Enhancer RNA m6A methylation facilitates transcriptional condensate formation and gene activation.

The mechanistic understanding of nascent RNAs in transcriptional control remains limited. Here, by a high sensitivity method methylation-inscribed nascent transcripts sequencing (MINT-seq), we characterized the landscapes of N6-methyladenosine (m6A) on nascent RNAs. We uncover heavy but selective m6A deposition on nascent RNAs produced by transcription regulatory elements, including promoter upstream antisense RNAs and enhancer RNAs (eRNAs), which positively correlates with their length, inclusion of m6A motif, and RNA abundances. m6A-eRNAs mark highly active enhancers, where they recruit nuclear m6A reader YTHDC1 to phase separate into liquid-like condensates, in a manner dependent on its C terminus intrinsically disordered region and arginine residues. The m6A-eRNA/YTHDC1 condensate co-mixes with and facilitates the formation of BRD4 coactivator condensate. Consequently, YTHDC1 depletion diminished BRD4 condensate and its recruitment to enhancers, resulting in inhibited enhancer and gene activation. We propose that chemical modifications of eRNAs together with reader proteins play broad roles in enhancer activation and gene transcriptional control.

Conserved Gsx2/Ind homeodomain monomer versus homodimer DNA binding defines regulatory outcomes in flies and mice.

How homeodomain proteins gain sufficient specificity to control different cell fates has been a long-standing problem in developmental biology. The conserved Gsx homeodomain proteins regulate specific aspects of neural development in animals from flies to mammals, and yet they belong to a large transcription factor family that bind nearly identical DNA sequences in vitro. Here, we show that the mouse and fly Gsx factors unexpectedly gain DNA binding specificity by forming cooperative homodimers on precisely spaced and oriented DNA sites. High-resolution genomic binding assays revealed that Gsx2 binds both monomer and homodimer sites in the developing mouse ventral telencephalon. Importantly, reporter assays showed that Gsx2 mediates opposing outcomes in a DNA binding site-dependent manner: Monomer Gsx2 binding represses transcription, whereas homodimer binding stimulates gene expression. In Drosophila, the Gsx homolog, Ind, similarly represses or stimulates transcription in a site-dependent manner via an autoregulatory enhancer containing a combination of monomer and homodimer sites. Integrating these findings, we test a model showing how the homodimer to monomer site ratio and the Gsx protein levels defines gene up-regulation versus down-regulation. Altogether, these data serve as a new paradigm for how cooperative homeodomain transcription factor binding can increase target specificity and alter regulatory outcomes.

The Dynamics of Transcriptional Activation by Hepatic Reprogramming Factors.

Specific combinations of two transcription factors (Hnf4α plus Foxa1, Foxa2, or Foxa3) can induce direct conversion of mouse fibroblasts into hepatocyte-like cells. However, the molecular mechanisms underlying hepatic reprogramming are largely unknown. Here, we show that the Foxa protein family members and Hnf4α sequentially and cooperatively bind to chromatin to activate liver-specific gene expression. Although all Foxa proteins bind to and open regions of closed chromatin as pioneer factors, Foxa3 has the unique potential of transferring from the distal to proximal regions of the transcription start site of target genes, binding RNA polymerase II, and co-traversing target genes. These distinctive characteristics of Foxa3 are essential for inducing the hepatic fate in fibroblasts. Similar functional coupling of transcription factors to RNA polymerase II may occur in other contexts whereby transcriptional activation can induce cell differentiation.

The SET oncoprotein promotes estrogen-induced transcription by facilitating establishment of active chromatin.

SET is a multifunctional histone-binding oncoprotein that regulates transcription by an unclear mechanism. Here we show that SET enhances estrogen-dependent transcription. SET knockdown abrogates transcription of estrogen-responsive genes and their enhancer RNAs. In response to 17ß-estradiol (E2), SET binds to the estrogen receptor α (ERα) and is recruited to ERα-bound enhancers and promoters at estrogen response elements (EREs). SET functions as a histone H2 chaperone that dynamically associates with H2A.Z via its acidic C-terminal domain and promotes H2A.Z incorporation, ERα, MLL1, and KDM3A loading and modulates histone methylation at EREs. SET depletion diminishes recruitment of condensin complexes to EREs and impairs E2-dependent enhancer-promoter looping. Thus, SET boosts E2-induced gene expression by establishing an active chromatin structure at ERα-bound enhancers and promoters, which is essential for transcriptional activation.

Glutamine-rich regions of the disordered CREB transactivation domain mediate dynamic intra- and intermolecular interactions.

The cyclic AMP response element (CRE) binding protein (CREB) is a transcription factor that contains a 280-residue N-terminal transactivation domain and a basic leucine zipper that mediates interaction with DNA. The transactivation domain comprises three subdomains, the glutamine-rich domains Q1 and Q2 and the kinase inducible activation domain (KID). NMR chemical shifts show that the isolated subdomains are intrinsically disordered but have a propensity to populate local elements of secondary structure. The Q1 and Q2 domains exhibit a propensity for formation of short ß-hairpin motifs that function as binding sites for glutamine-rich sequences. These motifs mediate intramolecular interactions between the CREB Q1 and Q2 domains as well as intermolecular interactions with the glutamine-rich Q1 domain of the TATA-box binding protein associated factor 4 (TAF4) subunit of transcription factor IID (TFIID). Using small-angle X-ray scattering, NMR, and single-molecule Förster resonance energy transfer, we show that the Q1, Q2, and KID regions remain dynamically disordered in a full-length CREB transactivation domain (CREBTAD) construct. The CREBTAD polypeptide chain is largely extended although some compaction is evident in the KID and Q2 domains. Paramagnetic relaxation enhancement reveals transient long-range contacts both within and between the Q1 and Q2 domains while the intervening KID domain is largely devoid of intramolecular interactions. Phosphorylation results in expansion of the KID domain, presumably making it more accessible for binding the CBP/p300 transcriptional coactivators. Our study reveals the complex nature of the interactions within the intrinsically disordered transactivation domain of CREB and provides molecular-level insights into dynamic and transient interactions mediated by the glutamine-rich domains.

Robust miniature Cas-based transcriptional modulation by engineering Un1Cas12f1 and tethering Sso7d.

The miniature V-F CRISPR-Cas12f system has been repurposed for gene editing and transcription modulation. The small size of Cas12f satisfies the packaging capacity of adeno-associated virus (AAV) for gene therapy. However, the efficiency of Cas12f-mediated transcriptional activation varies among different target sites. Here, we developed a robust miniature Cas-based transcriptional activation or silencing system using Un1Cas12f1. We engineered Un1Cas12f1 and the cognate guide RNA and generated miniCRa, which led to a 1,319-fold increase in the activation of the ASCL1 gene. The activity can be further increased by tethering DNA-binding protein Sso7d to miniCRa and generating SminiCRa, which reached a 5,628-fold activation of the ASCL1 gene and at least hundreds-fold activation at other genes examined. We adopted these mutations of Un1Cas12f1 for transcriptional repression and generated miniCRi or SminiCRi, which led to the repression of ∼80% on average of eight genes. We generated an all-in-one AAV vector AIOminiCRi used to silence the disease-related gene SERPINA1. AIOminiCRi AAVs led to the 70% repression of the SERPINA1 gene in the Huh-7 cells. In summary, miniCRa, SminiCRa, miniCRi, and SminiCRi are robust miniature transcriptional modulators with high specificity that expand the toolbox for biomedical research and therapeutic applications.

A Slow Conformational Switch in the BMAL1 Transactivation Domain Modulates Circadian Rhythms.

The C-terminal transactivation domain (TAD) of BMAL1 (brain and muscle ARNT-like 1) is a regulatory hub for transcriptional coactivators and repressors that compete for binding and, consequently, contributes to period determination of the mammalian circadian clock. Here, we report the discovery of two distinct conformational states that slowly exchange within the dynamic TAD to control timing. This binary switch results from cis/trans isomerization about a highly conserved Trp-Pro imide bond in a region of the TAD that is required for normal circadian timekeeping. Both cis and trans isomers interact with transcriptional regulators, suggesting that isomerization could serve a role in assembling regulatory complexes in vivo. Toward this end, we show that locking the switch into the trans isomer leads to shortened circadian periods. Furthermore, isomerization is regulated by the cyclophilin family of peptidyl-prolyl isomerases, highlighting the potential for regulation of BMAL1 protein dynamics in period determination.

A UTX-MLL4-p300 Transcriptional Regulatory Network Coordinately Shapes Active Enhancer Landscapes for Eliciting Transcription.

Enhancer activation is a critical step for gene activation. Here we report an epigenetic crosstalk at enhancers between the UTX (H3K27 demethylase)-MLL4 (H3K4 methyltransferase) complex and the histone acetyltransferase p300. We demonstrate that UTX, in a demethylase activity-independent manner, facilitates conversion of inactive enhancers in embryonic stem cells to an active (H3K4me1+/H3K27ac+) state by recruiting and coupling the enzymatic functions of MLL4 and p300. Loss of UTX leads to attenuated enhancer activity, characterized by reduced levels of H3K4me1 and H3K27ac as well as impaired transcription. The UTX-MLL4 complex enhances p300-dependent H3K27 acetylation through UTX-dependent stimulation of p300 recruitment, while MLL4-mediated H3K4 monomethylation, reciprocally, requires p300 function. Importantly, MLL4-generated H3K4me1 further enhances p300-dependent transcription. This work reveals a previously unrecognized cooperativity among enhancer-associated chromatin modulators, including a unique function for UTX, in establishing an "active enhancer landscape" and defines a detailed mechanism for the joint deposition of H3K4me1 and H3K27ac.

CircMAN1A2_009 facilitates YBX1 nuclear localization to induce GLO1 activation for cervical adenocarcinoma cell growth.

The molecular mechanisms driving the development of cervical adenocarcinoma (CADC) and optimal patient management strategies remain elusive. In this study, we have identified circMAN1A2_009 as an oncogenic circular RNA (circRNA) in CADC. Clinically, circMAN1A2_009 showed significant upregulation in CADC tissues, with an impressive area under the curve value of 0.8075 for detecting CADC. Functional studies, involving both gain-of-function and loss-of-function experiments, revealed that circMAN1A2_009 suppressed reactive oxygen species accumulation and apoptosis, and boosted cell viability in CADC cells. Conversely, silencing circMAN1A2_009 reversed these effects. Further mechanistic investigations indicated that circMAN1A2_009 interacted with YBX1, facilitating the phosphorylation levels of YBX1 at serine 102 (p-YBX1S102) and facilitating YBX1 nuclear localization through sequence 245-251. This interaction subsequently increased the activity of the glyoxalase 1 (GLO1) promoter, leading to the activation of GLO1 expression. Consistently, inhibition of either YBX1 or GLO1 mirrored the biological effects of circMAN1A2_009 in CADC cells. Additionally, knockdown of YBX1 or GLO1 partially reversed the oncogenic behaviors induced by circMAN1A2_009. In conclusion, our findings propose circMAN1A2_009 as a potential oncogene and a promising indicator for diagnosing and guiding therapy in CADC patients.

ZNF131 facilitates the growth of hepatocellular carcinoma by acting as a transcriptional activator of SMC4 expression.

ZNF131 is a Zinc finger protein that acts as a transcription factor with oncogenic effects in multiple cancers. In this study, we aimed to explore the alternative splicing profile of ZNF131 in hepatocellular carcinoma (HCC), its regulatory effects on cell-cycle progression, and the downstream effectors. ZNF131 transcriptional profile and HCC survival analysis were conducted using data from the Cancer Genome Atlas (TCGA)-Liver Hepatocellular Cancer (LIHC) dataset. Chromatin immunoprecipitation (ChIP)-qPCR and dual-luciferase reporter assays were utilized to explore transcriptional regulation. CCK-8, colony formation and xenograft tumor models were used to study HCC tumor growth. Results showed that ZNF131 isoform 2 is upregulated in HCC tissues and its upregulation was associated with unfavorable overall survival (OS) and progression-free interval (PFI). Knockdown of endogenous ZNF131 inhibits HCC cell growth and induces G2/M cell-cycle arrest. ZNF131 binds to the SMC4 promoter by interacting with ZBTB33 and the ZBTB33 recognizing motif. ZNF131 transcriptionally activates SMC4 expression in HCC cells. The tumor-suppressive effects of ZNF131 shRNA could be partially reversed by enforced SMC4 overexpression. In summary, this study highlights the ZNF131/ZBTB33/SMC4 axis as a driver of pathological cell cycling and proliferation in HCC.

Transcription factor TaNF-YB2 interacts with partners TaNF-YA7/YC7 and transcriptionally activates distinct stress-defensive genes to modulate drought tolerance in T. Aestivum.

BACKGROUND: Drought stress limits significantly the crop productivity. However, plants have evolved various strategies to cope with the drought conditions by adopting complex molecular, biochemical, and physiological mechanisms. Members of the nuclear factor Y (NF-Y) transcription factor (TF) family constitute one of the largest TF classes and are involved in plant responses to abiotic stresses. RESULTS: TaNF-YB2, a NY-YB subfamily gene in T. aestivum, was characterized in this study focusing on its role in mediating plant adaptation to drought stress. Yeast two-hybrid (Y-2 H), biomolecular fluoresence complementation (BiFC), and Co-immunoprecipitation (Co-IP) assays indicated that TaNF-YB2 interacts with the NF-YA member TaNF-YA7 and NF-YC family member TaNF-YC7, which constitutes a heterotrimer TaNF-YB2/TaNF-YA7/TaNF-YC7. The TaNF-YB2 transcripts are induced in roots and aerial tissues upon drought signaling; GUS histochemical staining analysis demonstrated the roles of cis-regulatory elements ABRE and MYB situated in TaNF-YB2 promoter to contribute to target gene response to drought. Transgene analysis on TaNF-YB2 confirmed its functions in regulating drought adaptation via modulating stomata movement, osmolyte biosynthesis, and reactive oxygen species (ROS) homeostasis. TaNF-YB2 possessed the abilities in transcriptionally activating TaP5CS2, the P5CS family gene involving proline biosynthesis and TaSOD1, TaCAT5, and TaPOD5, the genes encoding antioxidant enzymes. Positive correlations were found between yield and the TaNF-YB2 transcripts in a core panel constituting 45 wheat cultivars under drought condition, in which two types of major haplotypes including TaNF-YB2-Hap1 and -Hap2 were included, with the former conferring more TaNF-YB2 transcripts and stronger plant drought tolerance. CONCLUSIONS: TaNF-YB2 is transcriptional response to drought stress. It is an essential regulator in mediating plant drought adaptation by modulating the physiological processes associated with stomatal movement, osmolyte biosynthesis, and reactive oxygen species (ROS) homeostasis, depending on its role in transcriptionally regulating stress response genes. Our research deepens the understanding of plant drought stress underlying NF-Y TF family and provides gene resource in efforts for molecular breeding the drought-tolerant cultivars in T. aestivum.

Epigenome editing based on CRISPR/dCas9p300 facilitates transdifferentiation of human fibroblasts into Leydig-like cells.

Recently, Leydig cell (LCs) transplantation has a promising potential to treat male hypogonadism. However, the scarcity of seed cells is the actual barrier impeding the application of LCs transplantation. Utilizing the cutting-edge CRISPR/dCas9VP64 technology, human foreskin fibroblasts (HFFs) were transdifferentiated into Leydig-like cells（iLCs） in previous study, but the efficiency of transdifferentiation is not very satisfactory. Therefore, this study was conducted to further optimize the CRISPR/dCas9 system for obtaining sufficient iLCs. First, the stable CYP11A1-Promoter-GFP-HFFs cell line was established by infecting HFFs with CYP11A1-Promoter-GFP lentiviral vectors, and then co-infected with dCas9p300 and the combination of sgRNAs targeted to NR5A1, GATA4 and DMRT1. Next, this study adopted quantitative reverse transcription polymerase chain reaction (qRT-PCR), Western blot, and immunofluorescence to determine the efficiency of transdifferentiation, the generation of testosterone, the expression levels of steroidogenic biomarkers. Moreover, we utilized chromatin immuno-precipitation (ChIP) followed by quantitative polymerase chain reaction (ChIP-qPCR) to measure the levels of acetylation of targeted H3K27. The results revealed that advanced dCas9p300 facilitated generation of iLCs. Moreover, the dCas9p300-mediated iLCs significantly expressed the steroidogenic biomarkers and produced more testosterone with or without LH treatment than the dCas9VP64-mediated. Additionally, preferred enrichment in H3K27ac at the promoters was detected only with dCas9p300 treatment. The data provided here imply that the improved version of dCas9 can aid in the harvesting of iLCs, and will provide sufficient seed cells for cell transplantation treatment of androgen deficiency in the future.

Short cell-penetration peptide conjugated bioreducible polymer enhances gene editing of CRISPR system.

CRISPR-based gene therapy offers precise targeting and specific editing of disease-related gene sequences, potentially yielding long-lasting treatment effects. However, efficient delivery remains a significant challenge for its widespread application. In this study, we design a novel short peptide-conjugated bioreducible polymer named TSPscp as a safe and effective delivery vector for the CRISPR system. Our results show that TSPscp markedly boosts transcriptional activation and genome editing activities of multiple CRISPR systems as confirmed by decomposition-seq and Deep-seq, which is resulted from its capability in facilitating delivery of plasmid DNA by promoting cellular uptake and lysosomal escape. Additionally, TSPscp further enhances genome editing of CRISPR by delivery of minicircle DNA, a condensed form of regular plasmid DNA. More importantly, TSPscp significantly improves delivery and genome editing of CRISPR system in vivo. In summary, our study highlights TSPscp as a promising delivery tool for CRISPR applications in vivo.

GhCOL2 Positively Regulates Flowering by Activating the Transcription of GhHD3A in Upland Cotton (Gossypium hirsutum L.).

Plants have evolved sophisticated signaling networks to adjust flowering time, ensuring successful reproduction. Two crucial flowering regulators, FLOWERING LOCUS T (FT) and CONSTANS (CO), play pivotal roles in regulating flowering across various species. Previous studies have indicated that suppressing Gossypium hirsutum CONSTANS-LIKE 2 (GhCOL2), a homolog of Arabidopsis CO, leads to delayed flowering in cultivated cotton. However, the underlying regulatory mechanisms remain unknown. In this study, a yeast one-hybrid and dual-LUC expression assays were used to elucidate the molecular mechanism through which GhCOL2 regulates the transcription of GhHD3A. RT-qPCR was used to examine the expression of GhCOL2 and GhHD3A. Our findings reveal that GhCOL2 directly binds to CCACA cis-elements and atypical CORE (TGTGTATG) cis-elements in the promoter regions of HEADING DATE 3 A (HD3A), thereby activating GhHD3A transcription. Notably, GhCOL2 and GhHD3A exhibited high expression levels in the adult stage and low levels in the juvenile stage. Interestingly, the expression of GhCOL2 and GhHD3A varied significant between the two cotton varieties (Tx2094 and Maxxa). In summary, our study enhances the understanding of the molecular mechanism by which cotton GhCOL2-GhHD3A regulates flowering at the molecular level. Furthermore, it contributes to a broader comprehension of the GhCOL2-GhHD3A model in G. hirsutum.

STAT5A modulates gastric cancer progression via upregulation of CD44.

OBJECTIVE: Signal transduction and transcriptional activator 5A (STAT5A), which has been reported to be frequently phosphorylated in tumors, plays pivotal roles in tumor progression. However, the role of STAT5A in gastric cancer (GC) progression and the downstream targets of STAT5A remain largely unknown. METHODS: The expression of STAT5A and CD44 were assessed. GC cells were treated with altered STAT5A and CD44 to evaluate their biological functions. Nude mice were given injections of genetically manipulated GC cells and growth of xenograft tumors and metastases was measured. RESULTS: The increased level of p-STAT5A is associated with tumor invasion and poor prognosis in GC. STAT5A promoted GC cell proliferation by upregulating CD44 expression. STAT5A directly binds to the CD44 promoter and promotes its transcription. CONCLUSIONS: The STAT5A/CD44 pathway plays a critical role in GC progression, promising potential clinical applications for improving treatment of GC.

Genome-Wide Identification of the DOF Gene Family in Kiwifruit (Actinidia chinensis) and Functional Validation of AcDOF22 in Response to Drought Stress.

DNA-binding one zinc finger (DOF) transcription factors are crucial plant-specific regulators involved in growth, development, signal transduction, and abiotic stress response generation. However, the genome-wide identification and characterization of AcDOF genes and their regulatory elements in kiwifruit (Actinidia chinensis) has not been thoroughly investigated. In this study, we screened the kiwifruit genome database and identified 42 AcDOF genes (AcDOF1 to AcDOF42). Phylogenetic analysis facilitated the categorization of these genes into five subfamilies (DOF-a, DOF-b, DOF-c, DOF-d, and DOF-e). We further analyzed the motifs, conserved domains, gene structures, and collinearity of the AcDOFgene family. Gene ontology (GO) enrichment analysis indicated significant enrichment in the "flower development" term and the "response to abiotic stress" category. Promoter prediction analysis revealed numerous cis-regulatory elements related to responses to light, hormones, and low-temperature and drought stress in AcDOF promoters. RNA-seq expression profiles demonstrated the tissue-specific expression of AcDOF genes. Quantitative real-time PCR results showed that six selected genes (AcDOF04, AcDOF09, AcDOF11, AcDOF13, AcDOF21, and AcDOF22) were differentially induced by abscisic acid (ABA), methyl jasmonate (MeJA), and cold, salt, and drought stresses, with AcDOF22 specifically expressed at high levels in drought-tolerant cultivars. Further experiments indicated that transient AcDOF22 overexpression in kiwifruit leaf disks reduced water loss and chlorophyll degradation. Additionally, AcDOF22 was localized to the nucleus and exhibited transcriptional activation, enhancing drought resistance by activating the downstream drought marker gene AcDREB2A. These findings lay the foundation for elucidating the molecular mechanisms of drought resistance in kiwifruit and offer new insights into drought-resistant breeding.

Comprehensive Characterization of fucAO Operon Activation in Escherichia coli.

Wildtype Escherichia coli cells cannot grow on L-1,2-propanediol, as the fucAO operon within the fucose (fuc) regulon is thought to be silent in the absence of L-fucose. Little information is available concerning the transcriptional regulation of this operon. Here, we first confirm that fucAO operon expression is highly inducible by fucose and is primarily attributable to the upstream operon promoter, while the fucO promoter within the 3'-end of fucA is weak and uninducible. Using 5'RACE, we identify the actual transcriptional start site (TSS) of the main fucAO operon promoter, refuting the originally proposed TSS. Several lines of evidence are provided showing that the fucAO locus is within a transcriptionally repressed region on the chromosome. Operon activation is dependent on FucR and Crp but not SrsR. Two Crp-cAMP binding sites previously found in the regulatory region are validated, where the upstream site plays a more critical role than the downstream site in operon activation. Furthermore, two FucR binding sites are identified, where the downstream site near the first Crp site is more important than the upstream site. Operon transcription relies on Crp-cAMP to a greater degree than on FucR. Our data strongly suggest that FucR mainly functions to facilitate the binding of Crp to its upstream site, which in turn activates the fucAO promoter by efficiently recruiting RNA polymerase.

Neuroinflammation and the role of epigenetic-based therapies for Huntington's disease management: the new paradigm.

Huntington's disease (HD) is an inherited, autosomal, neurodegenerative ailment that affects the striatum of the brain. Despite its debilitating effect on its patients, there is no proven cure for HD management as of yet. Neuroinflammation, excitotoxicity, and environmental factors have been reported to influence the regulation of gene expression by modifying epigenetic mechanisms. Aside focusing on the etiology, changes in epigenetic mechanisms have become a crucial factor influencing the interaction between HTT protein and epigenetically transcribed genes involved in neuroinflammation and HD. This review presents relevant literature on epigenetics with special emphasis on neuroinflammation and HD. It summarizes pertinent research on the role of neuroinflammation and post-translational modifications of chromatin, including DNA methylation, histone modification, and miRNAs. To achieve this about 1500 articles were reviewed via databases like PubMed, ScienceDirect, Google Scholar, and Web of Science. They were reduced to 534 using MeSH words like 'epigenetics, neuroinflammation, and HD' coupled with Boolean operators. Results indicated that major contributing factors to the development of HD such as mitochondrial dysfunction, excitotoxicity, neuroinflammation, and apoptosis are affected by epigenetic alterations. However, the association between neuroinflammation-altered epigenetics and the reported transcriptional changes in HD is unknown. Also, the link between epigenetically dysregulated genomic regions and specific DNA sequences suggests the likelihood that transcription factors, chromatin-remodeling proteins, and enzymes that affect gene expression are all disrupted simultaneously. Hence, therapies that target pathogenic pathways in HD, including neuroinflammation, transcriptional dysregulation, triplet instability, vesicle trafficking dysfunction, and protein degradation, need to be developed.

The complexity of p53-mediated metabolic regulation in tumor suppression.

Although the classic activities of p53 including induction of cell-cycle arrest, senescence, and apoptosis are well accepted as critical barriers to cancer development, accumulating evidence suggests that loss of these classic activities is not sufficient to abrogate the tumor suppression activity of p53. Numerous studies suggest that metabolic regulation contributes to tumor suppression, but the mechanisms by which it does so are not completely understood. Cancer cells rewire cellular metabolism to meet the energetic and substrate demands of tumor development. It is well established that p53 suppresses glycolysis and promotes mitochondrial oxidative phosphorylation through a number of downstream targets against the Warburg effect. The role of p53-mediated metabolic regulation in tumor suppression is complexed by its function to promote both cell survival and cell death under different physiological settings. Indeed, p53 can regulate both pro-oxidant and antioxidant target genes for complete opposite effects. In this review, we will summarize the roles of p53 in the regulation of glucose, lipid, amino acid, nucleotide, iron metabolism, and ROS production. We will highlight the mechanisms underlying p53-mediated ferroptosis, AKT/mTOR signaling as well as autophagy and discuss the complexity of p53-metabolic regulation in tumor development.

Temperature modulation of CAMTA3 gene induction activity is mediated through the DNA binding domain.

The calmodulin-binding transcription activator (CAMTA) proteins of Arabidopsis thaliana play a major role in cold acclimation, contributing to the rapid induction of the C-REPEAT BINDING FACTOR (CBF) genes and other genes that impart freezing tolerance in plants exposed to cold temperature (4°C). The goal of this study was to better understand how the gene induction activity of CAMTA3 is modulated by temperature. Our results indicate that a severely truncated version of CAMTA3, CAMTA3334 , which includes the N-terminal CG-1 DNA binding domain and a newly identified transcriptional activation domain (TAD), was able to rapidly induce the expression of CBF2 and two newly identified target genes, EXPANSIN-LIKE A1 (EXPL1) and NINE-CIS-EPOXYCAROTENOID DIOXYGENASE 3 (NCED3), in response to cold temperature. Additionally, CAMTA3334 was able to restore freezing tolerance when expressed in a camta23 double mutant. The ability of CAMTA3 and CAMTA3334 to induce target genes at cold temperature did not involve increased levels of these proteins or increased binding of these proteins to target gene promoters in cold-treated plants. Rather, domain-swapping experiments indicated that the CAMTA3 CG-1 domain conferred temperature dependence to the ability of the CAMTA3 TAD to induce gene expression. The CG-1 domain also enabled the TAD to induce the expression of target genes at a moderate temperature (22°C) in response to cycloheximide treatment, consistent with the TAD activity not being intrinsically temperature dependent. We propose a working model in which the temperature modulation of CAMTA3 gene induction activity occurs independently from the C-terminal calmodulin-binding domains that previously have been proposed to activate CAMTA3 transcriptional activity in response to cold temperature.