Direct Transcriptional Activation Research Articles

TP53 R249S mutation in hepatic organoids captures the predisposing cancer risk.

Major genomic drivers of hepatocellular carcinoma (HCC) are nowadays well recognized, although models to establish their roles in human HCC initiation remain scarce. Here, we used human liver organoids in experimental systems to mimic the early stages of human liver carcinogenesis from the genetic lesions of TP53 loss and L3 loop R249S mutation. In addition, chromatin immunoprecipitation sequencing (ChIP-seq) of HCC cell lines shed important functional insights into the initiation of HCC consequential to the loss of tumor-suppressive function from TP53 deficiency and gain-of-function activities from mutant p53. Human liver organoids were generated from surgical nontumor liver tissues. CRISPR knockout of TP53 in liver organoids consistently demonstrated tumor-like morphological changes, increased in stemness and unrestricted in vitro propagation. To recapitulate TP53 status in human HCC, we overexpressed mutant R249S in TP53 knockout organoids. A spontaneous increase in tumorigenic potentials and bona fide HCC histology in xenotransplantations were observed. ChIP-seq analysis of HCC cell lines underscored gain-of-function properties from L3 loop p53 mutants in chromatin remodeling and overcoming extrinsic stress. More importantly, direct transcriptional activation of PSMF1 by mutant R249S could increase organoid resistance to endoplasmic reticulum stress, which was readily abrogated by PSMF1 knockdown in rescue experiments. In a patient cohort of primary HCC tumors and genome-edited liver organoids, quantitative polymerase chain reaction corroborated ChIP-seq findings and verified preferential genes modulated by L3 mutants, especially those enriched by R249S. We showed differential tumorigenic effects from TP53 loss and L3 mutations, which together confer normal hepatocytes with early clonal advantages and prosurvival functions.

MiR-585-5p impedes gastric cancer proliferation and metastasis by orchestrating the interactions among CREB1, MAPK1 and MITF.

BackgroundGastric cancer (GC) is one of the most malignant and lethal cancers worldwide. Multiple microRNAs (miRNAs) have been identified as key regulators in the progression of GC. However, the underlying pathogenesis that miRNAs govern GC malignancy remains uncertain. Here, we identified a novel miR-585-5p as a key regulator in GC development.MethodsThe expression of miR-585-5p in the context of GC tissue was detected by in situ hybridization for GC tissue microarray and assessed by H-scoring. The gain- and loss-of-function analyses comprised of Cell Counting Kit-8 assay and Transwell invasion and migration assay. The expression of downstream microphthalmia-associated transcription factor (MITF), cyclic AMP-responsive element-binding protein 1 (CREB1) and mitogen-activated protein kinase 1 (MAPK1) were examined by Immunohistochemistry, quantitative real-time PCR and western blot. The direct regulation between miR-585-5p and MITF/CREB1/MAPK1 were predicted by bioinformatic analysis and screened by luciferase reporter assay. The direct transcriptional activation of CREB1 on MITF was verified by luciferase reporter assay, chromatin immunoprecipitation (ChIP) and electrophoretic mobility shift assays (EMSAs). The interaction between MAPK1 and MITF was confirmed by co-immunoprecipitation (Co-IP) and immunofluorescent double-labelled staining.ResultsMiR-585-5p is progressively downregulated in GC tissues and low miR-585-5p levels were strongly associated with poor clinical outcomes. Further gain- and loss-of-function analyses showed that miR-585-5p possesses strong anti-proliferative and anti-metastatic capacities in GC. Follow-up studies indicated that miR-585-5p targets the downstream molecules CREB1 and MAPK1 to regulate the transcriptional and post-translational regulation of MITF, respectively, thus controlling its expression and cancer-promoting activity. MiR-585-5p directly and negatively regulates MITF together with CREB1 and MAPK1. According to bioinformatic analysis, promotor reporter gene assays, ChIP and EMSAs, CREB1 binds to the promotor region to enhance transcriptional expression of MITF. Co-IP and immunofluorescent double-labelled staining confirmed interaction between MAPK1 and MITF. Protein immunoprecipitation revealed that MAPK1 enhances MITF activity via phosphorylation (Ser73). MiR-585-5p can not only inhibit MITF expression directly, but also hinder MITF expression and pro-cancerous activity in a CREB1-/MAPK1-dependent manner indirectly.ConclusionsIn conclusion, this study uncovered miR-585-5p impedes gastric cancer proliferation and metastasis by orchestrating the interactions among CREB1, MAPK1 and MITF.

MiR-200a-3p- and miR-181-5p-Mediated HOXB5 Upregulation Promotes HCC Progression by Transcriptional Activation of EGFR.

BackgroundHepatocellular carcinoma (HCC) remains a worldwide burden. However, the mechanisms behind the malignant biological behavior of HCC remain unclear. The homeobox (HOX) family could act as either promoters or suppressors in different kinds of malignancies. Our study discovered the role of HOXB5 in regulating HCC progression.MethodsThe HOXB5 expression was assessed by RT-PCR analysis in human HCC samples and cell lines. HOXB5 transcriptional regulation of the EGFR was verified by the luciferase reporter assay and chromatin immunoprecipitation experiment. The oncogenic role of HOXB5 in HCC progression was analyzed by CCK8, colony-forming, and transwell assays.ResultsUpregulation of HOXB5 was found in human HCC, and was strongly correlated with HCC tumor size, tumor-nodule metastasis, TNM stage, and relatively unfavorable OS and DFS. Ectopic expression of HOXB5 promoted the capacity of cell growth and clonogenicity, while the inhibition of HOXB5 decreased the proliferation and clonogenicity potential in vitro by CCK8 and colony-forming assays. In addition, HOXB5 also promoted cell migration by transwell experiment. Mechanism studies elucidated that HOXB5 triggers HCC progression via direct transcriptional activation of EGFR. The upregulation of HOXB5 is regulated by miR-200a-3p and miR-181-5p. Transfection of miR-200a-3p and miR-181-5p mimics blocked the cell proliferation and migration regulated by HOXB5, while overexpression of the 3′-UTR mutant HOXB5 abolished the suppressive effect of miR-200a-3p and miR-181-5p, but not the wild-type HOXB5.ConclusionHOXB5 is a promising prognostic factor in human HCC. Targeting miR-200a-3p and the miR-181-5p/HOXB5/EGFR signaling pathway may provide new options for the treatment strategies of HCC.

Modulating gene regulation function by chemically controlled transcription factor clustering

Recent studies have suggested that transcriptional protein condensates (or clusters) may play key roles in gene regulation and cell fate determination. However, it remains largely unclear how the gene regulation function is quantitatively tuned by transcription factor (TF) clustering and whether TF clustering may confer emergent behaviors as in cell fate control systems. Here, to address this, we construct synthetic TFs whose clustering behavior can be chemically controlled. Through single-parameter tuning of the system (i.e., TF clustering propensity), we provide lines of evidence supporting the direct transcriptional activation and amplification of target genes by TF clustering. Single-gene imaging suggests that such amplification results from the modulation of transcriptional dynamics. Importantly, TF clustering propensity modulates the gene regulation function by significantly tuning the effective TF binding affinity and to a lesser extent the ultrasensitivity, contributing to bimodality and sustained response behavior that are reminiscent of canonical cell fate control systems. Collectively, these results demonstrate that TF clustering can modulate the gene regulation function to enable emergent behaviors, and highlight the potential applications of chemically controlled protein clustering.

KLF15 cistromes reveal a hepatocyte pathway governing plasma corticosteroid transport and systemic inflammation.

Circulating corticosteroids orchestrate stress adaptation, including inhibition of inflammation. While pathways governing corticosteroid biosynthesis and intracellular signaling are well understood, less is known about mechanisms controlling plasma corticosteroid transport. Here, we show that hepatocyte KLF15 (Kruppel-like factor 15) controls plasma corticosteroid transport and inflammatory responses through direct transcriptional activation of Serpina6, which encodes corticosteroid-binding globulin (CBG). Klf15-deficient mice have profoundly low CBG, reduced plasma corticosteroid binding capacity, and heightened mortality during inflammatory stress. These defects are completely rescued by reconstituting CBG, supporting that KLF15 works primarily through CBG to control plasma corticosterone homeostasis. To understand transcriptional mechanisms, we generated the first KLF15 cistromes using newly engineered Klf153xFLAG mice. Unexpectedly, liver KLF15 is predominantly promoter enriched, including Serpina6, where it binds a palindromic GC-rich motif, opens chromatin, and transactivates genes with minimal associated direct gene repression. Overall, we provide critical mechanistic insight into KLF15 function and identify a hepatocyte-intrinsic transcriptional module that potently regulates systemic corticosteroid transport and inflammation.

Coordinate control of basal epithelial cell fate and stem cell maintenance by core EMT transcription factor Zeb1.

Maintenance of undifferentiated, long-lived, and often quiescent stem cells in the basal compartment is important for homeostasis and regeneration of multiple epithelial tissues, but the molecular mechanisms that coordinately control basal cell fate and stem cell quiescence are elusive. Here, we report an epithelium-intrinsic requirement for Zeb1, a core transcriptional inducer of epithelial-to-mesenchymal transition, for mammary epithelial ductal side branching and for basal cell regenerative capacity. Our findings uncover an evolutionarily conserved role of Zeb1 in promoting basal cell fate over luminal differentiation. We show that Zeb1 loss results in increased basal cell proliferation at the expense of quiescence and self-renewal. Moreover, Zeb1 cooperates with YAP to activate Axin2 expression, and inhibition of Wnt signaling partially restores stem cell function to Zeb1-deficient basal cells. Thus, Zeb1 is a transcriptional regulator that maintains both basal cell fate and stem cell quiescence, and it functions in part through suppressing Wnt signaling.

Phosphorylation of RAV1/2 by KIN10 is essential for transcriptional activation of CAT6/7, which underlies oxidative stress response in cassava.

Related to ABI3/VP1 (RAV) transcription factors have important roles in plant stress responses; however, it is unclear whether RAVs regulates oxidative stress response in cassava (Manihot esculenta). In this study, we report that MeRAV1/2 positively regulate oxidative stress resistance and catalase (CAT) activity in cassava. Consistently, RNA sequencing (RNA-seq) identifies three MeCATs that are differentially expressed in MeRAV1/2-silenced cassava leaves. Interestingly, MeCAT6 and MeCAT7 are identified as direct transcriptional targets of MeRAV1/2 via binding to their promoters. In addition, protein kinase MeKIN10 directly interacts with MeRAV1/2 to phosphorylate them at Ser45 and Ser44 residues, respectively, to promote their direct transcriptional activation on MeCAT6 and MeCAT7. Site mutation of MeRAV1S45A or MeRAV2S44A has no significant effect on the activities of MeCAT6 and MeCAT7 promoters or on oxidative stress resistance. In summary, this study demonstrates that the phosphorylation of MeRAV1/2 by MeKIN10 is essential for its direct transcriptional activation of MeCAT6/7 in response to oxidative stress.

Ets-2 Propagates IL-6 Trans-Signaling Mediated Osteoclast-Like Changes in Human Rheumatoid Arthritis Synovial Fibroblast.

Rheumatoid arthritis synovial fibroblasts (RASFs) contribute to synovial inflammation and bone destruction by producing a pleiotropic cytokine interleukin-6 (IL-6). However, the molecular mechanisms through which IL-6 propels RASFs to contribute to bone loss are not fully understood. In the present study, we investigated the effect of IL-6 and IL-6 receptor (IL-6/IL-6R)-induced trans-signaling in human RASFs. IL-6 trans-signaling caused a significant increase in tartrate-resistant acid phosphatase (TRAP)-positive staining in RASFs and enhanced pit formation by ~3-fold in the osteogenic surface in vitro. IL-6/IL-6R caused dose-dependent increase in expression and nuclear translocation of transcription factor Ets2, which correlated with the expression of osteoclast-specific signature proteins RANKL, cathepsin B (CTSB), and cathepsin K (CTSK) in RASFs. Chromatin immunoprecipitation (ChIP) analysis of CTSB and CTSK promoters showed direct Ets2 binding and transcriptional activation upon IL-6/IL-6R stimulation. Knockdown of Ets2 significantly inhibited IL-6/IL-6R-induced RANKL, CTSB, and CTSK expression and TRAP staining in RASFs and suppressed markers of RASF invasive phenotype such as Thy1 and podoplanin (PDPN). Mass spectrometry analysis of the secretome identified 113 proteins produced by RASFs uniquely in response to IL-6/IL-6R that bioinformatically predicted its impact on metabolic reprogramming towards an osteoclast-like phenotype. These findings identified the role of Ets2 in IL-6 trans-signaling induced molecular reprogramming of RASFs to osteoclast-like cells and may contribute to RASF heterogeneity.

HIF2α promotes tumour growth in clear cell renal cell carcinoma by increasing the expression of NUDT1 to reduce oxidative stress.

BackgroundThe key role of hypoxia‐inducible factor 2alpha (HIF2α) in the process of renal cancer has been confirmed. In the field of tumour research, oxidative stress is also considered to be an important influencing factor. However, the relationship and biological benefits of oxidative stress and HIF2α in ccRCC remain unclear. This research attempts to explore the effect of oxidative stress on the cancer‐promoting effect of HIF2α in ccRCC and reveal its mechanism of action.MethodsThe bioinformatics analysis for ccRCC is based on whole transcriptome sequencing and TCGA database. The detection of the expression level of related molecules is realised by western blot and PCR. The expression of Nucleoside diphosphate‐linked moiety X‐type motif 1 (NUDT1) was knocked down by lentiviral infection technology. The functional role of NUDT1 were further investigated by CCK8 assays, transwell assays and cell oxidative stress indicator detection. The exploration of related molecular mechanisms is realised by Luciferase assays and Chromatin immunoprecipitation (ChIP) assays.ResultsMolecular screening based on knockdown HIF2α sequencing data and oxidative stress related data sets showed that NUDT1 is considered to be an important molecule for the interaction of HIF2α with oxidative stress. Subsequent experimental results showed that NUDT1 can cooperate with HIF2α to promote the progression of ccRCC. And this biological effect was found to be caused by the oxidative stress regulated by NUDT1. Mechanistically, HIF2α transcription activates the expression of NUDT1, thereby inhibiting oxidative stress and promoting the progression of ccRCC.ConclusionsThis research clarified a novel mechanism by which HIF2α stabilises sirtuin 3 (SIRT3) through direct transcriptional activation of NUDT1, thereby inhibiting oxidative stress to promote the development of ccRCC. It provided the possibility for the selection of new therapeutic targets for ccRCC and the study of combination medication regimens.

167 Contribution of miRNAs as epigenetic regulators of Retinol efficacy in the skin dermis

Vitamin A and its derivatives, particularly retinol, slow both chronological and photo-induced aging processes. Retinol reduces the appearance of wrinkles and the decrease of the skin firmness and elasticity by protecting against collagen and elastin fiber changes. In addition to retinol’s rejuvenating skin benefits through direct transcriptional activation, recent studies also suggest an epigenetic regulation through micro-RNAs (miRNAs) modulation. We investigated how retinol supports the stimulation of type I collagen and elastin through proteomic, transcriptomic and epigenetic miRNA- expression changes in human skin fibroblasts.

ROS-Nrf2 pathway mediates the development of TGF-β1-induced epithelial-mesenchymal transition through the activation of Notch signaling

Epithelial-mesenchymal transition (EMT) is a cellular process by which epithelial cells transform to acquire mesenchymal phenotypes. Accumulating evidence indicate the involvement of EMT in the progression of malignant diseases. Notch signaling mediates TGF-β1-induced EMT through direct transcriptional activation of Snai1. The molecular mechanism how TGF-β1 activates Notch signaling, however, remains unknown. In this study, we show a pivotal role for reactive oxygen species (ROS)-Nrf2 pathway in TGF-β1-induced Notch signaling activation and EMT development. TGF-β1 induces Nrf2 activation through ROS production. Inhibiting Nrf2 activation either by reducing ROS levels by N-acetylcysteine or by knocking down of Nrf2 by small interfering RNA attenuated both Notch signaling activation and EMT development. TGF-β1 induced the transcription of Notch4 via Nrf2-dependent promoter activation. In conclusion, our study indicates the ROS-Nrf2 pathway mediates the development of TGF-β1-induced EMT through the activation of Notch signaling.

FOXA2 inhibits doxorubicin-induced apoptosis via transcriptionally activating HBP rate-limiting enzyme GFPT1 in HCC cells.

Apoptosis plays an important role in both carcinogenesis and cancer treatment. Understanding the mechanisms through which resistance to apoptosis occurs in cancer cells has huge implications for cancer treatment. Although pieces of evidence have shown that elevated levels of global O-GlcNAcylation play an anti-apoptotic role in myriad cancers, the underlying mechanism is still ambiguous. In this study, we demonstrated that FOXA2, an essential transcription factor for liver homeostasis and hepatocellular carcinoma (HCC) development, inhibits doxorubicin (DOX)-induced apoptosis through elevating cellular O-GlcNAcylation in HCC cells. In response to DOX treatment, elevated FOXA2 and global O-GlcNAcylation level was observed in HCC cells, and higher FOXA2 levels indicated lower levels of DOX-induced apoptosis. Subsequently, we demonstrated that FOXA2 is a direct transcriptional activator of the hexosamine biosynthetic pathway (HBP) rate-limiting enzyme GFPT1. The upregulation of FOXA2 expression induced the synthesis of intracellular UDP-GlcNAc, which is the sugar substrate of O-GlcNAcylation produced by the HBP. The flux through the HBP elevated the global O-GlcNAcylation level and led to the activation of survival signaling pathways in HCC cells. Furthermore, GFPT1 was proved to be an important downstream regulator of FOXA2-mediated apoptotic suppression. These results provide insights into the molecular mechanism by which FOXA2 inhibits DOX-induced HCC cell apoptosis and suggest that targeting FOXA2 might offer a new strategy for HCC treatment.

Abstract 2031: The PERK arm of the unfolded protein response induces CXCL8 and CCL20 through C/EBPδ

Abstract Cancer cells often experience endoplasmic reticulum (ER) stress due to either activated oncogenes, microenvironmental conditions or therapeutic interventions. ER stress initiates an adaptive unfolded protein response (UPR) program, mediated by PERK, IRE1 and ATF6 transmembrane sensors, which alleviates the stress and promotes cells survival. However, if unmitigated, the UPR proceeds to initiation of cell death. Cancer cells often usurp the UPR pathways that contribute to cell survival and metastasis through intra- and intercellular mechanisms. Intracellular mechanisms of cellular adaptation are well-characterized; however, the intercellular consequences of ER stress are less understood. Here, we show that in cell lines representing melanoma, breast and prostate cancer, the transcription factor C/EBPδ (CEBPD) is an early ER stress response factor that is induced by the PERK arm of the UPR. CEBPD induction occurs in part through activation of PERK-mediated non-canonical JAK/STAT3 signaling. Transcriptional profiling in MDA-MB-435 melanoma cells revealed that C/EBPδ supports the expression of genes involved in cell intrinsic adaptations such as chaperones, components of ER associated degradation, and apoptosis inhibitors. In addition, C/EBPδ promotes expression of the inflammatory mediators CXCL8 and CCL20, which are known to modulate the tumor microenvironment through the receptors present on various host cells. Depletion of CEBPD by siRNA or inhibition of PERK kinase reduced CXCL8 and CCL20 transcripts and secreted proteins upon ER stress induction by thapsigargin. Bioinformatic analysis and the ENCODE ChIP-seq data suggest direct interaction of C/EBPδ with CXCL8 and CCL20 promoter/enhancer, indicative of direct transcriptional activation of the genes. Indeed, ectopic expression of C/EBPδ, but not a mutant lacking the transactivation domain, was sufficient to induced CXCL8 and CCL20 transcription in HEK293 cells in the absence of ER stress. Consistent with these results, conditioned media from ER-stressed MDA-MB-435 cells induced the gene expression of proinflammatory cytokines IL-6, IL-12, TNFα and IL-1RA in HL-60 promyelocytic cells while suppressing IL-10 expression. Taken together, these data show that the PERK arm of the UPR activates JAK/STAT3 pathway to induce expression of C/EBPδ and thereby enhance secretion of immunomodulators and transmit UPR-induced inflammatory signals to immune cells in the tumor microenvironment. This pathway may contribute to the overall outcome of the UPR on cancer cell survival, metastasis and response to anticancer therapies. Citation Format: Dipak Kumar Poria, Namratha Sheshadri, Shikha Sharan, Esta Sterneck. The PERK arm of the unfolded protein response induces CXCL8 and CCL20 through C/EBPδ [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2031.

TCF3 is epigenetically silenced by EZH2 and DNMT3B and functions as a tumor suppressor in endometrial cancer

Endometrial cancer (EC) is the most common gynecological malignancy worldwide. However, the molecular mechanisms underlying EC progression are still largely unknown, and chemotherapeutic options for EC patients are currently very limited. In this study, we found that histone methyltransferase EZH2 and DNA methyltransferase DNMT3B were upregulated in EC samples from patients, and promoted EC cell proliferation as evidenced by assays of cell viability, cell cycle, colony formation. Mechanistically, we found that EZH2 promoted EC cell proliferation by epigenetically repressing TCF3, a direct transcriptional activator of CCKN1A (p21WAF1/Cip1), in vitro and in vivo. In addition, we found that DNMT3B specifically methylated the TCF3 promoter, repressing TCF3 expression and accelerating EC cell proliferation independently of EZH2. Importantly, elevated expression of EZH2 or DNMT3B in EC patients inversely correlated with expression of TCF3 and p21, and was associated with shorter overall survival. We show that combined treatment with GSK126 and 5-Aza-2d treatment wit synergistically inhibited methyltransferase activity of EZH2 and DNMT3B, resulting in a profound block of EC cell proliferation as well as EC tumor progression in cell line-derived xenograft (CDX) and patient-derived xenograft (PDX) mouse models. These findings reveal that TCF3 functions as a tumor suppressor epigenetically silenced by EZH2 and DNMT3B in EC, and support the notion that targeting the EZH2/DNMT3B/TCF3/p21 axis may be a novel and effective therapeutic strategy for treatment of EC.

Heteromeric HSFA2/HSFA3 complexes drive transcriptional memory after heat stress in Arabidopsis

Adaptive plasticity in stress responses is a key element of plant survival strategies. For instance, moderate heat stress (HS) primes a plant to acquire thermotolerance, which allows subsequent survival of more severe HS conditions. Acquired thermotolerance is actively maintained over several days (HS memory) and involves the sustained induction of memory-related genes. Here we show that FORGETTER3/ HEAT SHOCK TRANSCRIPTION FACTOR A3 (FGT3/HSFA3) is specifically required for physiological HS memory and maintaining high memory-gene expression during the days following a HS exposure. HSFA3 mediates HS memory by direct transcriptional activation of memory-related genes after return to normal growth temperatures. HSFA3 binds HSFA2, and in vivo both proteins form heteromeric complexes with additional HSFs. Our results indicate that only complexes containing both HSFA2 and HSFA3 efficiently promote transcriptional memory by positively influencing histone H3 lysine 4 (H3K4) hyper-methylation. In summary, our work defines the major HSF complex controlling transcriptional memory and elucidates the in vivo dynamics of HSF complexes during somatic stress memory.

Transcriptional enhancement of GBP-5 by BATF aggravates sepsis-associated liver injury via NLRP3 inflammasome activation.

Strong inflammatory response triggered by the activation of the innate immune system is one typical characteristic of sepsis-associated liver injury (SALI). Guanylate-binding protein 5 (GBP-5) is a component of cell-autonomous immunity and known to be associated with inflammation. Currently, whether GBP-5 participates in SALI and its roles in this disease are yet to be investigated. Using a lipopolysaccharide (LPS)-induced SALI mouse model, we found GBP-5 was highly expressed in LPS-treated mice, and its expression was tightly related to the serum concentrations of live injury markers and inflammatory cytokines, liver damage scores by H&E staining, and amounts of apoptotic hepatocytes by TUNEL staining. Moreover, GBP-5 overexpression was found to aggravate LPS-induced SALI by promoting the activation of NLR family pyrin domain containing 3 (NLRP3) inflammasome, then facilitated the production of pro-inflammatory cytokines, eventually induced hepatocyte cell death. Direct transcriptional activation of GBP-5 by basic leucine zipper ATF-like transcription factor (BATF) was identified and further validated. This study unveils a transcriptional upregulation of GBP-5 by interacting with BATF, which promotes the progression of LPS-induced SALI through NLRP3 inflammasome activation, and provides novel therapeutic insights for halting the progression of liver injury in various liver diseases.

Posttranslational regulation of multiple clock-related transcription factors triggers cold-inducible gene expression in Arabidopsis

Cold stress is an adverse environmental condition that affects plant growth, development, and crop productivity. Under cold stress conditions, the expression of numerous genes that function in the stress response and tolerance is induced in various plant species, and the dehydration-responsive element (DRE) binding protein 1/C-repeat binding factor (DREB1/CBF) transcription factors function as master switches for cold-inducible gene expression. Cold stress strongly induces these DREB1 genes. Therefore, it is important to elucidate the mechanisms of DREB1 expression in response to cold stress to clarify the perception and response of cold stress in plants. Previous studies indicated that the central oscillator components of the circadian clock, CIRCADIAN CLOCK-ASSOCIATED 1 (CCA1) and LATE ELONGATED HYPOCOTYL (LHY), are involved in cold-inducible DREB1 expression, but the underlying mechanisms are not clear. We revealed that the clock-related MYB proteins REVEILLE4/LHY-CCA1-Like1 (RVE4/LCL1) and RVE8/LCL5 are quickly and reversibly transferred from the cytoplasm to the nucleus under cold stress conditions and function as direct transcriptional activators of DREB1 expression. We found that CCA1 and LHY suppressed the expression of DREB1s under unstressed conditions and were rapidly degraded specifically in response to cold stress, which suggests that they act as transcriptional repressors and indirectly regulate the cold-inducible expression of DREB1s We concluded that posttranslational regulation of multiple clock-related transcription factors triggers cold-inducible gene expression. Our findings clarify the complex relationship between the plant circadian clock and the regulatory mechanisms of cold-inducible gene expression.

Estrogen receptor 2b is the major determinant of sex-typical mating behavior and sexual preference in medaka

Male and female animals typically display innate sex-specific mating behaviors, which, in vertebrates, are highly dependent on sex steroid signaling. While estradiol-17β (E2) signaling through estrogen receptor 2 (ESR2) serves to defeminize male mating behavior in rodents, the available evidence suggests that E2 signaling is not required in teleosts for either male or female mating behavior. Here, we report that female medaka deficient for Esr2b, a teleost ortholog of ESR2, are not receptive to males but rather court females, despite retaining normal ovarian function with an unaltered sex steroid milieu. Thus, contrary to both prevailing views in rodents and teleosts, E2/Esr2b signaling in the brain plays a decisive role in feminization and demasculinization of female mating behavior and sexual preference in medaka. Further behavioral testing showed that mutual antagonism between E2/Esr2b signaling and androgen receptor-mediated androgen signaling in adulthood induces and actively maintains sex-typical mating behaviors and preference. Our results also revealed that the female-biased sexual dimorphism in esr2b expression in the telencephalic and preoptic nuclei implicated in mating behavior can be reversed between males and females by altering the sex steroid milieu in adulthood, likely via mechanisms involving direct E2-induced transcriptional activation. In addition, Npba, a neuropeptide mediating female sexual receptivity, was found to act downstream of E2/Esr2b signaling in these brain nuclei. Collectively, these functional and regulatory mechanisms of E2/Esr2b signaling presumably underpin the neural mechanism for induction, maintenance, and reversal of sex-typical mating behaviors and sexual preference in teleosts, at least in medaka.

Early growth response 2 (EGR2) is a novel regulator of the senescence programme.

Senescence, a state of stable growth arrest, plays an important role in ageing and age‐related diseases in vivo. Although the INK4/ARF locus is known to be essential for senescence programmes, the key regulators driving p16 and ARF transcription remain largely underexplored. Using siRNA screening for modulators of the p16/pRB and ARF/p53/p21 pathways in deeply senescent human mammary epithelial cells (DS HMECs) and fibroblasts (DS HMFs), we identified EGR2 as a novel regulator of senescence. EGR2 expression is up‐regulated during senescence, and its ablation by siRNA in DS HMECs and HMFs transiently reverses the senescent phenotype. We demonstrate that EGR2 activates the ARF and p16 promoters and directly binds to both the ARF and p16 promoters. Loss of EGR2 down‐regulates p16 levels and increases the pool of p16− p21− ‘reversed’ cells in the population. Moreover, EGR2 overexpression is sufficient to induce senescence. Our data suggest that EGR2 is a direct transcriptional activator of the p16/pRB and ARF/p53/p21 pathways in senescence and a novel marker of senescence.

Unorthodox Transcriptional Mechanisms of Lipid-Sensing Nuclear Receptors in Macrophages: Are We Opening a New Chapter?

Work over the past 30 years has shown that lipid-activated nuclear receptors form a bridge between metabolism and immunity integrating metabolic and inflammatory signaling in innate immune cells. Ligand-induced direct transcriptional activation and protein-protein interaction-based transrepression were identified as the most common mechanisms of liganded-nuclear receptor-mediated transcriptional regulation. However, the integration of different next-generation sequencing-based methodologies including chromatin immunoprecipitation followed by sequencing and global run-on sequencing allowed to investigate the DNA binding and ligand responsiveness of nuclear receptors at the whole-genome level. Surprisingly, these studies have raised the notion that a major portion of lipid-sensing nuclear receptor cistromes are not necessarily responsive to ligand activation. Although the biological role of the ligand insensitive portion of nuclear receptor cistromes is largely unknown, recent findings indicate that they may play roles in the organization of chromatin structure, in the regulation of transcriptional memory, and the epigenomic modification of responsiveness to other microenvironmental signals in macrophages. In this review, we will provide an overview and discuss recent advances of our understanding of lipid-activated nuclear receptor-mediated non-classical or unorthodox actions in macrophages.