Browsing by Subject "Enhancer"

Now showing 1 - 6 of 6
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    Analysis of KLF4 regulated genes in cancer cells reveals a role of DNA methylation in promoter- enhancer interactions
    (Taylor & Francis, 2018) Oyinlade, Olutobi; Wei, Shuang; Kammers, Kai; Liu, Sheng; Wang, Shuyan; Ma, Ding; Huang, Zhi-yong; Qian, Jiang; Zhu, Heng; Wan, Jun; Xia, Shuli; Medical and Molecular Genetics, School of Medicine
    Recent studies have revealed an unexpected role of DNA methylation at promoter regions in transcription activation. However, whether DNA methylation at enhancer regions activates gene expression and influences cellular functions remains to be determined. In this study, by employing the transcription factor krÜppel-like factor 4 (KLF4) that binds to methylated CpGs (mCpGs), we investigated the molecular outcomes of the recruitment of KLF4 to mCpGs at enhancer regions in human glioblastoma cells. First, by integrating KLF4 ChIP-seq, whole-genome bisulfite sequence, and H3K27ac ChIP-seq datasets, we found 1,299 highly methylated (β >0.5) KLF4 binding sites, three-quarters of which were located at putative enhancer regions, including gene bodies and intergenic regions. In the meantime, by proteomics, we identified 16 proteins as putative targets upregulated by KLF4-mCpG binding at enhancer regions. By chromosome conformation capture (3C) analysis, we demonstrated that KLF4 bound to methylated CpGs at the enhancer regions of the B-cell lymphocyte kinase (BLK) and Lim domain only protein 7 (LMO7) genes, and activated their expression via 3D chromatin loop formation with their promoter regions. Expression of mutant KLF4, which lacks KLF4 ability to bind methylated DNA, or removal of DNA methylation in enhancer regions by a DNA methyltransferase inhibitor abolished chromatin loop formation and gene expression, suggesting the essential role of DNA methylation in enhancer-promoter interactions. Finally, we performed functional assays and showed that BLK was involved in glioblastoma cell migration. Together, our study established the concept that DNA methylation at enhancer regions interacts with transcription factors to activate gene expression and influence cellular functions.
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    C/EBPα is an essential collaborator in Hoxa9/Meis1-mediated leukemogenesis
    (PNAS, 2014-07-08) Collins, Cailin; Wang, Jingya; Miao, Hongzhi; Bronstein, Joel; Nawer, Humaira; Xu, Tao; Figueroa, Maria; Muntean, Andrew G.; Hess, Jay L.; Department of Medicine, IU School of Medicine
    Homeobox A9 (HOXA9) is a homeodomain-containing transcription factor that plays a key role in hematopoietic stem cell expansion and is commonly deregulated in human acute leukemias. A variety of upstream genetic alterations in acute myeloid leukemia (AML) lead to overexpression of HOXA9, almost always in association with overexpression of its cofactor meis homeobox 1 (MEIS1) . A wide range of data suggests that HOXA9 and MEIS1 play a synergistic causative role in AML, although the molecular mechanisms leading to transformation by HOXA9 and MEIS1 remain elusive. In this study, we identify CCAAT/enhancer binding protein alpha (C/EBPα) as a critical collaborator required for Hoxa9/Meis1-mediated leukemogenesis. We show that C/EBPα is required for the proliferation of Hoxa9/Meis1-transformed cells in culture and that loss of C/EBPα greatly improves survival in both primary and secondary murine models of Hoxa9/Meis1-induced leukemia. Over 50% of Hoxa9 genome-wide binding sites are cobound by C/EBPα, which coregulates a number of downstream target genes involved in the regulation of cell proliferation and differentiation. Finally, we show that Hoxa9 represses the locus of the cyclin-dependent kinase inhibitors Cdkn2a/b in concert with C/EBPα to overcome a block in G1 cell cycle progression. Together, our results suggest a previously unidentified role for C/EBPα in maintaining the proliferation required for Hoxa9/Meis1-mediated leukemogenesis.
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    Identification of Aedes aegypti cis-regulatory elements that promote gene expression in olfactory receptor neurons of distantly related dipteran insects
    (BMC, 2018-07-11) Mysore, Keshava; Li, Ping; Duman-Scheel, Molly; Medical and Molecular Genetics, School of Medicine
    BACKGROUND: Sophisticated tools for manipulation of gene expression in select neurons, including neurons that regulate sexually dimorphic behaviors, are increasingly available for analysis of genetic model organisms. However, we lack comparable genetic tools for analysis of non-model organisms, including Aedes aegypti, a vector mosquito which displays sexually dimorphic behaviors that contribute to pathogen transmission. Formaldehyde-assisted isolation of regulatory elements followed by sequencing (FAIRE-seq) recently facilitated genome-wide discovery of putative A. aegypti cis-regulatory elements (CREs), many of which could be used to manipulate gene expression in mosquito neurons and other tissues. The goal of this investigation was to identify FAIRE DNA elements that promote gene expression in the olfactory system, a tissue of vector importance. RESULTS: Eight A. aegypti CREs that promote gene expression in antennal olfactory receptor neurons (ORNs) were identified in a Drosophila melanogaster transgenic reporter screen. Four CREs identified in the screen were cloned upstream of GAL4 in a transgenic construct that is compatible with transformation of a variety of insect species. These constructs, which contained FAIRE DNA elements associated with the A. aegypti odorant coreceptor (orco), odorant receptor 1 (Or1), odorant receptor 8 (Or8) and fruitless (fru) genes, were used for transformation of A. aegypti. Six A. aegypti strains, including strains displaying transgene expression in all ORNs, subsets of these neurons, or in a sex-specific fashion, were isolated. The CREs drove transgene expression in A. aegypti that corresponded to endogenous gene expression patterns of the orco, Or1, Or8 and fru genes in the mosquito antenna. CRE activity in A. aegypti was found to be comparable to that observed in D. melanogaster reporter assays. CONCLUSIONS: These results provide further evidence that FAIRE-seq, which can be paired with D. melanogaster reporter screening to test FAIRE DNA element activity in select tissues, is a useful method for identification of mosquito cis-regulatory elements. These findings expand the genetic toolkit available for the study of Aedes neurobiology. Moreover, given that the CREs drive comparable olfactory neural expression in both A. aegypti and D. melanogaster, it is likely that they may function similarly in multiple dipteran insects, including other disease vector mosquito species.
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    In vivo analysis of human LHX3 enhancer regulation
    (2014-01-03) Park, Soyoung; Rhodes, Simon J.; Day, Richard N.; Harrington, Maureen A.; Herring, B. Paul; Skalnik, David Gordon
    The LHX3 transcription factor is essential for pituitary gland and nervous system development in mammals. In humans, mutations in the LHX3 gene underlie combined pituitary hormone deficiency (CPHD) disease featuring deficits in anterior pituitary hormones and defects in the nervous system. The mechanisms that control temporal and spatial expression of the LHX3 gene are poorly understood. The proximal promoters of the human LHX3 gene are insufficient to guide expression in vivo and downstream elements including a conserved 7.9 kilobase (kb) enhancer region appear to play a role in tissue-specific expression in the pituitary and nervous system. In this study, I characterized the activity of this downstream enhancer region in regulating gene expression at the cellular level during development. Human LHX3 enhancer-driven Cre reporter transgenic mice were generated to facilitate studies of enhancer actions. The downstream LHX3 enhancer primarily guides gene transcription in αGSU-expressing cells secreting the TSHβ, LHβ or FSHβ hormones and expressing the GATA2 and SF1 transcription factors. In the developing nervous system, the enhancer serves as a targeting module for expression specifically in V2a interneurons. These results demonstrate that the downstream LHX3 enhancer is important in specific endocrine and neural cell types but also indicate that additional regulatory elements are likely involved in LHX3 gene expression in other cell types. Further, these studies demonstrate significant gonadotrope cell heterogeneity during pituitary development, providing insights into the cellular physiology of this key reproductive regulatory cell. The human LHX3 enhancer-driven Cre reporter transgenic mice provide a valuable tool for further developmental studies of cell determination and differentiation in the pituitary and nervous system. Furthermore understanding the regulation of human LHX3 gene will help develop tools to better diagnose and treat pituitary CPHD disease.
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    Sost, independent of the non-coding enhancer ECR5, is required for bone mechanoadaptation
    (Elsevier, 2016-11) Robling, Alexander G.; Kang, Kyung Shin; Bullock, Whitney A.; Foster, William H.; Murugesh, Deepa; Loots, Gabriela G.; Genetos, Damian C.; Anatomy and Cell Biology, School of Medicine
    Sclerostin (Sost) is a negative regulator of bone formation that acts upon the Wnt signaling pathway. Sost is mechanically regulated at both mRNA and protein level such that loading represses and unloading enhances Sost expression, in osteocytes and in circulation. The non-coding evolutionarily conserved enhancer ECR5 has been previously reported as a transcriptional regulatory element required for modulating Sost expression in osteocytes. Here we explored the mechanisms by which ECR5, or several other putative transcriptional enhancers regulate Sost expression, in response to mechanical stimulation. We found that in vivo ulna loading is equally osteoanabolic in wildtype and Sost-/- mice, although Sost is required for proper distribution of load-induced bone formation to regions of high strain. Using Luciferase reporters carrying the ECR5 non-coding enhancer and heterologous or homologous hSOST promoters, we found that ECR5 is mechanosensitive in vitro and that ECR5-driven Luciferase activity decreases in osteoblasts exposed to oscillatory fluid flow. Yet, ECR5-/- mice showed similar magnitude of load-induced bone formation and similar periosteal distribution of bone formation to high-strain regions compared to wildtype mice. Further, we found that in contrast to Sost-/- mice, which are resistant to disuse-induced bone loss, ECR5-/- mice lose bone upon unloading to a degree similar to wildtype control mice. ECR5 deletion did not abrogate positive effects of unloading on Sost, suggesting that additional transcriptional regulators and regulatory elements contribute to load-induced regulation of Sost.
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    Transcription factors and cis-acting elements in T helper cell cytokine expression
    (2017-12-15) Koh, Byunghee; Kaplan, Mark H.; Zhou, Baohua; Blum, Janice S.; Harrington, Maureen A.
    The immune system provides resistance to the myriad of pathogens in the environment, but can also respond inappropriately causing allergic inflammation and autoimmune disease. CD4+ T cells, which play a crucial role in adaptive immune system, can be divided into several subsets based on their effector functions. T helper 9 (Th9) cells, derived by the IL-4/STAT6 and TGF-β signaling pathways, produce IL-9 as a hallmark cytokine, as well as IL-10. Through IL-9 production, Th9 cells protect against parasite infection but are also involved in allergic inflammation and autoimmune diseases. Transcription factors that promote Th9 development include STATs, PU.1, BATF, and IRF4. In this study, we identify ETV5 as a factor that promotes IL-9 and IL-10 production by binding to cis-acting regulatory elements in the respective genes. At the Il9 gene, ETV5 cooperates with PU.1 in regulating gene expression. At the Il10 gene, ETV5 facilitates binding of other transcription factors to the locus. These studies and others suggested that there may be additional cis-acting regulatory elements in the Il9 gene. We demonstrate that a conserved noncoding sequence (CNS) located 25 kb upstream of the Il9 transcription start site, termed Il9 CNS-25, is critical for regulating Il9 expression in Th cell subsets. Th9 cells derived from Il9 CNS-25 mutant (Il9 ΔCNS-25) mice produce significantly less IL-9. Il9 CNS-25 promoted chromatin modifications at the promoter and accessibility of the locus. Il9 ΔCNS-25 mice showed attenuated airway inflammation compared to control mice. The Il9 CNS-25 region in mice is conserved with an IL9 CNS-18 region in the human genome. We deleted CNS-18 in primary human Th9 cells and observed diminished IL-9 production. Thus, we have identified transcription factors that regulate multiple cytokines in Th cell lineages and have demonstrated that the Il9 CNS-25/IL9 CNS-18 elements are respectively critical for Il9/IL9 gene expression.