Transcriptional Regulation In Response Research Articles

Reconstruction and computer analysis of the structural and functional organization of the gene network regulating cholesterol biosynthesis in humans and the evolutionary characteristics of the genes involved in the network

Cholesterol is an essential structural component of cell membranes and a precursor of vitamin D, as well as steroid hormones. Humans and other animal species can absorb cholesterol from food. Cholesterol is also syn­thesized de novo in the cells of many tissues. We have previously reconstructed the gene network regulating intra­cellular cholesterol levels, which included regulatory circuits involving transcription factors from the SREBP (Sterol Regulatory Element-Binding Proteins) subfamily. The activity of SREBP transcription factors is regulated inversely depending on the intracellular cholesterol level. This mechanism is implemented with the participation of proteins SCAP, INSIG1, INSIG2, MBTPS1/S1P and MBTPS2/S2P. This group of proteins, together with the SREBP factors, is designated as “cholesterol sensor”. An elevated cholesterol level is a risk factor for the development of cardiovas­cular diseases and may also be observed in obesity, diabetes and other pathological conditions. Systematization of information about the molecular mechanisms controlling the activity of SREBP factors and cholesterol biosyn­thesis in the form of a gene network and building new knowledge about the gene network as a single object is extremely important for understanding the molecular mechanisms underlying the predisposition to diseases. With a computer tool, ANDSystem, we have built a gene network regulating cholesterol biosynthesis. The gene network included data on: (1) the complete set of enzymes involved in cholesterol biosynthesis; (2) proteins that function as part of the “cholesterol sensor”; (3) proteins that regulate the activity of the “cholesterol sensor”; (4) genes encod­ing proteins of these groups; (5) genes whose transcription is regulated by SREBP factors (SREBP target genes). The gene network was analyzed and feedback loops that control the activity of SREBP factors were identified. These feedback loops involved the PPARG, NR0B2/SHP1, LPIN1, and AR genes and the proteins they encode. Analysis of the phylostratigraphic age of the genes showed that the ancestral forms of most human genes encoding the enzymes of cholesterol biosynthesis and the proteins of the “cholesterol sensor” may have arisen at early evolutionary stages (Cellular organisms (the root of the phylostratigraphic tree) and the stages of Eukaryota and Metazoa divergence). However, the mechanism of gene transcription regulation in response to changes in cholesterol levels may only have formed at later evolutionary stages, since the phylostratigraphic age of the genes encoding the transcription factors SREBP1 and SREBP2 corresponds to the stage of Vertebrata divergence.

The Histone Lysine Demethylase KDM7A Contributes to Reward Memory via Fscn1-Induced Synaptic Plasticity in the Medial Prefrontal Cortex.

Lysine demethylase 7A (KDM7A) catalyzes the removal of dimethylation from histone H3 lysine 9 and lysine 27, both of which are associated with transcription repression. Previous study indicates that Kdm7a mRNA in the medial prefrontal cortex (mPFC) increases after drug exposure, yet its role in drug-related behaviors is largely unknown. In a morphine-conditioned place preference (CPP) paradigm, these findings reveal a specific increase of Kdm7a expression in the mPFC 7 days after drug withdrawal. Subsequently, these results demonstrate that knockdown of Kdm7a in the mPFC do not affect the acquisition of morphine-induced CPP, but it attenuate memory consolidation. To further explore Kdm7a-mediated transcriptomic changes, this work employs Nanopore direct RNA sequencing. Transcriptome profiling unveils several gene expression alterations impacted by KDM7A, which are enriched in relevant neural function categories. Notably, this work identifies and validates fascin actin-bundling protein 1 (Fscn1) as a downstream molecular target. Knockdown of Fscn1 has a similar impact on CPP to Kdm7a, along with corresponding decrease of dendritic spine density and neuronal activity in the mPFC. Additionally, silencing Kdm7a decreases enrichment of H3K9me2 and H3K27me2 at the Fscn1 promoter region, suggesting that KDM7A may act as a crucial regulator of transcriptional responses to morphine-related reward memory via Fscn1.

Transcription factors PHR1 and PHR1-like 1 regulate ABA-mediated inhibition of seed germination and stomatal opening in Arabidopsis.

Low phosphate (LP) availability significantly impacts crop yield and quality. PHOSPHATE STARVATION RESPONSE1 (PHR1) along with PHR1-like 1 (PHL1) act as a key transcriptional regulator in a plant's adaptive response to LP conditions. Abscisic acid (ABA) plays an important role in how plants respond to environmental stresses like salinity and drought. However, the involvement of PHR1 and PHL1 in ABA response and signalling mechanisms remains to be fully understood. Our findings reveal that PHR1 and PHR1/PHL1 knockout mutations enhance the responsiveness of seed germination, early seedling growth, and stomatal opening to ABA in Arabidopsis. Furthermore, these mutations increase sensitivity to combined LP and ABA stress. In contrast, overexpression of PHR1 or PHL1 reduces this sensitivity in Arabidopsis. Knockout mutations of PHR1 and PHR1/PHL1 also increase sensitivity to salt and osmotic stresses, as well as to combined LP and salinity/osmotic stress, while overexpression of PHR1 or PHL1 reduces their sensitivity in seed germination and early seedling development. Knockout mutations of SPX1 and SPX2, negative regulators of PHR1 and PHL1, decrease sensitivity to ABA and salt/osmotic stresses in Arabidopsis. A group of genes related to ABA metabolism and signalling is significantly affected by the knockout or overexpression of PHR1 and PHL1, with a large proportion of these genes containing PHR1 binding site (P1BS) in their promoters. Moreover, the ABA-sensitive phenotype of phr1 or phr1 phl1 mutants can be rescued by PHR1 homologs from chlorophyte algae, liverwort and rice, suggesting their conserved roles in ABA signalling. These results indicate that PHR1 and its homologs negatively regulate plant responses to ABA in seed germination and stomatal aperture. This study provides new insights into the interplay between Pi homeostasis, abiotic stress and ABA signaling. Moderately increasing the expression ofPHR1or its homologs in crops could be a potential strategy to enhance plant resistance to combined LP and osmotic stress.

Tissue-specific chromatin accessibility and transcriptional regulation in maize cold stress response.

Maize, a vital crop globally, faces significant yield losses due to its sensitivity to cold stress, especially in temperate regions. Understanding the molecular mechanisms governing maize response to cold stress is crucial for developing strategies to enhance cold tolerance. However, the precise chromatin-level regulatory mechanisms involved remain largely unknown. In this study, we employed DNase-seq and RNA-seq techniques to investigate chromatin accessibility and gene expression changes in maize root, stem, and leaf tissues subjected to cold treatment. We discovered widespread changes in chromatin accessibility and gene expression across these tissues, with strong tissue specificity. Cold stress-induced DNase I hypersensitive sites (coiDHSs) were associated with differentially expressed genes, suggesting a direct link between chromatin accessibility and gene regulation under cold stress. Motif enrichment analysis identified ERF transcription factors (TFs) as central regulators conserved across tissues, with ERF5 emerging as pivotal in the cold response regulatory network. Additionally, TF co-localization analysis highlighted six TF pairs (ERF115-SHN3, ERF9-LEP, ERF7-SHN3, LEP-SHN3, LOB-SHN3, and AS2-LOB) conserved across tissues but showing tissue-specific binding preferences. These findings indicate intricate regulatory networks in maize cold response. Overall, our study provides insights into the chromatin-level regulatory mechanisms underpinning maize adaptive response to cold stress, offering potential targets for enhancing cold tolerance in agricultural contexts.

Comparative Genomics and Epigenomics of Transcriptional Regulation.

Transcriptional regulation in response to diverse physiological cues involves complicated biological processes. Recent initiatives that leverage whole genome sequencing and annotation of regulatory elements significantly contribute to our understanding of transcriptional gene regulation. Advances in the data sets available for comparative genomics and epigenomics can identify evolutionarily constrained regulatory variants and shed light on noncoding elements that influence transcription in different tissues and developmental stages across species. Most epigenomic data, however, are generated from healthy subjects at specific developmental stages. To bridge the genotype-phenotype gap, future research should focus on generating multidimensional epigenomic data under diverse physiological conditions. Farm animal species offer advantages in terms of feasibility, cost, and experimental design for such integrative analyses in comparison to humans. Deep learning modeling and cutting-edge technologies in sequencing and functional screening and validation also provide great promise for better understanding transcriptional regulation in this dynamic field.

Structural basis of transcriptional regulation by UrtR in response to uric acid.

Uric acid (UA)-responsive transcriptional regulators (UrtRs), which belong to the multiple antibiotic resistance regulator (MarR) superfamily, transcriptionally coordinate virulence and metabolism in bacteria by modulating interactions with operator DNA in response to UA. To elucidate the transcriptional regulatory mechanism of UrtR, we structurally analyzed UrtR proteins, including PecS, MftR, and HucR, alone and in complex with UA or DNA. UrtR contains a dimerization domain (DD) and a winged helix-turn-helix domain (wHTHD) and forms a homodimer primarily via the DD, as observed for other MarR superfamily proteins. However, UrtRs are characterized by a unique N-terminal α-helix, which contributes to dimerization and UA recognition. In the absence of UA, the UrtR dimer symmetrically binds to the operator double-stranded DNA (dsDNA) by inserting its α4 recognition helix and β-stranded wing within the wHTHD into the major and minor grooves of dsDNA, respectively. Upon exposure to UA, UrtR accommodates UA in the intersubunit pocket between the DD and wHTHD. UA binding induces a conformational change in the major groove-binding core element of the UrtR wHTHD, generating a DNA binding-incompatible structure. This local allosteric mechanism of UrtR completely differs from that generally observed in other MarR superfamily members, in which the entire wHTHD undergoes effector-responsive global shifts.

Integrated transcriptional and biochemical profiling suggests mechanisms associated with rapid cold hardening in adult Liriomyza trifolii (Burgess)

The leafminer Liriomyza trifolii causes severe economic damage on ornamental and horticultural crops in China. Rapid cold hardening (RCH) is a phenomenon where cold tolerance in insects can be significantly enhanced after a short-term acclimation to low temperatures. In this study, the regulation of transcription in response to cold hardening was investigated in L. trifolii adults, and fatty acids and cryoprotectant levels were measured. The composition of fatty acids changed after RCH treatment, and glucose and trehalose levels showed significant accumulation after acclimation, thus indicating that changes in fatty acids and cryoprotectants contribute to RCH in L. trifolii. RNA-seq was used to analyze transcriptional regulation after a 4 h hardening period and showed that differentially expressed genes clustered in multiple metabolic pathways, which indicates the importance of transcriptional regulation in RCH. This study expands our knowledge of biochemical and transcriptional changes in L. trifolii during cold hardening and provides a basis for further investigations aimed at understanding thermal adaptation in insects.

Transcriptomic Identification of Potential C2H2 Zinc Finger Protein Transcription Factors in Pinus massoniana in Response to Biotic and Abiotic Stresses.

Biotic and abiotic stresses have already seriously restricted the growth and development of Pinus massoniana, thereby influencing the quality and yield of its wood and turpentine. Recent studies have shown that C2H2 zinc finger protein transcription factors play an important role in biotic and abiotic stress response. However, the members and expression patterns of C2H2 TFs in response to stresses in P. massoniana have not been performed. In this paper, 57 C2H2 zinc finger proteins of P. massoniana were identified and divided into five subgroups according to a phylogenetic analysis. In addition, six Q-type PmC2H2-ZFPs containing the plant-specific motif 'QALGGH' were selected for further study under different stresses. The findings demonstrated that PmC2H2-ZFPs exhibit responsiveness towards various abiotic stresses, including drought, NaCl, ABA, PEG, H2O2, etc., as well as biotic stress caused by the pine wood nematode. In addition, PmC2H2-4 and PmC2H2-20 were nuclear localization proteins, and PmC2H2-20 was a transcriptional activator. PmC2H2-20 was selected as a potential transcriptional regulator in response to various stresses in P. massoniana. These findings laid a foundation for further study on the role of PmC2H2-ZFPs in stress tolerance.

Physical cell-cell contact elicits specific transcriptomic responses in wine yeast species.

Fermenting grape juice provides a habitat for a well-mapped and evolutionarily relevant microbial ecosystem consisting of many natural or inoculated strains of yeasts and bacteria. The molecular nature of many of the ecological interactions within this ecosystem remains poorly understood, with the partial exception of interactions of a metabolic nature such as competition for nutrients and production of toxic metabolites/peptides. Data suggest that physical contact between species plays a significant role in the phenotypic outcome of interspecies interactions. However, the molecular nature of the mechanisms regulating these phenotypes remains unknown. Here, we present a transcriptomic analysis of physical versus metabolic contact between two wine relevant yeast species, Saccharomyces cerevisiae and Lachancea thermotolerans. The data show that these species respond to the physical presence of the other species. In S. cerevisiae, physical contact results in the upregulation of genes involved in maintaining cell wall integrity, cell wall structural components, and genes involved in the production of H2S. In L. thermotolerans, HSP stress response genes were the most significantly upregulated gene family. Both yeasts downregulated genes belonging to the FLO family, some of which play prominent roles in cellular adhesion. qPCR analysis indicates that the expression of some of these genes is regulated in a species-specific manner, suggesting that yeasts adjust gene expression to specific biotic challenges or interspecies interactions. These findings provide fundamental insights into yeast interactions and evolutionary adaptations of these species to the wine ecosystem.IMPORTANCEWithin the wine ecosystem, yeasts are the most relevant contributors to alcoholic fermentation and wine organoleptic characteristics. While some studies have described yeast-yeast interactions during alcoholic fermentation, such interactions remain ill-defined, and little is understood regarding the molecular mechanisms behind many of the phenotypes observed when two or more species are co-cultured. In particular, no study has investigated transcriptional regulation in response to physical interspecies cell-cell contact, as opposed to the generally better understood/characterized metabolic interactions. These data are of direct relevance to our understanding of microbial ecological interactions in general while also creating opportunities to improve ecosystem-based biotechnological applications such as wine fermentation. Furthermore, the presence of competitor species has rarely been considered an evolutionary biotic selection pressure. In this context, the data reveal novel gene functions. This, and further such analysis, is likely to significantly enlarge the genome annotation space.

High expression of 3-hydroxy-3-methylglutaryl-CoA synthase 2 (HMGCS2) associated with Diquat-induced damage

Diquat (DQ) is a commonly used bipyridine herbicide known for its toxic properties and adverse effects on individuals. However, the mechanism underlying DQ-induced damage remain elusive. Our research aimed to uncover the regulatory network involved in DQ-induced damage. We analyzed publicly accessible gene expression patterns and performed research using a DQ-induced damage animal model. The GSE153959 dataset from the Gene Expression Omnibus collection and the animal model of DQ-induced kidney injury were used to identify differentially expressed genes (DEGs). Pathways including the regulation of DNA-templated transcription in response to stress, RNA polymerase II transcription regulator complex and transcription coregulatory activity were shown to be enriched in 21 DEGs. We used least absolute shrinkage and selection operator (LASSO) regression analysis to find possible diagnostic biomarkers for DQ-induced damage. Then, we used an HK-2 cell model to confirm these results. Additionally, we confirmed that 3-hydroxy-3-methylglutaryl-CoA synthase 2 (HMGCS2) was the major gene associated with DQ-induced damage using multi-omics screening. The sample validation strongly suggested that HMGCS2 has promise as a diagnostic marker and may provide new targets for therapy in the context of DQ-induced damage.

Functional analysis of the Aspergillus fumigatus kinome identifies a druggable DYRK kinase that regulates septal plugging

More than 10 million people suffer from lung diseases caused by the pathogenic fungus Aspergillus fumigatus. Azole antifungals represent first-line therapeutics for most of these infections but resistance is rising, therefore the identification of antifungal targets whose inhibition synergises with the azoles could improve therapeutic outcomes. Here, we generate a library of 111 genetically barcoded null mutants of Aspergillus fumigatus in genes encoding protein kinases, and show that loss of function of kinase YakA results in hypersensitivity to the azoles and reduced pathogenicity. YakA is an orthologue of Candida albicans Yak1, a TOR signalling pathway kinase involved in modulation of stress responsive transcriptional regulators. We show that YakA has been repurposed in A. fumigatus to regulate blocking of the septal pore upon exposure to stress. Loss of YakA function reduces the ability of A. fumigatus to penetrate solid media and to grow in mouse lung tissue. We also show that 1-ethoxycarbonyl-beta-carboline (1-ECBC), a compound previously shown to inhibit C. albicans Yak1, prevents stress-mediated septal spore blocking and synergises with the azoles to inhibit A. fumigatus growth.

D-SPIN constructs gene regulatory network models from multiplexed scRNA-seq data revealing organizing principles of cellular perturbation response.

Gene regulatory networks within cells modulate the expression of the genome in response to signals and changing environmental conditions. Reconstructions of gene regulatory networks can reveal the information processing and control principles used by cells to maintain homeostasis and execute cell-state transitions. Here, we introduce a computational framework, D-SPIN, that generates quantitative models of gene regulatory networks from single-cell mRNA-seq datasets collected across thousands of distinct perturbation conditions. D-SPIN models the cell as a collection of interacting gene-expression programs, and constructs a probabilistic model to infer regulatory interactions between gene-expression programs and external perturbations. Using large Perturb-seq and drug-response datasets, we demonstrate that D-SPIN models reveal the organization of cellular pathways, sub-functions of macromolecular complexes, and the logic of cellular regulation of transcription, translation, metabolism, and protein degradation in response to gene knockdown perturbations. D-SPIN can also be applied to dissect drug response mechanisms in heterogeneous cell populations, elucidating how combinations of immunomodulatory drugs can induce novel cell states through additive recruitment of gene expression programs. D-SPIN provides a computational framework for constructing interpretable models of gene-regulatory networks to reveal principles of cellular information processing and physiological control.

Regulation of eukaryotic transcription initiation in response to cellular stress

Transcription initiation is a vital step in the regulation of eukaryotic gene expression. It can be dysregulated in response to various cellular stressors which is associated with numerous human diseases including cancer. Transcription initiation is facilitated via many gene-specific trans-regulatory elements such as transcription factors, activators, and coactivators through their interactions with transcription pre-initiation complex (PIC). These trans-regulatory elements can uniquely facilitate PIC formation (hence, transcription initiation) in response to cellular nutrient stress. Cellular nutrient stress also regulates the activity of other pathways such as target of rapamycin (TOR) pathway. TOR pathway exhibits distinct regulatory mechanisms of transcriptional activation in response to stress. Like TOR pathway, the cell cycle regulatory pathway is also found to be linked to transcriptional regulation in response to cellular stress. Several transcription factors such as p53, C/EBP Homologous Protein (CHOP), activating transcription factor 6 (ATF6α), E2F, transforming growth factor (TGF)-β, Adenomatous polyposis coli (APC), SMAD, and MYC have been implicated in regulation of transcription of target genes involved in cell cycle progression, apoptosis, and DNA damage repair pathways. Additionally, cellular metabolic and oxidative stressors have been found to regulate the activity of long non-coding RNAs (lncRNA). LncRNA regulates transcription by upregulating or downregulating the transcription regulatory proteins involved in metabolic and cell signaling pathways. Numerous human diseases, triggered by chronic cellular stressors, are associated with abnormal regulation of transcription. Hence, understanding these mechanisms would help unravel the molecular regulatory insights with potential therapeutic interventions. Therefore, here we emphasize the recent advances of regulation of eukaryotic transcription initiation in response to cellular stress.

A time-resolved multi-omics atlas of transcriptional regulation in response to high-altitude hypoxia across whole-body tissues

High-altitude hypoxia acclimatization requires whole-body physiological regulation in highland immigrants, but the underlying genetic mechanism has not been clarified. Here we use sheep as an animal model for low-to-high altitude translocation. We generate multi-omics data including whole-genome sequences, time-resolved bulk RNA-Seq, ATAC-Seq and single-cell RNA-Seq from multiple tissues as well as phenotypic data from 20 bio-indicators. We characterize transcriptional changes of all genes in each tissue, and examine multi-tissue temporal dynamics and transcriptional interactions among genes. Particularly, we identify critical functional genes regulating the short response to hypoxia in each tissue (e.g., PARG in the cerebellum and HMOX1 in the colon). We further identify TAD-constrained cis-regulatory elements, which suppress the transcriptional activity of most genes under hypoxia. Phenotypic and transcriptional evidence indicate that antenatal hypoxia could improve hypoxia tolerance in offspring. Furthermore, we provide time-series expression data of candidate genes associated with human mountain sickness (e.g., BMPR2) and high-altitude adaptation (e.g., HIF1A). Our study provides valuable resources and insights for future hypoxia-related studies in mammals.

Role of transcriptional regulation in auxin-mediated response to abiotic stresses.

Global climate change (GCC) is posing a serious threat to organisms, particularly plants, which are sessile. Drought, salinity, and the accumulation of heavy metals alter soil composition and have detrimental effects on crops and wild plants. The hormone auxin plays a pivotal role in the response to stress conditions through the fine regulation of plant growth. Hence, rapid, tight, and coordinated regulation of its concentration is achieved by auxin modulation at multiple levels. Beyond the structural enzymes involved in auxin biosynthesis, transport, and signal transduction, transcription factors (TFs) can finely and rapidly drive auxin response in specific tissues. Auxin Response Factors (ARFs) such as the ARF4, 7, 8, 19 and many other TF families, such as WRKY and MADS, have been identified to play a role in modulating various auxin-mediated responses in recent times. Here, we review the most relevant and recent literature on TFs associated with the regulation of the biosynthetic, transport, and signalling auxin pathways and miRNA-related feedback loops in response to major abiotic stresses. Knowledge of the specific role of TFs may be of utmost importance in counteracting the effects of GCC on future agriculture and may pave the way for increased plant resilience.

Imparting As(III) Responsiveness to the Choline Response Transcriptional Regulator BetI.

The development of a low-cost and user-friendly sensor using microorganisms to monitor the presence of As(III) on earth has garnered significant attention. In conventional research on microbial As(III) sensors, the focus has been on transcription factor ArsR, which plays a role in As(III) metabolism. However, we recently discovered that LuxR, a quorum-sensing control factor in Vibrio fischeri that contains multiple cysteine residues, acted as an As(III) sensor despite having no role in As(III) metabolism. This finding suggested that any protein could be an As(III) sensor if cysteine residues were incorporated. In this study, we aimed to confer As(III) responsiveness to BetI, a transcriptional repressor of the TetR family involved in osmotic regulation of the choline response, unrelated to As(III) metabolism. Based on the BetI structure constructed using molecular dynamics calculations, we generated a series of mutants in which each of the three amino acids not critical for function was substituted with cysteine. Subsequent examination of their response to As(III) revealed that the cysteine-substituted mutant, incorporating all three substitutions, demonstrated As(III) responsiveness. This was evidenced by the fluorescence intensity of the downstream reporter superfolder green fluorescent protein expression regulated by the operator region. Intriguingly, the BetI cysteine mutant maintained its binding responsiveness to the natural ligand choline. We successfully engineered an OR logic gate capable of responding to two orthogonal ligands using a single protein.

Abstract A076: The role of PARP-1-mediated FoxA1 ADP-ribosylation in breast cancer biology

Abstract Breast cancer is the most common cancer diagnosed in women. At the time of diagnosis, approximately 70% of breast cancers express Estrogen Receptor alpha (ERα), a key signature of luminal subtype tumors. Equally important in these cases is the expression of FoxA1, a subtype-specific transcription factor (TF) essential for ERα-mediated cell proliferation. FoxA1 is a key pioneer TF that assists ERα with the assembly of enhancers and the transcription of estrogen (E2)-regulated target genes. Together, the FoxA1-ERα axis is a major driver of cancer cell growth. Our recently published work implicates poly(ADP-ribose) polymerase 1 (PARP-1) and ADP-ribosylation (ADPRylation) in the regulation of E2-dependent transcriptional responses in ERα+ breast cancer cells. To determine the underlying mechanisms, we identified FoxA1 as a substrate of PARP-1-mediated ADPRylation. We hypothesize that (1) PARP-1 elicits its effects on E2 signaling and ERα enhancer function, in part, by ADPRylating FoxA1 at specific residues and (2) Loss of these regulatory ADPRylation events alters FoxA1 activity, which in turn can alter the assembly of ERα enhancers and transform the breast cancer cell transcriptome. Indeed, the sites of FoxA1 ADPRylation are recurrently mutated in breast cancers and are highly enriched in metastatic cases. Using an integrated set of biochemical, molecular, cell based, genomic and proteomic approaches, we have validated the ADPRylation of FoxA1 by PARP-1 in vitro and in vivo. We selected two FoxA1 ADPRylation sites, D226 and E255, for further analysis due to their positions in a mutation ‘hot spot’ region corresponding to the C-terminal forkhead DNA-binding domain. Mutation of these sites blocks FoxA1 ADPRylation and enhances cell proliferation in MCF-7 human breast cancer cells. Additionally, preliminary ChIP-seq analysis indicates that the binding of both ERα and FoxA1 are globally reduced at ERα enhancers in MCF-7 cells expressing the FoxA1 ADPRylation site mutants compared to wild-type but may also increase both at selected enhancers. While there is a global reduction in mutant FoxA1 binding near the canonical estrogen-regulated genes, interestingly, we observe an overall enhanced binding of FoxA1 D226N compared to wild-type outside of this canonical program. Collectively, these studies will (1) reveal new molecular endpoints modulated by PARP-1, providing new insights about the use of PARPi to treat ERα+ breast cancers, and (2) suggest new therapeutic strategies to disrupt the aberrant signaling pathways associated with mutant FoxA1 luminal breast cancers, and more importantly metastatic cases, which is of utmost clinical significance. Citation Format: Cristel V. Camacho, Aishwarya Sundaresan, Tulip Nandu, W. Lee Kraus. The role of PARP-1-mediated FoxA1 ADP-ribosylation in breast cancer biology [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Advances in Breast Cancer Research; 2023 Oct 19-22; San Diego, California. Philadelphia (PA): AACR; Cancer Res 2024;84(3 Suppl_1):Abstract nr A076.

Comparative functional analyses of PHR1, PHL1, and PHL4 transcription factors in regulating Arabidopsis responses to phosphate starvation.

To cope with phosphate (Pi) starvation, plants trigger an array of adaptive responses to sustain their growth and development. These responses are largely controlled at transcriptional levels. In Arabidopsis (Arabidopsis thaliana), PHOSPHATE RESPONSE 1 (PHR1) is a key regulator of plant physiological and transcriptional responses to Pi starvation. PHR1 belongs to a MYB-CC-type transcription factor family which contains 15 members. In this PHR1 family, PHR1/PHR1-like 1(PHL1) and PHL2/PHL3 form two distinct modules in regulating plant development and transcriptional responses to Pi starvation. PHL4 is the most closely related member to PHR1. Previously, using the phr1phl4 mutant, we showed that PHL4 is also involved in regulating plant Pi responses. However, the precise roles of PHL1 and PHL4 in regulating plant Pi responses and their functional relationships with PHR1 have not been clearly defined. In this work, we further used the phl1phl4 and phr1phl1phl4 mutants to perform comparative phenotypic and transcriptomic analyses with phr1, phr1phl1, and phr1phl4. The results showed that both PHL1 and PHL4 act redundantly and equally with PHR1 to regulate leaf senescence, Pi starvation induced-inhibition of primary root growth, and accumulation of anthocyanins in shoots. Unlike PHR1 and PHL1, however, the role of PHL4 in maintaining Pi homeostasis is negligible. In regulating transcriptional responses to Pi starvation at genomic levels, both PHL1 and PHL4 play minor roles when acts alone, however, they act synergistically with PHR1. In regulating Pi starvation-responsive genes, PHL4 also function less than PHL1 in terms of the number of the genes it regulates and the magnitude of gene transcription it affects. Furthermore, no synergistic interaction was found between PHL1 and PHL4 in regulating plant response to Pi starvation. Therefore, our results clarified the roles of PHL1 and PHL4 in regulating plant responses to Pi starvation. In addition, this work revealed a new function of these three transcription factors in regulating flowering time.

Linking HIF oxygen-sensing system diversity to hypoxia fitness in Eleutheronema: Molecular characterization and transcriptional response to hypoxia exposure

Hypoxia is a mounting environmental problem affecting coastal waters globally, posing severe consequences for biodiversity and marine life. Metazoans respond to hypoxia stress via the hypoxia-inducible factor (HIF) pathway, but few studies have addressed the gene diversity of the functionally important HIF-pathway. Understanding whether functional diversity exists in the HIF-pathway is a key first step in identifying genes that may impact hypoxia fitness. Here, we leveraged whole-genome resequencing data and bioinformatics tools to identify the key members of the HIF-pathway (HIFα/β, EGLN, and VHL) and genetic diversity in the threatened Eleutheronema. Phylogenetic analysis revealed that teleost-specific duplicates of epas1 (epas1a/b) were followed by the loss of one of each hif1α and hif1αl in Eleutheronema species. Strong collinearity and similarity of gene characteristics suggested the functional conservation of the HIF-pathway during Eleutheronema evolution. Purifying selection was the major theme in HIF-pathway evolution, leading to a reduction in genetic diversity. Substantially low nucleotide diversity of the HIF-pathway was observed among populations, which might indicate the loss of hypoxia fitness in Eleutheronema. Additionally, the normoxic presence of the HIF-pathway differed among tissues and was species-dependent, indicating their diverse roles during development. Significant regulation of HIF-pathway expression levels was observed across tissues under hypoxic conditions, suggesting critical roles in the hypoxia stress response. Moreover, variant molecular characters suggested different roles in response to hypoxia of the HIF-pathway, which were reflected in the different expression patterns across tissues. Our present study provides novel insights into the interplay between gene diversity within the HIF-pathway and hypoxia fitness in threatened Eleutheronema. We highlighted the importance of HIF-pathway-mediated transcriptional regulation in response to hypoxia stress, which provided valuable information for the genetic mechanisms underlying hypoxia adaptation in fish. The bioinformatic methods developed here have broad applications for other species.

Characterization of TREM-1 Signaling in Human Neutrophils By Kinome Array and RNA-Seq

The Triggering Receptor Expressed on Myeloid Cells (TREM)-1 is a member of the Immunoglobulin superfamily and an activating receptor mainly expressed on myeloid cells. TREM-1 ligation leads to immediate activation of polymorphonuclear neutrophils (PMN) resulting in degranulation and release of reactive oxygen species as well as various effector molecules. Beyond its role in acute and chronic inflammatory processes, TREM-1 is also involved in cancer emergence and progression probably by alteration of the tumor associated neutrophils (TAN) and macrophages (TAM). Advanced information about the TREM-1 signaling cascade may reveal novel therapeutic approaches, as various kinases can specifically be targeted with kinase inhibitors. Therefore, we investigated the protein tyrosine kinome (PTK) and serine threonine kinome (STK) of human PMN after TREM-1 activation. To gain further insights beyond the signaling cascade, we performed RNAseq to validate the TREM-1 mediated activation and to reveal the TREM-1 mediated transcriptome. Purified PMN from healthy donors were stimulated with a monoclonal TREM-1 antibody compared to an isotype matched control for 20 minutes (kinome activity) or 60 minutes (RNAseq). For kinomics, PamChip arrays (PamGene) were used to quantify the kinome activity and upstream kinase analysis (UKA) was used to predict the kinase activity. The final data analysis and the UKA was performed by PamGene. A median final score (depending on the significance and the specificity of the kinase activity compared to the control group) > 1.2 was defined as specific kinase activity according to the manufacturer's recommendations. For transcriptomics, Novogene performed library preparation and sequencing, and data analysis was performed with CLC. Differentially expressed genes were defined as FDR adjusted p value ≤ 0.05 and fold change ≥2. Gene set enrichment analysis was performed with Enrichr using the libraries Reactome 2022, WikiPathway 2021 Human, and GO Biological Process 2023 and significance was defined as adjusted p value ≤ 0.05. We identified the activation of 31 PTKs and 38 STKs upon TREM-1 ligation. The kinase statistic revealed changes in the activation state of the kinases present in the TREM-1 treated cells compared to the untreated control group ranging from 2.4 to 5.5 (PTK) and from 1.2 to 1.6 (STK) suggesting more TREM-1 mediated activity alteration of the PTKs. Interestingly, there were no downregulated kinase activities after receptor ligation. Out of the PTK family, we identified ten Src family kinases as well as the Syk family member ZAP70 and other kinases such as BTK, already previously shown to abrogate TREM-1 mediated PMN activation. The RNAseq after TREM-1 ligation revealed 542 differentially expressed genes from which 102 genes were upregulated and 440 genes were downregulated. Gene set enrichment analysis revealed upregulation of genes related to nuclear events (kinase and transcription factor activaton, R-HSA-198725) and cell chemotaxis (GO:0060326) validating the activation of several kinases after TREM-1 ligation. We found increased expressions of genes involved in adipogenesis (WP236) and lung fibrosis (WP3624) suitably to the observed increased activity of Src family kinases that are described to be involved in lung fibrosis. Moreover, some upregulated genes related to NGF-stimulated transcription (R-HSA-9031628) or regulation of transcription in response to stress (GO:0043618) in which the MAPK are known to be involved. However, we did not observe clearly increased MAPK activity. Besides upregulation, TREM-1 ligation led to decreased expression of several genes involved in (positive) regulation of cytokine production involved in inflammatory response (GO:1900017 and GO:1900015) and signaling by interleukins (R-HSA-449147). Taken together, the results contribute to the knowledge about TREM-1 in inflammatory disorders and reveal approaches of its role in cancer. This study revealed various kinases possibly involved in early TREM-1 signaling of neutrophils that may be suitable targets to inhibit TREM-1 mediated cell activation. Moreover, we linked the TREM-1 mediated kinase activity to the resulting gene expression. Further studies are needed to validate the results in vitro and in vivo and and to identify suitable pharmacological inhibitors to modulate TREM-1 mediated activities in inflammatory conditions and cancer.