Expanding the repertoire of glucocorticoid receptor target genes by engineering genomic response elements

The glucocorticoid receptor (GR), a hormone-activated transcription factor, binds to a myriad of genomic binding sites yet seems to regulate a much smaller number of genes. Genome-wide analysis of GR binding and gene regulation has shown that the likelihood of GR-dependent regulation increases with decreased distance of its binding to the transcriptional start site of a gene. To test if we can adopt this knowledge to expand the repertoire of GR target genes, we used homology directed repair (HDR)-mediated genome editing to add a single GR binding site directly upstream of the transcriptional start site of four genes. To our surprise, we found that the addition of a single GR binding site can be enough to convert a gene into a GR target. The gain of GR-dependent regulation was observed for two out of four genes analyzed and coincided with acquired GR binding at the introduced binding site. However, the gene-specific gain of GR-dependent regulation could not be explained by obvious differences in chromatin accessibility between converted genes and their non-converted counterparts. Further, by introducing GR binding sequences with different nucleotide compositions, we show that activation can be facilitated by distinct sequences without obvious differences in activity between the GR binding sequence variants we tested. The approach to use genome engineering to build genomic response elements facilitates the generation of cell lines with tailored repertoires of GR-responsive genes and a framework to test and refine our understanding of the cis-regulatory logic of gene regulation by testing if engineered response elements behave as predicted.

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Expanding the repertoire of glucocorticoid receptor target genes by engineering genomic response elements

Life Science Alliance ◽

10.26508/lsa.201800283 ◽

2019 ◽

Vol 2 (2) ◽

pp. e201800283 ◽

Cited By ~ 6

Author(s):

Verena Thormann ◽

Laura V Glaser ◽

Maika C Rothkegel ◽

Marina Borschiwer ◽

Melissa Bothe ◽

...

Keyword(s):

Gene Regulation ◽

Glucocorticoid Receptor ◽

Binding Site ◽

Genome Engineering ◽

Target Genes ◽

Transcriptional Start Site ◽

Chromatin Accessibility ◽

Start Site ◽

Response Elements ◽

Genomic Response

The glucocorticoid receptor (GR), a hormone-activated transcription factor, binds to a myriad of genomic binding sites yet seems to regulate a much smaller number of genes. Genome-wide analysis of GR binding and gene regulation has shown that the likelihood of GR-dependent regulation increases with decreased distance of its binding to the transcriptional start site of a gene. To test if we can adopt this knowledge to expand the repertoire of GR target genes, we used CRISPR/Cas-mediated homology-directed repair to add a single GR-binding site directly upstream of the transcriptional start site of each of four genes. To our surprise, we found that the addition of a single GR-binding site can be enough to convert a gene into a GR target. The gain of GR-dependent regulation was observed for two of four genes analyzed and coincided with acquired GR binding at the introduced binding site. However, the gene-specific gain of GR-dependent regulation could not be explained by obvious differences in chromatin accessibility between converted genes and their non-converted counterparts. Furthermore, by introducing GR-binding sequences with different nucleotide compositions, we show that activation can be facilitated by distinct sequences without obvious differences in activity between the GR-binding sequence variants we tested. The approach to use genome engineering to build genomic response elements facilitates the generation of cell lines with tailored repertoires of GR-responsive genes and a framework to test and refine our understanding of the cis-regulatory logic of gene regulation by testing if engineered response elements behave as predicted.

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Characterization of PmfR, the Transcriptional Activator of the pAO1-Borne purU-mabO-folD Operon of Arthrobacter nicotinovorans

Journal of Bacteriology ◽

10.1128/jb.187.9.3062-3070.2005 ◽

2005 ◽

Vol 187 (9) ◽

pp. 3062-3070 ◽

Cited By ~ 14

Author(s):

Calin B. Chiribau ◽

Cristinel Sandu ◽

Gabor L. Igloi ◽

Roderich Brandsch

Keyword(s):

Binding Site ◽

Transcriptional Start Site ◽

Transcriptional Activator ◽

Open Reading Frames ◽

Start Site ◽

Gene Encoding ◽

Chloramphenicol Resistance ◽

Nuclease Digestion ◽

Arthrobacter Nicotinovorans

ABSTRACT Nicotine catabolism by Arthrobacter nicotinovorans is linked to the presence of the megaplasmid pAO1. Genes involved in this catabolic pathway are arranged on the plasmid into gene modules according to function. During nicotine degradation γ-N-methylaminobutyrate is formed from the pyrrolidine ring of nicotine. Analysis of the pAO1 open reading frames (ORF) resulted in identification of the gene encoding a demethylating γ-N-methylaminobutyrate oxidase (mabO). This gene was shown to form an operon with purU- and folD-like genes. Only in bacteria grown in the presence of nicotine could transcripts of the purU-mabO-folD operon be detected, demonstrating that this operon constitutes part of the pAO1 nicotine regulon. Its transcriptional start site was determined by primer extension analysis. Transcription of the operon was shown to be controlled by a new transcriptional regulator, PmfR, the product of a gene that is transcribed divergently from the purU, mabO, and folD genes. PmfR was purified, and electromobility shift assays and DNase I-nuclease digestion experiments were used to determine that its DNA binding site is located between −48 and −88 nucleotides upstream of the transcriptional start site of the operon. Disruption of pmfR by homologous recombination with a chloramphenicol resistance cassette demonstrated that PmfR acts in vivo as a transcriptional activator. Mutagenesis of the PmfR target DNA suggested that the sequence GTTT-14 bp-AAAC is the core binding site of the regulator upstream of the −35 promoter region of the purU-mabO-folD operon.

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Independent Regulation of MucD, an HtrA-Like Protease in Pseudomonas aeruginosa, and the Role of Its Proteolytic Motif in Alginate Gene Regulation

Journal of Bacteriology ◽

10.1128/jb.188.8.3134-3137.2006 ◽

2006 ◽

Vol 188 (8) ◽

pp. 3134-3137 ◽

Cited By ~ 39

Author(s):

Lynn F. Wood ◽

Dennis E. Ohman

Keyword(s):

Pseudomonas Aeruginosa ◽

Gene Regulation ◽

Transcriptional Start Site ◽

Single Copy ◽

Start Site ◽

Temperature Resistance

ABSTRACT Expression of mucD, encoding a homologue of the HtrA(DegP) family of endoserine proteases, was investigated in Pseudomonas aeruginosa. Expressed from the algT-mucABCD operon, MucD was detected in mucoid (FRD1) and nonmucoid (PAO1) parental strains and also when polar insertions were placed upstream in algT or mucB. A transcriptional start site for a mucD promoter (PmucD) was mapped within mucC. Expression of single-copy mucD217, encoding MucD altered in the protease motif (S217A), was defective in temperature resistance and alginate gene regulation.

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Transcriptional Regulation ofFucosyltransferase 1Gene Expression in Colon Cancer Cells

The Scientific World JOURNAL ◽

10.1155/2013/105464 ◽

2013 ◽

Vol 2013 ◽

pp. 1-9 ◽

Cited By ~ 9

Author(s):

Fumiko Taniuchi ◽

Koji Higai ◽

Tomomi Tanaka ◽

Yutaro Azuma ◽

Kojiro Matsumoto

Keyword(s):

Gene Expression ◽

Colon Cancer ◽

Transcriptional Regulation ◽

Binding Site ◽

Transcriptional Start Site ◽

Dominant Negative ◽

Site Directed Mutagenesis ◽

Luciferase Assay ◽

Start Site ◽

Lewis Y

Theα1,2-fucosyltransferase I (FUT1) enzyme is important for the biosynthesis of H antigens, Lewis B, and Lewis Y. In this study, we clarified the transcriptional regulation of FUT1 in the DLD-1 colon cancer cell line, which has high expression of Lewis B and Lewis Y antigens, expresses theFUT1gene, and showsα1,2-fucosyltransferase (FUT) activity. 5′-rapid amplification of cDNA ends revealed a FUT1 transcriptional start site −10 nucleotides upstream of the site registered at NM_000148 in the DataBase of Human Transcription Start Sites (DBTSS). Using the dual luciferase assay,FUT1gene expression was shown to be regulated at the region −91 to −81 nt to the transcriptional start site, which contains the Elk-1 binding site. Site-directed mutagenesis of this region revealed the Elk-1 binding site to be essential for FUT1 transcription. Furthermore, transfection of the dominant negative Elk-1 gene, and the chromatin immunoprecipitation (CHIp) assay, supported Elk-1-dependent transcriptional regulation ofFUT1gene expression in DLD-1 cells. These results suggest that a defined region in the 5′-flanking region of FUT1 is critical for FUT1 transcription and that constitutive gene expression ofFUT1is regulated by Elk-1 in DLD-1 cells.

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A Novel Negative Fe-Deficiency-Responsive Element and a TGGCA-Type-Like FeRE Control the Expression ofFTR1inChlamydomonas reinhardtii

Journal of Biomedicine and Biotechnology ◽

10.1155/2010/790247 ◽

2010 ◽

Vol 2010 ◽

pp. 1-9 ◽

Cited By ~ 5

Author(s):

Xiaowen Fei ◽

Mats Eriksson ◽

Yajun Li ◽

Xiaodong Deng

Keyword(s):

Iron Deficiency ◽

Chlamydomonas Reinhardtii ◽

Mutational Analysis ◽

Transcriptional Start Site ◽

Regulatory Elements ◽

Fe Deficiency ◽

Start Site ◽

Response Elements ◽

Responsive Element ◽

Necessary And Sufficient

We have reported three Fe-deficiency-responsive elements (FEREs),FOX1, ATX1,andFEA1, all of which are positive regulatory elements in response to iron deficiency inChlamydomonas reinhardtii. Here we describeFTR1, another iron regulated gene and mutational analysis of its promoter. Our results reveal that the FeREs ofFTR1distinguish itself from other iron response elements by containing bothnegativeandpositiveregulatory regions. InFTR1, the−291/−236 region from the transcriptional start site is necessary and sufficient for Fe-deficiency-inducible expression. This region contains two positive FeREs with a TGGCA-like core sequence: the FtrFeRE1 (ATGCAGGCT) at−287/−279 and the FtrFeRE2 (AAGCGATTGCCAGAGCGC) at−253/−236. Furthermore, we identified a novel FERE, FtrFeRE3 (AGTAACTGTTAAGCC) localized at−319/−292, which negatively influences the expression ofFTR1.

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Comparative analysis of dioxin response elements in human, mouse and rat genomic sequences

Nucleic Acids Research ◽

10.1093/nar/gkh782 ◽

2004 ◽

Vol 32 (15) ◽

pp. 4512-4523 ◽

Cited By ~ 151

Author(s):

Y. V. Sun ◽

D. R. Boverhof ◽

L. D. Burgoon ◽

M. R. Fielden ◽

T. R. Zacharewski

Keyword(s):

Gene Expression ◽

Transcriptional Start Site ◽

Genomic Sequences ◽

Mouse Tissue ◽

Start Site ◽

Orthologous Genes ◽

Multiple Sequence ◽

Response Elements

Abstract Comparative approaches were used to identify human, mouse and rat dioxin response elements (DREs) in genomic sequences unambiguously assigned to a nucleotide RefSeq accession number. A total of 13 bona fide DREs, all including the substitution intolerant core sequence (GCGTG) and adjacent variable sequences, were used to establish a position weight matrix and a matrix similarity (MS) score threshold to rank identified DREs. DREs with MS scores above the threshold were disproportionately distributed in close proximity to the transcription start site in all three species. Gene expression assays in hepatic mouse tissue confirmed the responsiveness of 192 genes possessing a putative DRE. Previously identified functional DREs in well-characterized AhR-regulated genes including Cyp1a1 and Cyp1b1 were corroborated. Putative DREs were identified in 48 out of 2437 human–mouse–rat orthologous genes between −1500 and the transcriptional start site, of which 19 of these genes possessed positionally conserved DREs as determined by multiple sequence alignment. Seven of these nineteen genes exhibited 2,3,7,8-tetrachlorodibenzo- p -dioxin-mediated regulation, although there were significant discrepancies between in vivo and in vitro results. Interestingly, of the mouse–rat orthologous genes with a DRE between −1500 and +1500, only 37% had an equivalent human ortholog. These results suggest that AhR-mediated gene expression may not be well conserved across species, which could have significant implications in human risk assessment.

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Cell-specific expression of the macrophage scavenger receptor gene is dependent on PU.1 and a composite AP-1/ets motif.

Molecular and Cellular Biology ◽

10.1128/mcb.14.7.4408 ◽

1994 ◽

Vol 14 (7) ◽

pp. 4408-4418 ◽

Cited By ~ 107

Author(s):

K S Moulton ◽

K Semple ◽

H Wu ◽

C K Glass

Keyword(s):

Gene Family ◽

Binding Site ◽

Transcriptional Control ◽

Transcriptional Start Site ◽

Receptor Gene ◽

Regulatory Elements ◽

Type I ◽

Start Site ◽

Macrophage Differentiation ◽

Ets Domain

The type I and II scavenger receptors (SRs) are highly restricted to cells of monocyte origin and become maximally expressed during the process of monocyte-to-macrophage differentiation. In this report, we present evidence that SR genomic sequences from -245 to +46 bp relative to the major transcriptional start site were sufficient to confer preferential expression of a reporter gene to cells of monocyte and macrophage origin. This profile of expression resulted from the combinatorial actions of multiple positive and negative regulatory elements. Positive transcriptional control was primarily determined by two elements, located 181 and 46 bp upstream of the major transcriptional start site. Transcriptional control via the -181 element was mediated by PU.1/Spi-1, a macrophage and B-cell-specific transcription factor that is a member of the ets domain gene family. Intriguingly, the -181 element represented a relatively low-affinity binding site for Spi-B, a closely related member of the ets domain family that has been shown to bind with relatively high affinity to other PU.1/Spi-1 binding sites. These observations support the idea that PU.1/Spi-1 and Spi-B regulate overlapping but nonidentical sets of genes. The -46 element represented a composite binding site for a distinct set of ets domain proteins that were preferentially expressed in monocyte and macrophage cell lines and that formed ternary complexes with members of the AP-1 gene family. In concert, these observations suggest a model for how interactions between cell-specific and more generally expressed transcription factors function to dictate the appropriate temporal and cell-specific patterns of SR expression during the process of macrophage differentiation.

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Sequences flanking the core-binding site modulate glucocorticoid receptor structure and activity

Nature Communications ◽

10.1038/ncomms12621 ◽

2016 ◽

Vol 7 (1) ◽

Cited By ~ 24

Author(s):

Stefanie Schöne ◽

Marcel Jurk ◽

Mahdi Bagherpoor Helabad ◽

Iris Dror ◽

Isabelle Lebars ◽

...

Keyword(s):

Dna Binding ◽

Glucocorticoid Receptor ◽

Binding Site ◽

Structural Changes ◽

Target Genes ◽

Quaternary Structure ◽

Dimensional Structure ◽

Genomic Context ◽

The Core ◽

Induced Changes

Abstract The glucocorticoid receptor (GR) binds as a homodimer to genomic response elements, which have particular sequence and shape characteristics. Here we show that the nucleotides directly flanking the core-binding site, differ depending on the strength of GR-dependent activation of nearby genes. Our study indicates that these flanking nucleotides change the three-dimensional structure of the DNA-binding site, the DNA-binding domain of GR and the quaternary structure of the dimeric complex. Functional studies in a defined genomic context show that sequence-induced changes in GR activity cannot be explained by differences in GR occupancy. Rather, mutating the dimerization interface mitigates DNA-induced changes in both activity and structure, arguing for a role of DNA-induced structural changes in modulating GR activity. Together, our study shows that DNA sequence identity of genomic binding sites modulates GR activity downstream of binding, which may play a role in achieving regulatory specificity towards individual target genes.

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