepatocellular carcinoma (HCC). In this study, we found that the expression of STX6 was significantly increased in HCC tissues and was associated with poor survival. Gain- and loss-of-function experiments showed that STX6 promotes cell proliferation and metastasis of HCC cells both in vitro and in vivo. Mechanistically, STX6 was negatively regulated by the upstream stimulatory factor 2 (USF2). In addition, STX6 facilitates the association of autophagosomes with lysosomes. Importantly, we demonstrated that STX6 overexpression, despite enhanced resistance to lenvatinib, sensitizes HCC cells to the autophagy activator rapamycin. This study revealed that, under the control of USF2, STX6 accelerates the degradation of microtubule-associated protein 1 light chain 3 beta (LC3) by promoting autophagic flux, ultimately promoting HCC progression. Collectively, we suggest that the USF2-STX6-LC3B axis is a potential therapeutic target in liver cancer.

HIV-1 provirus expression is controlled by signaling pathways that are responsive to T cell receptor engagement, including those involving Ras and downstream protein kinases. The induction of transcription from the HIV-1 LTR in response to Ras signaling requires binding of the Ras-responsive element binding factor (RBF-2) to conserved cis elements flanking the enhancer region, designated RBE3 and RBE1. RBF-2 is composed minimally of the USF1, USF2, and TFII-I transcription factors. We recently determined that TFII-I regulates transcriptional elongation from the LTR through recruitment of the co-activator TRIM24. However, the function of USF1 and USF2 for this effect are uncharacterized. Here, we find that genetic deletion of USF2 but not USF1 in T cells inhibits HIV-1 expression. The loss of USF2 caused a reduction in expression of the USF1 protein, an effect that was not associated with decreased USF1 mRNA abundance. USF1 and USF2 were previously shown to exist predominately as heterodimers and to cooperatively regulate target genes. To examine cooperativity between these factors, we performed RNA-seq analysis of T cell lines bearing knockouts of the genes encoding these factors. In untreated cells, we found limited evidence of coordinated global gene regulation between USF1 and USF2. In contrast, we observed a high degree of genome-wide cooperative regulation of RNA expression between these factors in cells stimulated with the combination of PMA and ionomycin. In particular, we found that the deletion of USF1 or USF2 restricted T cell activation response. These observations indicate that USF2, but not USF1, is crucial for HIV-1 expression, while the combined function of these factors is required for a robust T cell inflammatory response.

Cellular senescence is a program of cell cycle arrest, apoptosis resistance, and cytokine release induced by stress exposure in metazoan cells. Landmark studies in laboratory mice have characterized a number of master senescence regulators, including p16INK4a, p21, NF-κB, p53, and C/EBPβ. To discover other molecular players in senescence, we developed a screening approach to harness the evolutionary divergence between mouse species. We found that primary cells from the Mediterranean mouse Mus spretus, when treated with DNA damage to induce senescence, produced less cytokine and had less-active lysosomes than cells from laboratory Mus musculus. We used allele-specific expression profiling to catalog senescence-dependent cis-regulatory variation between the species at thousands of genes. We then tested for correlation between these expression changes and interspecies sequence variants in the binding sites of transcription factors. Among the emergent candidate senescence regulators, we chose a little-studied cell cycle factor, upstream stimulatory factor 2 (USF2), for molecular validation. In acute irradiation experiments, cells lacking USF2 had compromised DNA damage repair and response. Longer-term senescent cultures without USF2 mounted an exaggerated senescence regulatory program-shutting down cell cycle and DNA repair pathways, and turning up cytokine expression, more avidly than wild-type. We interpret these findings under a model of pro-repair, anti-senescence regulatory function by USF2. Our study affords new insights into the mechanisms by which cells commit to senescence, and serves as a validated proof of concept for natural variation-based regulator screens.

The nuclear factor-κB (NF-κB) signalling pathway plays a critical role in the pathogenesis of multiple vascular diseases. However, in endothelial cells (ECs), the molecular mechanisms responsible for the negative regulation of the NF-κB pathway are poorly understood. In this study, we investigated a novel role for protein tyrosine phosphatase type IVA1 (PTP4A1) in NF-κB signalling in ECs.
In human tissues, human umbilical artery ECs, and mouse models for loss of function and gain of function of PTP4A1, we conducted histological analysis, immunostaining, laser-captured microdissection assay, lentiviral infection, small interfering RNA transfection, quantitative real-time PCR and reverse transcription-PCR, as well as luciferase reporter gene and chromatin immunoprecipitation assays. Short hairpin RNA-mediated knockdown of PTP4A1 and overexpression of PTP4A1 in ECs indicated that PTP4A1 is critical for inhibiting the expression of cell adhesion molecules (CAMs). PTP4A1 increased the transcriptional activity of upstream stimulatory factor 1 (USF1) by dephosphorylating its S309 residue and subsequently inducing the transcription of tumour necrosis factor-alpha-induced protein 3 (TNFAIP3/A20) and the inhibition of NF-κB activity. Studies on Ptp4a1 knockout or transgenic mice demonstrated that PTP4A1 potently regulates the interleukin 1β-induced expression of CAMs in vivo. In addition, we verified that PTP4A1 deficiency in apolipoprotein E knockout mice exacerbated high-fat high-cholesterol diet-induced atherogenesis with upregulated expression of CAMs.
Our data indicate that PTP4A1 is a novel negative regulator of vascular inflammation by inducing USF1/A20 axis-mediated NF-κB inactivation. Therefore, the expression and/or activation of PTP4A1 in ECs might be useful for the treatment of vascular inflammatory diseases.

Angiotensin II type 1 receptor-associated protein (ATRAP) is widely expressed in different tissues and organs, although its mechanistic role in breast cancer remains unclear. Here, we show that ATRAP is highly expressed in breast cancer tissues. Its aberrant upregulation promotes breast cancer aggressiveness and is positively correlated with poor prognosis. Functional assays revealed that ATRAP participates in promoting cell growth, metastasis, and aerobic glycolysis, while microarray analysis showed that ATRAP can activate the AKT/mTOR signaling pathway in cancer progression. In addition, ATRAP was revealed to direct Ubiquitin-specific protease 14 (USP14)-mediated deubiquitination and stabilization of Pre-B cell leukemia homeobox 3 (PBX3). Importantly, ATRAP is a direct target of Upstream stimulatory factor 1 (USF1), and that ATRAP overexpression reverses the inhibitory effects of USF1 knockdown. Our study demonstrates the broad contribution of the USF1/ATRAP/PBX3 axis to breast cancer progression and provides a strong potential therapeutic target.

Glioblastoma stem cells (GSCs) and their interplay with tumor-associated macrophages (TAMs) are responsible for malignant growth and tumor recurrence of glioblastoma multiforme (GBM), but the underlying mechanisms are largely unknown.
Cell viability, stemness, migration, and invasion were measured in GSCs after the knockdown of upstream stimulating factor 1 (USF1). Luciferase assay and chromatin immunoprecipitation qPCR were performed to determine the regulation of CD90 by USF1. Immunohistochemistry and immunofluorescent staining were used to examine the expression of USF1 and GSC markers, as well as the crosstalk between GSCs and TAMs. In addition, the interaction between GSCs and TAMs was confirmed using in vivo GBM models.
We show that USF1 promotes malignant glioblastoma phenotypes and GSCs-TAMs physical interaction by inducing CD90 expression. USF1 predicts a poor prognosis for glioma patients and is upregulated in patient-derived GSCs and glioblastoma cell lines. USF1 overexpression increases the proliferation, invasion, and neurosphere formation of GSCs and glioblastoma cell lines, while USF1 knockdown exerts an opposite effect. Further mechanistic studies reveal that USF1 promotes GSC stemness by directly regulating CD90 expression. Importantly, CD90 of GSCs functions as an anchor for physical interaction with macrophages. Additionally, the USF1/CD90 signaling axis supports the GSCs and TAMs adhesion and immunosuppressive feature of TAMs, which in turn enhance the stemness of GSCs. Moreover, the overexpression of CD90 restores the stemness property in USF1 knockdown GSCs and its immunosuppressive microenvironment.
Our findings indicate that the USF1/CD90 axis might be a potential therapeutic target for the treatment of glioblastoma.