Transcriptional Regulatory Networks Research Articles

Xbp1 promotes odontoblastic differentiation through modulating mitochondrial homeostasis.

Odontoblast differentiation depends on the orderly recruitment of transcriptional factors (TFs) in the transcriptional regulatory network. The depletion of crucial TFs disturbs dynamic alteration of the chromatin landscape and gene expression profile, leading to developmental defects. Our previous studies have revealed that the basic leucine zipper (bZIP) TF family is crucial in odontoblastic differentiation, but the function of bZIP TF family member XBP1 is still unknown. Here, we showed the stage-specific expression patterns of the spliced form Xbp1s during tooth development. Elevated Xbp1 expression and nuclear translocation of XBP1S in mesenchymal stem cells (MSCs) were induced by differentiation medium invitro. Diminution of Xbp1 expression impaired the odontogenic differentiation potential of MSCs. The further integration of ATAC-seq and RNA-seq identified Hspa9 as a direct downstream target, an essential mitochondrial chaperonin gene that modulated mitochondrial homeostasis. The amelioration of mitochondrial dysfunction rescued the impaired odontogenic differentiation potential of MSCs caused by the diminution of Xbp1. Furthermore, the overexpression of Hspa9 rescued Xbp1-deficient defects in odontoblastic differentiation. Our study illustrates the crucial role of Xbp1 in odontoblastic differentiation via modulating mitochondrial homeostasis and brings evidence to the therapy of mitochondrial diseases caused by genetic defects.

Correction to: INO80-Dependent Remodeling of Transcriptional Regulatory Network Underlies the Progression of Heart Failure.

Correction to: INO80-Dependent Remodeling of Transcriptional Regulatory Network Underlies the Progression of Heart Failure.

Analysis of genes related to xylem cell wall development based on transcriptomics in Populus alba ‘Berolinensis’ tension wood

Populus alba ‘Berolinensis’ is a fast-growing, high-yielding species with strong biotic and abiotic stress resistance, and widely planted for timber, shelter belts and aesthetic purposes. In this study, molecular development is explored and the important genes regulating xylem formation in P. alba ‘Berolinensis’ under artificial bending treatments was identified. Anatomical investigation indicated that tension wood (TW) was characterized by eccentric growth of xylem and was enriched in cellulose; the degree of lignification was lower than for normal wood (NW) and opposite wood (OW). RNA-Seq-based transcriptome analysis was performed using developing xylem from three wood types (TW, OW and NW). A large number of differentially expressed genes (DEGs) were screened and 4889 counted. In GO and KEGG enrichment results, genes involved in plant hormone signal transduction, phenylpropanoid biosynthesis, and cell wall and secondary cell wall biogenesis play major roles in xylem development under artificial bending. Eight expansin (PalEXP) genes were identified from the RNA-seq data; four were differentially expressed during tension wood formation. Phylogenetic analysis indicated that PalEXLB1 belongs to the EXPB subfamily and that the other PalEXPs are members of the EXPA subfamily. A transcriptional regulatory network construction showed 10 transcription factors located in the first and second layers upstream of EXP, including WRKY, ERF and bHLH. RT‒qPCR analysis in leaves, stems and roots combined with transcriptome analysis suggests that PalEXPA2, PalEXPA4 and PalEXPA15 play significant regulatory roles in cell wall formation during tension wood development. The candidate genes involved in xylem cell wall development during tension wood formation marks an important step toward identifying the molecular regulatory mechanism of xylem development and wood property improvement in P. alba ‘Berolinensis’.

Unveiling the regulatory mechanisms of nodules development and quality formation in Panax notoginseng using multi-omics and MALDI-MSI

IntroductionRenowned for its role in traditional Chinese medicine, Panax notoginseng exhibits healing properties including bidirectional regulatory effects on hematological system diseases. However, the presence of nodular structures near the top of the main root, known as nail heads, may impact the quality of the plant's valuable roots. ObjectivesIn this paper, we aim to systematically analyze nail heads to identify their potential correlation with P. notoginseng quality. Additionally, we will investigate the molecular mechanisms behind nail head development. MethodsMorphological characteristics and anatomical features were analyzed to determine the biological properties of nail heads. Active component analysis and MALDI mass spectrometry imaging (MALDI-MSI) were performed to determine the correlation between nail heads and P. notoginseng quality. Phytohormone quantitation, MALDI-MSI, RNA-seq, and Arabidopsis transformation were conducted to elucidate the mechanisms of nail head formation. Finally, protein-nucleic acid and protein–protein interactions were investigated to construct a transcriptional regulatory network of nodule development and quality formation. ResultsOur analyses have revealed that nail heads originate from an undeveloped lateral root. The content of ginsenosides was found to be positively associated with the amount of nail heads. Ginsenoside Rb1 specifically accumulated in the cortex of nail heads, while IAA, tZR and JAs also showed highest accumulation in the nodule. RNA-seq analysis identified PnIAA14 and PnCYP735A1 as inhibitors of lateral root development. PnMYB31 and PnMYB78 were found to form binary complexes with PnbHLH31 to synergistically regulate the expression of PnIAA14, PnCYP735A1, PnSS, and PnFPS. ConclusionOur study details the major biological properties of nodular structures in P. notoginseng and outlines their impact on the quality of the herb. It was also determined that PnMYB31- and PnMYB78-PnbHLH31 regulate phytohormones and ginsenosides accumulation, further affecting plant development and quality. This research provides insights for quality evaluation and clinical applications of P. notoginseng.

Integrative Analysis of Transcriptome and Metabolome Sheds Light on Flavonoid Biosynthesis in the Fruiting Body of Stropharia rugosoannulata

Flavonoids are a diverse family of natural compounds that are widely distributed in plants and play a critical role in plant growth, development, and stress adaptation. In recent years, the biosynthesis of flavonoids in plants has been well-researched, with the successive discovery of key genes driving this process. However, the regulation of flavonoid biosynthesis in fungi remains unclear. Stropharia rugosoannulata is an edible mushroom known for its high nutritional and pharmacological value, with flavonoids being one of its main active components. To investigate the flavonoid content of S. rugosoannulata, a study was conducted to extract and determine the total flavonoids at four stages: young mushroom (Ym), gill (Gi), maturation (Ma), and parachute-opening (Po). The findings revealed a gradual increase in total flavonoid concentration as the fruiting body developed, with significant variations observed between the Ym, Gi, and Ma stages. Subsequently, we used UPLC-MS/MS and transcriptome sequencing (RNA-seq) to quantify the flavonoids and identify regulatory genes of Ym, Gi, and Ma. In total, 53 flavonoid-related metabolites and 6726 differentially expressed genes (DEGs) were identified. Through KEGG pathway enrichment analysis, we identified 59 structural genes encoding flavonoid biosynthesis-related enzymes, most of which were up-regulated during the development of the fruiting body, consistent with the accumulation of flavonoids. This research led to the establishment of a comprehensive transcriptional metabolic regulatory network encompassing flavonoids, flavonoid synthases, and transcription factors (TFs). This represents the first systematic exploration of the molecular mechanism of flavonoids in the fruiting of fungi, offering a foundation for further research on flavonoid mechanisms and the breeding of high-quality S. rugosoannulata.

Genome-wide identification and functional characterization of trans-IDS genes give new insights into terpene biosynthesis in Syringa oblata

Floral scents play a vital role in plant pollination and ornamental plant industry. Syringa oblata, a typical aromatic and medicinal plant, holds a high terpene concentration as the main fragrant volatiles and plays a crucial role in the breeding in the Syringa genus. The trans-isopentenyl diphosphate synthases (trans-IDSs) were critical enzymes in terpenoid diversity and accumulation. However, genome-wide characterization and systematic analysis of the lilac trans-IDS family are still under-explored. In this study, we identified sixteen trans-IDS genes belonging to five subfamilies on 10 chromosomes of the lilac genome. Each subfamily shared similar exon, intron, and motif distributions. Synteny analysis suggested that the lilac trans-IDS gene family expanded through segmental and tandem duplication events. We also analyzed their expression levels in various organs and flower development stages for terpene biosynthesis functional insights. Furthermore, spatiotemporal gene expression, virus-induced gene silencing (VIGS), transient overexpression, and transgenic Arabidopsis results collectively showed SoGPPS2 enhances terpene volatile synthesis. SoGPPS2 also impacted plant growth and flowering. And potential upstream transcription regulatory networks for SoGPPS2 were identified. In summary, this study enhances our comprehensive understanding of the lilac trans-IDS gene family for the first time, providing crucial insights into terpene synthesis and laying the groundwork for future floral aromatic breeding, medicinal and essential oil industries in lilac.

The accessible chromatin landscape of lipopolysaccharide-induced systemic inflammatory response identifying epigenome signatures and transcription regulatory networks in chickens

Lipopolysaccharide (LPS) can induce systemic inflammatory response (SIR) in animals. Understanding the regulatory mechanism of SIR and therapies to ensure healthy growth is urgently needed. Chromatin remodeling plays a crucial role in the expression of genes involved in immune diseases. In the present study, the ATAC-seq analysis revealed 3491 differential open chromatin sites in the spleen of chicks with SIR induced by LPS challenge, and we presented the motifs on these sites and the associated transcription factors. The regulatory network was presented by combining the differential open chromatin data with the mRNAs and exploded cytokines. Interestingly, the LPS challenge could regulate the mRNA expression of 202 genes through chromatin reprogramming, including critical genes such as TLE1 and JUN, which regulate signaling pathways such as I-κB kinase/NF-κB, Toll-like receptor, and downstream cytokine genes. Furthermore, dietary daidzein could inhibit DNA topoisomerase II, which reprograms the spatial conformation of chromatin in the inflammatory response and attenuates SIR. In conclusion, we successfully identified key genes directly regulated by chromatin reprogramming in SIR and demonstrated the chromatin epigenome signatures and transcriptional regulatory network, which provides an important reference for further research on avian epigenetics. There is great potential for alleviating SIR using dietary daidzein.

Linc00673-V3 positively regulates autophagy by promoting Smad3-mediated LC3B transcription in NSCLC.

Since its first discovery, long noncoding RNA Linc00673 has been linked to carcinogenesis and metastasis of various human cancers. Linc00673 had five transcriptional isoforms and their biological functions remained to be explored. Here we have reported that Linc00673-V3, one of the isoforms of Linc00673, promoted non-small cell lung cancer chemoresistance, and increased Linc00673-V3 expression level was associated with enhanced autophagy. Mechanistically, we discerned the existence of a stem-loop configuration engendered by the 1-100-nt and 2200-2275-nt fragments within Linc00673-V3. This structure inherently interacted with Smad3, thereby impeding its ubiquitination and subsequent degradation orchestrated by E3 ligase STUB1. The accumulation of Smad3 contributed to autophagy via up-regulation of LC3B transcription and ultimately conferred chemoresistance in NSCLC. Our results revealed a novel transcriptional regulation network between Linc00673-V3, Smad3, and LC3B, which provided an important insight into the interplay between autophagy regulation and non-canonical function of Smad3. Furthermore, the results from in vivo experiments suggested Linc00673-V3 targeted antisense oligonucleotide as a promising therapeutic strategy to overcome chemotherapy resistance in NSCLC.

Genome-wide analysis of the KNOX gene family in Moso bamboo: insights into their role in promoting the rapid shoot growth

BackgroundKNOTTED1-like homeobox (KNOX) genes, plant-specific homologous box transcription factors (TFs), play a central role in regulating plant growth, development, organ formation, and response to biotic and abiotic stresses. However, a comprehensive genome-wide identification of the KNOX genes in Moso bamboo (Phyllostachys edulis), the fastest growing plant, has not yet been conducted, and the specific biological functions of this family remain unknown.ResultsThe expression profiles of 24 KNOX genes, divided into two subfamilies, were determined by integrating Moso bamboo genome and its transcriptional data. The KNOX gene promoters were found to contain several light and stress-related cis-acting elements. Synteny analysis revealed stronger similarity with rice KNOX genes than with Arabidopsis KNOX genes. Additionally, several conserved structural domains and motifs were identified in the KNOX proteins. The expansion of the KNOX gene family was primarily regulated by tandem duplications. Furthermore, the KNOX genes were responsive to naphthaleneacetic acid (NAA) and gibberellin (GA) hormones, exhibiting distinct temporal expression patterns in four different organs of Moso bamboo. Short Time-series Expression Miner (STEM) analysis and quantitative real-time PCR (qRT-PCR) assays demonstrated that PeKNOX genes may play a role in promoting rapid shoot growth. Additionally, Gene Ontology (GO) and Protein–Protein Interaction (PPI) network enrichment analyses revealed several functional annotations for PeKNOXs. By regulating downstream target genes, PeKNOXs are involved in the synthesis of AUX /IAA, ultimately affecting cell division and elongation.ConclusionsIn the present study, we identified and characterized a total of 24 KNOX genes in Moso bamboo and investigated their physiological properties and conserved structural domains. To understand their functional roles, we conducted an analysis of gene expression profiles using STEM and RNA-seq data. This analysis successfully revealed regulatory networks of the KNOX genes, involving both upstream and downstream genes. Furthermore, the KNOX genes are involved in the AUX/IAA metabolic pathway, which accelerates shoot growth by influencing downstream target genes. These results provide a theoretical foundation for studying the molecular mechanisms underlying the rapid growth and establish the groundwork for future research into the functions and transcriptional regulatory networks of the KNOX gene family.

Abstract 4883: NFE2L3 regulates the expression of BHLHE40 in triple-negative breast cancer

Abstract Introduction: Breast Cancer (BrCA) is one of the most common cancers, causing mortality in women worldwide. Among the subtypes of breast cancer, triple-negative breast cancer (TNBC) is the most aggressive due to the absence of three major receptors: estrogen receptor (ER), progesterone receptor (PR), and human epidermal growth factor receptor-2 (HER-2). Treatment options for TNBC are limited because of the lack of these molecular targets. We hypothesized that the identification and characterization of novel transcriptional regulatory relationships in TNBC will provide etiologic insights, enhance therapeutic outcomes, and improve patient survival. Overexpression of NFE2L3 in BrCA has inhibited cell proliferation and metastasis while overexpression of BHLHE40 has produced an inverse effect. This study aims to characterize the relationship between NFE2L3 and BHLHE40 in TNBC cells. Methods: Patient data from the two groups of our focus from the Cancer Genome Atlas BrCA RNAseq gene expression dataset were examined: triple-positive breast cancer (TPBC) and TNBC. Differentially expressed genes (DEGs) between these groups were identified using DESeq2. Pathways over-represented among the DEGs were determined using enrichR. For their impact on biological interactions, those DEGs were mapped to curate protein-protein interactions - the STRING database. Additionally, DEGs were superposed on two network inference algorithm-generated transcriptional regulatory networks (TRNs). Virtual Inference of protein activity by Enriched Regulon analysis was applied to the generated TRN to identify master regulators driving gene expression differences between TPBC and TNBC. Of the master regulators observed, the activity of NFE2L3 was explored in TNBC cells. The TRNs indicate that NFE2L3 regulates the expression of BHLHE40. This regulatory relationship was explored in TNBC cells by RNA interference, qRT-PCR, immunoblotting, and co-immunoprecipitation assays to investigate NFE2L3 and BHLHE40 gene and protein expression and interaction. Results: Among 12,000 DEGs (5% FDR), over-represented pathways included “mitotic signaling” and “G1/S transition”. Master regulators with higher expression in TNBC included NFE2L3, HMGA1, TP53, FOXC1, and SOX9; those with lower expression included FOXA1, BHLHE40, AR, XBP1, and ESR1. On silencing the cells of NFE2L3 and performing qRTPCR analysis, the expression of BHLHE40 was decreased in the TNBC cells. The protein-protein interaction between NFE2L3 and BHLHE40 was also validated. Conclusion: DEGs up-regulated in TNBC participate in cell cycle signaling. And there is a novel transcriptional regulatory relationship between NFE2L3 and BHLHE40 in TNBC cells. Citation Format: Shail Rakesh Modi, Terrick Andey, George Acquaah-Mensah. NFE2L3 regulates the expression of BHLHE40 in triple-negative breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4883.

Abstract 1691: p300 catalytic inhibition selectively targets IRF4 oncogenic activity in multiple myeloma

Abstract Multiple myeloma (MM), an incurable plasma cell malignancy, is addicted to oncogenic transcription factor (TF) IRF4 signaling. Lineage TFs like IRF4 are difficult to drug but represent high value oncology targets owing to the depth and context selectivity of their genetic dependency. Using a transcription regulatory network (TRN) based approach to integrate a high dimensional multi-platform dataset, we defined the key regulatory activity of IRF4 and its druggable co-factors. This unbiased analysis identified p300 lysine acetyl transferase (KAT) function as the nearest selective neighbor for therapeutic intervention, with more potent and selective effects than historically observed with p300/CBP bromodomain inhibitors. To further probe this biology, we developed a highly selective inhibitor of the KAT domain of p300 and its close homolog CBP. p300 KAT inhibition led to selective downregulation of IRF4 at the mRNA and protein level as well as reduction in acetylation at highly specific sites co-localized with IRF4, in contrast to more general agents like BET inhibitors. Gene expression reduction was most pronounced at IRF4/p300 co-bound genes, in particular IRF4 itself and MYC. IRF4 and p300 also directly bind to each other and other key MM nodes regulating them at the chromatin, mRNA, and protein levels. p300 KAT inhibition exhibits strong antiproliferative activity across in vitro MM models regardless of cytogenetics or resistance to other therapies. Moreover, this relationship is mediated by the KAT domain and not its acetyl-lysine recognizing bromodomain. Comparative pharmacology studies found that a p300 KAT inhibitor downregulated the IRF4 TRN and induced apoptosis in MM cells more effectively than existing standard of care agents, including IMiDs and glucocorticoid receptor agonists, or p300/CBP bromodomain inhibitors. Combinations of a p300 KAT inhibitor with clinically active molecules such as IMiDs and glucocorticoid agonists showed synergistic impacts on reducing key MM TFs and cell line viability. Ex vivo treatment of primary patient samples demonstrated broad anti-proliferative activity with loss of IRF4 and induction of apoptosis in relapsed-refractory MM patient samples, independent of prior lines of therapy. Importantly, the induction of MM cell apoptosis occurs in the milieu of CD138 negative bone marrow stromal cells while also sparing them from toxicity, supporting a potential therapeutic index. Finally, p300 KAT inhibition demonstrated activity in in vivo MM models with robust tumor growth inhibition at tolerated doses and exposures correlated to reduced p300-dependent substrate acetylation. IRF4 dependency is a hallmark of MM that has been challenging to target with existing therapies, motivating further development of p300 KAT targeting therapeutics. Citation Format: Michael R. McKeown, Giulia Giorgetti, Walter F. Lenoir, Marek J. Kobylarz, Tamara D. Hopkins, Wayne L. Glore, Michelle G. Shum, Yare Calderon, Kameron R. Mori, Hua Gao, Luis A. Carvajal, Nikolaus D. Obholzer, Benjamin W. Trotter, Christopher D. Dinsmore, Nikhil C. Munshi, Peter B. Rahl, Mariateresa Fulciniti, Charles Y. Lin. p300 catalytic inhibition selectively targets IRF4 oncogenic activity in multiple myeloma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1691.

Abstract 6883: Investigation of tumor-immune interactions in pediatric sarcomas using single-cell multi-omics

Abstract Background: Pediatric sarcoma represents the second most common solid tumor in children. Despite advances in multimodal therapies, one-third of sarcoma patients do not survive the disease. Over the past few decades, the development of novel therapeutic strategies for sarcoma has been impeded by an insufficient understanding of the tumor microenvironment (TME). In particular, the potential of immunotherapy remains constrained by the gap of knowledge about the molecular and functional phenotypes of the immune cells in the sarcoma TME. Methods: To address this knowledge gap, we collected treatment-naïve tumor samples from 32 patients with five pediatric sarcoma subtypes. We employed a single-nuclei RNA+ATAC-Seq multi-omics assay to comprehensively characterize the transcriptomic and epigenomic heterogeneity of tumor and immune cells within the sarcoma TME. Results: Our analysis revealed that tumor-associated macrophages (TAMs) were the most abundant immune cell type in the sarcoma TME. We identified seven distinct TAM subpopulations, each exhibiting unique gene expression and chromatin accessibility patterns. Among these, TAM subsets with higher expression of SPP1 and C1QC genes displayed the strongest immunosuppressive potential. Transcriptional regulatory network (TRN) analysis identified transcription factors that were most likely to drive the phenotypes of SPP1+ and C1QC+ TAMs, including ELF2, JUND and RUNX1. Through cell-cell communication analysis, we discovered that SPP1+ TAMs interact with T cells via SPP1-CD44 signaling, while C1QC+ TAMs interact with NK cells through HLA-E-KLRC1 signaling, suggesting the potential involvement of these signaling pathways in TAM-mediated suppression of these cytotoxic lymphocytes. We prioritized ligand-receptor gene pairs, such as SEMA3D-NRP1 and LTBP3-ITGB5, which likely mediate the interaction between malignant sarcoma cells and SPP1+/C1QC+ TAMs based on their dominance across sarcoma subtypes and association with poorer clinical outcomes. On the other hand, our analysis revealed that sarcoma subtype-specific interactions between TAMs and malignant cells are common, highlighting the heterogeneity of signaling events across sarcoma subtypes. Conclusion and Significance: Our study presents a comprehensive single-cell multi-omics characterization of pediatric sarcomas and sheds light on its tumor microenvironment. By identifying unique markers for immunosuppressive TAM subpopulations and elucidating intercellular interactions promoting tumor development, this study proposes potential therapeutic targets for sarcoma treatment based on the role of TAMs in sarcoma TME. Citation Format: Zhan Zhang, Kyung Jin Ahn, Changya Chen, Rumeysa Biyik Sit, Hesham Mohei, Brian Lockhart, Malay Haldar, Kathrin Bernt, Vinodh Pillai, Kai Tan. Investigation of tumor-immune interactions in pediatric sarcomas using single-cell multi-omics [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6883.

Abstract 5636: CUX1 transcriptionally regulates intestinal epithelial homeostasis

Abstract The small intestine digests and absorbs nutrients, a role made possible by specialized secretory and absorptive cells that constantly replenish from the intestinal stem cell niche. Disruption in intestinal epithelium homeostasis has been linked to pathologies including inflammatory bowel disease (IBD) and cancer. However, the processes regulating differentiation in healthy and disease setting are incompletely understood. CUX1 is a highly conserved homeodomain-containing transcription factor that is expressed in small intestinal crypts and has been previously shown to be essential in mice and Drosophila. CUX1 is upregulated upon inflammatory stress conditions, including intestinal damage and in IBD. In addition, CUX1 is predicted to be protective against IBD from genome-wide association studies. The aim of the current work is to determine the mechanistic role of CUX1 in secretory intestinal cells differentiation and epithelial homeostasis. To this end, we have generated transgenic mouse models that modulate CUX1 levels in an inducible manner. Mice with near-null CUX1 levels exhibit rapid weight loss requiring euthanasia while mice with ~50% residual CUX1 halt normal weight gain. Histologic examination of tissues from mice with CUX1 knock-down reveals a lack of mature secretory cells, including goblet and Paneth cells, the latter playing a supportive role to intestinal stem cells. Moreover, mouse intestinal tissues from our knock-down models demonstrated decreased proliferation with increased apoptosis. In transcriptome profiling, we observed downregulation of proliferation, intestinal stem cell, and digestive system developmental gene signatures and decreased levels of key secretory cell regulators, pointing towards a role for CUX1 in stem cell maintenance and differentiation. Indeed, reducing CUX1 levels decreased β-catenin protein levels, while Wnt/β-catenin signaling is required for the organization of the crypt-villus axis. Finally, CUX1 knock-down compromises the ability to form budding organoids, demonstrating the cell intrinsic role for CUX1 in stem cell differentiation. In ongoing studies, we are identifying CUX1 genome-wide binding targets and the epigenetic impact of CUX1 in intestinal epithelium cells. Our findings indicate that CUX1 is a critical regulator of intestinal epithelium homeostasis that has a role in differentiation and potential role in injury-induced de-differentiation in healthy as well as IBD patients. We propose that CUX1 regulates stem cell maintenance and crypt composition by regulating Wnt/β-catenin signaling and inducing fetal-like transcriptomic repair program shown to be coopted by colon cancers. Resolving an intricate network of transcriptional regulation of differentiation in the intestinal crypt holds the potential to successfully target inflammation-related damage in human intestines. Citation Format: Katarzyna Zawieracz, Ningfei An, Megan McNerney. CUX1 transcriptionally regulates intestinal epithelial homeostasis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5636.

Hi-C, a chromatin 3D structure technique advancing the functional genomics of immune cells.

The functional performance of immune cells relies on a complex transcriptional regulatory network. The three-dimensional structure of chromatin can affect chromatin status and gene expression patterns, and plays an important regulatory role in gene transcription. Currently available techniques for studying chromatin spatial structure include chromatin conformation capture techniques and their derivatives, chromatin accessibility sequencing techniques, and others. Additionally, the recently emerged deep learning technology can be utilized as a tool to enhance the analysis of data. In this review, we elucidate the definition and significance of the three-dimensional chromatin structure, summarize the technologies available for studying it, and describe the research progress on the chromatin spatial structure of dendritic cells, macrophages, T cells, B cells, and neutrophils.

Abstract 6236: Systems biology modeling identifies SMARCD3 as a master regulator facilitating everolimus resistance in ER+ breast cancer

Abstract Breast cancer is the most common malignancy in women and estrogen receptor positive (ER+) breast cancers represent nearly 75% of all breast tumors. Everolimus, an mTORC1 inhibitor, in combination with exemestane has been approved for patients with metastatic ER+ breast cancer. However, many patients eventually develop resistance to everolimus and leads to poor survival outcomes. Characterizing everolimus-resistant cells can reveal targetable molecular phenotypes that can eventually lead to new therapeutic targets. In this study, we elucidate systems-level phenotypic distinctions between everolimus sensitive and refractory cells. We generated several isogenic ER+ breast cancer cell lines resistant or sensitive to everolimus. We then leveraged transcriptomic profiles from sensitive and resistant cells before or after everolimus treatment to decipher the underlying mechanisms essential to the resistant state. Using linear mixed effect models, we identified several meta-phenotypes that were distinct between sensitive and resistant cell states. These included growth-factor signaling, cell cycle, metabolism, stemness, and apoptosis meta-phenotypes. Our analyses also identified strong activation of growth-factor receptors including IGF1R/INSR and ESR1 in the resistant cells, which were maintained despite everolimus treatment. Next, we adopted a transcriptional regulatory network reconstruction framework to identify the master regulators responsible for the activation of resistance meta-phenotypes. We identified SMARCD3 as the key master regulator controlling expression of genes across diverse resistance meta-phenotypes, including growth-factor signaling. Further, using transcriptomic profiles from a neoadjuvant trial of 23 post-menopausal ER+ breast cancers treated with everolimus, we found that SMARCD3 expression was elevated in resistant tumors. To confirm the role of SMARCD3 in everolimus resistance, we repressed transcription of the SMARCD3 gene using CRISPR interference to re-sensitize resistant cells to everolimus treatment. In conclusion, we used context-specific cell transcriptomic profiles and integrative systems-biology approaches to elucidate mechanisms of resistance in response to everolimus treatment in ER+ breast cancer. Our findings suggest SMARCD3 upregulation promotes resistance to everolimus treatment and may serve as a novel therapeutic target in ER+ breast cancer. Citation Format: Eric F. Medina, Patrick A. Cosgrove, Eleni Farmaki, Vince K. Grolmusz, Feng Chi, Jason I. Griffiths, Andrea H. Bild, Aritro Nath. Systems biology modeling identifies SMARCD3 as a master regulator facilitating everolimus resistance in ER+ breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6236.

PTGS2: A potential immune regulator and therapeutic target for chronic spontaneous urticaria

AimsChronic spontaneous urticaria (CSU) is a common and debilitating skin disease that is difficult to control with existing treatments, and the pathogenesis of CSU has not been fully revealed. The aim of this study was to explore the underlying mechanisms of CSU and identify potential treatments. Materials and methodsMicroarray datasets of CSU were obtained from Gene Expression Omnibus database. Differentially expressed genes between skin lesions of CSU and normal controls (LNS-DEGs) were identified, and the enrichment analyses of LNS-DEGs were performed. Hub genes of LNS-DEGs were selected by protein–protein interaction analysis. The co-expression and transcriptional regulatory networks of hub genes were conducted using GeneMANIA and TRRUST database, respectively. CIBERSORT was utilized for immune cell infiltration analysis. Experimental validation was performed by β-hexosaminidase release examination and passive cutaneous anaphylaxis (PCA) mouse model. Key findingsA total of 247 LNS-DEGs were identified, which were enriched in cell migration, cell chemotaxis, and inflammatory pathways such as TNF and interleukin (IL) -17 signaling pathway. Among LNS-DEGs, seven upregulated (PTGS2, CCL2, IL1B, CXCL1, IL6, VCAM1, ICAM1) and one downregulated hub gene (PECAM1) were selected. Immune infiltration analysis identified eight different immune cells, such as activated/resting mast cells and neutrophils. Furthermore, PTGS2, encoding cyclooxygenase 2 (COX2), was selected for further validation. COX2 inhibitor, celecoxib, significantly inhibited mast cell degranulation, and reduced vascular permeability and inflammatory cytokine expression in PCA mouse model. SignificancePTGS2 may be a potential regulator of immunity and inflammation in CSU. Targeting PTGS2 is a new perspective for CSU treatment.

Whole-transcriptome profiling and identification of cold tolerance-related ceRNA networks in japonica rice varieties.

Low-temperature stress negatively impacts rice yield, posing a significant risk to food security. While previous studies have explored the physiological and linear gene expression alterations in rice under low-temperature conditions, the changes in competing endogenous RNA (ceRNA) networks remain largely unexamined. We conducted RNA sequencing on two japonica rice varieties with differing cold-tolerance capabilities to establish ceRNA networks. This enabled us to investigate the transcriptional regulatory network and molecular mechanisms that rice employs in response to low-temperature stress. We identified 364 differentially expressed circular RNAs (circRNAs), 224 differentially expressed microRNAs (miRNAs), and 12,183 differentially expressed messenger RNAs (mRNAs). WRKY family was the most prominent transcription factor family involved in cold tolerance. Based on the expression patterns and targeted relationships of these differentially expressed RNAs, we discerned five potential ceRNA networks related to low-temperature stress in rice: osa-miR166j-5p from the miR166 family was associated with cold tolerance; osa-miR528-3p and osa-miR156j-3p were linked to stress response; and osa-miR156j-3p was involved in the antioxidant system. In addition, Os03g0152000 in the antioxidant system, as well as Os12g0491800 and Os05g0381400, correlated with the corresponding stress response and circRNAs in the network. A gene sequence difference analysis and phenotypic validation of Os11g0685700 (OsWRKY61) within the WRKY family suggested its potential role in regulating cold tolerance in rice. We identified Os11g0685700 (OsWRKY61) as a promising candidate gene for enhancing cold tolerance in japonica rice. The candidate miRNAs, mRNAs, and circRNAs uncovered in this study are valuable targets for researchers and breeders. Our findings will facilitate the development of cold-tolerant rice varieties from multiple angles and provide critical directions for future research into the functions of cold-tolerance-related miRNAs, mRNAs, and circRNAs in rice.

RNA cis-regulators are important for Streptococcus pneumoniae in vivo success.

Bacteria have evolved complex transcriptional regulatory networks, as well as many diverse regulatory strategies at the RNA level, to enable more efficient use of metabolic resources and a rapid response to changing conditions. However, most RNA-based regulatory mechanisms are not well conserved across different bacterial species despite controlling genes important for virulence or essential biosynthetic processes. Here, we characterize the activity of, and assess the fitness benefit conferred by, twelve cis-acting regulatory RNAs (including several riboswitches and a T-box), in the opportunistic pathogen Streptococcus pneumoniae TIGR4. By evaluating native locus mutants of each regulator that result in constitutively active or repressed expression, we establish that growth defects in planktonic culture are associated with constitutive repression of gene expression, while constitutive activation of gene expression is rarely deleterious. In contrast, in mouse nasal carriage and pneumonia models, strains with either constitutively active and repressed gene expression are significantly less fit than matched control strains. Furthermore, two RNA-regulated pathways, FMN synthesis/transport and pyrimidine synthesis/transport display exceptional sensitivity to mis-regulation or constitutive gene repression in both planktonic culture and in vivo environments. Thus, despite lack of obvious phenotypes associated with constitutive gene expression in vitro, the fitness benefit conferred on bacteria via fine-tuned metabolic regulation through cis-acting regulatory RNAs is substantial in vivo, and therefore easily sufficient to drive the evolution and maintenance of diverse RNA regulatory mechanisms.

Flexible gold standards for transcription factor regulatory interactions in Escherichia coli K-12: architecture of evidence types.

Post-genomic implementations have expanded the experimental strategies to identify elements involved in the regulation of transcription initiation. Here, we present for the first time a detailed analysis of the sources of knowledge supporting the collection of transcriptional regulatory interactions (RIs) of Escherichia coli K-12. An RI groups the transcription factor, its effect (positive or negative) and the regulated target, a promoter, a gene or transcription unit. We improved the evidence codes so that specific methods are incorporated and classified into independent groups. On this basis we updated the computation of confidence levels, weak, strong, or confirmed, for the collection of RIs. These updates enabled us to map the RI set to the current collection of HT TF-binding datasets from ChIP-seq, ChIP-exo, gSELEX and DAP-seq in RegulonDB, enriching in this way the evidence of close to one-quarter (1329) of RIs from the current total 5446 RIs. Based on the new computational capabilities of our improved annotation of evidence sources, we can now analyze the internal architecture of evidence, their categories (experimental, classical, HT, computational), and confidence levels. This is how we know that the joint contribution of HT and computational methods increase the overall fraction of reliable RIs (the sum of confirmed and strong evidence) from 49% to 71%. Thus, the current collection has 3912 reliable RIs, with 2718 or 70% of them with classical evidence which can be used to benchmark novel HT methods. Users can selectively exclude the method they want to benchmark, or keep for instance only the confirmed interactions. The recovery of regulatory sites in RegulonDB by the different HT methods ranges between 33% by ChIP-exo to 76% by ChIP-seq although as discussed, many potential confounding factors limit their interpretation. The collection of improvements reported here provides a solid foundation to incorporate new methods and data, and to further integrate the diverse sources of knowledge of the different components of the transcriptional regulatory network. There is no other genomic database that offers this comprehensive high-quality architecture of knowledge supporting a corpus of transcriptional regulatory interactions.

Key role of hormone signal transduction and lipid metabolism in the development of Nitraria sibirica leaves: An integrated metabolomic and transcriptomic analysis

Plant growth is a complex process controlled by a variety of genetic and environmental variables. Nitraria sibirica Pall. is a multi-purpose euhalophyte with economic and medicinal value. However, the molecular mechanisms underlying the changes at different growth stages in N. sibirica remain unreported. In this study, the morphology and physiological characteristics of N. sibirica were compared at the vegetative, full flowering, and fruiting stages. Metabolomics and transcriptomics were used to describe metabolite accumulation and transcriptome expression at different stages, and the relationship between gene expression patterns and metabolite accumulation was studied based on a joint analysis of the two approaches. The results indicated significant variations in the morphological and physiological characteristics of N. sibirica at different growth stages (P < 0.05). Transcriptome and metabolome samples corresponding to the three stages were clearly distinguished, and the metabolite and gene module showed high consistency. The amount of DEMs and DEGs decreased as leaf growth progressed. Differentially expressed genes (DEGs) predominantly belonged to the MYB transcription family, whereas differentially expressed metabolites (DEMs) were dominated by lipids and lipid-like molecules. DEGs were mainly enriched in plant hormone signal transduction, cutin, suberine, and wax biosynthesis pathways, whereas DEMs were mainly enriched in linoleic acid, alpha-linolenic acid metabolism, and phenylpropane biosynthesis pathways. Combined transcriptomic and metabolomic analyses revealed that plant hormone signal transduction, linoleic acid, alpha-linolenic acid, cutin, suberine, and wax biosynthesis were the most significant key pathways for the co-enrichment of DEMs and DEGs. Pearson correlation analysis showed that physiological traits were strongly correlated with DEGs, whereas morphological traits were strongly correlated with DEMs. Moreover, a strong correlation was also observed between DEMs and DEGs. In conclusion, this research provides insights into the metabolic and transcriptional regulatory networks involved in plant development.