The nucleolus is a subnuclear compartment critical in ribosome biogenesis and cellular stress responses. These mechanisms are governed by a complex interplay of proteins, including NOC1, a member of the NOC family of nucleolar proteins responsible for controlling rRNA processing and ribosomal maturation. This study reveals a novel relationship between NOC1 and MYC transcription factor, known for its crucial role in controlling ribosomal biogenesis, cell growth, and proliferation. Here, we demonstrate that NOC1 functions as a direct target of MYC, as it is transcriptionally induced through a functional MYC-binding E-box sequence in the NOC1 promoter region. Furthermore, protein interactome analysis reveals that NOC1-complex includes the nucleolar proteins NOC2 and NOC3 and other nucleolar components such as Nucleostemin1 Ns1 transporters of ribosomal subunits and components involved in rRNA processing and maturation. In response to MYC, NOC1 expression and localization within the nucleolus significantly increase, suggesting a direct functional link between MYC activity and NOC1 function. Notably, NOC1 over-expression leads to the formation of large nuclear granules and enlarged nucleoli, which co-localize with nucleolar fibrillarin and Ns1. Additionally, we demonstrate that NOC1 expression is necessary for Ns1 nucleolar localization, suggesting a role for NOC1 in maintaining nucleolar structure. Finally, the co-expression of NOC1 and MYC enhances nucleolus size and maintains their co-localization, outlining another aspect of the cooperation between NOC1 and MYC in nucleolar dynamics. This study also reveals an enrichment with NOC1 with few proteins involved in RNA processing, modification, and splicing. Moreover, proteins such as Ythdc1, Flacc, and splenito are known to mediate N6-methyladenosine (m6A) methylation of mRNAs in nuclear export, revealing NOC1\'s potential involvement in coordinating RNA splicing and nuclear mRNA export. In summary, we uncovered novel roles for NOC1 in nucleolar homeostasis and established its direct connection with MYC in the network governing nucleolar structure and function. These findings also highlight NOC1\'s interaction with proteins relevant to specific RNA functions, suggesting a broader role in addition to its control of nucleolar homeostasis and providing new insight that can be further investigated.

E-boxes are important regulatory elements in the eukaryotic genome. Transcription factors can bind to E-boxes through their basic helix-loop-helix or zinc finger domain to regulate gene transcription. E-box-binding transcription factors (EBTFs) are important regulators of development and essential for physiological activities of the cell. The fundamental role of EBTFs in cancer has been highlighted by studies on the canonical oncogene MYC, yet many EBTFs exhibit common features, implying the existence of shared molecular principles of how they are involved in tumorigenesis. A comprehensive analysis of TFs that share the basic function of binding to E-boxes has been lacking. Here, we review the structure of EBTFs, their common features in regulating transcription, their physiological functions, and their mutual regulation. We also discuss their converging functions in cancer biology, their potential to be targeted as a regulatory network, and recent progress in drug development targeting these factors in cancer therapy.

The fleshy fruit of tomato (Solanum lycopersicum) are climacteric and, as such, ethylene plays a pivotal role in their ripening and quality traits. In this study, a basic helix-loop-helix transcription factor, EMB1444-like, was found to induce the expression of YELLOW-FRUITED TOMATO 1 (YFT1), which encodes the SlEIN2 protein, a key element in the ethylene signaling pathway. Yeast one-hybrid and EMSA analyses revealed that EMB1444-like binds to the E-box motif (CACTTG, -1295 bp to -1290 bp upstream of the ATG start codon) of the YFT1 promoter (pYFT1). Suppression of EMB1444-like expression in tomato lines (sledl) using RNAi reduced ethylene production by lowering the expression of 1-AMINOCYCLOPROPANE-1-CARBOXYLATE SYNTHASE 2/4 (ACS2/4) and ACC OXIDASE1 (ACO1) in a positive feedback loop. sledl tomato also showed differences in numerous quality traits related to fruit ripening, compared with the wild type, such as delayed chromoplast differentiation, a decrease in carotenoid accumulation, and delayed fruit ripening in an ethylene-independent manner, or at least upstream of ripening mediated by YFT1/SlEIN2. This study elucidates the regulatory framework of fruit ripening in tomato, providing information that may be used to breed tomato hybrid cultivars with an optimal balance of shelf-life, durability, and high quality.

TWIST1 (TW) is a pro-oncogenic basic helix-loop-helix (bHLH) transcription factor and promotes the hallmark features of malignancy (e.g., cell invasion, cancer cell stemness, and treatment resistance), which contribute to poor prognoses of glioblastoma (GBM). We previously reported that specific TW dimerization motifs regulate unique cellular phenotypes in GBM. For example, the TW:E12 heterodimer increases periostin (POSTN) expression and promotes cell invasion. TW dimer-specific transcriptional regulation requires binding to the regulatory E-box consensus sequences, but alternative bHLH dimers that balance TW dimer activity in regulating pro-oncogenic TW target genes are unknown. We leveraged the ENCODE DNase I hypersensitivity data to identify E-box sites and tethered TW:E12 and TW:TW proteins to validate dimer binding to E-boxes in vitro. Subsequently, TW knockdown revealed a novel TCF4:TCF12 bHLH dimer occupying the same TW E-box site that, when expressed as a tethered TCF4:TCF12 dimer, markedly repressed POSTN expression and extended animal survival. These observations support TCF4:TCF12 as a novel dimer with tumor-suppressor activity in GBM that functions in part through displacement of and/or competitive inhibition of pro-oncogenic TW dimers at E-box sites.

The organization of a circadian system includes an endogenous pacemaker system, input pathways for environmental synchronizing (entraining) stimuli, and output pathways through which the clock regulates physiological and behavioral processes, for example, the glucose-sensing mechanism in the liver. The liver is the central regulator of metabolism and one of our peripherals clocks. In mammals, central to this pacemaker are the transcription factors Circadian Locomotor Output Cycles Kaput (CLOCK) and BMAL1 (Brain and Muscle ARNT-Like 1). BMAL1 dimerizes with CLOCK, and this heterodimer then binds to the E-box promoter elements (CACGTG) present in clock and clock-controlled genes (CCGs). However, we are just beginning to understand how output pathways and regulatory mechanisms of CCGs are involved in rhythmic physiological processes. Glucokinase (GCK) is a fundamental enzyme in glucose homeostasis, catalyzing the high Km phosphorylation of glucose and allowing its storage. Moreover, gck is a dependent circadian gene. This study aims to determine the contribution of clock genes to hepatic gck expression and to define the specific role of E-box sequences on the circadian regulation of hepatic gck. Results showed that gck expression follows a circadian rhythm in rat hepatocytes in vitro. Accordingly, bmal1 expression induces the glucokinase circadian rhythmic expression in hepatocytes and the analysis of human and rat gck promoters, indicating the presence of E-box regions. Moreover, the basal activity of gck promoter was increased by clock/bmal1 co-transfection but inhibited by Period1/Period2 (per1/per2) co-transfection. Thus, the data suggest that the clock proteins tightly regulate the transcriptional activity of the gck promoter.

Injury triggers a genetic program that induces gene expression for regeneration. Recent studies have identified regeneration-response enhancers (RREs); however, it remains unclear whether a common mechanism operates in these RREs. We identified three RREs from the zebrafish fn1b promoter by searching for conserved sequences within the surrounding genomic regions of regeneration-induced genes and performed a transgenic assay for regeneration response. Two regions contained in the transposons displayed RRE activity when combined with the -0.7 kb fn1b promoter. Another non-transposon element functioned as a stand-alone enhancer in combination with a minimum promoter. By searching for transcription factor-binding motifs and validation by transgenic assays, we revealed that the cooperation of E-box and activator protein 1 motifs is necessary and sufficient for regenerative response. Such RREs respond to variety of tissue injuries, including those in the zebrafish heart and Xenopus limb buds. Our findings suggest that the fidelity of regeneration response is ensured by the two signals evoked by tissue injuries. It is speculated that a large pool of potential enhancers in the genome has helped shape the regenerative capacities during evolution.

The population sizes of different retinal cell types vary between different strains of mice, and that variation can be mapped to genomic loci in order to identify its polygenic origin. In some cases, controlling genes act independently, whereas in other instances, they exhibit epistasis. Here, we identify an epistatic interaction revealed through the mapping of quantitative trait loci from a panel of recombinant inbred strains of mice. The population of retinal horizontal cells exhibits a twofold variation in number, mapping to quantitative trait loci on chromosomes 3 and 13, where these loci are shown to interact epistatically. We identify a prospective genetic interaction underlying this, mediated by the bHLH transcription factor Neurog2, at the chromosome 3 locus, functioning to repress the LIM homeodomain transcription factor Isl1, at the chromosome 13 locus. Using single and double conditional knockout mice, we confirm the countervailing actions of each gene, and validate in vitro a crucial role for two single nucleotide polymorphisms in the 5\'UTR of Isl1, one of which yields a novel E-box, mediating the repressive action of Neurog2.

The intrinsically disordered protein MYC belongs to the family of basic helix-loop-helix leucine zipper (bHLH-LZ) transcription factors (TFs). In complex with its cognate binding partner MAX, MYC preferentially binds to E-Box promotor sequences where it controls fundamental cellular processes such as cell cycle progression, metabolism, and apoptosis. Intramolecular regulation of MYC:MAX has not yet been investigated in detail. In this work, we use Nuclear Magnetic Resonance (NMR) spectroscopy to identify and map interactions between the disordered MAX N-terminus and the MYC:MAX DNA binding domain (DBD). We find that this binding event is mainly driven by electrostatic interactions and that it is competitive with DNA binding. Using NMR spectroscopy and Surface Plasmon Resonance (SPR), we demonstrate that the MAX N-terminus serves to accelerate DNA binding kinetics of MYC:MAX and MAX:MAX dimers, while it simultaneously provides specificity for E-Box DNA. We also establish that these effects are further enhanced by Casein Kinase 2-mediated phosphorylation of two serine residues in the MAX N-terminus. Our work provides new insights how bHLH-LZ TFs are regulated by intramolecular interactions between disordered regions and the folded DNA binding domain.

The photoperiodic system is concealed in the highly complex black-box, comprising four functional subunits: 1) a photo/thermo-sensitive input unit, 2) a photoperiodic clock based on a circadian system, 3) a condenser unit counting the number of inductive signals, and 4) a neuroendocrine switch that triggers a phenotypic shift. This review aims to summarize the research history and current reach of our understanding on this subject to connect it with the molecular mechanism of the circadian clock rapidly being unveiled. The review also focuses on the mode of intersubunit information transduction. It will scan the recent advancement in research on each functional subunit, but special attention will be given to the circadian clock-endocrine conjunct and the role of melatonin signaling in the regulation of insect photoperiodism. Prothoracicotropic hormone (PTTH) probably plays the most crucial role in the regulation of pupal diapause, which is the simplest model system of diapause regulation by hormones investigated so far, particularly in the Chinese oak silkmoth (Antheraea pernyi). A search for the trigger to release the PTTH found some candidates, that is, indoleamines. Indolamine metabolism is controlled by arylalkylamine N-acetyltransferase (aaNAT). Indolamine dynamics and aaNAT enzymatic activity changed according to photoperiods. aaNAT activity and melatonin content in the brain showed not only a photoperiodic response but also a circadian fluctuation. aaNAT had multiple E-boxes, suggesting that it is a clock-controlled gene (ccg), which implies that cycle (cyc, or brain-muscle Arnt-like 1 = Bmal1)/Clock (Clk) heterodimer binds to E-box and stimulates the transcription of aaNAT, which causes the synthesis of melatonin. RNAi against transcription modulators, cyc, or Clk downregulated aaNAT transcription, while RNAi against repressor of cyc/Clk, per upregulated aaNAT transcription. Immunohistochemical localization showed that the circadian neurons carry epitopes of melatonin-producing elements such as aaNAT, the precursor serotonin, HIOMT, and melatonin as well as clock gene products such as cyc-ir, Per-ir, and dbt-ir, while PTTH-producing neurons juxtaposed against the clock neurons showed hMT2-ir in A. pernyi brain. Melatonin probably binds to the putative melatonin receptor (MT) that stimulates Ca2+ influx, which in turn activates PKC. This induces Rab 8 phosphorylation and exocytosis of PTTH, leading to termination of diapause. All the PTTH-expressing neurons have PKC-ir, and Rab8-ir. When diapause is induced and maintained under short days, serotonin binding to 5HTR1B suppresses PTTH release in a yet unknown way. RNAi against this receptor knocked out photoperiodism; short day response is blocked and diapause was terminated even under the short day condition. The result showed that a relatively simple system controls both induction and termination in pupal diapause of A. pernyi: the circadian system regulates the transcription of aaNAT as a binary switch, the enzyme produces a melatonin rhythm that gates PTTH release, and 5HTR1B and MT are probably also under photoperiodic regulation. Finally, we listed the remaining riddles which need to be resolved, to fully understand this highly complex system in future studies.

Retinoids are essential in balancing proliferation, differentiation and apoptosis, and they exert their effects through retinoic acid receptors (RARs) and retinoid X receptors (RXRs). RARβ is a tumor-suppressor gene silenced by epigenetic mechanisms such as DNA methylation in breast, cervical and non-small cell lung cancers. An increased expression of RARβ has been associated with improved breast cancer-specific survival. The PAH2 domain of the scaffold protein SIN3A interacts with the specific Sin3 Interaction Domain (SID) of several transcription factors, such as MAD1, bringing chromatin-modifying proteins such as histone deacetylases, and it targets chromatin for specific modifications. Previously, we have established that blocking the PAH2-mediated Sin3A interaction with SID-containing proteins using SID peptides or small molecule inhibitors (SMI) increased RARβ expression and induced retinoic acid metabolism in breast cancer cells, both in in vitro and in vivo models. Here, we report studies designed to understand the mechanistic basis of RARβ induction and function. Using human breast cancer cells transfected with MAD1 SID or treated with the MAD SID peptide, we observed a dissociation of MAD1, RARα and RARβ from Sin3A in a coimmunoprecipitation assay. This was associated with increased RARα and RARβ expression and function by a luciferase assay, which was enhanced by the addition of AM580, a specific RARα agonist; EMSA showed that MAD1 binds to E-Box, similar to MYC, on the RARβ promoter, which showed a reduced enrichment of Sin3A and HDAC1 by ChIP and was required for the AM580-enhanced RARβ activation in MAD1/SID cells. These data suggest that the Sin3A/HDAC1/2 complex co-operates with the classical repressors in regulating RARβ expression. These data suggest that SIN3A/MAD1 acts as a second RARβ repressor and may be involved in fine-tuning retinoid sensitivity.