E-box Motifs Research Articles

GhEXL3 participates in brassinosteroids regulation of fiber elongation in Gossypium hirsutum.

Cotton fiber (Gossypium hirsutum) serves as an ideal model for investigating the molecular mechanisms of plant cell elongation at the single-cell level. Brassinosteroids (BRs) play a crucial role in regulating plant growth and development. However, the mechanism by which BR influences cotton fiber elongation remains incompletely understood. In this study, we identified EXORDIUM-like (GhEXL3) through transcriptome analysis of fibers from BR-deficient cotton mutant pagoda 1 (pag1) and BRI1-EMS-SUPPRESSOR 1 (GhBES1.4, encoding a central transcription factor of BR signaling) overexpression cotton lines. Knockout of GhEXL3 using CRISPR/Cas9 was found to impede cotton fiber elongation, while its overexpression promoted fiber elongation, suggesting a positive regulatory function for GhEXL3 in fiber elongation. Furthermore, in vitro ovule culture experiments revealed that the overexpression of GhEXL3 partially counteracted the inhibitory effects of brassinazole (BRZ) on cotton fiber elongation, providing additional evidence of GhEXL3 involvement in BR signaling pathways. Moreover, our findings demonstrate that GhBES1.4 directly binds to the E-box (CACGTG) motif in the GhEXL3 promoter region and enhances its transcription. RNA-seq analysis revealed that overexpression of GhEXL3 upregulated the expression of EXPs, XTHs, and other genes associated with fiber cell elongation. Overall, our study contributes to understanding the mechanism by which BR regulates the elongation of cotton fibers through the direct modulation of GhEXL3 expression by GhBES1.4.

NtMYB27 acts downstream of NtBES1 to modulate flavonoids accumulation in response to UV-B radiation in tobacco.

UV-B radiation can induce the accumulation of many secondary metabolites, including flavonoids, in plants to protect them from oxidative damage. BRI1-EMS-SUPPRESSOR1 (BES1) has been shown to mediate the biosynthesis of flavonoids in response to UV-B. However, the detailed mechanism by which it acts still needs to be further elucidated. Here, we revealed that UV-B significantly inhibited the transcription of multiple transcription factor genes in tobacco, including NtMYB27, which was subsequently shown to be a repressor of flavonoids synthesis in tobacco. We further demonstrated that NtBES1 directly binds to the E-box motifs present in the promoter of NtMYB27 to mediate its transcriptional repression upon UV-B exposure. The UV-B-repressed NtMYB27 could bind to the ACCT-containing element (ACE) in the promoters of Nt4CL and NtCHS and served as a modulator that promoted the biosynthesis of lignin and chlorogenic acid (CGA) but inhibited the accumulation of flavonoids in tobacco. The expression of NtMYB27 was also significantly repressed by heat stress, suggesting its putative roles in regulating heat-induced flavonoids accumulation. Taken together, our results revealed the role of NtBES1 and NtMYB27 in regulating the synthesis of flavonoids during the plant response to UV-B radiation in tobacco.

Remodeling Ca2+ dynamics by targeting a promising E-box containing G-quadruplex at ORAI1 promoter in triple-negative breast cancer

ORAI1 is an intrinsic component of store-operated calcium entry (SOCE) that strictly regulates Ca2+ influx in most non-excitable cells. ORAI1 is overexpressed in a wide variety of cancers, and its signal transduction has been associated with chemotherapy resistance. There is extensive proteomic interaction of ORAI1 with other channels and effectors, resulting in various altered phenotypes. However, the transcription regulation of ORAI1 is not well understood. We have found a putative G-quadruplex (G4) motif, ORAI1-Pu, in the upstream promoter region of the gene, having regulatory functions. High-resolution 3-D NMR structure elucidation suggests that ORAI1-Pu is a stable parallel-stranded G4, having a long 8-nt loop imparting dynamics without affecting the structural stability. The protruded loop further houses an E-box motif that provides a docking site for transcription factors like Zeb1. The G4 structure was also endogenously observed using Chromatin Immunoprecipitation (ChIP) with anti-G4 antibody (BG4) in the MDA-MB-231 cell line overexpressing ORAI1. Ligand-mediated stabilization suggested that the stabilized G4 represses transcription in cancer cell line MDA-MB-231. Downregulation of transcription further led to decreased Ca2+ entry by the SOCE pathway, as observed by live-cell Fura-2 Ca2+ imaging.

Interpretable deep learning reveals the role of an E-box motif in suppressing somatic hypermutation of AGCT motifs within human immunoglobulin variable regions.

Somatic hypermutation (SHM) of immunoglobulin variable (V) regions by activation induced deaminase (AID) is essential for robust, long-term humoral immunity against pathogen and vaccine antigens. AID mutates cytosines preferentially within WRCH motifs (where W=A or T, R=A or G and H=A, C or T). However, it has been consistently observed that the mutability of WRCH motifs varies substantially, with large variations in mutation frequency even between multiple occurrences of the same motif within a single V region. This has led to the notion that the immediate sequence context of WRCH motifs contributes to mutability. Recent studies have highlighted the potential role of local DNA sequence features in promoting mutagenesis of AGCT, a commonly mutated WRCH motif. Intriguingly, AGCT motifs closer to 5' ends of V regions, within the framework 1 (FW1) sub-region1, mutate less frequently, suggesting an SHM-suppressing sequence context. Here, we systematically examined the basis of AGCT positional biases in human SHM datasets with DeepSHM, a machine-learning model designed to predict SHM patterns. This was combined with integrated gradients, an interpretability method, to interrogate the basis of DeepSHM predictions. DeepSHM predicted the observed positional differences in mutation frequencies at AGCT motifs with high accuracy. For the conserved, lowly mutating AGCT motifs in FW1, integrated gradients predicted a large negative contribution of 5'C and 3'G flanking residues, suggesting that a CAGCTG context in this location was suppressive for SHM. CAGCTG is the recognition motif for E-box transcription factors, including E2A, which has been implicated in SHM. Indeed, we found a strong, inverse relationship between E-box motif fidelity and mutation frequency. Moreover, E2A was found to associate with the V region locale in two human B cell lines. Finally, analysis of human SHM datasets revealed that naturally occurring mutations in the 3'G flanking residues, which effectively ablate the E-box motif, were associated with a significantly increased rate of AGCT mutation. Our results suggest an antagonistic relationship between mutation frequency and the binding of E-box factors like E2A at specific AGCT motif contexts and, therefore, highlight a new, suppressive mechanism regulating local SHM patterns in human V regions.

Clock-dependent chromatin accessibility rhythms regulate circadian transcription.

Chromatin organization plays a crucial role in gene regulation by controlling the accessibility of DNA to transcription machinery. While significant progress has been made in understanding the regulatory role of clock proteins in circadian rhythms, how chromatin organization affects circadian rhythms remains poorly understood. Here, we employed ATAC-seq (Assay for Transposase-Accessible Chromatin with Sequencing) on FAC-sorted Drosophila clock neurons to assess genome-wide chromatin accessibility at dawn and dusk over the circadian cycle. We observed significant oscillations in chromatin accessibility at promoter and enhancer regions of hundreds of genes, with enhanced accessibility either at dusk or dawn, which correlated with their peak transcriptional activity. Notably, genes with enhanced accessibility at dusk were enriched with E-box motifs, while those more accessible at dawn were enriched with VRI/PDP1-box motifs, indicating that they are regulated by the core circadian feedback loops, PER/CLK and VRI/PDP1, respectively. Further, we observed a complete loss of chromatin accessibility rhythms in per01 null mutants, with chromatin consistently accessible at both dawn and dusk, underscoring the critical role of Period protein in driving chromatin compaction during the repression phase at dawn. Together, this study demonstrates the significant role of chromatin organization in circadian regulation, revealing how the interplay between clock proteins and chromatin structure orchestrates the precise timing of biological processes throughout the day. This work further implies that variations in chromatin accessibility might play a central role in the generation of diverse circadian gene expression patterns in clock neurons.

Transcriptional and post-translational regulation of MITF mediated by bHLH domain during the melanogenesis and melanocyte proliferation in Crassostrea gigas

Melanocyte differentiation is orchestrated by the master regulator transcription factor MITF. However, its ability to discern distinct binding sites linked to effective gene regulation remains poorly understood. This study aims to assess how co-activator acetyltransferase interacts with MITF to modulate their related lysine action, thereby mediating downstream gene regulation, including DNA affinity, stability, transcriptional activity, particularly in the process of shell pigmentation. Here, we have demonstrated that the CgMITF protein can be acetylated, further enabling selective amplification of the melanocyte maturation program. Collaboration with transcriptional co-regulator p300 advances MITF dynamically interplay with downstream targeted gene promoters. We have established that MITF activation was partially dependent on the bHLH domain, which was well conserved across species. The bHLH domain contained conserved lysine residues, including K6 and K43, which interacted with the E-box motif of downstream targeted-genes. Mutations at K6 and K43 lead to a decrease in the binding affinity of the E-box motif. CgMITF protein bound to the E-box motif within the promoter regions of the tyrosinase-related genes, contributing to melanogenesis, and also interacted with the E-box motif within the TBX2 promoter regions, associated with melanocyte proliferation. We elucidated how the bHLH domain links the transcriptional regulation and acetylation modifications in the melanocyte development in C. gigas.

Identification of transcription factor genes responsive to MeJA and characterization of a LaMYC2 transcription factor positively regulates lycorine biosynthesis in Lycoris aurea

Jasmonates (JAs) are among the main phytohormones, regulating plant growth and development, stress responses, and secondary metabolism. As the major regulator of the JA signaling pathway, MYC2 also plays an important role in plant secondary metabolite synthesis and accumulation. In this study, we performed a comparative transcriptome analysis of Lycoris aurea seedlings subjected to methyl jasmonate (MeJA) at different treatment times. A total of 31,193 differentially expressed genes (DEGs) were identified by RNA sequencing. Among them, 732 differentially expressed transcription factors (TFs) comprising 51 TF families were characterized. The most abundant TF family was WRKY proteins (80), followed by AP2/ERF-EFR (67), MYB (59), bHLH (52), and NAC protein (49) families. Subsequently, by calculating the Pearson's correlation coefficient (PCC) between the expression level of TF DEGs and the lycorine contents, 41 potential TF genes (|PCC| >0.8) involved in lycorine accumulation were identified, including 36 positive regulators and 5 negative regulators. Moreover, a MeJA-inducible MYC2 gene (namely LaMYC2) was cloned on the basis of transcriptome sequencing. Bioinformatic analyses revealed that LaMYC2 proteins contain the bHLH-MYC_N domain and bHLH-AtAIB_like motif. LaMYC2 protein is localized in the cell nucleus, and can partly rescue the MYC2 mutant in Arabidopsis thaliana. LaMYC2 protein could interact with most LaJAZs (especially LaJAZ3 and LaJAZ4) identified previously. Transient overexpression of LaMYC2 increased lycorine contents in L. aurea petals, which might be associated with the activation of the transcript levels of tyrosine decarboxylase (TYDC) and phenylalanine ammonia lyase (PAL) genes. By isolating the 887-bp-length promoter fragment upstream of the start codon (ATG) of LaTYDC, we found several different types of E-box motifs (CANNTG) in the promoter of LaTYDC. Further study demonstrated that LaMYC2 was indeed able to bind the E-box (CACATG) present in the LaTYDC promoter, verifying that the pathway genes involved in lycorine biosynthesis could be regulated by LaMYC2, and that LaMYC2 has positive roles in the regulation of lycorine biosynthesis. These findings demonstrate that LaMYC2 is a positive regulator of lycorine biosynthesis and may facilitate further functional research of the LaMYC2 gene, especially its potential regulatory roles in Amaryllidaceae alkaloid accumulation in L. aurea.

Genetic insights into Tietz albinism-deafness syndrome: A new dominant-negative mutation in MITF.

Tietz albinism-deafness syndrome (TADS) is a rare and severe manifestation of Waardenburg syndrome that is primarily linked to mutations in MITF. In this report, we present a case of TADS resulting from a novel c.637G>C mutation in MITF (p.Glu213Gln; GenBank Accession number: NM_000248). A 3-year-old girl presented with congenital generalized hypopigmentation of the hair, skin, and irides along with complete sensorineural hearing loss. Histopathological and electron microscopy investigations indicated that this variant did not alter the number of melanocytes in the skin but significantly impaired melanosome maturation within melanocytes. Comprehensive melanin analysis revealed marked reductions in both eumelanin (EM) and pheomelanin (PM) rather than changes in the EM-to-PM ratio observed in oculocutaneous albinism. We conducted an electrophoretic mobility shift assay to investigate the binding capability of the identified variant to DNA sequences containing the E-box motif along with other known variants (p.Arg217del and p.Glu213Asp). Remarkably, all three variants exhibited dominant-negative effects, thus providing novel insights into the pathogenesis of TADS. This study sheds light on the genetic mechanisms underlying TADS and offers a deeper understanding of this rare condition and its associated mutations in MITF.

Identification of two MEF2s and their role in inhibiting the transcription of the mstn2a gene in the yellowfin seabream, Acanthopagrus latus (Hottuyn, 1782)

Myocyte-specific enhancer binding factor 2 (MEF2), which belongs to the MADS superfamily, is a pivotal and conserved transcription factor that combines with the E-box motif to control the expression of muscle genes. Myostatin (mstn), a muscle growth inhibitor, is a vital member of the TGF-β superfamily. Currently, an understanding of the mechanisms of A. latus mstn (Almstn) transcriptional regulation mediated by MEF2 in fish muscle development is lacking. In the present study, two AlMEF2s (AlMEF2A and AlMEF2B) and Almstn2a were characterized from Acanthopagrus latus. AlMEF2A and AlMEF2B had 456 and 315 amino acid (aa) residues, respectively. Two typical regions, a MADS-box, MEF2, and transcriptionally activated (TAD) domains, are present in both AlMEF2s. The expression profiles of the two AlMEF2 genes were similar. The AlMEF2 genes were mainly expressed in the brain, white muscle, and liver, while Almstn2a expression was higher in the brain than in other tissues. Moreover, the expression trends of AlMEF2s and Almstn2a were significantly changed after starvation and refeeding in the five groups. Additionally, truncation experiments showed that −987 to +168 and −105 to +168 were core promoters of Almstn2a that responded to AlMEF2A and AlMEF2B, respectively. The point mutation experiment confirmed that Almstn2a transcription relies on the mutation binding sites 1 or 5 (M1/5) and mutation binding sites 4 or 5 (M4/5) for AlMEF2A and AlMEF2B regulation, respectively. The electrophoretic mobile shift assay (EMSA) further verified that M1 (−527 to −512) was a pivotal site where AlMEF2A acted on the Almstn2a gene. Furthermore, a siRNA interference gene expression experiment showed that reduced levels of AlMEF2A or AlMEF2B could prominently increase Almstn2a transcription. These results provide new information about the regulation of Almstn2a transcriptional activity by AlMEF2s and a theoretical basis for the regulatory mechanisms involved in muscle development in fish.

Active oxygen generation induced by the glucose sensor TaHXK7-1A decreased the drought resistance of transgenic Arabidopsis and wheat (Triticum aestivum L.)

Improving wheat drought resistance is of great significance for grain production and food security. Hexokinases (HXKs) play a role in sugar signal transduction and are involved in abiotic stress responses in wheat. To clarify the relationship between HXKs and drought stress in wheat, we used the rice active oxygen induction gene OsHXK1 as a reference sequence and the homologously cloned wheat TaHXK7-1A gene. TaHXK7-1A was localized in the nucleus and cell membrane. Under drought stress, over-expression of TaHXK7-1A increased the contents of O2·－ and malondialdehyde (MDA) and significantly up-regulated the respiratory burst oxidative homologue (RBOHs) genes in transgenic Arabidopsis. In addition, the over-expression of TaHXK7-1A inhibited the growth of Arabidopsis seedlings and increased ROS accumulation under 6 % exogenous glucose treatment. Gene silencing of TaHXK7-1 decreased the contents of O2·－ and MDA in wheat leaves under drought stress, and the RBOHs was significantly down-regulated, which improved the drought resistance of wheat. The results of yeast one-hybrid, EMSA, and dual-luciferase assays showed that TabHLH148-5A bound to the E-box motif of the TaHXK7-1A promoter and inhibited the expression of TaHXK7-1A. In addition, yeast two-hybrid and luciferase complementation imaging assays showed that TaHXK7-1A interacted with TaGRF3-4A. These results indicate that the glucose sensor TaHXK7-1A was negatively regulated by TabHLH148-5A, interacted with TaGRF3-4A, and negatively regulated wheat drought resistance by regulating RBOHs expression and inducing ROS production, thus providing a theoretical basis for revealing the molecular mechanism of wheat drought resistance.

Molecular module GmPTF1a/b-GmNPLa regulates rhizobia infection and nodule formation in soybean.

Nodulation begins with the initiation of infection threads (ITs) in root hairs. Though mutual recognition and early symbiotic signaling cascades in legumes are well understood, molecular mechanisms underlying bacterial infection processes and successive nodule organogenesis remain largely unexplored. We functionally investigated a novel pectate lyase enzyme, GmNPLa, and its transcriptional regulator GmPTF1a/b in soybean (Glycine max), where their regulatory roles in IT development and nodule formation were elucidated through investigation of gene expression patterns, bioinformatics analysis, biochemical verification of genetic interactions, and observation of phenotypic impacts in transgenic soybean plants. GmNPLa was specifically induced by rhizobium inoculation in root hairs. Manipulation of GmNPLa produced remarkable effects on IT and nodule formation. GmPTF1a/b displayed similar expression patterns as GmNPLa, and manipulation of GmPTF1a/b also severely influenced nodulation traits. LI soybeans with low nodulation phenotypes were nearly restored to HI nodulation level by complementation of GmNPLa and/or GmPTF1a. Further genetic and biochemical analysis demonstrated that GmPTF1a can bind to the E-box motif to activate transcription of GmNPLa, and thereby facilitate nodulation. Taken together, our findings potentially reveal novel mediation of cell wall gene expression involving the basic helix-loop-helix transcription factor GmPTF1a/b acts as a key early regulator of nodulation in soybean.

Thousands of oscillating LncRNAs in the mouse testis

The long noncoding RNAs (lncRNAs) are involved in numerous fundamental biological processes, including circadian regulation. Although recent studies have revealed insights into the functions of lncRNAs, how the lncRNAs regulate circadian rhythms still requires a deeper investigation. In this study, we generate two datasets of RNA-seq profiles of the mouse (Mus musculus) testis under light-dark (LD) cycle. The first dataset included 18,613 unannotated transcripts measured at 12 time points, each with duplicate samples, under LD conditions; while the second dataset included 21,414 unannotated transcripts measured at six time points, each with three replicates, under desynchronized and control conditions. We identified 5964 testicular lncRNAs in each dataset by BLASTing these transcripts against the known mouse lncRNAs from the NONCODE database. MetaCycle analyses were performed to identify 519, 475, and 494 rhythmically expressed mouse testicular lncRNAs in the 12-time-point dataset, the six-time-point control dataset, and the six-time-point desynchronized dataset, respectively. A comparison of the expression profiles of the lncRNAs under desynchronized and control conditions revealed that 427 rhythmically expressed lncRNAs from the control condition became arrhythmic under the desynchronized condition, suggesting a possible loss of rhythmicity. In contrast, 446 arrhythmic lncRNAs from the control condition became rhythmic under the desynchronized condition, suggesting a possible gain of rhythmicity. Interestingly, 48 lncRNAs were rhythmically expressed under both desynchronized and control conditions. These oscillating lncRNAs were divided into morning lncRNAs, evening lncRNAs, and night lncRNAs based on their time-course expression patterns. We interrogated the promoter regions of these rhythmically expressed mouse testicular lncRNAs to predict their possible regulation by the E-box, D-box, or RORE promoter motifs. GO and KEGG analyses were performed to identify the possible biological functions of these rhythmically expressed mouse testicular lncRNAs. Further, we conducted conservation analyses of the rhythmically expressed mouse testicular lncRNAs with lncRNAs from humans, rats, and zebrafish, and uncovered three mouse testicular lncRNAs conserved across these four species. Finally, we computationally predicted the conserved lncRNA-encoded peptides and their 3D structures from each of the four species. Taken together, our study revealed thousands of rhythmically expressed lncRNAs in the mouse testis, setting the stage for further computational and experimental validations.

Circadian clock gene BMAL1 regulates STAR expression in goose ovarian preovulatory granulosa cells

The ovarian circadian clock plays a regulatory role in the avian ovulation-oviposition cycle. However, little is known regarding the ovarian circadian clock of geese. In this study, we investigated rhythmic changes in clock genes over a 48-h period and identified potential clock-controlled genes involved in progesterone synthesis in goose ovarian preovulatory granulosa cells. The results showed that BMAL1, CRY1, and CRY2, as well as 4 genes (LHR, STAR, CYP11A1, and HSD3B) involved in progesterone synthesis exhibited rhythmic expression patterns in goose ovarian preovulatory granulosa cells over a 48-h period. Knockdown of BMAL1 decreased the progesterone concentration and downregulated STAR mRNA and protein levels in goose ovarian preovulatory granulosa cells. Overexpression of BMAL1 increased the progesterone concentration and upregulated the STAR mRNA level in goose ovarian preovulatory granulosa cells. Moreover, we demonstrated that the BMAL1/CLOCK complex activated the transcription of goose STAR gene by binding to an E-box motif. These results suggest that the circadian clock is involved in the regulation of progesterone synthesis in goose ovarian preovulatory granulosa cells by orchestrating the transcription of steroidogenesis-related genes.

The transcription factor EMB1444-like affects tomato fruit ripening by regulating YELLOW-FRUITED TOMATO 1, a core component of ethylene signaling transduction.

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.

Prediction of mammalian tissue-specific CLOCK–BMAL1 binding to E-box DNA motifs

The Brain and Muscle ARNTL-Like 1 protein (BMAL1) forms a heterodimer with either Circadian Locomotor Output Cycles Kaput (CLOCK) or Neuronal PAS domain protein 2 (NPAS2) to act as a master regulator of the mammalian circadian clock gene network. The dimer binds to E-box gene regulatory elements on DNA, activating downstream transcription of clock genes. Identification of transcription factor binding sites and genomic features that correlate to DNA binding by BMAL1 is a challenging problem, given that CLOCK–BMAL1 or NPAS2–BMAL1 bind to several distinct binding motifs (CANNTG) on DNA. Using three different types of tissue-specific machine learning models with features based on (1) DNA sequence, (2) DNA sequence plus DNA shape, and (3) DNA sequence and shape plus histone modifications, we developed an interpretable predictive model of genome-wide BMAL1 binding to E-box motifs and dissected the mechanisms underlying BMAL1–DNA binding. Our results indicated that histone modifications, the local shape of the DNA, and the flanking sequence of the E-box motif are sufficient predictive features for BMAL1–DNA binding. Our models also provide mechanistic insights into tissue specificity of DNA binding by BMAL1.

Dynamic modulation of genomic enhancer elements in the suprachiasmatic nucleus, the site of the mammalian circadian clock.

The mammalian suprachiasmatic nucleus (SCN), located in the ventral hypothalamus, synchronizes and maintains daily cellular and physiological rhythms across the body, in accordance with environmental and visceral cues. Consequently, the systematic regulation of spatiotemporal gene transcription in the SCN is vital for daily timekeeping. So far, the regulatory elements assisting circadian gene transcription have only been studied in peripheral tissues, lacking the critical neuronal dimension intrinsic to the role of the SCN as central brain pacemaker. By using histone-ChIP-seq, we identified SCN-enriched gene regulatory elements that associated with temporal gene expression. Based on tissue-specific H3K27ac and H3K4me3 marks, we successfully produced the first-ever SCN gene-regulatory map. We found that a large majority of SCN enhancers not only show robust 24-h rhythmic modulation in H3K27ac occupancy, peaking at distinct times of day, but also possess canonical E-box (CACGTG) motifs potentially influencing downstream cycling gene expression. To establish enhancer-gene relationships in the SCN, we conducted directional RNA-seq at six distinct times across the day and night, and studied the association between dynamically changing histone acetylation and gene transcript levels. About 35% of the cycling H3K27ac sites were found adjacent to rhythmic gene transcripts, often preceding the rise in mRNA levels. We also noted that enhancers encompass noncoding, actively transcribing enhancer RNAs (eRNAs) in the SCN, which in turn oscillate, along with cyclic histone acetylation, and correlate with rhythmic gene transcription. Taken together, these findings shed light on genome-wide pretranscriptional regulation operative in the central clock that confers its precise and robust oscillation necessary to orchestrate daily timekeeping in mammals.

The RNA-Binding and RNA-Melting Activities of the Multifunctional Protein Nucleobindin 1

Nucleobindin 1 (NUCB1) is a ubiquitous multidomain protein that belongs to the EF-hand Ca2+-binding superfamily. NUCB1 interacts with Galphai3 protein, cyclooxygenase, amyloid precursor protein, and lipids. It is involved in stress response and human diseases. In addition, this protein is a transcription factor that binds to the DNA E-box motif. Using surface plasmon resonance and molecular beacon approaches, we first showed the RNA binding and RNA melting activities of NUCB1. We suggest that NUCB1 could induce local changes in structured RNAs via binding to the GGAUAU loop sequence. Our results demonstrate the importance of the multidomain structure of NUCB1 for its RNA-chaperone activity in vitro.

Splashed E-box and AP-1 motifs cooperatively drive regeneration response and shape regeneration abilities.

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.

Dynamic chromatin accessibility deploys heterotypic cis/trans-acting factors driving stomatal cell-fate commitment

Chromatin architecture and transcription factor (TF) binding underpin cell-fate specification during development, but their mutual regulatory relationships remain unclear. Here we report an atlas of dynamic chromatin landscapes during stomatal cell-lineage progression, in which sequential cell-state transitions are governed by lineage-specific bHLH TFs. Major reprogramming of chromatin accessibility occurs at the proliferation-to-differentiation transition. We discover novel co-cis regulatory elements (CREs) signifying the early precursor stage, BBR/BPC (GAGA) and bHLH (E-box) motifs, where master-regulatory bHLH TFs, SPEECHLESS and MUTE, consecutively bind to initiate and terminate the proliferative state, respectively. BPC TFs complex with MUTE to repress SPEECHLESS expression through a local deposition of repressive histone marks. We elucidate the mechanism by which cell-state-specific heterotypic TF complexes facilitate cell-fate commitment by recruiting chromatin modifiers via key co-CREs.

Targeting MYC with modular synthetic transcriptional repressors derived from bHLH DNA-binding domains.

Despite unequivocal roles in disease, transcription factors (TFs) remain largely untapped as pharmacologic targets due to the challenges in targeting protein-protein and protein-DNA interactions. Here we report a chemical strategy to generate modular synthetic transcriptional repressors (STRs) derived from the bHLH domain of MAX. Our synthetic approach yields chemically stabilized tertiary domain mimetics that cooperatively bind the MYC/MAX consensus E-box motif with nanomolar affinity, exhibit specificity that is equivalent to or beyond that of full-length TFs and directly compete with MYC/MAX protein for DNA binding. A lead STR directly inhibits MYC binding in cells, downregulates MYC-dependent expression programs at the proteome level and inhibits MYC-dependent cell proliferation. Co-crystallization and structure determination of a STR:E-box DNA complex confirms retention of DNA recognition in a near identical manner as full-length bHLH TFs. We additionally demonstrate structure-blind design of STRs derived from alternative bHLH-TFs, confirming that STRs can be used to develop highly specific mimetics of TFs targeting other gene regulatory elements.