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Retinal Ganglion Cells (RGCs) lose their ability to grow axons during development. Adult RGCs thus fail to regenerate their axons after injury, leading to vision loss. To uncover mechanisms that promote regeneration of RGC axons, we identified transcription factors (TF) and open chromatin regions that are enriched in rat embryonic RGCs (high axon growth capacity) compared to postnatal RGCs (low axon growth capacity). We found that developmental stage-specific gene expression changes correlated with changes in promoter chromatin accessibility. Binding motifs for TFs such as CREB, CTCF, JUN and YY1 were enriched in the regions of the chromatin that were more accessible in embryonic RGCs. Proteomic analysis of purified rat RGC nuclei confirmed the expression of TFs with potential role in axon growth such as CREB, CTCF, YY1, and JUND. The CREB/ATF binding motif was widespread at the open chromatin region of known pro-regenerative TFs, supporting a role of CREB in regulating axon regeneration. Consistently, overexpression of CREB fused to the VP64 transactivation domain in mouse RGCs promoted axon regeneration after optic nerve injury. Our study provides a map of the chromatin accessibility during RGC development and highlights that TF associated with developmental axon growth can stimulate axon regeneration in mature RGC.

B-Myb is an important transcription factor that plays a critical role in gene expression regulation and tumorigenesis. However, its functional implication in colorectal cancer remains elusive. In this study, we found that B-Myb was significantly upregulated at both mRNA and protein levels in colorectal cancer samples compared to non-tumor counterparts. B-Myb overexpression accelerated cell proliferation, cell cycle progression and cell motility in colorectal cancer cells, and promoted tumor growth in orthotopic nude mouse models in vivo. In contrast, B-Myb depletion inhibited these malignant phenotypes. Mechanistic investigations revealed that E2F2 was a novel transcriptional target of B-Myb and is essential to B-Myb-induced malignant phenotypes. Notably, B-Myb and E2F2 exhibited positive expression correlation, and interacted with each other in colorectal cancer cells. In addition to their autoregulatory mechanisms, B-Myb and E2F2 can also directly transactivate each other, thus constituting consolidated reciprocal feed-forward transactivation loops. Moreover, both B-Myb and E2F2 are required for the activation of ERK and AKT signaling pathways in colorectal cancer cells. Taken together, our data clarified a critical role for B-Myb in colorectal cancer and unraveled an exquisite mutual collaboration and reciprocal cross regulation between B-Myb and E2F2 that contribute to the malignant progression of human colorectal cancer.

Ribonucleotide reductase (RR) is a unique enzyme for the reduction of NDPs to dNDPs, the building blocks for DNA synthesis and thus essential for cell proliferation. Pan-cancer profiling studies showed that RRM2, the small subunit M2 of RR, is abnormally overexpressed in multiple types of cancers; however, the underlying regulatory mechanisms in cancers are still unclear. In this study, through searching in cancer-omics databases and immunohistochemistry validation with clinical samples, we showed that the expression of MYBL2, a key oncogenic transcriptional factor, was significantly upregulated correlatively with RRM2 in colorectal cancer (CRC). Ectopic expression and knockdown experiments indicated that MYBL2 was essential for CRC cell proliferation, DNA synthesis, and cell cycle progression in an RRM2-dependent manner. Mechanistically, MYBL2 directly bound to the promoter of RRM2 gene and promoted its transcription during S-phase together with TAF15 and MuvB components. Notably, knockdown of MYBL2 sensitized CRC cells to treatment with MK-1775, a clinical trial drug for inhibition of WEE1, which is involved in a degradation pathway of RRM2. Finally, mouse xenograft experiments showed that the combined suppression of MYBL2 and WEE1 synergistically inhibited CRC growth with a low systemic toxicity in vivo. Therefore, we propose a new regulatory mechanism for RRM2 transcription for CRC proliferation, in which MYBL2 functions by constituting a dynamic S-phase transcription complex following the G1/early S-phase E2Fs complex. Doubly targeting the transcription and degradation machines of RRM2 could produce a synthetic inhibitory effect on RRM2 level with a novel potential for CRC treatment.

The immediate early protein 1 (IE1) acts as a transcriptional activator and is essential for viral gene transcription and viral DNA replication. However, the key regulatory domains of IE1 remain poorly understood. Here, we analyzed the sequence characteristics of Bombyx mori nucleopolyhedrovirus (BmNPV) IE1 and identified the key functional domains of BmNPV IE1 by stepwise truncation. Our results showed that BmNPV IE1 was highly similar to Autographa californica nucleopolyhedrovirus (AcMNPV) IE1, but was less conserved with IE1 of other baculoviruses, the C-terminus of IE1 was more conserved than the N-terminus, and BmNPV IE1 was also necessary for BmNPV proliferation. Moreover, we found that IE1158–208 was a major nuclear localization element, and IE11–157 and IE1539–559 were minor nuclear localization elements, but the combination of these two minor elements was equally sufficient to fully mediate the nuclear entry of IE1. Meanwhile, IE11–258, IE1560–584, and the association of amino acids 258 and 259 were indispensable for the transactivation activity of BmNPV IE1. These results systematically resolve the functional domains of BmNPV IE1, which contribute to the understanding of the mechanism of baculovirus infection and provide a possibility to synthesize a small molecule IE1-truncated mutant as an agonist or antagonist.

The androgen receptor (AR) plays an important role in the progression of prostate cancer and is the most important therapeutic target. However, androgen deprivation therapy will finally lead patients to progress to castration-resistant prostate cancer (CRPC). Here, we confirmed that GAS5, a long noncoding RNA, could interact and suppress AR transactivation in CRPC C4-2 cells. Knockdown GAS5 by short hairpin RNA would enhance the transcription of AR via promote AR recruitment to the promoter of its downstream target genes. Functionally, GAS5 overexpression inhibits cell proliferation partially through inhibiting AR transactivation in C4-2 cells. Moreover, knocking down GAS5 protects C4-2 cells from the docetaxel-induced cell apoptosis. In return, the suppressed AR was found to downregulate the GAS5 expression, which forms a feedback loop resulted in AR high transcription activity in CRPC. Collectively, our findings revealed the important role of GAS5 in AR axis activity regulation and CRPC progression. Targeting GAS5 to intervene the feedback loop might be a new potential therapeutic approach for the patients at CRPC stage.

A novel proteasome deubiquitinase inhibitor, VLX1570, has been highlighted as a promising therapeutic agent mainly for lymphoid neoplasms and solid tumors. We examined in vitro effects of VLX1570 on eight myeloid and three lymphoid leukemia cell lines. From cell culture studies, 10 out of 11 cell lines except K562 were found to be susceptible to VLX1570 treatment and it inhibited cell growth mainly by apoptosis. Next, to identify the signaling pathways associated with apoptosis, we performed gene expression profiling using HL‐60 with or without 50 nmol/L of VLX1570 for 3 hours and demonstrated that VLX1570 induced the genetic pathway involved in “heat shock transcription factor 1 (HSF1) activation”, “HSF1 dependent transactivation”, and “Regulation of HSF1 mediated heat shock response”. VLX1570 increased the amount of high molecular weight polyubiquitinated proteins and the expression of HSP70 as the result of the suppression of ubiquitin proteasome system, the expression of heme oxygenase‐1, and the amount of phosphorylation in JNK and p38 associated with the generation of reactive oxygen species (ROS) induced apoptosis and the amount of phosphorylation in eIF2α, inducing the expression of ATF4 and endoplasmic reticulum (ER) stress dependent apoptosis protein, CHOP, and the amount of phosphorylation slightly in IRE1α, leading to increased expression of XBP‐1s in leukemia cell lines. In the present study, we demonstrate that VLX1570 induces apoptosis and exerts a potential anti‐leukemic effect through the generation of ROS and induction of ER stress in leukemia cell lines., VLX1570 induces apoptosis mainly through reactive oxygen (ROS) stress signaling and unfolded protein response pathway under endoplasmic reticulum (ER) stress. Regarding ER stress, VLX1570 activated the protein kinase RNA‐like endoplasmic reticulum kinase signaling pathway, leading to ER stress‐dependent apoptosis. As another pathway, excess accumulation of polyubiquitinated proteins on the mitochondria induces ROS generation, followed by phosphorylation of JNK and p38, leading to caspases‐dependent apoptosis.

Postmeiotic germ cells require the lactate produced by the adjacent Sertoli cells (SCs) as their sole energy fuels. Lactate production in SCs is elaborately regulated by monitoring the transcription of the lactate dehydrogenase A (Ldha) gene. However, the transcription factors that are responsible for the control of Ldha transcription in SCs remain ill defined. Herein, the expression of forkhead box Q1 (FOXQ1), a central modulator of glucose metabolism in liver, was demonstrated in mouse testis throughout postnatal development, with maximum levels in adult specimens. At this age, FOXQ1 was immunolocalized in the nuclei of the functionally mature SCs. Testicular levels of FOXQ1 were overtly modulated by germ cells (GCs)-derived IL-1α, in a dose- and time-dependent manner. To further clarify the biological functions of FOXQ1, we disrupted the mouse Foxq1 gene using a Cas9/RNA-mediated gene targeting strategy. Foxq1-/- males were subfertile and showed oligoasthenozoospermia due to lactate deficiency. Moreover, we provided the molecular evidence that FOXQ1 may regulate lactate production by directly targeting the transactivation of the Ldha gene in SCs. From a functional standpoint, overexpression of the exogenous Ldha ameliorated Foxq1 deficiency-impaired lactate synthesis in the SCsFoxq1-/- cells. Thus, these findings collectively underscore a reproductive facet of this recently characterized transcription factor, which may operate as a novel transcriptional integrator linking energy homeostasis and nursery function in SCs.

NAC (NAM, ATAF1/2 and CUC2) transcription factors play critical roles in plant development and abiotic stress responses, and aquaporins have diverse functions in environmental stress responses. In this study, we described the salt-induced transcriptional responses of ThNAC12 and ThPIP2;5 in Tamarix hispida, and their regulatory mechanisms in response to salt stress. Using yeast one-hybrid (Y1H), chromatin immunoprecipitation (ChIP) and dual-luciferase reporter assays, we identified that ThNAC12 directly binds to the NAC recognition sequence (NACRS) of the ThPIP2;5 promoter and then activates the ThPIP2;5 expression. Subcellular localization and transcriptional activation assays demonstrated that ThNAC12 was a nuclear protein with a C-terminal transactivation domain. Compared with the corresponding control plants, transgenic plants overexpressing ThNAC12 exhibited enhanced salt tolerance and displayed increased reactive oxygen species (ROS) scavenging capability and antioxidant enzyme activity levels under salt stress. All results suggested that overexpression of ThNAC12 in plants enhanced salt tolerance through modulation of ROS scavenging via direct regulation of ThPIP2;5 expression in T. hispida.

Background The regulation of the transcription factor sex-determining region Y-box transcription factor 9 (SOX9) in lung development has been described in mouse, but the same principles apply to human lung development is unknown due to a lack of appropriate experimental approaches and models. Methods Here, we used gene editing technology to inactivate SOX9 in human embryonic stem cells that were then induced to differentiate into lung organoids to investigate the role of SOX9 in human lung epithelium development. Results Complete knockout of the transactivation domain of SOX9 by gene editing resulted in indels in both alleles of SOX9. SOX9−/− hESCs could be induced to differentiate into lung progenitor organoids. In vitro long-term expansion showed that SOX9 inactivation did not affect the differentiation of pulmonary epithelial cells, but promoted apoptosis and reduced proliferative capacity in the organoids. When lung progenitor organoids were transplanted under the kidney capsule of immunodeficient mice, expression of the club cell marker secretoglobin family 1A member 1 (SCGB1A1) was detected in SOX9−/− transplants but was absent in wild-type (WT) transplants. The maturation of goblet cells was also affected by SOX9 inactivation, as evidenced by the presence of mucin 5 AC (MUC5AC) in the cytoplasm of SOX9−/− grafts as compared to WT grafts in which most MUC5AC was secreted into the lumen. In vivo lung orthotopic transplantations showed that SOX9 inactivation had a limited effect on the differentiation of alveolar cells and lung regeneration in injured mice. Conclusions SOX9 modulates the proliferative capacity of lung epithelium but is not an indispensable transcription factor in the regulation of human lung epithelium development.

Heavy metals, both toxic and essential, have long been an important research focus in life science. To investigate the intracellular actions of heavy metals at the molecular level, I have been exploring protein factors involved in induction of metallothionein (MT) genes by heavy metals that specifically bind to a metal responsive element (MRE) in the region upstream of the human MT-IIA gene. Purification of a zinc-dependent MRE-binding factor, and cloning of its cDNA identified a sequence identical to that of metal-responsive transcription factor-1 (MTF-1). MTF-1, which is characterized by six tandem repeats of the C2H2 type zinc finger motif, is indispensable for induction of MT gene expression by multiple types of heavy metal, but zinc is the only metal that can directly activate MTF-1 binding to the MRE, indicating that other heavy metal signals act through zinc as a second messenger. Functional analysis of various MTF-1 point mutants revealed several cysteine (Cys) residues critical for DNA binding and/or transactivation activity. Interestingly, six finger motifs seem to mediate several MTF-1 functions other than DNA binding. Immunohistochemical analyses of various mouse tissues revealed selective expression of MTF-1 in spermatocytes among the testicular cells, suggesting roles relevant to spermatogenesis. The zinc regulon, under the control of MTF-1, will likely provide good clues to aid in unraveling novel functions of intracellular zinc ions.

Long non-coding RNAs (lncRNAs) regulate the development of follicles and reproductive diseases, but the mechanisms by which lncRNAs regulate ovarian functions and fertility remain elusive. We profiled the expression of lncRNAs in ovarian tissues of Hu sheep with different prolificacy and identified 21,327 lncRNAs. Many of the lncRNAs were differentially expressed in different groups. We further characterized an lncRNA that was predominantly expressed in the ovaries of the low prolificacy FecB+ (LPB+) group and mainly present in granulosa cells (GCs), and the expression of this lncRNA decreased during follicular development, which we named follicular development-associated lncRNA (FDNCR). Next, we found that FDNCR directly binds miR-543-3p, and decorin (DCN) was identified as a target of miR-543-3p. FDNCR overexpression promoted GC apoptosis through increased expression of DCN, which could be attenuated by miR-543-3p. Furthermore, miR-543-3p increased and FDNCR reduced the expression of transforming growth factor-β (TGF-β) pathway-related genes, including TGF-β1 and inhibin beta A (INHBA), which were upregulated upon DCN silencing. Our results demonstrated that FDNCR sponges miR-543-3p in GCs and prevents miR-543-3p from binding to the DCN 3′ UTR, resulting in DCN transactivation and TGF-β pathway inhibition and promotion of GC apoptosis in Hu sheep. These findings provide insights into the mechanisms underlying prolificacy in sheep.

Mitochondrial dynamics play vital roles in the tumorigenicity and malignancy of various types of cancers by promoting the tumor-initiating potential of cancer cells, suggesting that targeting crucial factors that drive mitochondrial dynamics may lead to promising anticancer therapies. In the current study, we report that overexpression of mitochondrial fission factor (MFF), which is upregulated significantly in liver cancer–initiating cells (LCIC), promotes mitochondrial fission and enhances stemness and tumor-initiating capability in non-LCICs. MFF-induced mitochondrial fission evoked mitophagy and asymmetric stem cell division and promoted a metabolic shift from oxidative phosphorylation to glycolysis that decreased mitochondrial reactive oxygen species (ROS) production, which prevented ROS-mediated degradation of the pluripotency transcription factor OCT4. CRISPR affinity purification in situ of regulatory elements showed that T-box transcription factor 19 (TBX19), which is overexpressed uniquely in LCICs compared with non-LCICs and liver progenitor cells, forms a complex with PRMT1 on the MFF promoter in LCICs, eliciting epigenetic histone H4R3me2a/H3K9ac-mediated transactivation of MFF. Targeting PRMT1 using furamidine, a selective pharmacologic inhibitor, suppressed TBX19-induced mitochondrial fission, leading to a profound loss of self-renewal potential and tumor-initiating capacity of LCICs. These findings unveil a novel mechanism underlying mitochondrial fission–mediated cancer stemness and suggest that regulation of mitochondrial fission via inhibition of PRMT1 may be an attractive therapeutic option for liver cancer treatment. Significance: These findings show that TBX19/PRMT1 complex–mediated upregulation of MFF promotes mitochondrial fission and tumor-initiating capacity in liver cancer cells, identifying PRMT1 as a viable therapeutic target in liver cancer.

The squalene synthase inhibitor squalestatin 1 (Squal1) is a potent and efficacious inducer of CYP2B expression in primary cultured rat hepatocytes and rat liver. To determine whether Squal1 is also an inducer of human CYP2B, the effects of Squal1 treatment were evaluated in primary cultured human hepatocytes, differentiated HepaRG cells, and humanized mouse livers. Squal1 treatment did not increase CYP2B6 mRNA levels in human hepatocytes or HepaRG cells and only slightly and inconsistently increased CYP2B6 mRNA content in humanized mouse liver. However, treatment with farnesol, which mediates Squal1’s effect on rat CYP2B expression, increased CYP2B6 mRNA levels in HepaRG cells expressing the constitutive androstane receptor (CAR), but not in cells with knocked-down CAR. To determine the impact of cholesterol biosynthesis inhibition on CAR activation, the effects of pravastatin (Prava) were determined on CITCO-mediated gene expression in primary cultured human hepatocytes. Prava treatment abolished CITCO-inducible CYP2B6 expression, but had less effect on rifampicin-mediated CYP3A4 induction, and CITCO treatment did not affect Prava-inducible HMG-CoA reductase (HMGCR) expression. Treatment with inhibitors of different steps of cholesterol biosynthesis attenuated CITCO-mediated CYP2B6 induction in HepaRG cells, and Prava treatment increased HMGCR expression and inhibited CYP2B6 induction with comparable potency. Transfection of HepG2 cells with transcriptionally active sterol regulatory element binding proteins (SREBPs) reduced CAR-mediated transactivation, and inducible expression of transcriptionally active SREBP2 attenuated CITCO-inducible CYP2B6 expression in HepaRG cells. These findings suggest that Squal1 does not induce CYP2B6 in human hepatocytes because Squal1’s inhibitory effect on cholesterol biosynthesis interferes with CAR activation. SIGNIFICANCE STATEMENT The cholesterol biosynthesis inhibitor squalestatin 1 induces rat hepatic CYP2B expression indirectly by causing accumulation of an endogenous isoprenoid that activates the constitutive androstane receptor (CAR). This study demonstrates that squalestatin 1 does not similarly induce CYP2B6 expression in human hepatocytes. Rather, inhibition of cholesterol biosynthesis interferes with CAR activity, likely by activating sterol regulatory element binding proteins. These findings increase our understanding of the endogenous processes that modulate human drug-metabolizing gene expression.

Detection of cytosolic DNA triggers an innate immune response that leads to neuroinflammation. The sources of such DNA include viral or bacterial pathogens as well as nuclear and mitochondrial DNA (mtDNA) released in pathologic conditions.1 Sensing of cytosolic DNA triggers a signal mediated by cyclic GMP-AMP synthase (cGAS) and the stimulator of interferon (IFN) genes (STING) protein, resulting in activation of type I IFNs (IFN-I) and secretion of proinflammatory cytokines.2-5 Whereas the cGAS–STING pathway mediates immunosurveillance that is often neuroprotective, for example against herpes simplex virus type 1 infection, its excessive engagement can be deleterious and promote age-related neuroinflammation and neurodegeneration.1,6 For example, mitochondrial stress due to impaired mitophagy stimulates STING-mediated IFN-I responses.7 Detection of mtDNA released from mitochondria in response to accumulation of transactivation DNA binding protein of 43 kD (TDP-43) activates the STING pathway, leading to neuroinflammation and neurodegeneration in amyotrophic lateral sclerosis (ALS).8 The cGAS-STING pathway is thus a potential target for neuroprotective therapy.1,9

DNA double-strand breaks (DSBs) induced by ionizing radiation are the major cause of cell death, leading to tissue/organ injuries, which is a fundamental mechanism underlying the development of tissue reaction. Since unscheduled senescence, predominantly induced among epithelial tissues/organs, is one of the major modes of cell death in response to radiation exposure, its role in tissue reaction has been extensively studied, and it has become clear that senescence-mediated secretion of soluble factors is an indispensable component of the manifestation of tissue reaction. Recently, an unexpected link between cytoplasmic DSBs and innate immunity was discovered. The activation of cyclic GMP-AMP (cGAMP) synthase (cGAS) results in the stimulation of the cGAS–stimulator of interferon genes (STING) pathway, which has been shown to regulate the transactivation of a variety of secretory factors that are the same as those secreted from senescent cells. Furthermore, it has been proven that cGAS–STING pathway also mediates execution of the senescence process by itself. Hence, an autocrine/paracrine feedback loop has been discussed in previous literature in relation to its effect on the tissue microenvironment. As the tissue microenvironment plays a crucial role in cancer development, tissue reaction could be involved in the late health effects caused by radiation exposure. In this paper, the novel findings in radiation biology, which should provide a better understanding of the mechanisms underlying radiation-induced carcinogenesis, are overviewed., Journal of Radiation Research, 62(S1), pp. i30-i35; 2021

Human adipogenesis is the process through which uncommitted human mesenchymal stem cells (hMSCs) differentiate into adipocytes. Through a siRNA-based high-throughput screen that identifies adipogenic regulators whose expression knockdown leads to enhanced adipogenic differentiation of hMSCs, two new regulators, SUV39H1, a histone methyltransferase that catalyzes H3K9Me3, and CITED2, a CBP/p300-interacting transactivator with Glu/Asp-rich carboxy-terminal domain 2 were uncovered. Both SUV39H1 and CITED2 are normally downregulated during adipogenic differentiation of hMSCs. Further expression knockdown induced by siSUV39H1 or siCITED2 at the adipogenic initiation stage significantly enhanced adipogenic differentiation of hMSCs as compared with siControl treatment, with siSUV39H1 acting by both accelerating fat accumulation in individual adipocytes and increasing the total number of committed adipocytes, whereas siCITED2 acting predominantly by increasing the total number of committed adipocytes. In addition, both siSUV39H1 and siCITED2 were able to redirect hMSCs to undergo adipogenic differentiation in the presence of osteogenic inducing media, which normally only induces osteogenic differentiation of hMSCs in the absence of siSUV39H1 or siCITED2. Interestingly, simultaneous knockdown of both SUV39H1 and CITED2 resulted in even greater levels of adipogenic differentiation of hMSCs and expression of CEBPα and PPARγ, two master regulators of adipogenesis, as compared with those elicited by single gene knockdown. Furthermore, the effects of co-knockdown were equivalent to the additive effect of individual gene knockdown. Taken together, this study demonstrates that SUV39H1 and CITED2 are both negative regulators of human adipogenesis, and downregulation of both genes exerts an additive effect on promoting adipogenic differentiation of hMSCs through augmented commitment.

Background & Aims Epithelial cells form a monolayer at mucosal surface that functions as a highly selective barrier. Lysophosphatidic acid (LPA) is a bioactive lipid that elicits a broad range of biological effects via cognate G protein-coupled receptors. LPA receptor 5 (LPA5) is highly expressed in intestinal epithelial cells, but its role in the intestine is not well-known. Here we determined the role of LPA5 in regulation of intestinal epithelial barrier. Methods Epithelial barrier integrity was determined in mice with intestinal epithelial cell (IEC)-specific LPA5 deletion, Lpar5ΔIEC. LPA was orally administered to mice, and intestinal permeability was measured. Dextran sulfate sodium (DSS) was used to induce colitis. Human colonic epithelial cell lines were used to determine the LPA5-mediated signaling pathways that regulate epithelial barrier. Results We observed increased epithelial permeability in Lpar5ΔIEC mice with reduced claudin-4 expression. Oral administration of LPA decreased intestinal permeability in wild-type mice, but the effect was greatly mitigated in Lpar5ΔIEC mice. Serum lipopolysaccharide level and bacterial loads in the intestine and liver were elevated in Lpar5ΔIEC mice. Lpar5ΔIEC mice developed more severe colitis induced with DSS. LPA5 transcriptionally regulated claudin-4, and this regulation was dependent on transactivation of the epidermal growth factor receptor, which induced localization of Rac1 at the cell membrane. LPA induced the translocation of Stat3 to the cell membrane and promoted the interaction between Rac1 and Stat3. Inhibition of Stat3 ablated LPA-mediated regulation of claudin-4. Conclusions This study identifies LPA5 as a regulator of the intestinal barrier. LPA5 promotes claudin-4 expression in IECs through activation of Rac1 and Stat3., Graphical abstract

Aims/Introduction Peroxisome proliferator‐activated receptor (PPAR)‐γ2 is a transcription factor crucial for regulating adipogenesis and glucose/lipid metabolism, and synthetic PPARγ ligands, such as thiazolidinediones, are effective oral medication for type 2 diabetes. Sirtuin 7 (SIRT7), a nicotinamide adenine dinucleotide‐dependent deacetylase, also controls metabolism. However, it is not known whether SIRT7 regulates the function of PPARγ2 by its deacetylation. Materials and Methods Physical interaction between SIRT7 and PPARγ2, the effect of SIRT7 on PPARγ2 acetylation, and the deacetylation residue targeted by SIRT7 were investigated. The effects of PPARγ2 K382 acetylation on lipid accumulation, gene expression in C3H10T1/2 cell‐derived adipocytes, and ligand‐dependent transactivation activity were also evaluated. Results We demonstrated that SIRT7 binds to PPARγ2 and deacetylates PPARγ2 at K382. C3H10T1/2‐derived adipocytes expressing PPARγ2K382Q (a mimic of acetylated K) accumulated much less fat than adipocytes expressing wild‐type PPARγ2 or PPARγ2K382R (a mimic of nonacetylated K). Global gene expression analysis of adipocytes expressing PPARγ2K382Q revealed that K382Q caused the dysregulation of a set of genes involved in lipogenesis, including Srebp1c, Acaca, Fasn, and Scd1. The rosiglitazone‐dependent transcriptional activity of PPARγ2K382Q was reduced compared with that of PPARγ2K382R. Conclusion Our findings indicate that SIRT7‐dependent PPARγ2 deacetylation at K382 controls lipogenesis in adipocytes., SIRT7 regulates lipogenesis in adipocytes through deacetylation of PPARgamma2.

(E/Z)-3-(4-((E)-1-(4-Hydroxyphenyl)-2-phenylbut-1-enyl)phenyl)acrylic acid (GW7604) as a derivative of (Z)-4-hydroxytamoxifen (4-OHT) was linked by diaminoalkane spacers to molecules that are known binders to the coactivator binding site (benzimidazole or thioxo-quinazolinone scaffolds). With this modification, an optimization of the pharmacological profile was achieved. The most active thioxo-quinazolinone derivative 16 showed extraordinarily high affinity to the estrogen receptor (ER) β (RBA = 110%), inhibited effectively the coactivator recruitment (IC50 = 20.88 nM (ERα) and 28.34 nM (ERβ)), acted as a pure estradiol (E2) antagonist in a transactivation assay (IC50 = 18.5 nM (ERα) and 7.5 nM (ERβ)), and downregulated the ERα content in MCF-7 cells with an efficacy of 60% at 1 μM. The cytotoxicity was restricted to hormone-dependent MCF-7 (IC50 = 4.2 nM) and tamoxifen-resistant MCF-7TamR cells (IC50 = 476.6 nM). The compounds bearing a thioxo-quinazolinone moiety can therefore be assigned as pure E2-antagonistic selective ER degraders/downregulators. By contrast, the benzimidazole derivatives acted solely as pure antagonists without degradation of the ER.

Human endogenous retroviruses (HERVs) are remnants of ancient retroviral germline infections. Most HERV sequences are silenced in somatic cells, but interest is emerging on the involvement of HERV derived transcripts and proteins in human physiology and disease. A HERV-W encoded protein, syncytin-1, has been co-opted into fetal physiology, where it plays a role in trophoblast formation. Altered HERV transcription and expression of HERV derived proteins are associated with various cancer types and neurological diseases such as multiple sclerosis (MS). The implication of HERVs as potential mediators of both health and disease suggests important roles of regulatory mechanisms and alterations of these in physiological and pathological processes. The regulation of HERV sequences is mediated by a wide variety of mechanisms, and the focus of this review is on selected aspects of these, including epigenetic mechanisms such as CpG methylation and histone modifications of the HP1-H3K9me axis, viral transactivation events, and regulatory perspectives of transient stimuli in the microenvironment. Increasing knowledge of the regulation of HERV sequences will not only contribute to the understanding of complex pathogeneses, but also may pinpoint potential targets for better diagnosis and treatment in complex diseases as MS.

Conventional T cells are selected by peptide-MHC expressed by cortical epithelial cells in the thymus, and not by cortical thymocytes themselves that do not express MHC I or MHC II. Instead, cortical thymocytes express non-peptide presenting MHC molecules like CD1d and MR1, and promote the selection of PLZF+ iNKT and MAIT cells, respectively. Here, we report an inducible class-I transactivator mouse that enables the expression of peptide presenting MHC I molecules in different cell types. We show that MHC I expression in DP thymocytes leads to expansion of peptide specific PLZF+ innate-like (PIL) T cells. Akin to iNKT cells, PIL T cells differentiate into three functional effector subsets in the thymus, and are dependent on SAP signaling. We demonstrate that PIL and NKT cells compete for a narrow niche, suggesting that the absence of peptide-MHC on DP thymocytes facilitates selection of non-peptide specific lymphocytes., Conventional T cell subsets are selected in the thymus by peptide bearing MHC expressed by cortical epithelial cells, in contrast cortical thymocytes express non-peptide bearing MHC molecules including CD1d and MR1 and select iNKT and MAIT cell populations respectively. Here, the authors generate a novel inducible MHC class-I trasnactivator murine system and suggest the absence of peptide-MHC on thymocytes is involved in the selection of non-peptide specific lymphocytes.

In a search for effective PPAR-γ agonists, 110 clinical drugs were screened via molecular docking, and 9 drugs, including parecoxib, were selected for subsequent biological evaluation. Molecular docking of parecoxib to the ligand-binding domain of PPAR-γ showed high binding affinity and relevant binding conformation compared with the PPAR-γ ligand/antidiabetic drug rosiglitazone. Per the docking result, parecoxib showed the best PPAR-γ transactivation in Ac2F rat liver cells. Further docking simulation and a luciferase assay suggested parecoxib would be a selective (and partial) PPAR-γ agonist. PPAR-γ activation by parecoxib induced adipocyte differentiation in 3T3-L1 murine preadipocytes. Parecoxib promoted adipogenesis in a dose-dependent manner and enhanced the expression of adipogenesis transcription factors PPAR-γ, C/EBPα, and C/EBPβ. These data indicated that parecoxib might be utilized as a partial PPAR-γ agonist for drug repositioning study.

Better understanding of the transcriptional regulatory network in acute promyelocytic leukemia (APL) cells is critical to illustrate the pathogenesis of other types of acute myeloid leukemia. Previous studies have primarily focused on the retinoic acid signaling pathway and how it is interfered with by promyelocytic leukemia/retinoic acid receptor-α (PML/RARα) fusion protein. However, this hardly explains how APL cells are blocked at the promyelocytic stage. Here, we demonstrated that C/EBPα bound and transactivated the promoter of long non-coding RNA NEAT1, an essential element for terminal differentiation of APL cells, through C/EBP binding sites. More importantly, PML/RARα repressed C/EBPα-mediated transactivation of NEAT1 through binding to NEAT1 promoter. Consistently, mutation of the C/EBP sites or deletion of retinoic acid responsive elements (RAREs) and RARE half motifs abrogated the PML/RARα-mediated repression. Moreover, silencing of C/EBPα attenuated ATRA-induced NEAT1 upregulation and APL cell differentiation. Finally, simultaneous knockdown of C/EBPα and C/EBPβ reduces ATRA-induced upregulation of C/EBPe and dramatically impaired NEAT1 activation and APL cell differentiation. In sum, C/EBPα binds and transactivates NEAT1 whereas PML/RARα represses this process. This study describes an essential role for C/EBPα in PML/RARα-mediated repression of NEAT1 and suggests that PML/RARα could contribute to the pathogenesis of APL through suppressing C/EBPα targets.

The histone acetyltransferase MOF (KAT8) is mainly involved in the acetylation of histone H4 at lysine 16 (H4K16) and some non‐histone proteins. The MOF expression level is significantly reduced in many cancers, however the biological function of MOF and its underlying mechanism are still elusive in hepatocellular carcinoma (HCC). Estrogen receptor α (ERα) has been considered as a tumor suppressor in HCC. Here, we demonstrated that MOF expression is significantly reduced in HCC samples, and is positively correlated with that of ERα. MOF interacts with ERα, and participates in acetylation of ERα at K266, K268, K299, thereby inhibiting ERα ubiquitination to maintain the stability of ERα. In addition, MOF participates in the upregulation of ERα‐mediated transactivation. Depletion of MOF significantly promotes cell growth, migration, and invasion in HCC cell lines. Taken together, our results provide new insights to understand the mechanism underlying the modulation function of MOF on ERα action in HCC, suggesting that MOF might be a potential therapeutic target for HCC., In summary, we have demonstrated that MOF as a crucial histone acetylase participates in the acetylation of ERα, thereby inhibiting the ubiquitination of ERα to stabilize ERα protein in HCC. MOF upregulates ERα‐induced transactivation. MOF depletion promotes the cell proliferation, migration, and invasion in HCC cells.

A key transcriptional activator, activating transcription factor 5 (ATF5), is aberrantly overexpressed in glioma and supports both poor prognosis and antiapototic potential. Unfortunately, data on ATF5 is largely based on its regulatory mechanism. Further investigation of the upstream regulatory factor for ATF5 transcription in glioma is required. Clinical data for patients with diagnosed glioma were obtained from The Cancer Genome Atlas (TCGA). Additionally, transcription factors potentially regulating the ATF5 promoter in glioma were screened with bioinformatics. A further experimental study was performed to investigate both the role of E74-like factor 1 (ELF1) and the binding of ELF1 and the ATF5 promoter in glioma. We show that ATF5 expression is upregulated in glioma tissues and associated with tumor malignancy and worse prognosis. As a putative upstream regulator, silencing ELF1 inhibits glioma cell growth and migration with ATF5 involvement. Moreover, ELF1 upregulation is also associated with poor prognosis in glioma. Importantly, the luciferase assay and chromatin immunoprecipitation (ChIP) reveal that the ATF5 gene promoter is essential for ELF1-dependent activation of ATF5 gene transcription. These results indicate that a high expression of ELF1 may be related to the malignant behavior of human glioma and ELF1 promotes glioma development mediated by transactivation of the ATF5 gene.

Biosynthesis of testosterone occurs mainly in the testicular Leydig cells. Nur77, an orphan nuclear receptor that is expressed in response to the luteinizing hormone/cyclic adenosine monophosphate (LH/cAMP) signaling pathway, is one of the key factors that regulate steroidogenesis in Leydig cells. The function of Nur77 is modulated through interaction with other proteins. FOXA3, a transcription factor that is crucial for male fertility, is also expressed in Leydig cells. Here, we sought to elucidate the role of FOXA3 in testicular steroidogenesis by focusing on its interaction with Nur77. LH/cAMP signaling induces the onset of steroidogenesis in Leydig cells but has a repressive effect on the expression of FOXA3. Overexpression of FOXA3 in MA-10 Leydig cells repressed cAMP-induced expression of Nur77 and its target steroidogenic genes (StAR, P450c17, and Hsd3β). Furthermore, FOXA3 suppressed Nur77 transactivation of the promoter of steroidogenic genes. In mouse primary Leydig cells, adenovirus-mediated overexpression of FOXA3 had similar effects and resulted in decreased production of testosterone. Taken together, these results suggest the role of FOXA3 in the regulation of steroidogenic genes in Leydig cells and fine-tuning steroidogenesis in the testis.

Pyruvate kinase (PK) catalyzes the last irreversible reaction of glycolysis pathway, generating pyruvate and ATP, from Phosphoenol Pyruvate (PEP) and ADP precursors. In mammals, four different tissue-specific isoforms (M1, M2, L and R) of PK exist, which are translated from two genes (PKL and PKR). PKM2 is the highly expressed isoform of PK in cancers, which regulates the aerobic glycolysis via reprogramming cancer cell’s metabolic pathways to provide an anabolic advantage to the tumor cells. In addition to the established role of PKM2 in aerobic glycolysis of multiple cancer types, various recent findings have highlighted the non-metabolic functions of PKM2 in brain tumor development. Nuclear PKM2 acts as a co-activator and directly regulates gene transcription. PKM2 dependent transactivation of various oncogenic genes is instrumental in the progression and aggressiveness of Glioblastoma Multiforme (GBM). Also, PKM2 acts as a protein kinase in histone modification which regulates gene expression and tumorigenesis. Ongoing research has explored novel regulatory mechanisms of PKM2 and its association in GBM progression. This review enlists and summarizes the metabolic and non-metabolic roles of PKM2 at the cellular level, and its regulatory function highlights the importance of the nuclear functions of PKM2 in GBM progression, and an emerging role of PKM2 as novel cancer therapeutics.

Aberrant leptin signaling and overexpression of fibroblast growth factor receptor 1 (FGFR1) are both implicated in the pathogenesis of letrozole resistance in breast cancer (BCa), but it remains unknown whether these two pathways are involved in letrozole resistance in a coordinated manner. Here, we demonstrate that expression levels of the pre-B-cell leukemia homeobox transcription factor 3 (PBX3), a pioneer factor that governs divergent biological processes, were significantly upregulated in letrozole-resistant BCa cells and tissues, and this upregulation correlated to a poorer progression-free survival in patients. By leveraging a patient-derived xenograft model with pharmacological approaches, we demonstrated that leptin activated PBX3 expression in a STAT3 (signal transducer and activator of transcription 3)-dependent manner. Our loss- and gain-of-function study further showed that PBX3 attenuated response to letrozole by potentiating BCa cell survival and anchorage-independent growth in BCa cells. By profiling BCa cells with ectopic PBX3 expression, we revealed that PBX3 conferred letrozole resistance via transactivation of the FGFR1 signaling, and this molecular event must coordinate a synergistic transcription activation programs through interacting with MTA1-HDAC2 (metastasis-associated 1-histone deacetylase 2) complex. Overall, the available data reveal a novel role of leptin/PBX3 cascade linking energy homeostasis (i.e. hyperleptinemia) and endocrine therapy failure (i.e. letrozole resistance) in BCa.

The factors that determine the outcomes of host-pathogen interactions, such as host specificity, tissue specificity, and transition from asymptomatic to symptomatic behavior of a pathogen, are yet to be deciphered. The initial interaction of a pathogen with host and host-associated factors play a crucial role in deciding such outcomes. One of the several host-factors that contribute to bacterial adhesion and the outcome of an infection is the activation of early growth response 1 (EGR1). EGR1 is an initial response transcriptional regulator that plays a vital role in regulating cell growth, differentiation, and survival. EGR1 expression is seen in most of the mammalian tissues. Multiple post-translational modifications occur, which modulate the EGR1 transcriptional activity. Upon activation, EGR1 can transactivate several genes with diverse cellular functions, including transcriptional regulatory proteins and cell proliferation. EGR1 has also been identified as a potential mediator of inflammatory gene expression. Recent studies have highlighted the role of EGR1 as a potent signaling molecule that facilitates bacterial adhesion to host epithelial cells, thus modulating colonization pathways. The pathways for the regulation of EGR1 during host-pathogen interaction remain yet unidentified. The review focuses on the role and regulation of EGR1 during host-pathogen interaction.

Renal arginine vasopressin receptor 2 (AVPR2) plays a crucial role in osmoregulation. Engagement of ligand with AVPR2 results in aquaporin 2 movement to the apical membrane and water reabsorption from the urinary filtrate. Despite this essential role, little is known about transcriptional regulation of Avpr2. Here, we identify novel roles for PAX2, a transcription factor crucial for kidney development, and its adaptor protein, Pax transcription interacting protein (PTIP), for epigenetic regulation of Avpr2 and thus body water balance. Chromatin immunoprecipitation (ChIP) from murine inner medulla cells (IMCD-3) identified the minimal DNA-binding region of PAX2 on the Avpr2 promoter. Regulation of Avpr2 by PAX2 was confirmed using a heterologous DNA expression system. PAX2 recruits the adaptor protein PTIP and its associated histone methyltransferase (HMT) complex to Avpr2 promoter, imposing epigenetic marks on this region and throughout the coding sequence that modulate Avpr2 gene transcription. Reduction of PAX2 or PTIP protein levels by siRNA prevented histone lysine methylation and expression of Avpr2. ChIP using mouse or human kidneys determined that PAX2 is highly enriched in the AVPR2 promoter alongside PTIP and HMT proteins, leading to high levels of histone H3 lysine trimethylation within the promoter and throughout the gene. In conclusion, PAX2 provides locus specificity for PTIP, allowing the HMT complex to impart epigenetic changes at the Avpr2 locus and regulate Avpr2 transcription. These finding have major implications for understanding regulation of body water balance.NEW & NOTEWORTHY The transcription factor PAX2 plays an indispensable role in kidney development. In the adult kidney, we identified the first described protein this protein regulates. PAX2 and its interacting partner Pax transcription interacting protein recruit a histone methyltransferase complex to the promoter and epigentically regulate the expression of arginine vasopressin receptor 2, a protein that plays a crucial role in osmoregulation in the distal tubule.

Aberrant expression of the transcriptional modulator and early B-cell factor 1 (EBF1) antagonist ZNF423 has been implicated in B-cell leukemogenesis, but its impact on transcriptional circuitries in lymphopoiesis has not been elucidated in a comprehensive manner. Herein, in silico analyses of multiple expression data sets on 1354 acute leukemia samples revealed a widespread presence of ZNF423 in various subtypes of acute lymphoblastic leukemia (ALL). Average expression of ZNF423 was highest in ETV6-RUNX1, B-other, and TCF3-PBX1 ALL followed by BCR-ABL, hyperdiploid ALL, and KMT2A-rearranged ALL. In a KMT2A-AFF1 pro-B ALL model, a CRISPR-Cas9–mediated genetic ablation of ZNF423 decreased cell viability and significantly prolonged survival of mice upon xenotransplantation. For the first time, we characterized the genome-wide binding pattern of ZNF423, its impact on the chromatin landscape, and differential gene activities in a B-lineage context. In general, chromatin-bound ZNF423 was associated with a depletion of activating histone marks. At the transcriptional level, EBF1-dependent transactivation was disrupted by ZNF423, whereas repressive and pioneering activities of EBF1 were not discernibly impeded. Unexpectedly, we identified an enrichment of ZNF423 at canonical EBF1-binding sites also in the absence of EBF1, which was indicative of intrinsic EBF1-independent ZNF423 activities. A genome-wide motif search at EBF1 target gene loci revealed that EBF1 and ZNF423 co-regulated genes often contain SMAD1/SMAD4-binding motifs as exemplified by the TGFB1 promoter, which was repressed by ZNF423 outcompeting EBF1 by depending on its ability to bind EBF1 consensus sites and to interact with EBF1 or SMADs. Overall, these findings underscore the wide scope of ZNF423 activities that interfere with B-cell lymphopoiesis and contribute to leukemogenesis.

Improving photosynthesis is a major approach to increasing crop yield potential. Here we identify a transcription factor as a negative regulator of photosynthesis, which can be manipulated to increase rice photosynthesis and plant biomass in the field. This transcription factor, named negative regulator of photosynthesis 1 (NRP1; Os07g0471900), was identified through a co-expression analysis using rice leaf RNA sequencing data. NRP1 expression showed significantly negative correlation with the expression of many genes involved in photosynthesis. Knocking out NRP1 led to greater photosynthesis and increased biomass in the field, while overexpression of NRP1 decreased photosynthesis and biomass. Transcriptomic data analysis shows that NRP1 can negatively regulate the expression of photosynthetic genes. Protein transactivation experiments show that NRP1 is a transcription activator, implying that NRP1 may indirectly regulate photosynthetic gene expression through an unknown regulator. This study shows that combination of bioinformatics analysis with transgenic testing can be used to identify new regulators to improve photosynthetic efficiency in crops.

M-cadherin is a skeletal muscle-specific transmembrane protein mediating the cell-cell adhesion of myoblasts during myogenesis. It is expressed in the proliferating satellite cells and highly induced by myogenic regulatory factors (MRFs) during terminal myogenic differentiation. Several conserved cis-elements, including 5 E-boxes, 2 GC boxes, and 1 conserved downstream element (CDE) were identified in the M-cadherin proximal promoter. We found that E-box-3 and -4 close to the transcription initiation site (TIS) mediated most of its transactivation by MyoD, the strongest myogenic MRF. Including of any one of the other E-boxes restored the full activation by MyoD, suggesting an essential collaboration between E-boxes. Stronger activation of M-cadherin promoter than that of muscle creatine kinase (MCK) by MyoD was observed regardless of culture conditions and the presence of E47. Furthermore, MyoD/E47 heterodimer and MyoD ∼ E47 fusion protein achieved similar levels of activation in differentiation medium (DM), suggesting high affinity of MyoD/E47 to E-boxes 3/4 under DM. We also found that GC boxes and CDE positively affected MyoD mediated activation. The CDE element was predicted to be the target of the chromatin-modifying factor Meis1/Pbx1 heterodimer. Knockdown of Pbx1 significantly reduced the expression level of M-cadherin, but increased that of N-cadherin. Using ChIP assay, we further found significant reduction in MyoD recruitment to M-cadherin promoter when CDE was deleted. Taken together, these observations suggest that the chromatin-modifying function of Pbx1/Meis1 is critical to M-cadherin promoter activation before MyoD is recruited to E-boxes to trigger transcription.

Background Increased hypoxia‐inducible factor 2α (HIF2α) activation is a common event in clear cell renal cell carcinoma (ccRCC) progression. However, the function and underlying mechanism of HIF2α in ccRCC remains uninvestigated. We conducted this study to access the potential link between junction plakoglobin (JUP) and HIF2α in ccRCC. Methods Affinity purification and mass spectrometry (AP‐MS) screening, glutathione‐s‐transferase (GST) pull‐down and co‐immunoprecipitation (Co‐IP) assays were performed to detect the interacting proteins of HIF2α. Quantitative PCR (qPCR) and Western blotting were used to detect the expression of JUP in human ccRCC samples. Luciferase reporter assays, chromatin immunoprecipitation (ChIP), cycloheximide chase assays, and ubiquitination assays were conducted to explore the regulation of JUP on the activity of HIF2α. Cell Counting Kit‐8 (CCK‐8) assays, colony formation assays, transwell assays, and xenograft tumor assays were performed to investigate the effect of JUP knockdown or overexpression on the tumorigenicity of renal cancer cells. Results We identified JUP as a novel HIF2α‐binding partner and revealed an important role of JUP in recruiting von Hippel‐Lindau (VHL) and histone deacetylases 1/2 (HDAC1/2) to HIF2α to regulate its stability and transactivation. JUP knockdown promoted and overexpression suppressed the tumorigenicity of renal cell carcinoma in vitro and in vivo. Importantly, the low expression of JUP was found in clinical ccRCC samples and correlated with enhanced hypoxia scores and poor treatment outcomes. Conclusion Taken together, these data support a role of JUP in modulating HIF2α signaling during ccRCC progression and identify JUP as a potential therapeutic target., We provide a mechanism by which the tumor suppressor JUP interact with the HIF2α transcription factor in ccRCC cells. JUP downregulation is likely to trigger the aberrant upregulation of HIF2α stability and transactivity, which further exerts effects on tumorigenesis in ccRCC. These results have important implications in both the diagnosis and treatment of RCC.

Summary Artemisinin, a sesquiterpene lactone widely used in malaria treatment, was discovered in the medicinal plant Artemisia annua. The biosynthesis of artemisinin is efficiently regulated by jasmonate (JA) and abscisic acid (ABA) via regulatory factors. However, the mechanisms linking JA and ABA signalling with artemisinin biosynthesis through an associated regulatory network of downstream transcription factors (TFs) remain enigmatic. Here we report AaTCP15, a JA and ABA dual‐responsive teosinte branched1/cycloidea/proliferating (TCP) TF, which is essential for JA and ABA‐induced artemisinin biosynthesis by directly binding to and activating the promoters of DBR2 and ALDH1, two genes encoding enzymes for artemisinin biosynthesis. Furthermore, AaORA, another positive regulator of artemisinin biosynthesis responds to JA and ABA, interacts with and enhances the transactivation activity of AaTCP15 and simultaneously activates AaTCP15 transcripts. Hence, they form an AaORA‐AaTCP15 module to synergistically activate DBR2, a crucial gene for artemisinin biosynthesis. More importantly, AaTCP15 expression is activated by the multiple reported JA and ABA‐responsive TFs that promote artemisinin biosynthesis. Among them, AaGSW1 acts at the nexus of JA and ABA signalling to activate the artemisinin biosynthetic pathway and directly binds to and activates the AaTCP15 promoter apart from the AaORA promoter, which further facilitates formation of the AaGSW1‐AaTCP15/AaORA regulatory module to integrate JA and ABA‐mediated artemisinin biosynthesis. Our results establish a multilayer regulatory network of the AaGSW1‐AaTCP15/AaORA module to regulate artemisinin biosynthesis through JA and ABA signalling, and provide an interesting avenue for future research exploring the special transcriptional regulation module of TCP genes associated with specialized metabolites in plants.

Objective Periodontitis is one the most common chronic inflammatory conditions, resulting in destruction of tooth-supporting tissues and leading to tooth loss. Porphyromonas gingivalis activates host macrophages to secrete pro-inflammatory cytokines and elicit tissue damage, in part by inducing NF-kappa-B transactivation. Since NFκB transactivation is negatively regulated by the Nicotinamide adenine dinucleotide (NAD)-dependent deacetylase enzyme Sirt1, we sought to assess if RAW264.7 macrophages exposed to P. gingivalis demonstrate impaired Sirt1 activity, to ultimately induce a pro-inflammatory response. Methods RAW264.7 macrophages were incubated with heat- killed P. gingivalis for 2, 4, 8, and 24 h. Stimulated RAW264.7 were assessed for TNFα expression via PCR, ELISA, and ChIP analysis. Following the activation of RAW264.7 macrophages, immunoblot analysis was executed to detect modifications in Sirt1 and the NFκB subunit RelA that is essential for NFκB transcriptional activity. Results TNFα expression was elevated 4 h after exposure to P. gingivalis. ChIP confirmed that RelA was enriched in the mouse TNFα promoter 4 h following stimulation, which correlated with the increased TNFα mRNA levels. Preceding TNFα expression, we detected Phosphoserine 536 and acetylated lysine 310 of RelA after 2 hours exposure with P. gingivalis. Moreover, reduced Sirt1 activity was associated with its cleavage in RAW264.7 protein extracts, after 2 hours of P. gingivalis exposure. Blocking TLR2/4 signaling prevented Sirt1 cleavage, loss of deacetylase activity, and TNFα secretion, while co-administering CA074Me (a cathepsin B inhibitor) with P. gingivalis reduced RelA promoter enrichment, resulting in impaired TNFα expression. Conclusions Together, the results suggest that P. gingivalis induces TNFα expression, at least in part, by enhancing cleavage of Sirt1 via a TLR-dependent signaling circuit.

HIV-1 transactivator of transcription (Tat) has a great impact on the development of HIV-1 associated neurocognitive disorders through disrupting dopamine transmission. This study determined the mutational effects of human dopamine transporter (hDAT) on basal and Tat-induced inhibition of dopamine transport. Compared to wild-type hDAT, the maximal velocity (Vmax) of [3H]dopamine uptake was decreased in D381L and Y88F/D206L/H547A, increased in D206L/H547A, and unaltered in D206L. Recombinant TatR1 − 86 inhibited dopamine uptake in wild-type hDAT, which was attenuated in either DAT mutants (D206L, D206L/H547A, and Y88F/D206L/H547A) or mutated TatR1 − 86 (K19A and C22G), demonstrating perturbed Tat-DAT interaction. Mutational effects of hDAT on the transporter conformation were evidenced by attenuation of zinc-induced increased [3H]WIN35,428 binding in D206L/H547A and Y88F/D206A/H547A and enhanced basal MPP+ efflux in D206L/H547A. H547A-induced outward-open transport conformational state was further validated by enhanced accessibility to MTSET ([2-(trimethylammonium)ethyl]-methanethiosulfonate) of an inserted cysteine (I159C) on a hDAT background.. Furthermore, H547A displayed an increase in palmitoylation inhibitor-induced inhibition of dopamine uptake relative to wide-type hDAT, indicating a change in basal palmitoylation in H547A. These results demonstrate that Y88F, D206L, and H547A attenuate Tat inhibition while preserving DA uptake, providing insights into identifying targets for improving DAT-mediated dopaminergic dysregulation. Graphical Abstract HIV-1 Tat inhibits dopamine uptake through human dopamine transporter (hDAT) on the presynaptic terminal through a direct allosteric interaction. Key hDAT residues D-H547, D-Y88, and D-D206 are predicted to be involved in the HIV-1 Tat-DAT binding. Mutating these residues attenuates this inhibitory effect by disrupting the Tat-hDAT interaction

Maize (Zea mays L.) is one of the most important food crops in the world, and starch is the main component of its endosperm. Transcriptional regulation plays a vital role in starch biosynthesis. However, it is not well understood in maize. We report the identification of the transcription factor ZmNAC126 and its role in regulation of starch synthesis in maize. Transcriptional expression of ZmNAC126 was higher in maize endosperm and kernels than in roots or stems. ZmNAC126 shared a similar expression pattern with starch synthesis genes during seed development, and its expression pattern was also consistent with the accumulation of starch. ZmNAC126 is a typical transcription factor with a transactivation domain between positions 201 and 227 of the amino acid sequence, is located in the nucleus, and binds to CACG repeats in vitro. Yeast one-hybrid assay revealed that ZmNAC126 bound the promoters of ZmGBSSI, ZmSSIIa, ZmSSIV, ZmISA1, and ZmISA2. Transient overexpression of ZmNAC126 in maize endosperm increased the activities of promoters pZmSh2, pZmBt2, pZmGBSSI, pZmSSIIIa, and pZmBT1 but inhibited the activities of pZmISA1 and pZmISA2. ZmNAC126 thus acts in starch synthesis by transcriptionally regulating targeted starch synthesis-related genes in maize kernels.

Maize is one of the three major crops worldwide based on its yield and quality. Starch is crucial to both the yield and quality of maize as it accounts more than 60% of the seed weight, and its structure influences the quality of the crop. Starch synthase I (SSI) contributes to the majority of the starch synthase activity in the maize endosperm. An in-depth understanding of the starch synthesis regulatory mechanism would provide opportunities for improving the yield and quality of maize. In this study, ZmPLATZ2, a plant AT-rich sequence and zinc-binding protein (PLATZ) transcription factor related to starch synthesis, was selected based on co-expression analysis. The semiquantitative RT-PCR and qRT-PCR assays revealed that ZmPLATZ2 had a high expression in the endosperm, and reached the peak at 12 days after pollination (DAP). Different treatments demonstrated that ZmPLATZ2 was downregulated by the presence of sucrose. Subsequent transactivation and subcellular localization analyses showed that ZmPLATZ2 was localized in the nuclei without transactivation. Yeast one-hybrid and transient expression in maize endosperm indicated that ZmPLATZ2 could bind to the promoters of ZmSSI, ZmISA1, and ZmISA2 and increase their gene expression. After ZmPLATZ2 overexpression in rice, four starch synthesis genes were significantly upregulated in the transgenic plant, including the OsSSI gene. In vitro DAP-seq data showed that ZmPLATZ2 could bind to the CAAAAAAA element. In conclusion, our data support that ZmPLATZ2 binds to the CAAAAAAA element in the ZmSSI promoter and mediates the Glu signal pathway.

Carotenoids provide precursors for the biosynthesis of strigolactones, which are a new class of hormones that are essential in phosphate (Pi) signaling during plant development. Carotenoid metabolism is a finely tuned pathway, but our understanding of the regulation mechanisms is still limited. In this study, we isolated a protein designated as CsPHL3 from citrus. CsPHL3 belonged to the Pi starvation response factor (PHR)-like subclade and was upregulated by low Pi. Acting as a nucleus-localized protein with transactivation activity, CsPHL3 bound directly to activate the promoter of a key metabolic gene, lycopene β-cyclase1 (LCYb1). Transgenic analysis revealed that the CsPHL3-overexpressing tomato plants exhibited abnormal growth, like the plants grew under limited Pi conditions. The transgenic lines showed reduced carotenoid contents and elevated expression of LCYb genes but downregulation of other key carotenogenic genes, including phytoene synthase (PSY). Moreover, CsPHL3 induced anthocyanin biosynthesis and affected Pi signaling in the transgenic plants. We further demonstrated that the expression of PSY was negatively regulated by CsPHL3 and high Pi. It is concluded that CsPHL3 is a Pi starvation response factor that negatively regulates carotenoid metabolism by modulating the expression of carotenogenic genes. Establishment of the CsPHL3-CsLCYb1 network provides new valuable knowledge of the function and underlying mechanism of PHR transcription factors and expands our understanding of the complex regulation mechanisms of carotenoid biosynthesis.

The transactivator of transcription (Tat) is a key HIV regulatory protein. We aimed to identify the frequency of key polymorphisms in HIV-1C compared with HIV-1B Tat protein, chiefly in the cysteine-, arginine-, and glutamine-rich domains and identify novel point mutations in HIV-1B and C sequences from Southern Brazil. This study was the first to investigate the genetic diversity and point mutations within HIV-1 Tat C in a Brazilian cohort. This was an observational, cross-sectional study, which included sequences of HIV-1B (n = 20) and HIV-1C (n = 21) from Southern Brazil. Additionally, 344 HIV-1C sequences were obtained from the Los Alamos database: 29 from Brazil and 315 from Africa, Asia, and Europe. The frequency of C31S substitution on HIV-1 Tat C in Brazil was 82% vs. 10% in the HIV-1B group (p < 0.0001). The frequency of the R57S substitution among the HIV-1C sequences from Brazil was 74% vs. 20% in HIV-1B (p = 0.004), and that of substitution Q63E in HIV-1C was 80% and 20% in HIV-1B (p < 0.0001). The mutation P60Q was more frequent in HIV-1B than in HIV-1C (55% and 6.12%, respectively, p < 0.0001)). Novel point mutations in the HIV-1C and B Tat functional domains were described. The frequency of C31S and other key point mutations in HIV-1 Tat C in Brazil were similar to those described in Africa, although lower than those in India. The Tat-B and C sequences found in Southern Brazil are consistent with biological differences and have potential implications for HIV-1 subtype pathogenesis.

Tetrazanbigen (TNBG) is a novel sterol isoquinoline derivative with poor water solubility and moderate inhibitory effects on human cancer cell lines via lipoapoptosis induction. Herein, we developed a series of novel TNBG analogues with improved water solubility and antiproliferative activities. The CCK-8 assay enabled us to identify a novel compound, 14g, which strongly inhibited HepG2 and A549 cell growth with IC50 values of 0.54 and 0.47 μM, respectively. The anticancer effects might be explained by the partial activation and upregulation of PPARγ expression, as indicated by the transactivation assay and western blotting evaluation. Furthermore, the in vitro antiproliferative activity was verified in an in vivo xenograft model in which 14g strongly reduced tumor growth at a dose of 10 mg/kg. In line with these positive observations, 14g exhibited an excellent water solubility of 31.4 mg/mL, which was more than 1000-fold higher than that of TNBG (4 μg/mL). Together, these results suggest that 14g is a promising anticancer therapeutic that deserves further investigation.

Although inhibitors of the kinases CHK1, ATR, and WEE1 are undergoing clinical testing, it remains unclear how these three classes of agents kill susceptible cells and whether they utilize the same cytotoxic mechanism. Here we observed that CHK1 inhibition induces apoptosis in a subset of acute leukemia cell lines in vitro, including TP53-null acute myeloid leukemia (AML) and BCR/ABL–positive acute lymphoid leukemia (ALL), and inhibits leukemic colony formation in clinical AML samples ex vivo. In further studies, downregulation or inhibition of CHK1 triggered signaling in sensitive human acute leukemia cell lines that involved CDK2 activation followed by AP1-dependent TNF transactivation, TNFα production, and engagement of a TNFR1- and BID-dependent apoptotic pathway. AML lines that were intrinsically resistant to CHK1 inhibition exhibited high CHK1 expression and were sensitized by CHK1 downregulation. Signaling through this same CDK2–AP1–TNF cytotoxic pathway was also initiated by ATR or WEE1 inhibitors in vitro and during CHK1 inhibitor treatment of AML xenografts in vivo. Collectively, these observations not only identify new contributors to the antileukemic cell action of CHK1, ATR, and WEE1 inhibitors, but also delineate a previously undescribed pathway leading from aberrant CDK2 activation to death ligand–induced killing that can potentially be exploited for acute leukemia treatment. Significance: This study demonstrates that replication checkpoint inhibitors can kill AML cells through a pathway involving AP1-mediated TNF gene activation and subsequent TP53-independent, TNFα-induced apoptosis, which can potentially be exploited clinically.

Identification of chemicals that affect hormone-regulated systems will help to predict endocrine disruption. In our previous study, a 46 gene biomarker was found to be an accurate predictor of estrogen receptor (ER) α modulation in chemically treated MCF-7 cells. Here, potential ERα modulators were identified using the biomarker by screening a microarray compendium consisting of ∼1600 gene expression comparisons representing exposure to ∼1200 chemicals. A total of ∼170 chemicals were identified as potential ERα modulators. In the Connectivity Map 2.0 collection, 75 and 39 chemicals were predicted to activate or suppress ERα, and they included 12 and six known ERα agonists and antagonists/selective ERα modulators, respectively. Nineteen and eight of the total number were also identified as active in an ERα transactivation assay carried out in an MCF-7-derived cell line used to screen the Tox21 10K chemical library in agonist or antagonist modes, respectively. Chemicals predicted to modulate ERα in MCF-7 cells were examined further using global and targeted gene expression in wild-type and ERα-null cells, transactivation assays, and cell-free ERα coregulator interaction assays. Environmental chemicals classified as weak and very weak agonists were confirmed to activate ERα including apigenin, kaempferol, and oxybenzone. Novel activators included digoxin, nabumetone, ivermectin, and six progestins. Novel suppressors included emetine, mifepristone, niclosamide, and proscillaridin. Our strategy will be useful to identify environmentally relevant ERα modulators in future high-throughput transcriptomic screens.