Multiple Regulatory Elements Research Articles

BMP7 is a candidate gene for reproductive traits in Yorkshire sows

Bone morphogenetic protein 7 (BMP7) is of the BMP subfamily, and has effects on female fertility by regulating steroidogenesis, granulosa cell states, and follicular development. In the present study, there was assessment of the combined genotypes formed by the three variants within the 3′-UTR of BMP7 gene as associations with sow reproductive functions. The 3′-UTR of the BMP7 gene of pigs was identified using the 3′ RACE assay, and its full-length sequence was found to be 1538 bp in length. Multiple RNA regulatory elements were detected in this region, luciferase activity assays were performed and results indicated miR-22−3p affects BMP7 by directly binding to the miRNA response element in the 3′-UTR (c.2358–2382). In addition, two novel complete linkage variants, c.2256 G > C and a 7-bp indel (c.2259–2265), were identified within the 3′-UTR of the BMP7 gene of pigs. Importantly, combined genotypes with these two novel variants and c.1569A > G, a variant previously identified in the BMP7 3′-UTR of pigs, were associated with sow reproductive traits, including the total number of piglets born, number of dead piglets at birth, and litter weight in the Yorkshire pig population studies. Results from the present study confirm that BMP7 is a candidate gene for the reproductive traits in Yorkshire sows.

MiR-130a/TGF-β1 axis is involved in sow fertility by controlling granulosa cell apoptosis

TGF-β1 is a ligand of the TGF-β superfamily and an important cytokine that regulates ovarian functions including follicular development, steroid production, ovulation, luteinization, and female fertility. However, little is known about the regulation of TGF-β1 expression in ovary. Here, we identified that TGF-β1 is a functional target of miR-130a in porcine ovarian granulosa cells (GCs). The 3′-UTR sequence of TGF-β1 gene (1137 bp in length) in Large White (LW) pig was isolated, and multiple RNA regulatory elements (RREs), including several binding motifs of different miRNAs, were identified in this region. Luciferase activity assay showed that miR-130a dramatically suppresses the 3′-UTR luciferase activity of TGF-β1 gene, and further inhibits the expression of TGF-β1 in porcine GCs. FACS revealed that miR-130a acts as a pro-apoptotic factor and promotes GC apoptosis by inhibiting TGF-β1. Two novel linked mutations (−573G > A and −540T > C) were identified in the promoter region of ssc-miR-130a, but their polymorphisms are not associated with sow reproductive traits. Importantly, combined genotype analysis with a known mutation (c.1583 A > G) in the 3′-UTR of porcine TGF-β1 gene showed a significant association with reproductive performance in LW sow population. Overall, our findings defined a novel regulatory axis, miR-130a/TGF-β1 axis, which is involved in regulating sow fertility.

Genomewide Meta-Analysis Validates a Role for S1PR1 in Microtubule Targeting Agent-Induced Sensory Peripheral Neuropathy.

Microtubule targeting agents (MTAs) are anticancer therapies commonly prescribed for breast cancer and other solid tumors. Sensory peripheral neuropathy (PN) is the major dose-limiting toxicity for MTAs and can limit clinical efficacy. The current pharmacogenomic study aimed to identify genetic variations that explain patient susceptibility and drive mechanisms underlying development of MTA-induced PN. A meta-analysis of genomewide association studies (GWAS) from two clinical cohorts treated with MTAs (Cancer and Leukemia Group B (CALGB) 40502 and CALGB 40101) was conducted using a Cox regression model with cumulative dose to first instance of grade 2 or higher PN. Summary statistics from a GWAS of European subjects (n=469) in CALGB 40502 that estimated cause-specific risk of PN were meta-analyzed with those from a previously published GWAS of European ancestry (n=855) from CALGB 40101 that estimated the risk of PN. Novel single nucleotide polymorphisms in an enhancer region downstream of sphingosine-1-phosphate receptor 1 (S1PR1 encoding S1PR1 ; e.g., rs74497159, βCALGB 40101 per allele log hazard ratio (95% confidence interval (CI))=0.591 (0.254-0.928), βCALGB 40502 per allele log hazard ratio (95% CI)=0.693 (0.334-1.053); PMETA =3.62×10-7 ) were the most highly ranked associations based on P values with risk of developing grade 2 and higher PN. In silico functional analysis identified multiple regulatory elements and potential enhancer activity for S1PR1 within this genomic region. Inhibition of S1PR1 function in induced pluripotent stem cell-derived human sensory neurons shows partial protection against paclitaxel-induced neurite damage. These pharmacogenetic findings further support ongoing clinical evaluations to target S1PR1 as a therapeutic strategy for prevention and/or treatment of MTA-induced neuropathy.

Trait-associated noncoding variant regions affect TBX3 regulation and cardiac conduction.

Genome-wide association studies have implicated common genomic variants in the gene desert upstream of TBX3 in cardiac conduction velocity. Whether these noncoding variants affect expression of TBX3 or neighboring genes and how they affect cardiac conduction is not understood. Here, we use high-throughput STARR-seq to test the entire 1.3 Mb human and mouse TBX3 locus, including two cardiac conduction-associated variant regions, for regulatory function. We identified multiple accessible and functional regulatory DNA elements that harbor variants affecting their activity. Both variant regions drove gene expression in the cardiac conduction tissue in transgenic reporter mice. Genomic deletion from the mouse genome of one of the regions caused increased cardiac expression of only Tbx3, PR interval shortening and increased QRS duration. Combined, our findings address the mechanistic link between trait-associated variants in the gene desert, TBX3 regulation and cardiac conduction.

Postadmixture Selection on Chileans Targets Haplotype Involved in Pigmentation, Thermogenesis and Immune Defense against Pathogens.

Detection of positive selection signatures in populations around the world is helping to uncover recent human evolutionary history as well as the genetic basis of diseases. Most human evolutionary genomic studies have been performed in European, African, and Asian populations. However, populations with Native American ancestry have been largely underrepresented. Here, we used a genome-wide local ancestry enrichment approach complemented with neutral simulations to identify postadmixture adaptations underwent by admixed Chileans through gene flow from Europeans into local Native Americans. The top significant hits (P = 2.4×10−7) are variants in a region on chromosome 12 comprising multiple regulatory elements. This region includes rs12821256, which regulates the expression of KITLG, a well-known gene involved in lighter hair and skin pigmentation in Europeans as well as in thermogenesis. Another variant from that region is associated with the long noncoding RNA RP11-13A1.1, which has been specifically involved in the innate immune response against infectious pathogens. Our results suggest that these genes were relevant for adaptation in Chileans following the Columbian exchange.

Intrinsic and Extrinsic Factors Governing the Transcriptional Regulation of ESR1.

Transcriptional regulation of ESR1, the gene that encodes for estrogen receptor α (ER), is critical for regulating the downstream effects of the estrogen signaling pathway in breast cancer such as cell growth. ESR1 is a large and complex gene that is regulated by multiple regulatory elements, which has complicated our understanding of how ESR1 expression is controlled in the context of breast cancer. Early studies characterized the genomic structure of ESR1 with subsequent studies focused on identifying intrinsic (chromatin environment, transcription factors, signaling pathways) and extrinsic (tumor microenvironment, secreted factors) mechanisms that impact ESR1 gene expression. Currently, the introduction of genomic sequencing platforms and additional genome-wide technologies has provided additional insight on how chromatin structures may coordinate with these intrinsic and extrinsic mechanisms to regulate ESR1 expression. Understanding these interactions will allow us to have a clearer understanding of how ESR1 expression is regulated and eventually provide clues on how to influence its regulation with potential treatments. In this review, we highlight key studies concerning the genomic structure of ESR1, mechanisms that affect the dynamics of ESR1 expression, and considerations towards affecting ESR1 expression and hormone responsiveness in breast cancer.

Human transcription factor YY1 could upregulate the HIV-1 gene expression

Gene expression in HIV-1 is regulated by the promoters in 5’ long-terminal repeat (LTR) element, which contain multiple DNA regulatory elements that serve as binding sites for cellular transcription factors. YY1 could repress HIV-1 gene expression and latent infection. Here, however, we observed that virus production can be increased by YY1 over-expression and decreased under YY1 depleted condition by siRNA treatment. To identify functional domain(s) of YY1 activation, we constructed a number of YY1 truncated mutants. Our data show that full-length YY1 enhances the viral transcription both through U3 and U3RU5 promoters. Moreover, the C-terminal region (296-414 residues) of YY1 is responsible for the transcriptional upregulation, which could be enhanced further in the presence of the viral Tat protein. The central domain of YY1 (155-295 residues) does not affect LTR activity but has a negative effect on HIV-1 gene expression. Taken together, our study shows that YY1 could act as a transcriptional activator in HIV-1 replication, at least in the early stages of infection.

Gene regulatory landscape of the sonic hedgehog locus in embryonic development.

The organs of vertebrate species display a wide variety of morphology. A remaining challenge in evolutionary developmental biology is to elucidate how vertebrate lineages acquire distinct morphological features. Developmental programs are driven by spatiotemporal regulation of gene expression controlled by hundreds of thousands of cis-regulatory elements. Changes in the regulatory elements caused by the introduction of genetic variants can confer regulatory innovation that may underlie morphological novelties. Recent advances in sequencing technology have revealed a number of potential regulatory variants that can alter gene expression patterns. However, a limited number of studies demonstrate causal dependence between genetic and morphological changes. Regulation of Shh expression is a good model to understand how multiple regulatory elements organize tissue-specific gene expression patterns. This model also provides insights into how evolution of molecular traits, such as gene regulatory networks, lead to phenotypic novelty.

Increased BnaMFT-transcript level is associated with secondary dormancy in oilseed rape (Brassica napus L.)

Brassica napus cultivars have little or no primary dormancy; however, they are prone to secondary dormancy induction. Secondary dormant seeds can produce volunteer plants, which can result in genetic contamination, reduced quality and biosafety issues. However, information regarding the molecular mechanism underlying secondary dormancy is limited. The MOTHER OF FT AND TFL1 (MFT) gene, which is evolutionarily conserved in the plant kingdom, acts in a complex gene network in the seed dormancy or germination processes. In this study, we identified four B. napus genes that share high homology with AtMFT, named as BnaMFT. Analyses of cis-acting elements showed that BnaMFT promoters contain multiple seed-specific regulatory elements, and various stress- and hormone-responsive elements. Further experiments validated that BnaMFTs were specifically expressed during seed maturation and in the dry seed, with peaks at 35–42 days after pollination. BnaMFTs were not sufficient for primary dormancy; however, they were significantly enhanced by secondary dormancy induction with PEG6000 treatment. Moreover, BnaMFT transcripts were elevated by treatment with abscisic acid (ABA), which is known to be accumulated during secondary dormancy. These results collectively suggest that increased BnaMFT transcription levels are associated with secondary dormancy induction in an ABA-dependent manner in B. napus.

Engineering Multiple Abiotic Stress Tolerance in Canola, Brassica napus.

Impacts of climate change like global warming, drought, flooding, and other extreme events are posing severe challenges to global crop production. Contribution of Brassica napus towards the oilseed industry makes it an essential component of international trade and agroeconomics. Consequences from increasing occurrences of multiple abiotic stresses on this crop are leading to agroeconomic losses making it vital to endow B. napus crop with an ability to survive and maintain yield when faced with simultaneous exposure to multiple abiotic stresses. For an improved understanding of the stress sensing machinery, there is a need for analyzing regulatory pathways of multiple stress-responsive genes and other regulatory elements such as non-coding RNAs. However, our understanding of these pathways and their interactions in B. napus is far from complete. This review outlines the current knowledge of stress-responsive genes and their role in imparting multiple stress tolerance in B. napus. Analysis of network cross-talk through omics data mining is now making it possible to unravel the underlying complexity required for stress sensing and signaling in plants. Novel biotechnological approaches such as transgene-free genome editing and utilization of nanoparticles as gene delivery tools are also discussed. These can contribute to providing solutions for developing climate change resilient B. napus varieties with reduced regulatory limitations. The potential ability of synthetic biology to engineer and modify networks through fine-tuning of stress regulatory elements for plant responses to stress adaption is also highlighted.

Decoding an organ regeneration switch by dissecting cardiac regeneration enhancers.

Heart regeneration in regeneration-competent organisms can be accomplished through the remodeling of gene expression in response to cardiac injury. This dynamic transcriptional response relies on the activities of tissue regeneration enhancer elements (TREEs); however, the mechanisms underlying TREEs are poorly understood. We dissected a cardiac regeneration enhancer in zebrafish to elucidate the mechanisms governing spatiotemporal gene expression during heart regeneration. Cardiac lepb regeneration enhancer (cLEN) exhibits dynamic, regeneration-dependent activity in the heart. We found that multiple injury-activated regulatory elements are distributed throughout the enhancer region. This analysis also revealed that cardiac regeneration enhancers are not only activated by injury, but surprisingly, they are also actively repressed in the absence of injury. Our data identified a short (22 bp) DNA element containing a key repressive element. Comparative analysis across Danio species indicated that the repressive element is conserved in closely related species. The repression mechanism is not operational during embryogenesis and emerges when the heart begins to mature. Incorporating both activation and repression components into the mechanism of tissue regeneration constitutes a new paradigm that might be extrapolated to other regeneration scenarios.

New insights into the evolution of the SBP-box family and expression analysis of genes in the growth and development of Brassica juncea

The SQUAMOSA promoter binding protein (SBP)-box consists of a family of transcription factors unique to plants and mediates important roles in plant growth and development. We identified 57 SBP genes in the Brassica juncea (swollen-stem mustard) genome, and analyzed the structural characteristics, evolutionary relationships, as well as their involvement in regulation and response to environmental stress. By the help of phylogenetic analysis, the SBP genes were divided into seven subgroups (G1-G7). Interestingly, most members of the same group shared conserved gene structure and motifs as well as gene duplications. This suggests that their functions are redundant highlighting evolutionary relatedness. Identification of the cis-acting elements of the promoter revealed multiple regulatory elements that responded to phytohormone or abiotic stresses. In addition, data from GO annotation indicated that BjuSBPs play a role in biological processes. Further interrogation of the expression of BjuSBPs in different stages of swollen-stem mustard showed spatiotemporal expression patterns. On the other hand, quantitative real-time polymerase chain reaction (qRT-PCR) showed that the expression levels of SBP genes at the development stage correlated with their transcriptome data but treatment with phytohormones affected their expression levels. Our study showed that the SBP-box responds to phytohormone signals and the genes might confer different functions in the development of the mustard plant. Our data lay the foundation for further studies evaluating the regulatory mechanisms of SBP-box family of genes.

A revised model for promoter competition based on multi-way chromatin interactions at the \u03b1-globin locus

Specific communication between gene promoters and enhancers is critical for accurate regulation of gene expression. However, it remains unclear how specific interactions between multiple regulatory elements contained within a single chromatin domain are coordinated. Recent technological advances which can detect multi-way chromatin interactions at single alleles can provide insights into how multiple regulatory elements cooperate or compete for transcriptional activation. Here, we use such an approach to investigate how interactions of the α-globin enhancers are distributed between multiple promoters in a mouse model in which the α-globin domain is extended to include several additional genes. Our data show that gene promoters do not form mutually exclusive interactions with enhancers, but all interact simultaneously in a single complex. These findings suggest that promoters do not structurally compete for interactions with enhancers, but form a regulatory hub structure, which is consistent with recent models of transcriptional activation occurring in non-membrane bound nuclear compartments.

The first enhancer in an enhancer chain safeguards subsequent enhancer-promoter contacts from a distance

BackgroundRobustness and evolutionary stability of gene expression in the human genome are established by an array of redundant enhancers.ResultsUsing Hi-C data in multiple cell lines, we report a comprehensive map of promoters and active enhancers connected by chromatin contacts, spanning 9000 enhancer chains in 4 human cell lines associated with 2600 human genes. We find that the first enhancer in a chain that directly contacts the target promoter is commonly located at a greater genomic distance from the promoter than the second enhancer in a chain, 96 kb vs. 45 kb, respectively. The first enhancer also features higher similarity to the promoter in terms of tissue specificity and higher enrichment of loop factors, suggestive of a stable primary contact with the promoter. In contrast, a chain of enhancers which connects to the target promoter through a neutral DNA segment instead of an enhancer is associated with a significant decrease in target gene expression, suggesting an important role of the first enhancer in initiating transcription using the target promoter and bridging the promoter with other regulatory elements in the locus.ConclusionsThe widespread chained structure of gene enhancers in humans reveals that the primary, critical enhancer is distal, commonly located further away than other enhancers. This first, distal enhancer establishes contacts with multiple regulatory elements and safeguards a complex regulatory program of its target gene.

Genome-wide mapping of DNA-binding sites identifies stemness-related genes as directly repressed targets of SNAIL1 in colorectal cancer cells

At the molecular level, epithelial-to-mesenchymal transition (EMT) necessitates extensive transcriptional reprogramming which is orchestrated by a small group of gene-regulatory factors that include the zinc-finger DNA-binding protein SNAIL1. Although SNAIL1 is a well-known master regulator of EMT, knowledge of its immediate target genes is incomplete. Here, we used ChIP-seq to identify genes directly regulated by SNAIL1 in colorectal adenocarcinoma cells. When comparing the genomic distribution of SNAIL1 to that of the intestinal stem cell (ISC) transcription factors ASCL2 and TCF7L2, we observed a significant overlap. Furthermore, SNAIL1 ChIP-seq peaks are associated with a substantial fraction of ISC signature genes. In two colorectal cancer cell lines, we verified that SNAIL1 decreases ISC marker expression. Likewise, SNAIL1 directly represses the proto-oncogene MYB, and the long noncoding RNA (lncRNA) WiNTRLINC1, a recently described regulator of ASCL2. SNAIL1 targets multiple regulatory elements at the MYB and WiNTRLINC1 loci, and displaces ASCL2 and TCF7L2 from their binding regions at a MYB downstream regulatory element. Correlation analyses and expression profiling showed antiparallel expression of SNAIL1 and MYB in colorectal and breast cancer cell lines and tumor transcriptomes, suggesting that SNAIL1 controls MYB expression in different tissues. MYB loss-of-function attenuated proliferation and impaired clonogenicity in two- and three-dimensional cell cultures. Therefore, SNAIL1-mediated downregulation of MYB and ISC markers like WiNTRLINC1 likely contributes to the decrease in proliferation known to be associated with EMT, while simultaneously abrogating stemness features of colorectal cancer cells. Apparently, the relationship between EMT and stemness varies in different tumor entities.

Two modes of transvection at the eyes absent gene of Drosophila demonstrate plasticity in transcriptional regulatory interactions in cis and in trans

For many genes, proper gene expression requires coordinated and dynamic interactions between multiple regulatory elements, each of which can either promote or silence transcription. In Drosophila, the complexity of the regulatory landscape is further complicated by the tight physical pairing of homologous chromosomes, which can permit regulatory elements to interact in trans, a phenomenon known as transvection. To better understand how gene expression can be programmed through cis- and trans-regulatory interactions, we analyzed transvection effects for a collection of alleles of the eyes absent (eya) gene. We find that trans-activation of a promoter by the eya eye-specific enhancers is broadly supported in many allelic backgrounds, and that the availability of an enhancer to act in trans can be predicted based on the molecular lesion of an eya allele. Furthermore, by manipulating promoter availability in cis and in trans, we demonstrate that the eye-specific enhancers of eya show plasticity in their promoter preference between two different transcriptional start sites, which depends on promoter competition between the two potential targets. Finally, we show that certain alleles of eya demonstrate pairing-sensitive silencing resulting from trans-interactions between Polycomb Response Elements (PREs), and genetic and genomic data support a general role for PcG proteins in mediating transcriptional silencing at eya. Overall, our data highlight how eya gene regulation relies upon a complex but plastic interplay between multiple enhancers, promoters, and PREs.

Chromatin modification factors in plant pathogenic fungi: Insights from Ustilago maydis

Adaptation to the environment is a requirement for the survival of every organism. For pathogenic fungi this also implies coping with the different conditions that occur during the infection cycle. After detecting changes to external media, organisms must modify their gene expression patterns in order to accommodate the new circumstances. Control of gene expression is a complex process that involves the coordinated action of multiple regulatory elements. Chromatin modification is a well-known mechanism for controlling gene expression in response to environmental changes in all eukaryotes. In pathogenic fungi, chromatin modifications are known to play crucial roles in controlling host interactions and their virulence capacity, yet little is known about the specific genes they directly target and to which signals they respond. The smut fungus Ustilago maydis is an excellent model system in which multiple molecular and cellular approaches are available to study biotrophic interactions. Many target genes regulated during the infection process have been well studied, however, how they are controlled and specifically how chromatin modifications affect gene regulation in the context of infection is not well known in this organism. Here, we analyse the presence of chromatin modifying enzymes and complexes in U. maydis and discuss their putative roles in this plant pathogen in the context of findings from other organisms, including other plant pathogens such as Magnaporthe oryzae and Fusarium graminearum. We propose U. maydis as a remarkable organism with interesting chromatin features, which would allow finding new functions of chromatin modifications during plant pathogenesis.

Effects of Fibroblast Growth Factor 1 (FGF‐1) on CCN2 Gene Expression in Chondrocytic Cells

Cartilage and bone are developed by chondrocytes and osteoblasts differentiated from mesenchymal stem cells under the multiple interactions of extracellular signaling molecules. Among such molecules, CCN family protein 2 (CCN2) plays a critical role in the development and regeneration of the cartilage and bone to promote proliferation, differentiation and chondrocyte maturation as well as matrix remodeling during chondrogenesis. CCN2 interacts with several growth factors involved in endochondral ossification, which include fibroblast growth factor 1 (FGF‐1) showing a specific binding with this CCN2. However, the role of FGF‐1 in chondrocyte metabolism has not been investigated well. Therefore, in this study, we evaluated the effect of FGF‐1 on chondrocytes and its role on CCN2 regulation. The effect of FGF‐1 on CCN2 in human chondrocytic HCS‐2/8 cells and the cellular phenotype was estimated by the gene expression of chondrocytic markers. Addition of FGF‐1 to chondrocytic cell culture drastically repressed the mRNA levels of CCN2, with significant repression of other chondrocytic markers in those cells. The ELISA experiments revealed consistent results, in which the protein level of CCN2 was drastically down‐regulated by the addition of FGF‐1. Next, reporter gene assay was used to examine whether the CCN2 regulation by FGF‐1 is mediated by genetic elements involved in the proximal promoter, or not. However, the negative effect of FGF‐1 on CCN2 promoter activity appears to have been too weak to account for the observed decrease at mRNA and protein levels. Another reporter gene assay was performed with a plasmid containing multiple post‐transcriptional regulatory elements in the 3′‐untranslated region of the CCN2 gene. Surprisingly, FGF‐1 has no effect on these elements. Degradation fate of CCN2 mRNA after the addition of actinomycin D was tested, in the presence or absence of FGF‐1, showing unexpected results. Suspecting a regulation at a chromatin level, the effect of a histone deacetylase inhibitor, valproic acid (VPA), on the FGF‐1 function was evaluated, but VPA did not prevent the repressive effect of FGF‐1 on CCN2 gene expression. Further investigation is underway, suspecting a transcriptional regulation through a silencer located outside of the proximal promoter region.Support or Funding InformationJapan Society for the Promotion of Science (JSPS)This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Capturing Complex Enhancer Regulatory Hubs? Try Tri-C

A recent study in Nature Genetics (Oudelaar etal., 2018) has developed a new method to study multi-way chromatin interactions at the allelic level from regulatory elements with high sensitivity and resolution. The method, termed 'Tri-C' is used to explore the combinatorial interactions between multiple regulatory elements underlying precise gene regulation of the globin locus.

A conserved enhancer regulates Il9 expression in multiple lineages

Cytokine genes are regulated by multiple regulatory elements that confer tissue-specific and activation-dependent expression. The cis-regulatory elements of the gene encoding IL-9, a cytokine that promotes allergy, autoimmune inflammation and tumor immunity, have not been defined. Here we identify an enhancer (CNS-25) upstream of the Il9 gene that binds most transcription factors (TFs) that promote Il9 gene expression. Deletion of the enhancer in the mouse germline alters transcription factor binding to the remaining Il9 regulatory elements, and results in diminished IL-9 production in multiple cell types including Th9 cells, and attenuates IL-9-dependent immune responses. Moreover, deletion of the homologous enhancer (CNS-18) in primary human Th9 cultures results in significant decrease of IL-9 production. Thus, Il9 CNS-25/IL9 CNS-18 is a critical and conserved regulatory element for IL-9 production.