Transcriptional Enhancer Research Articles

Abstract LB153: Spatial transcriptomics of mouse parathyroid gland cells expressing an activating variant of gcm2

Abstract Tumors of one or multiple parathyroid glands and elevated serum calcium and parathyroid hormone (PTH) are the common characteristics of primary hyperparathyroidism (PHPT). Genes associated with familial PHPT (hereditary parathyroid tumors) include - MEN1, CDKN1B, CDC73, RET, MAX and GCM2. The glial cells missing 2 (GCM2) gene functions as a transcription factor that is essential for parathyroid gland development. Heterozygous germline missense variants located within the C-terminal conserved inhibitory domain (CCID) of GCM2 have been reported in a subset of patients with familial PHPT. In functional assays, these missense variants enhance the transcriptional activity of GCM2, suggesting that GCM2 could act as a proto-oncogene in parathyroid neoplasia. A recurrent germline activating missense variant of GCM2, p.Y394S in patients with familial PHPT has been reported in various publications. To study the direct contribution of this missense variant in parathyroid neoplasia, we used the CRISPR/Cas9 system to generate a heterozygous germline knock-in of p.Y392S in the mouse Gcm2 gene (which corresponds to the human variant p.Y394S in GCM2). In GCM2-binding site mediated luciferase reporter assays, the mouse Gcm2 variant p.Y392S also exhibited the transcription enhancing effect that was observed for the human GCM2 p.Y394S variant. However, mice with this variant (Gcm2 +/Y392S) did not show obvious parathyroid tumors. Because the mouse Gcm2 variant p.Y392S exhibited enhanced transcriptional activity, we performed spatial transcriptomics of parathyroid glands from wildtype (WT) and Gcm2 +/Y392S mice to examine differentially expressed transcripts in parathyroid cells with the p.Y392S variant of Gcm2. Using the 10X Genomics Visium platform, spatially resolved RNA-seq analysis was performed on parathyroid gland cells of WT and Gcm2 +/Y392S mice specifically marked by the expression of Pth and Gcm2. Spatial transcriptomics and Pathway analysis was performed to evaluate transcripts with more than 2-fold increased expression in parathyroid cells from the p.Y392S variant of Gcm2 compared to WT. Further investigation of these genes and pathways in mouse and human parathyroid cells can provide insights into the functional consequences of GCM2 activating variants and the pathophysiology of parathyroid tumorigenesis. Citation Format: Priyanka Bolel, Jeremie O. Pina, Fabio R. Faucz, Lee S. Weinstein, Sunita K. Agarwal. Spatial transcriptomics of mouse parathyroid gland cells expressing an activating variant of gcm2 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr LB153.

Xenopus tropicalis osteoblast-specific open chromatin regions reveal promoters and enhancers involved in human skeletal phenotypes and shed light on early vertebrate evolution

While understanding the genetic underpinnings of osteogenesis has far-reaching implications for skeletal diseases and evolution, a comprehensive characterization of the osteoblastic regulatory landscape in non-mammalian vertebrates is still lacking. Here, we compared the ATAC-Seq profile of Xenopus tropicalis (Xt) osteoblasts to a variety of non mineralizing control tissues, and identified osteoblast-specific nucleosome free regions (NFRs) at 527 promoters and 6747 distal regions. Sequence analyses, Gene Ontology, RNA-Seq and ChIP-Seq against four key histone marks confirmed that the distal regions correspond to bona fide osteogenic transcriptional enhancers exhibiting a shared regulatory logic with mammals. We report 425 regulatory regions conserved with human and globally associated to skeletogenic genes. Of these, 35 regions have been shown to impact human skeletal phenotypes by GWAS, including one trps1 enhancer and the runx2 promoter, two genes which are respectively involved in trichorhinophalangeal syndrome type I and cleidocranial dysplasia. Intriguingly, 60 osteoblastic NFRs also align to the genome of the elephant shark, a species lacking osteoblasts and bone tissue. To tackle this paradox, we chose to focus on dlx5 because its conserved promoter, known to integrate regulatory inputs during mammalian osteogenesis, harbours an osteoblast-specific NFR in both frog and human. Hence, we show that dlx5 is expressed in Xt and elephant shark odontoblasts, supporting a common cellular and genetic origin of bone and dentine. Taken together, our work (i) unravels the Xt osteogenic regulatory landscape, (ii) illustrates how cross-species comparisons harvest data relevant to human biology and (iii) reveals that a set of genes including bnc2, dlx5, ebf3, mir199a, nfia, runx2 and zfhx4 drove the development of a primitive form of mineralized skeletal tissue deep in the vertebrate lineage.

Exploring transient global transcriptional changes induced by ascorbic acid revealed via atKAS-seq profiling.

Transcription initiates the formation of single-stranded DNA (ssDNA) regions within the genome, delineating transcription bubbles, a highly dynamic genomic process. Kethoxal-assisted single-stranded DNA sequencing (KAS-seq) utilizing N3-kethoxal has emerged as a potent tool for mapping specific guanine positions in ssDNA on a genome-wide scale. However, the original KAS-seq method required the costly Accel-NGS Methyl-seq DNA library kit. This study introduces an optimized iteration of the KAS-seq technique, referred to as adapter-tagged KAS-seq (atKAS-seq), incorporating an adapter tagging strategy. This modification involves integrating sequencing adapters via complementary strand synthesis using random N9 tagging. Additionally, by harnessing the potential of ascorbic acid (ASC), recognized for inducing global epigenetic changes, we employed the atKAS-seq methodology to elucidate critical pathways influenced by short-term, high-dose ASC treatment. Our findings underscore that atKAS-seq enables rapid and precise analyses of transcription dynamics and enhancer activities concurrently. This method offers a streamlined, cost-efficient, and low-input approach, affirming its utility in probing intricate genomic regulatory mechanisms.

Abstract 5831: The TLE1 transcriptional and epigenetic machinery contributes to EGFR targeted therapy resistance in human lung adenocarcinoma

Abstract In human lung adenocarcinoma that carry Epidermal Growth Factor Receptor (EGFR) sensitizing mutations, targeting the EGFR pathway with the use of EGFR tyrosine kinase inhibitors (EGFR-TKIs) has emerged as a promising therapeutic strategy providing initial clinical benefit to many patients. Unfortunately, resistance to EGFR-TKIs eventually develops leading to disease progression. In addition to genetic mutations, transcriptional and epigenetic mediated changes in gene expression remain an important mechanism contributing to EGFR-TKI resistance. Here, we report a novel function of the transcriptional corepressor Transducin-Like Enhancer of Split-1 (TLE1) in mediating EGFR-TKI resistance in EGFR mutant LUAD cells through its survival promoting gene transcriptional program. In EGFR mutant, EGFR TKI sensitive LUAD cells, sole activation of the TLE1 nuclear function attenuates EGFR TKI sensitivity with concomittant reduction of EGFR-TKI-mediated apoptosis. Conversely, downregulation of endogenous TLE1 expression via siRNAs/shRNAs and deletion of TLE1 gene via CRISPR-Cas9 in EGFR-mutant, EGFR-TKI resistant cells partially restore EGFR-TKI sensitivity via induction of EGFR-TKI apoptosis. Mechanistically, the anti-apoptosis promoting gene transcriptional program of TLE1 is dependent on its functional interaction with the transcriptional repressor HES-1, a downstream Notch target which has been associated with relapse in LUAD patients following EGFR-TKI treatment. Collectively, these studies indicate the TLE1 corepressor as a determinant of resistance to EGFR targeted therapy and raise the possibility that inhibiting TLE1 nuclear function may serve as an adjuct therapy to circumvent EGFR-TKI resistance. Citation Format: Hector Ramos Biliran, Xin Yao, Nasir Roberts, Prince Iheukwumere, Renwei Chen, Ma. Carmela Dela Cruz. The TLE1 transcriptional and epigenetic machinery contributes to EGFR targeted therapy resistance in human lung adenocarcinoma [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 5831.

Loss of ERβ Disrupts Gene Regulation in Primordial and Primary Follicles.

Loss of ERβ increases primordial follicle growth activation (PFGA), leading to premature ovarian follicle reserve depletion. We determined the expression and gene regulatory functions of ERβ in dormant primordial follicles (PdFs) and activated primary follicles (PrFs) using mouse models. PdFs and PrFs were isolated from 3-week-old Erβ knockout (Erβnull) mouse ovaries, and their transcriptomes were compared with those of control Erβfl/fl mice. We observed a significant (≥2-fold change; FDR p-value ≤ 0.05) deregulation of approximately 5% of genes (866 out of 16,940 genes, TPM ≥ 5) in Erβnull PdFs; ~60% (521 out of 866) of the differentially expressed genes (DEGs) were upregulated, and 40% were downregulated, indicating that ERβ has both transcriptional enhancing as well as repressing roles in dormant PdFs. Such deregulation of genes may make the Erβnull PdFs more susceptible to increased PFGA. When the PdFs undergo PFGA and form PrFs, many new genes are activated. During PFGA of Erβfl/fl follicles, we detected a differential expression of ~24% genes (4909 out of 20,743; ≥2-fold change; FDR p-value ≤ 0.05; TPM ≥ 5); 56% upregulated and 44% downregulated, indicating the gene enhancing and repressing roles of Erβ-activated PrFs. In contrast, we detected a differential expression of only 824 genes in Erβnull follicles during PFGA (≥2-fold change; FDR p-value ≤ 0.05; TPM ≥ 5). Moreover, most (~93%; 770 out of 824) of these DEGs in activated Erβnull PrFs were downregulated. Such deregulation of genes in Erβnull activated follicles may impair their inhibitory role on PFGA. Notably, in both Erβnull PdFs and PrFs, we detected a significant number of epigenetic regulators and transcription factors to be differentially expressed, which suggests that lack of ERβ either directly or indirectly deregulates the gene expression in PdFs and PrFs, leading to increased PFGA.

Nrf2 pre-recruitment at Enhancer 2 is a hallmark of H2O2-induced epigenetic transcriptional memory in the HMOX1 gene in human umbilical artery endothelial cells.

Maternal obesity (MO) is a significant cause of increased cardiometabolic risk in offspring, who present endothelial dysfunction at birth. Alterations in physiologic and cellular redox status are strongly associated with altered gene regulation in arterial endothelium. However, specific mechanisms by which the pro-oxidant fetal environment in MO could modulate the vascular gene expression and function during the offspring's postnatal life are elusive. We tested if oxidative stress could reprogram the antioxidant-coding gene's response to a pro-oxidant challenge through an epigenetic transcriptional memory (ETM) mechanism. A pro-oxidant double-hit protocol was applied to human umbilical artery endothelial cells (HUAECs) and EA.hy 926 endothelial cell lines. The ETM acquisition in the HMOX1 gene was analyzed by RT-qPCR. HMOX1 mRNA decay was evaluated by Actinomycin-D treatment and RT-qPCR. To assess the chromatin accessibility and the enrichment of NRF2, RNAP2, and phosphorylation at serin-5 of RNAP2, at HMOX1 gene regulatory regions, were used DNase HS-qPCR and ChIP-qPCR assays, respectively. The CpG methylation pattern at the HMOX1 core promoter was analyzed by DNA bisulfite conversion and Sanger sequencing. Data were analyzed using two-way ANOVA, and p < 0.05 was statistically significant. Using a pro-oxidant double-hit protocol, we found that the Heme Oxygenase gene (HMOX1) presents an ETM response associated with changes in the chromatin structure at the promoter and gene regulatory regions. The ETM response was characterized by a paused-RNA Polymerase 2 and NRF2 enrichment at the transcription start site and Enhancer 2 of the HMOX1 gene, respectively. Changes in DNA methylation pattern at the HMOX1 promoter were not a hallmark of this oxidative stress-induced ETM. These data suggest that a pro-oxidant milieu could trigger an ETM at the vascular level, indicating a potential epigenetic mechanism involved in the increased cardiovascular risk in the offspring of women with obesity.

Weakened tanning ability is an important mechanism for evolutionary skin lightening in East Asians

The evolution of light-skin pigmentation among Eurasians is considered as an adaptation to the high-latitude environments. East Asians are ideal populations for studying skin color evolution because of the complex environment of East Asia. Here, we report a strong selection signal for the pigmentation gene phenylalanine hydroxylase (PAH) in light-skinned Han Chinese individuals. The intron mutation rs10778203 in PAH is enriched in East Asians and is significantly associated with skin color of the back of the hand in Han Chinese males (P < 0.05). In vitro luciferase and transcription factor binding assays show that the ancestral allele of rs10778203 could bind to SMAD2 and has a significant enhancer activity for PAH. However, the derived T allele (the major allele in East Asians) of rs10778203 decreases the binding activity of transcription factors and enhancer activity. Meanwhile, the derived T allele of rs10778203 shows a weaker ultraviolet radiation response in A375 cells and zebrafish embryos. Furthermore, rs10778203 decreases melanin production in transgenic zebrafish embryos after ultraviolet B (UVB) treatment. Collectively, PAH is a potential pigmentation gene that regulates skin tanning ability. Natural selection has enriched the adaptive allele, resulting in weakened tanning ability in East Asians, suggesting a unique genetic mechanism for evolutionary skin lightening in East Asians.

Dynamic enhancer landscapes in human craniofacial development

The genetic basis of human facial variation and craniofacial birth defects remains poorly understood. Distant-acting transcriptional enhancers control the fine-tuned spatiotemporal expression of genes during critical stages of craniofacial development. However, a lack of accurate maps of the genomic locations and cell type-resolved activities of craniofacial enhancers prevents their systematic exploration in human genetics studies. Here, we combine histone modification, chromatin accessibility, and gene expression profiling of human craniofacial development with single-cell analyses of the developing mouse face to define the regulatory landscape of facial development at tissue- and single cell-resolution. We provide temporal activity profiles for 14,000 human developmental craniofacial enhancers. We find that 56% of human craniofacial enhancers share chromatin accessibility in the mouse and we provide cell population- and embryonic stage-resolved predictions of their in vivo activity. Taken together, our data provide an expansive resource for genetic and developmental studies of human craniofacial development.

Abstract B008: Alternative PER2 (Period2) enhancer usage drives epithelial-mesenchymal transition (EMT) in epithelial ovarian cancer (OC)

Abstract B008: Alternative <i>PER2</i> (Period2) enhancer usage drives epithelial-mesenchymal transition (EMT) in epithelial ovarian cancer (OC)

Histone H3K4ac, as a marker of active transcription start sites and enhancers, plays roles in histone eviction and RNA transcription

The lysine 4 of histone H3 (H3K4) can be methylated or acetylated into four states: H3K4me1, H3K4me2, H3K4me3, or H3K4ac. Unlike H3K4 methylation, the genome-wide distribution and functional roles of H3K4ac remain unclear. To understand the relationship of acetylation with methylation at H3K4 and to explore the roles of H3K4ac in the context of chromatin, we analyzed H3K4ac across the human genome and compared it with H3K4 methylation in K562 cells. H3K4ac was positively correlated with H3K4me1/2/3 in reciprocal analysis. A decrease in H3K4ac through the mutation of the histone acetyltransferase p300 reduced H3K4me1 and H3K4me3 at the H3K4ac peaks. H3K4ac was also impaired by H3K4me depletion in the histone methyltransferase MLL3/4-mutated cells. H3K4ac peaks were enriched at enhancers in addition to the transcription start sites (TSSs) of genes. H3K4ac of TSSs and enhancers was positively correlated with mRNA and eRNA transcription. A decrease in H3K4ac reduced H3K4me3 and H3K4me1 in TSSs and enhancers, respectively, and inhibited the eviction of histone H3 from them. The mRNA transcription of highly transcribed genes was affected by the reduced H3K4ac. Interestingly, H3K4ac played a redundant role with regard to H3K27ac in eRNA transcription. These results indicate that H3K4ac serves as a marker of both active TSSs and enhancers and plays a role in histone eviction and RNA transcription by leading to H3K4me1/3.

CHD7 and SOX2 act in a common gene regulatory network during mammalian semicircular canal and cochlear development

Inner ear morphogenesis requires tightly regulated epigenetic and transcriptional control of gene expression. CHD7, an ATP-dependent chromodomain helicase DNA-binding protein, and SOX2, an SRY-related HMG box pioneer transcription factor, are known to contribute to vestibular and auditory system development, but their genetic interactions in the ear have not been explored. Here, we analyzed inner ear development and the transcriptional regulatory landscapes in mice with variable dosages of Chd7 and/or Sox2. We show that combined haploinsufficiency for Chd7 and Sox2 results in reduced otic cell proliferation, severe malformations of semicircular canals, and shortened cochleae with ectopic hair cells. Examination of mice with conditional, inducible Chd7 loss by Sox2CreER reveals a critical period (~E9.5) of susceptibility in the inner ear to combined Chd7 and Sox2 loss. Data from genome-wide RNA-sequencing and CUT&Tag studies in the otocyst show that CHD7 regulates Sox2 expression and acts early in a gene regulatory network to control expression of key otic patterning genes, including Pax2 and Otx2. CHD7 and SOX2 directly bind independently and cooperatively at transcription start sites and enhancers to regulate otic progenitor cell gene expression. Together, our findings reveal essential roles for Chd7 and Sox2 in early inner ear development and may be applicable for syndromic and other forms of hearing or balance disorders.

Molecular dissection of an intronic enhancer governing cold-induced expression of the vacuolar invertase gene in potato.

Potato (Solanum tuberosum) is the third most important food crop in the world. Potato tubers must be stored at cold temperatures to minimize sprouting and losses due to disease. However, cold temperatures strongly induce the expression of the potato vacuolar invertase gene (VInv) and cause reducing sugar accumulation. This process, referred to as "cold-induced sweetening," is a major postharvest problem for the potato industry. We discovered that the cold-induced expression of VInv is controlled by a 200 bp enhancer, VInvIn2En, located in its second intron. We identified several DNA motifs in VInvIn2En that bind transcription factors involved in the plant cold stress response. Mutation of these DNA motifs abolished VInvIn2En function as a transcriptional enhancer. We developed VInvIn2En deletion lines in both diploid and tetraploid potato using clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated nuclease 9 (Cas9)-mediated gene editing. VInv transcription in cold-stored tubers was significantly reduced in the deletion lines. Interestingly, the VInvIn2En sequence is highly conserved among distantly related Solanum species, including tomato (Solanum lycopersicum) and other non-tuber-bearing species. We conclude that the VInv gene and the VInvIn2En enhancer have adopted distinct roles in the cold stress response in tubers of tuber-bearing Solanum species.

Novel Enhanced Mammalian Cell Transient Expression Vector via Promoter Combination.

During the emergence of infectious diseases, evaluating the efficacy of newly developed vaccines requires antigen proteins. Available methods enhance antigen protein productivity; however, structural modifications may occur. Therefore, we aimed to construct a novel transient overexpression vector capable of rapidly producing large quantities of antigenic proteins in mammalian cell lines. This involved expanding beyond the exclusive use of the human cytomegalovirus (CMV) promoter, and was achieved by incorporating a transcriptional enhancer (CMV enhancer), a translational enhancer (woodchuck hepatitis virus post-transcriptional regulatory element), and a promoter based on the CMV promoter. Twenty novel transient expression vectors were constructed, with the vector containing the human elongation factor 1-alpha (EF-1a) promoter showing the highest efficiency in expressing foreign proteins. This vector exhibited an approximately 27-fold higher expression of enhanced green fluorescent protein than the control vector containing only the CMV promoter. It also expressed the highest level of severe acute respiratory syndrome coronavirus 2 receptor-binding domain protein. These observations possibly result from the simultaneous enhancement of the transcriptional activity of the CMV promoter and the human EF-1a promoter by the CMV enhancer. Additionally, the synergistic effect between the CMV and human EF-1a promoters likely contributed to the further enhancement of protein expression.

NS1-mediated enhancement of MVC transcription and replication promoted by KAT5/H4K12ac.

Histone modifications function in both cellular and viral gene expression. However, the roles of acetyltransferases and histone acetylation in parvoviral infection remain poorly understood. In the current study, we found the histone deacetylase (HDAC) inhibitor, trichostatin A (TSA), promoted the replication and transcription of parvovirus minute virus of canines (MVC). Notably, the expression of host acetyltransferases KAT5, GTF3C4, and KAT2A was increased in MVC infection, as well as H4 acetylation (H4K12ac). KAT5 is not only responsible for H4K12ac but also crucial for viral replication and transcription. The viral nonstructural protein NS1 interacted with KAT5 and enhanced its expression. Further study showed that Y44 in KAT5, which may be tyrosine-phosphorylated, is indispensable for NS1-mediated enhancement of KAT5 and efficient MVC replication. The data demonstrated that NS1 interacted with KAT5, which resulted in an enhanced H4K12ac level to promote viral replication and transcription, implying the epigenetic addition of H4K12ac in viral chromatin-like structure by KAT5 is vital for MVC replication.IMPORTANCEParvoviral genomes are chromatinized with host histones. Therefore, histone acetylation and related acetyltransferases are required for the virus to modify histones and open densely packed chromatin structures. This study illustrated that histone acetylation status is important for MVC replication and transcription and revealed a novel mechanism that the viral nonstructural protein NS1 hijacks the host acetyltransferase KAT5 to enhance histone acetylation of H4K12ac, which relies on a potential tyrosine phosphorylation site, Y44 in KAT5. Other parvoviruses share a similar genome organization and coding potential and may adapt a similar strategy for efficient viral replication and transcription.

CHIP suppresses the proliferation and migration of A549 cells by mediating the ubiquitination of eIF2α and upregulation of tumor suppressor RBM5

The PERK-eIF2α pathway plays an essential role in endoplasmic reticulum (ER) stress. When the PERK-eIF2α pathway is activated, PERK phosphorylates eIF2α (p-eIF2α) at Ser51 and quenches global protein synthesis. In this study, we verified eIF2α as a bona fide substrate of the E3 ubiquitin ligase CHIP both in vitro and in cells. CHIP mediated the ubiquitination and degradation of nonphosphorylated eIF2α in a chaperone-independent manner and promoted the upregulation of the transcription factor ATF4 under ER stress conditions. ATF4 induced the transcriptional enhancement of the tumor suppressor genes PTEN and RBM5. Although transcription was enhanced, the PTEN protein was subsequently degraded by CHIP, but the expression of the RBM5 protein was upregulated, thereby suppressing the proliferation and migration of A549 cells. Overall, our study established a new mechanism that deepened the understanding of the PERK-eIF2α pathway through the ubiquitination and degradation of eIF2α. The crosstalk between the phosphorylation and ubiquitination of eIF2α shed light on a new perspective for tumor progression.

Vitamin D inhibits tamoxifen-induced non-alcoholic fatty liver disease through a nonclassical estrogen receptor/liver X receptor pathway

Non-alcoholic Fatty Liver Disease (NAFLD) is one of the common side effects of tamoxifen treatment for estrogen receptor-positive breast cancer, and is representative of disorders of energy metabolism. Fatty liver is induced after tamoxifen (TAM) inhibition of estrogen receptor activity, but the exact mechanism is not clear. This study investigated the effects and mechanisms of TAM-induced steatosis in the liver. The effects and mechanisms of TAM on hepatocyte lipid metabolism were assessed using C57BL/6 female mice and human hepatoma cells. TAM promoted fat accumulation in the liver by upregulation of Srebp-1c expression. Regarding the molecular mechanism, TAM promoted the recruitment of the auxiliary transcriptional activator, p300, and dissociated the auxiliary transcriptional repressor, nuclear receptor corepressor (NCOR), of the complexes, which led to enhancement of Srebp-1c transcription and an increase of triglyceride (TG) synthesis. Vitamin D (VD), a common fat-soluble vitamin, can decrease TAM-induced NAFLD by promoting p300 dissociation and NCOR recruitment. Tamoxifen promoted the recruitment and dissociation of co-transcription factors on the LXR/ER/RXR receptor complex, leading to a disorder of liver lipid metabolism. VD interfered with TAM-induced liver lipid metabolism disorders by reversing this process.

Tissue-specific RNA Polymerase II promoter-proximal pause release and burst kinetics in a Drosophila embryonic patterning network

BackgroundFormation of tissue-specific transcriptional programs underlies multicellular development, including dorsoventral (DV) patterning of the Drosophila embryo. This involves interactions between transcriptional enhancers and promoters in a chromatin context, but how the chromatin landscape influences transcription is not fully understood.ResultsHere we comprehensively resolve differential transcriptional and chromatin states during Drosophila DV patterning. We find that RNA Polymerase II pausing is established at DV promoters prior to zygotic genome activation (ZGA), that pausing persists irrespective of cell fate, but that release into productive elongation is tightly regulated and accompanied by tissue-specific P-TEFb recruitment. DV enhancers acquire distinct tissue-specific chromatin states through CBP-mediated histone acetylation that predict the transcriptional output of target genes, whereas promoter states are more tissue-invariant. Transcriptome-wide inference of burst kinetics in different cell types revealed that while DV genes are generally characterized by a high burst size, either burst size or frequency can differ between tissues.ConclusionsThe data suggest that pausing is established by pioneer transcription factors prior to ZGA and that release from pausing is imparted by enhancer chromatin state to regulate bursting in a tissue-specific manner in the early embryo. Our results uncover how developmental patterning is orchestrated by tissue-specific bursts of transcription from Pol II primed promoters in response to enhancer regulatory cues.

Identification of eQTLs using different sets of single nucleotide polymorphisms associated with carcass and body composition traits in pigs

BackgroundMapping expression quantitative trait loci (eQTLs) in skeletal muscle tissue in pigs is crucial for understanding the relationship between genetic variation and phenotypic expression of carcass traits in meat animals. Therefore, the primary objective of this study was to evaluate the impact of different sets of single nucleotide polymorphisms (SNP), including scenarios removing SNPs pruned for linkage disequilibrium (LD) and SNPs derived from SNP chip arrays and RNA-seq data from liver, brain, and skeletal muscle tissues, on the identification of eQTLs in the Longissimus lumborum tissue, associated with carcass and body composition traits in Large White pigs. The SNPs identified from muscle mRNA were combined with SNPs identified in the brain and liver tissue transcriptomes, as well as SNPs from the GGP Porcine 50 K SNP chip array. Cis- and trans-eQTLs were identified based on the skeletal muscle gene expression level, followed by functional genomic analyses and statistical associations with carcass and body composition traits in Large White pigs.ResultsThe number of cis- and trans-eQTLs identified across different sets of SNPs (scenarios) ranged from 261 to 2,539 and from 29 to 13,721, respectively. Furthermore, 6,180 genes were modulated by eQTLs in at least one of the scenarios evaluated. The eQTLs identified were not significantly associated with carcass and body composition traits but were significantly enriched for many traits in the “Meat and Carcass” type QTL. The scenarios with the highest number of cis- (n = 304) and trans- (n = 5,993) modulated genes were the unpruned and LD-pruned SNP set scenarios identified from the muscle transcriptome. These genes include 84 transcription factor coding genes.ConclusionsAfter LD pruning, the set of SNPs identified based on the transcriptome of the skeletal muscle tissue of pigs resulted in the highest number of genes modulated by eQTLs. Most eQTLs are of the trans type and are associated with genes influencing complex traits in pigs, such as transcription factors and enhancers. Furthermore, the incorporation of SNPs from other genomic regions to the set of SNPs identified in the porcine skeletal muscle transcriptome contributed to the identification of eQTLs that had not been identified based on the porcine skeletal muscle transcriptome alone.

MdASMT9-mediated melatonin biosynthesis enhances basal thermotolerance in apple plants.

High temperatures negatively impact the yield and quality of fruit crops. Exogenous melatonin (MT) application has been shown to enhance heat tolerance, but the response of endogenous MT to heat stress, particularly in perennial fruit trees, remains unclear. The present study investigated the effects of high temperatures on transgenic apple plants overexpressing the MT biosynthesis gene N-acetylserotonin methyltransferase 9 (MdASMT9). Endogenous MT protectedtransgenic plants from heat stress by increasing antioxidant enzyme activity and scavenging reactive oxygen species (ROS), and protecting the chloroplasts from damage. Application of MT and overexpression of MdASMT9 also reduced abscisic acid accumulation through promoting MdWRKY33-mediated transcriptional inhibition of MdNCED1 and MdNCED3, thus inducing stomatal opening for better heat dissipation. Furthermore, MT-enhanced autophagic activity through promoting MdWRKY33-mediated transcriptional enhancement of MdATG18a under heat stress. These findings provide new insights into the regulation of endogenous MT and its role in improving basal thermotolerance in perennial fruit trees.

A Bioinformatically Initiated Approach to Evaluate GATA1 Regulatory Regions in Samples with Weak D, Del, or D– Phenotypes Despite Normal RHD Exons

Introduction: With over 360 blood group antigens in systems recognized, there are antigens, such as RhD, which demonstrate a quantitative reduction in antigen expression due to nucleotide variants in the non-coding region of the gene that result in aberrant splicing or a regulatory mechanism. This study aimed to evaluate bioinformatically predicted GATA1-binding regulatory motifs in the RHD gene for samples presenting with weak or apparently negative RhD antigen expression but showing normal RHD exons. Methods: Publicly available open chromatin region data were overlayed with GATA1 motif candidates in RHD. Genomic DNA from weak D, Del or D– samples with normal RHD exons (n = 13) was used to confirm RHD zygosity by quantitative PCR. Then, RHD promoter, intron 1, and intron 2 regions were amplified for Sanger sequencing to detect potential disruptions in the GATA1 motif candidates. Electrophoretic mobility shift assay (EMSA) was performed to assess GATA1-binding. Luciferase assays were used to assess transcriptional activity. Results: Bioinformatic analysis identified five of six GATA1 motif candidates in the promoter, intron 1 and intron 2 for investigation in the samples. Luciferase assays showed an enhancement in transcription for GATA1 motifs in intron 1 and for intron 2 only when the R2 haplotype variant (rs675072G>A) was present. GATA1 motifs were intact in 12 of 13 samples. For one sample with a Del phenotype, a novel RHD c.1–110A>C variant disrupted the GATA1 motif in the promoter which was supported by a lack of a GATA1 supershift in the EMSA and 73% transcriptional activity in the luciferase assay. Two samples were D+/D– chimeras. Conclusion: The bioinformatic predictions enabled the identification of a novel DEL allele, RHD c.1–110A>C, which disrupted the GATA1 motif in the proximal promoter. Although the majority of the samples investigated here remain unexplained, we provide GATA1 targets which may benefit future RHD regulatory investigations.