Transcription Factor MYC Research Articles

Penfluridol suppresses MYC-driven ANLN expression and liver cancer progression by disrupting the KEAP1–NRF2 interaction

Hepatocellular carcinoma (HCC) comprises the majority of primary liver cancers and possesses a low 5-year survival rate when in the advanced stages. Anillin (ANLN), a key player in cell growth and cytokinesis, is implicated in HCC development. Currently, no treatment agents are known to suppress ANLN. Analysis of The Cancer Genome Atlas data showed that high ANLN expression is associated with poor prognosis and survival in HCC patients. ANLN knockdown was shown to inhibit proliferation, cell cycle progression, and PD-L1 expression in liver cancer cells. The antipsychotic drug penfluridol was identified to suppress ANLN expression in the Connectivity Map analysis. Penfluridol downregulated ANLN at both the mRNA and protein levels, leading to G2/M cell cycle arrest and reduced colony formation in liver cancer cells. Mechanistically, penfluridol inhibited the transcription factor MYC from binding to an E-box motif in the ANLN promoter. This process was mediated by penfluridol-induced upregulation of NRF2, which competitively bound and sequestered MYC away from the ANLN promoter. Penfluridol inhibited the interaction between NRF2 and KEAP1, increasing NRF2. In a syngeneic mouse model, penfluridol suppressed liver tumour growth accompanied by increased NRF2 and decreased MYC and ANLN expression. These findings suggest penfluridol can be applied as the first ANLN blocker to modulate the MYC/NRF2/KEAP1 axis.

Crispr-Cas9-based long non-coding RNA interference and activation identified that the aberrant expression of Myc-regulated ST8SIA6 antisense RNA 1 promotes tumorigenesis and metastasis in hepatocellular carcinoma

Objective: Long non-coding RNAs (lncRNAs) participate in the formation, progression, and metastasis of cancer. This study aimed to explore the roles of the lncRNA ST8SIA6 antisense RNA 1 (ST8SIA6-AS1) in tumorigenesis and elucidate the underlying molecular mechanism of its upregulation in hepatocellular carcinoma (HCC). Material and Methods: A total of 56 in-house pairs of HCC tissues were examined, and ST8SIA6-AS1 levels were determined through real-time polymerase chain reaction (PCR). The biological behavior of ST8SIA6-AS1 by Crispr-Cas9-based gene repression and activation was determined in vitro and in vivo. The binding sites and biological behavior of Myc proto-oncogene and forkhead box A on chromatin were investigated through luciferase reporter assays, chromatin immunoprecipitation–quantitative PCR, and co-immunoprecipitation (co-IP) assays. The regulatory mechanisms of ST8SIA6-AS1 expression were analyzed with encyclopedia of DNA elements and gene expression profiling interactive analysis. Results: The expression of ST8SIA6-AS1 significantly increased in multiple HCC cell lines and the 56 in-house pairs of HCC tissues (P = 0.0018). Functionally, high-efficiency Crispr-Cas9-based knockdown of ST8SIA6-AS1 revealed that ST8SIA6-AS1 knockdown attenuated the proliferation, migration, and infiltration of HCC cells and considerably reduced the growth rate of subcutaneous and orthotopic HCC tumors. Conversely, ST8SIA6-AS1 overexpression considerably improved the oncogenic characteristics of the HCC cells. Furthermore, ST8SIA6-AS1 upregulation was regulated by the direct binding of transcription factor Myc to the −260 bp to+155 bp and +1003 bp to +1312 bp regions of the ST8SIA6-AS1 transcription start site, which is a segment with high level of H3K27 acetylation. Myc knockdown or treatment with the BET bromodomain inhibitor JQ-1 considerably reduced ST8SIA6-AS1 RNA expression in the HCC cells. Conclusion: Our study has established the oncogenic role of ST8SIA6-AS1 and elucidated the Myc-dependent upregulation mechanism of ST8SIA6-AS1 in HCC, providing a profound theoretical molecular basis for the carcinogenic function of ST8SIA6-AS1 in HCC.

The MYC Gene RrbHLH105 Contributes to Salt Stress-Induced Geraniol in Rose by Regulating Trehalose-6-Phosphate Signalling.

Rose (Rosa rugosa) is an important perfume plant, but its cultivation is significantly constrained by salt stress. Terpenes represent the most abundant volatile aromatic compounds in roses, yet little is known about how terpene metabolism responds to salt stress. In this study, salt-treated rose petals presented significant accumulation of monoterpenes, including geraniol, due to the disruption of jasmonic acid (JA) biosynthesis and signalling. Overexpression and silencing analyses revealed a MYC transcription factor involved in JA signalling (RrbHLH105) as a repressor of geraniol biosynthesis. RrbHLH105 was shown to activate the trehalose-6-phosphate synthase genes RrTPS5 and RrTPS8 by binding to the E-box (5'-CANNTG-3'). The increased trehalose-6-phosphate content and decreased geraniol content in rose petals overexpressing TPS5 or RrTPS8, along with the high accumulation of geraniol in petals where both RrbHLH105 and TPSs were cosilenced, indicate that trehalose signalling plays a role in the negative regulation of geraniol accumulation via the RrbHLH105-TPS module. In summary, the suppression of RrbHLH105 by salt stress leads to excessive geraniol accumulation through the inhibition of both RrbHLH105-mediated JA signalling and RrTPS-mediated trehalose signalling in rose petals. Additionally, this study highlights the emerging role of RrbHLH105 as a critical integrator of JA and trehalose signalling crosstalk.

Development of DuoMYC: a synthetic cell penetrant miniprotein that efficiently inhibits the oncogenic transcription factor MYC.

The master regulator transcription factor MYC is implicated in numerous human cancers, and its targeting is a long-standing challenge in drug development. MYC is a typical 'undruggable' target, with no binding pockets on its DNA binding domain and extensive intrinsically disordered regions. Rather than trying to target MYC directly with classical modalities, here we engineer synthetic miniproteins that can bind to MYC's target DNA, the enhancer box (E-Box), and potently inhibit MYC-driven transcription. We crafted the miniproteins via structure-based design and a combination of solid phase peptide synthesis and site-specific crosslinking. Our lead variant, DuoMYC, binds to E-Box DNA with high affinity (KD ~0.1 μM) and is able to enter cells and inhibit MYC-driven transcription with submicromolar potency (IC50=464 nM) as shown by reporter gene assay and confirmed by RNA sequencing. Notably, DuoMYC surpasses the efficacy of several other recently developed MYC inhibitors. Our results highlight the potential of engineered synthetic protein therapeutics for addressing challenging intracellular targets.

Development of DuoMYC: a synthetic cell penetrant miniprotein that efficiently inhibits the oncogenic transcription factor MYC

AbstractThe master regulator transcription factor MYC is implicated in numerous human cancers, and its targeting is a long‐standing challenge in drug development. MYC is a typical ‘undruggable’ target, with no binding pockets on its DNA binding domain and extensive intrinsically disordered regions. Rather than trying to target MYC directly with classical modalities, here we engineer synthetic miniproteins that can bind to MYC's target DNA, the enhancer box (E‐Box), and potently inhibit MYC‐driven transcription. We crafted the miniproteins via structure‐based design and a combination of solid phase peptide synthesis and site‐specific crosslinking. Our lead variant, DuoMYC, binds to E‐Box DNA with high affinity (KD ~0.1 μM) and is able to enter cells and inhibit MYC‐driven transcription with submicromolar potency (IC50=464 nM) as shown by reporter gene assay and confirmed by RNA sequencing. Notably, DuoMYC surpasses the efficacy of several other recently developed MYC inhibitors. Our results highlight the potential of engineered synthetic protein therapeutics for addressing challenging intracellular targets.

Jasmonate induces translation of the Arabidopsis tRNA-binding protein YUELAO1, which activates MYC2 in jasmonate signaling.

Jasmonate is ubiquitous in the plant kingdom and regulates multiple physiological processes. Although jasmonate signaling has been thoroughly investigated in Arabidopsis thaliana, most studies have focused on the transcriptional mechanisms underlying various jasmonate responses. It remains unclear whether (and how) translation-related pathways help improve transcription efficiency to modulate jasmonate signaling, which may enable plants to respond to stressful conditions effectively. Here, we demonstrate that jasmonate induces translation of the tRNA-binding protein YUELAO 1 (YL1) via a specific region in its 3' untranslated region (3' UTR). YL1 and its homolog YL2 redundantly stimulate jasmonate responses such as anthocyanin accumulation and root growth inhibition, with the YL1 3' UTR being critical for YL1-promoted jasmonate responses. Once translated, YL1 acts as an activator of the MYC2 transcription factor through direct interaction, and disrupting YL1 3' UTR impairs the YL1-mediated transcriptional activation of MYC2. YL1 enhances jasmonate responses mainly in a MYC2-dependent manner. Together, these findings reveal a translational mechanism involved in jasmonate signaling and advance our understanding of the transcriptional regulation of jasmonate signaling. The YL1 3' UTR acts as a crucial signal transducer that integrates translational and transcriptional regulation, allowing plants to respond to jasmonate in a timely fashion.

Identification of the sweet orange (Citrus sinensis) bHLH gene family and the role of CsbHLH55 and CsbHLH87 in regulating salt stress.

Salt stress is one of the primary environmental stresses limiting plant growth and production and adversely affecting the growth, development, yield, and fruit quality of Citrus sinensis. bHLH (basic helix-loop-helix) genes are involved in many bioregulatory processes in plants, including growth and development, phytohormone signaling, defense responses, and biosynthesis of specific metabolites. In this study, by bioinformatics methods, 120 CsbHLHgenes were identified, and phylogenetic analysis classified them into 18 subfamilies that were unevenly distributed on nine chromosomes. The cis-acting elements of the CsbHLH genes were mainly hormone-related cis-acting elements. Seventeen CsbHLH genes exhibited significant differences in expression under salt stress. Six CsbHLH genes with significant differences in expression were randomly selected for quantitative real-time polymerase chain reaction (qRT-PCR) validation. The qRT-PCR results showed a strong correlation with the transcriptome data. Phytohormones such as jasmonic acid (JA) are essential for biotic and abiotic stress responses in plants, and CsbHLH55 and CsbHLH87 are considered candidate target genes for sweet orange MYC2 transcription factors involved in the JA signaling pathway. These genes are the main downstream effectors in the JA signaling pathway and can be activated to participate in the JA signaling pathway. Activation of the JA signaling pathway inhibits the production of reactive oxygen species and improves the salt tolerance of sweet orange plants. The CsbHLH55 and CsbHLH87 genes could be candidate genes for breeding new transgenic salt-resistant varieties of sweet orange.

The MYC2 and MYB43 transcription factors cooperate to repress HMA2 and HMA4 expression, altering cadmium tolerance in Arabidopsis thaliana

Cadmium (Cd) represents a hazardous heavy metal, prevalent in agricultural soil due to industrial and agricultural expansion. Its propensity for being absorbed by edible plants, even at minimal concentrations, and subsequently transferred along the food chain poses significant risks to human health. Accordingly, it is imperative to investigate novel genes and mechanisms that govern Cd tolerance and detoxification in plants. Here, we discovered that the transcription factor MYC2 directly binds to the promoters of HMA2 and HMA4 to repress their expression, thereby altering the distribution of Cd in plant tissues and negatively regulating Cd stress tolerance. Additionally, molecular, biochemical, and genetic analyses revealed that MYC2 interacts and cooperates with MYB43 to negatively regulate the expression of HMA2 and HMA4 and Cd stress tolerance. Notably, under Cd stress conditions, MYC2 undergoes degradation, thereby alleviating its inhibitory effect on HMA2 and HMA4 expression and plant tolerance to Cd stress. Thus, our study highlights the dynamic regulatory role of MYC2, in concert with MYB43, in regulating the expression of HMA2 and HMA4 under both normal and Cd stress conditions. These findings present MYC2 as a promising target for directed breeding efforts aimed at mitigating Cd accumulation in edible plant roots.

5-Aminoimidazole-4-carboxamide ribonucleotide formyltransferase/inosine monophosphate cyclohydrolase promotes pulmonary arterial smooth muscle cell proliferation via the Ras signaling pathway.

Pulmonary arterial hypertension (PAH) is a debilitating vascular disorder characterized by abnormal pulmonary artery smooth muscle cell (PASMC) proliferation and collagen synthesis, contributing to vascular remodeling and elevated pulmonary vascular resistance. This study investigated the critical role of 5-aminoimidazole-4-carboxamide ribonucleotide formyltransferase/inosine monophosphate cyclohydrolase (ATIC) in cell proliferation and collagen synthesis in PASMCs in PAH. Here we show that ATIC levels are significantly increased in the lungs of monocrotaline (MCT)-induced PAH rat model, hypoxia-induced PAH mouse model, and platelet-derived growth factor (PDGF)-stimulated PASMCs. Inhibition of ATIC attenuated PDGF-induced cell proliferation and collagen I synthesis in PASMCs. Conversely, overexpression or knockdown of ATIC causes a significant promotion or inhibition of Ras and ERK activation, cell proliferation, and collagen synthesis in PASMCs. Moreover, ATIC deficiency attenuated Ras activation in the lungs of hypoxia-induced PAH mice. Furthermore, Ras inhibition attenuates ATIC overexpression- and PDGF-induced collagen synthesis and PASMC proliferation. Notably, we identified that transcription factors MYC, early growth response protein 1 (EGR1), and specificity protein 1 (SP1) directly binds to promoters of Atic gene and regulate ATIC expression. These results provide the first evidence that ATIC promotes PASMC proliferation in pulmonary vascular remodeling through the Ras signaling pathway.NEW & NOTEWORTHY Our findings highlight the important role of ATIC in the PASMC proliferation of pulmonary vascular remodeling through its modulation of the Ras signaling pathway and its regulation by transcription factors MYC, EGR1, and SP1. ATIC's modulation of Ras signal pathway represents a novel mechanism contributing to PAH development.

The MYC transcription factor PbrMYC8 negatively regulates PbrMSL5 expression to promote pollen germination in Pyrus

The successful germination of pollen is essential for double fertilization in flowering plants. Mechanosensitive channels of small conductance (MscS-like, MSL) inhibit pollen germination and maintains cellular integrity of pollen during this process. Therefore, it is vital to carefully regulate the expression of MSL to promote successful pollen germination. Despite its importance, the molecular mechanisms governing MSL expression in plants remain poorly understood. Here, we had identified 15 MSL genes in the pear, among which PbrMSL5 was expressed in pollen development. Subcellular localization experiments revealed that PbrMSL5 was located in both plasma membrane and cytoplasm. Functional investigations, including complementation experiments using the atmsl8 mutant background, demonstrated the involvement of PbrMSL5 in preserving pollen cell integrity and inhibiting germination. Antisense oligonucleotide experiments further confirmed that PbrMSL5 suppressed pear pollen germination by reducing osmotic pressure and Cl− content. Yeast one-hybrid, electrophoretic mobility shift assays, and dual luciferase reporter assay elucidated that PbrMYC8 interacts directly with the N-box element, leading to the suppression of PbrMSL5 expression and promoted pollen germination. These results represented a significant advancement in unraveling the molecular mechanisms controlling plant MSL expression. This study showed valuable contribution to advancing our comprehension of the mechanism underlying pollen germination.

The MYC-MAF-SAGA axis drives oncogenic gene expression in multiple myeloma.

The SAGA complex is an evolutionarily conserved histone acetyltransferase complex and transcription coactivator essential for development and disease. Dysregulation of SAGA is implicated in various human diseases, including cancer. In this issue of Genes & Development, Chen et al. (doi:10.1101/gad.351789.124) uncover a critical role for SAGA in multiple myeloma wherein SAGA's ADA2B component is required for the expression of mTORC1 pathway genes and targets of the MYC, E2F, and MAF (musculoaponeurotic fibrosarcoma) transcription factors. SAGA cooperates with MYC and MAF to sustain oncogenic gene expression programs vital for multiple myeloma survival and thus may serve as a therapeutic target for future cancer therapies.

Genomic characterization of bZIP gene family and patterns of gene regulation on Cercospora beticola Sacc resistance in sugar beet (Beta vulgaris L.).

Sugar beet (Beta vulgaris L.) is one of the most important sugar crops, accounting for nearly 30% of the world's annual sugar production. And it is mainly distributed in the northwestern, northern, and northeastern regions of China. However, Cercospora leaf spot (CLS) is the most serious and destructive foliar disease during the cultivation of sugar beet. In plants, the bZIP gene family is one of important family of transcription factors that regulate many biological processes, including cell and tissue differentiation, pathogen defense, light response, and abiotic stress signaling. Although the bZIP gene family has been mentioned in previous studies as playing a crucial role in plant defense against diseases, there has been no comprehensive study or functional analysis of the bZIP gene family in sugar beet with respect to biotic stresses. In this study, we performed a genome-wide analysis of bZIP family genes (BvbZIPs) in sugar beet to investigate their phylogenetic relationships, gene structure and chromosomal localization. At the same time, we observed the stomatal and cell ultrastructure of sugar beet leaf surface during the period of infestation by Cercospora beticola Sacc (C. beticola). And identified the genes with significant differential expression in the bZIP gene family of sugar beet by qRT-PCR. Finally we determined the concentrations of SA and JA and verified the associated genes by qRT-PCR. The results showed that 48 genes were identified and gene expression analysis indicated that 6 BvbZIPs were significantly differential expressed in C. beticola infection. It is speculated that these BvbZIPs are candidate genes for regulating the response of sugar beet to CLS infection. Meanwhile, the observation stomata of sugar beet leaves infected with C. beticola revealed that there were also differences in the surface stomata of the leaves at different periods of infection. In addition, we further confirmed that the protein encoded by the SA signaling pathway-related gene BVRB_9g222570 in high-resistant varieties was PR1, which is closely related to systemic acquired resistance. One of the protein interaction modes of JA signal transduction pathway is the response of MYC2 transcription factor caused by JAZ protein degradation, and there is a molecular interaction between JA signal transduction pathway and auxin. Despite previous reports on abiotic stresses in sugar beet, this study provides very useful information for further research on the role of the sugar beet bZIP gene family in sugar beet through experiments. The above research findings can promote the development of sugar beet disease resistance breeding.

Real-Time Measurement of a Weak Interaction of a Transcription Factor Motif with a Protein Hub at Single-Molecule Precision.

Transcription factors often interact with other protein cofactors, regulating gene expression. Direct detection of these brief events using existing technologies remains challenging due to their transient nature. In addition, intrinsically disordered domains, intranuclear location, and lack of cofactor-dependent active sites of transcription factors further complicate the quantitative analysis of these critical processes. Here, we create a genetically encoded label-free sensor to identify the interaction between a motif of the MYC transcription factor, a primary cancer driver, and WDR5, a chromatin-associated protein hub. Using an engineered nanopore equipped with this motif, WDR5 is probed through reversible captures and releases in a one-by-one and time-resolved fashion. Our single-molecule kinetic measurements indicate a weak-affinity interaction arising from a relatively slow complex association and a fast dissociation of WDR5 from the tethered motif. Further, we validate this subtle interaction by determinations in an ensemble using single nanodisc-wrapped nanopores immobilized on a biolayer interferometry sensor. This study also provides the proof-of-concept for a sensor that reveals unique recognition signatures of different protein binding sites. Our foundational work may be further developed to produce sensing elements for analytical proteomics and cancer nanomedicine.

Jasmonate signaling modulates root growth by suppressing iron accumulation during ammonium stress.

Plants adapt to changing environmental conditions by adjusting their growth physiology. Nitrate (NO3-) and ammonium (NH4+) are the major inorganic nitrogen forms for plant uptake. However, high NH4+ inhibits plant growth, and roots undergo striking changes, such as inhibition of cell expansion and division, leading to reduced root elongation. In this work, we show that high NH4+ modulates nitrogen metabolism and root developmental physiology by inhibiting iron (Fe)-dependent Jasmonate (JA) signaling and response in Arabidopsis (Arabidopsis thaliana). Transcriptomic data suggested that NH4+ availability regulates Fe and JA-responsive genes. High NH4+ levels led to enhanced root Fe accumulation, which impaired nitrogen balance and growth by suppressing JA biosynthesis and signaling response. Integrating pharmacological, physiological, and genetic experiments revealed the involvement of NH4+ and Fe-derived responses in regulating root growth and nitrogen metabolism through modulation of the JA pathway during NH4+ stress. The JA signaling transcription factor MYC2 directly bound the promoter of the NITRATE TRANSPORTER 1.1 (NRT1.1) and repressed it to optimize the NH4+/Fe-JA balance for plant adaptation during NH4+ stress. Our findings illustrate the intricate balance between nutrient and hormone-derived signaling pathways that appear essential for optimizing plant growth by adjusting physiological and metabolic responses during NH4+/Fe stress.

Arabidopsis thaliana MYC2 and MYC3 Are Involved in Ethylene-Regulated Hypocotyl Growth as Negative Regulators.

The ethylene-regulated hypocotyl elongation of Arabidopsis thaliana involves many transcription factors. The specific role of MYC transcription factors in ethylene signal transduction is not completely understood. The results here revealed that two MYCs, MYC2 and MYC3, act as negative regulators in ethylene-suppressed hypocotyl elongation. Etiolated seedlings of the loss-of-function mutant of MYC2 or MYC3 were significantly longer than wild-type seedlings. Single- or double-null mutants of MYC2 and MYC3 displayed remarkably enhanced response to ACC(1-aminocyclopropane-1-carboxylate), the ethylene precursor, compared to wild-type seedlings. MYC2 and MYC3 directly bind to the promoter zone of ERF1, strongly suppressing its expression. Additionally, EIN3, a key component in ethylene signaling, interacts with MYC2 or MYC3 and significantly suppresses their binding to ERF1's promoter. MYC2 and MYC3 play crucial roles in the ethylene-regulated expression of functional genes. The results revealed the novel role and functional mechanism of these transcription factors in ethylene signal transduction. The findings provide valuable information for deepening our understanding of their role in regulating plant growth and responding to stress.

The MET Family of Receptor Tyrosine Kinases Promotes a Shift to Pro-Tumor Metabolism.

The development and growth of cancer is fundamentally dependent on pro-tumor changes in metabolism. Cancer cells generally shift away from oxidative phosphorylation as the primary source of energy and rely more heavily on glycolysis. Receptor tyrosine kinases (RTKs) are a type of receptor that is implicated in this shift to pro-tumor metabolism. RTKs are important drivers of cancer growth and metastasis. One such family of RTKs is the MET family, which consists of MET and RON (MST1R). The overexpression of either MET or RON has been associated with worse cancer patient prognosis in a variety of tumor types. Both MET and RON signaling promote increased glycolysis by upregulating the expression of key glycolytic enzymes via increased MYC transcription factor activity. Additionally, both MET and RON signaling promote increased cholesterol biosynthesis downstream of glycolysis by upregulating the expression of SREBP2-induced cholesterol biosynthesis enzymes via CTTNB1. These changes in metabolism, driven by RTK activity, provide potential targets in limiting tumor growth and metastasis via pharmacological inhibition or modifications in diet. This review summarizes pro-tumor changes in metabolism driven by the MET family of RTKs. In doing so, we will offer our unique perspective on metabolic pathways that drive worse patient prognosis and provide suggestions for future study.

CRISPR/dCas9-Mediated DNA Methylation Editing on emx2 in Chinese Tongue Sole (Cynoglossus semilaevis) Testis Cells.

DNA methylation is a key epigenetic mechanism orchestrating gene expression networks in many biological processes. Nonetheless, studying the role of specific gene methylation events in fish faces challenges. In this study, we validate the regulation of DNA methylation on empty spiracles homeobox 2 (emx2) expression with decitabine treatment in Chinese tongue sole testis cells. We used the emx2 gene as the target gene and developed a new DNA methylation editing system by fusing dnmt3a with catalytic dead Cas9 (dCas9) and demonstrated its ability for sequence-specific DNA methylation editing. Results revealed that utilizing dCas9-dnmt3a to target emx2 promoter region led to increased DNA methylation levels and decreased emx2 expression in Chinese tongue sole testis cells. More importantly, the DNA methylation editing significantly suppressed the expression of MYC proto-oncogene, bHLH transcription factor (myc), one target gene of emx2. Furthermore, we assessed the off-target effects of dCas9-dnmt3a and confirmed no significant impact on the predicted off-target gene expression. Taken together, we developed the first DNA methylation editing system in marine species and demonstrated its effective editing ability in Chinese tongue sole cells. This provides a new strategy for both epigenetic research and molecular breeding of marine species.

Transcriptomic and Metabolomic Insights into ABA-Related Genes in Cerasus humilis under Drought Stress.

Cerasus humilis, a small shrub of the Cerasus genus within the Rosaceae family, is native to China and renowned for its highly nutritious and medicinal fruits, robust root system, and remarkable drought resistance. This study primarily employed association transcriptome and metabolome analyses to assess changes in abscisic acid (ABA) levels and identify key regulatory genes in C. humilis subjected to varying degrees of drought stress. Notably, we observed distinct alterations in transcription factors across different drought intensities. Specifically, our transcriptome data indicated noteworthy shifts in GATA, MYB, MYC, WRKY, C2H2, and bHLH transcription factor families. Furthermore, combined transcriptomic and metabolomic investigations demonstrated significant enrichment of metabolic pathways, such as 'Carbon metabolism', 'Biosynthesis of amino acids', 'Biosynthesis of cofactors', 'Phenylpropanoid biosynthesis', 'Starch and sucrose metabolism', and 'Plant hormone signal transduction' under moderate (Mod) or severe (Sev) drought conditions. A total of 11 candidate genes involved in ABA biosynthesis and signaling pathways were identified. The down-regulated genes included secoisolariciresinol dehydrogenase-like and PYL2. Conversely, genes including FAD-dependent urate hydroxylase-like, cytochrome P450 97B2, carotenoid cleavage dioxygenase 4 (CCD4), SnRK2.2, ABI 5-like protein 5, PP2C 51, and SnRK2.3, were up-regulated under Mod or Sev drought stress. This study lays the genetic foundation for ABA biosynthesis to enhance drought tolerance and provides genetic resources for plant genetic engineering and breeding efforts.

The gapless genome assembly and multi-omics analyses unveil a pivotal regulatory mechanism of oil biosynthesis in the olive tree.

Olive is a valuable oil-bearing tree with fruits containing high levels of fatty acids. Oil production is a multifaceted process involving intricate interactions between fatty acid biosynthesis and other metabolic pathways that are affected by genetics and the developmental stages of the fruit. However, a comprehensive understanding of the underlying regulatory mechanisms is still lacking. Here, we generated a gap-free telomere-to-telomere assembly for Olea europaea cv. 'Leccino', representing an olive genome with the highest contiguity and completeness to date. The combination of time-course metabolomics and transcriptomics datasets revealed a negative correlation between fatty acid and flavonoid biosynthesis in the initial phase of olive fruit development, which was subject to an opposing regulatory mechanism mediated by the hub transcription factor MYC2. Multifaceted molecular assays demonstrated that MYC2 is a repressor of fatty acid biosynthesis by downregulating the expression of BCCP2 (biotin carboxylase carrier protein 2), while it acts as an activator of FLS (flavonol synthase), leading to an increase in flavonoid synthesis. Furthermore, the expression of MYC2 is regulated by fluctuations of methyl jasmonate content during olive fruit development. Our study completes a high-quality gapless genome of an olive cultivar, and provides new insight into the regulatory mechanisms underlying the biosynthesis of fatty acids and flavonoids in its fruit.

MDB-38. AN INTEGRATED MODEL FOR RESISTANCE TO BROMODOMAIN INHIBITORS IN CHILDHOOD MEDULLOBLASTOMA

Abstract BACKGROUND Bromodomain inhibitors (BETi) have emerged as a potential treatment option for children with medulloblastoma, where hyperactivation of gene transcription by the MYC and MYCN transcription factors may lead to a transcriptional dependency. Yet, drug resistance limits the utility of these agents when employed as a monotherapy. We therefore sought to identify mechanisms of BETi resistance that inform rational development of multimodal therapy options. METHODS We used CRISPR screening of D458 medulloblastoma cells that have acquired resistance to the JQ1 inhibitor. We subsequently used small molecule inhibition and knockdown experiments to validate Menin as a target. Finally, we used RNAseq, ribosome profiling, and mass spectrometry to characterize transcriptional and post-transcriptional changes in D458 cells compared to D458 with JQ1 resistance. RESULTS In the setting of BETi resistance, we observed dependency on key transcriptional actors, such as MLL (KMT2A) and the Mediator complex, which we are now validating in mini-pool experiments. We further characterized aberrant MLL complex function through knockdown, small molecule inhibition, and RNA-sequencing of Menin, a critical MLL cofactor. Next, we were surprised to observe ribosome and proteosome genes as CRISPR dependencies in our initial screening of BETi-resistant cells, suggesting post-transcriptional regulation. Indeed, MYC, whose transcription is potently blocked by BETi, remained robust at the protein level in BETi resistance. We therefore performed integrated transcriptional, translational, and proteomic profiling to characterize gene regulation in BETi-resistance. We found that the BETi-resistant state leads to an “uncoupling” of transcriptional and post-transcriptional regulation for a subset of genes, leading us to test small molecule inhibitors of post-transcriptional processes in BETi-resistant medulloblastoma. CONCLUSIONS Taken together, our data inform an integrated model of BETi resistance based on both transcriptional and post-transcriptional mechanisms, suggesting new therapeutic angles that may be effective in combination with BETi therapies.