Mus musculus) model of asthma applied to the Collaborative Cross population, we previously identified a lung gene expression quantitative trait locus (eQTL) for Zinc finger protein 30 (Zfp30) that was also a QTL for neutrophil recruitment and the hallmark neutrophil chemokine CXCL1. The Zfp30 eQTL is defined by three functionally distinct haplotypes. In this study, we searched for causal genetic variants that underlie the Zfp30 eQTL to gain a better understanding of this candidate repressor’s regulation. First, we identified a putative regulatory region spanning 500 bp upstream of Zfp30, which contains 10 SNPs that form five haplotypes. In reporter gene assays in vitro, these haplotypes recapitulated the three previously identified in vivo expression patterns. Second, using site-directed mutagenesis followed by reporter gene assays, we identified a single variant, rs51434084, which explained the majority of variation in expression between two out of three haplotype groups. Finally, using a combination of in silico predictions and electrophoretic mobility shift assays, we identified ZFP148 as a transcription factor that differentially binds to the Zfp30 promoter region harboring rs51434084. In conclusion, we provide evidence in support of rs51434084 being a causal variant for the Zfp30 eQTL, and have identified a mechanism by which this variant alters Zfp30 expression, namely differential binding of ZFP148.Neutrophil chemotaxis to the airways is a key aspect of host response to microbes and a feature of multiple pulmonary diseases including asthma. Tight regulation of this recruitment is critical to prevent unwanted host tissue damage and inflammation. Using a mouse ( Recruitment of neutrophils to the lung is a hallmark of innate immune responses to inhaled pathogens and air pollutants, and is associated with decreased lung function and disease susceptibility (Dpc1 (Zfp30) was also regulated by this locus , and Zfp30 expression was strongly correlated with CXCL1 and neutrophil counts . We also found that Zfp30 is expressed in airway epithelia and that knockdown of Zfp30 in an epithelial cell line resulted in increased CXCL1 production in response to endotoxin. These results led to development of a model in which ZFP30 negatively regulates CXCL1, and CXCL1 then affects neutrophil recruitment, all under the control of Dpc1.Previously, we applied a house dust mite model of asthma to incipient lines of the Collaborative Cross (CC) population to identify novel genes and pathways associated with neutrophil recruitment responses in the context of allergic inflammation. The CC is a new mouse genetics reference population composed of recombinant inbred lines that are derived from eight founder strains . We measZfp30 except that it encodes an as yet uncharacterized C2H2 zinc finger protein that contains a Krüppel-associated box domain, signifying a role in gene repression via heterochromatin formation  to interrogate the effect that specific variants have on Zfp30 expression. In aggregate, our data point to a causal variant responsible for the modulation of Zfp30 expression, and show that this variant alters transcription factor binding.These findings prompted us to seek to identify the specific variant(s) that regulate 2. Twenty-four-well plates were seeded at a density of 120,000 cells per well for MLE12 or 125,000 cells per well for LA4 24 hr prior to transfection for luciferase assays.The MLE12 mouse lung epithelia cell line (generated from FVB/N mouse strain) was cultured in a 1:1 mix of Dulbecco’s Modified Eagle Medium and Ham’s F12 supplemented with 5% fetal bovine serum (FBS). The LA4 mouse lung airway cell line (generated from the A/He mouse strain) was cultured in Ham’s F12K medium supplemented with 15% FBS. Both cell lines were grown at 37° with 5% COZfp30 promoter surrounding a euchromatic region containing a variant of interest was amplified from five of the eight CC founder strains. Amplified candidate promoters were subcloned into the multiple cloning site upstream of the firefly luciferase gene in the promoterless pGL4.10 vector  using a Gateway subcloning approach, and then verified by sequencing. MLE12 or LA4 cell lines were cotransfected in 24-well cell culture plates with 500 ng of candidate promoter constructs and 28 ng of Renilla control vector pRL-SV40 (Promega), using Lipofectamine 3000 , according to manufacturer’s protocol. Transfected cells were cultured 48 hr before cell lysates were collected. Luciferase activity was measured using the Dual Luciferase Assay System (Promega). Firefly luciferase activity was normalized to Renilla luciferase activity as a control for transfection efficiency. Data are reported as the ratio of firefly to Renilla luciferase activity, and these data are provided in Table S1. Assays include four replicates for each candidate promoter and two technical replicates for each lysate collected. Luciferase assays were performed in each cell line at least two times, and results were consistent across experiments. Welch’s two-sided t-tests were performed to compare luciferase activity between promoter constructs.A 500 bp genomic region of the Renilla luciferase was assayed in MLE12 RNA samples collected 48 hr post-transfection with candidate promoters and pRL-SV40. iTaq Universal SYBR Green Supermix  was used in conjunction with luc2 primers  or Renilla luciferase primers . We validated the qPCR primers for efficiency by performing assays with serial dilutions of template and verifying the expected changes in Cq values. In subsequent assays, Renilla luciferase levels were used to normalize luc2 Cq values, and fold change in expression of A/J and 129S1/SvImJ over C57BL/6J was calculated.mRNA expression of firefly and http://www.sanger.ac.uk/sanger/Mouse_SnpViewer/rel-1505) to identify variants within our cloned promoter region, which spans 29783622–29784093 bp on chromosome 7. These variants are shown in We utilized the Sanger Mouse Genomes Project variant database  according to manufacturer’s protocol. Primers  were designed to mutate the position corresponding to rs51434084 from C to G.Complementary 17 bp oligonucleotide probes centered on rs51434084 were purchased from  for use in EMSA experiments. Sequences for the moderate (C57BL/6J) and high (A/J) expression haplotypes, respectively, were as follows: Fwd: 5′-CCCTTCCCCTGACCAAGAG-3′; Rev: 5′-CTCTTGGTCAGGGGAAGGG-3′; and Fwd: 5′-CCCTTCCCGTGACCAAGAG-3′; Rev: 5′-CTCTTGGTCACGGGAAGGG-3′. Sets of complementary probes were ordered with and without biotin end-labeling (Integrated DNA Technologies). Complementary oligos were annealed into double-stranded DNA probes at a concentration of 2 pmol/μl by heating complimentary oligos to 95° in an annealing buffer  and cooling them to 25° in a thermocycler at a rate of 1° per minute. NE-PER Nuclear and Cytoplasmic Extraction Reagents  were used to collect nuclear protein lysates from MLE12 cells. Protein concentrations were quantified using the Pierce BCA Protein Assay (Thermo Scientific).Figure S3) antibody incubated for 30 min prior to addition of biotinylated probe. Antibodies for ZFP148  and KLF4  were ordered for use in EMSA supershifts. DNA–protein complexes were separated on 6% DNA retardation gels (Life Technologies) using 0.5× TBE buffer (Life Technologies). Complexes were transferred to Biodyne B Pre-Cut Modified Nylon Membranes (Thermo Scientific) and UV cross-linked before chemiluminescent detection.EMSA experiments were performed using the LightShift Chemiluminescent EMSA Kit (Thermo Scientific) according to the manufacturer’s protocol. DNA–protein binding reactions contained 1× binding buffer, 1 μg poly(dIdC), 4 μg nuclear protein lysate, 200 fmol biotinylated DNA probe, and water, for a total reaction volume of 20 μl. Reactions were incubated at room temperature for 30 min prior to gel loading. Reactions including nonbiotinylated probes for DNA competition were incubated with 45-fold excess unlabeled probe for 25 min prior to addition of biotinylated probe. ZFP148 supershift reactions contained either 10 μg  or 20 μghttp://gene-regulation.com/pub/programs.html#match) and HOCOMOCO (http://opera.autosome.ru/perfectosape/scan/new) prediction tools. Based on EMSA results indicating enhanced binding of nuclear proteins to the C57BL/6J allele (vs. 129S1/SvImJ), we limited our search to putative transcription factor binding sites in which the C57BL/6J allele is predicted to bind the transcription factor better than the 129S1/SvImJ allele. We then performed literature searches on the initial list of candidates to prioritize any transcription factors with a known connection to regulation of neutrophil recruitment.To identify transcription factor binding sites potentially affected by rs51434084, we used TRANSFAC  responsible for the er lines , indicatZfp30 expression, we examined ENCODE data for euchromatic regions marked by DNAse I hypersensitivity near the 5′ region of Zfp30 in mouse lung tissue, because our previous data indicated a strong correlation between 5′ region haplotypes and gene expression , which contains 10 SNPs. An examination of the strain distribution patterns within the identified region of open chromatin at the Zfp30 promoter revealed a single shared SNP (rs51434084) among the high expression strains and six shared SNPs among the low-expressing strains  and LA4 (P < 0.05) cell lines , and literature searches revealed that two candidates have established roles in immune regulation and have repressive transcriptional activity, rendering them priority candidates. ZFP148  has been shown to regulate CXCL5, a chemokine similar to CXCL1 in function , indicating that ZFP148 is among the DNA–protein complexes identified in EMSA experiments with a supershift condition did not reveal a supershift with KLF4 antibody (data not shown). In contrast, addition of ZFP148 antibody produced a supershift . The expression pattern seen in the LA4 cell line (generated from the A/He mouse strain) matched mRNA expression results seen in vivo, but the pattern seen in MLE12 (generated from FVB/N mouse strain) did not. This suggests that these cell lines differentially express a repressor protein that downregulates expression of Zfp30 in CAST/EiJ and PWK/PhJ. Future experiments will be directed toward identifying the causal variant(s) and protein(s) responsible for this modulation of Zfp30 expression.Our reporter assays with Zfp30 promoter region and thereby regulate Zfp30 expression. Thus, we propose that our initial model, in which Zfp30 expression regulates CXCL1 and neutrophil recruitment (of Cxcl5, a CXCR2 ligand similar to CXCL1, in human colonic epithelial cells (Cxcl5 and Zfp30. These functions may seem contradictory at first, as Zfp30 is proposed to downregulate Cxcl1, but decreased expression of Cxcl5 has actually been linked to increased neutrophil recruitment into the lungs in an Escherichia coli pneumonia mouse model (Our EMSA results indicate that ZFP148 is among the transcription factors that bind to the rs51434084 locus in the Cxcl1 is a priority locus of interest; however, KAP-1/zinc finger protein complexes could have multiple binding sites and can act over tens of kilobases (ZFP30 is a C2H2 zinc finger protein with domains that suggest DNA-binding and KAP-1–binding functions. Wherever ZFP30 binds in the genome, a complex of proteins recruited by KAP-1 are predicted to induce heterochromatin domains and silence nearby genes (Zfp30 is regulated as a function of an allelic variant, which we previously showed was linked to neutrophils in the airways in a mouse model. Future work examining the relevance of the genes/proteins studied here, namely ZFP148 and ZFP30, in neutrophil chemotaxis in other model systems and in human patients is a critical next step to evaluating the biomedical importance of our findings. Additionally, identification and characterization of additional functional variants that impact neutrophil recruitment will further develop our understanding of the complex regulatory networks that drive disease severity phenotypes in both model systems and human patients with asthma, acute lung injury, cystic fibrosis, or COPD.This work contributes to an increased understanding of how www.g3journal.org/lookup/suppl/doi:10.1534/g3.117.300507/-/DC1.Supplemental material is available online at Click here for additional data file.Click here for additional data file.Click here for additional data file.Click here for additional data file.Click here for additional data file.Click here for additional data file.


Bptf-deficient HSCs are defective in reconstituted hematopoiesis, and hematopoietic-specific knockout of Bptf caused profound defects including bone marrow failure and anemia. Genome-wide transcriptome profiling revealed that BPTF loss caused downregulation of HSC-specific gene-expression programs, which contain several master transcription factors  required for HSC maintenance and self-renewal. Furthermore, we show that BPTF potentiates the chromatin accessibility of key HSC “stemness” genes. These results demonstrate an essential requirement of the chromatin remodeler BPTF and NURF for activation of “stemness” gene-expression programs and proper function of adult HSCs.Self-renewal and differentiation of adult stem cells are tightly regulated partly through configuration of chromatin structure by chromatin remodelers. Using knockout mice, we here demonstrate that bromodomain PHD finger transcription factor (BPTF), a component of the nucleosome remodeling factor (NURF) chromatin-remodeling complex, is essential for maintaining the population size of hematopoietic stem/progenitor cells (HSPCs), including long-term hematopoietic stem cells (HSCs).  •Bptf shows preferential expression in primitive HSPCsThe chromatin remodeler gene •Bptf ensures maintenance and functionality of HSCs in a cell-autonomous manner•Bptf is required for the activation of HSC “stemness” genes including master TFs•Hematopoietic-specific loss of Bptf results in bone marrow failure and anemia Wang and colleagues show that a chromatin remodeler, BPTF, sustains appropriate functions of hematopoietic stem/progenitor cells (HSPCs). BPTF loss causes bone marrow failure and anemia. The authors further define a BPTF-dependent gene-expression program in HSPCs, which contains key HSC stemness factors. These results demonstrate an essential requirement of the BPTF-associated chromatin remodelers for HSC functionality and adult hematopoiesis. Appropriate self-renewal and differentiation of adult stem cells are essential for tissue homeostasis and are tightly controlled by various cellular and molecular mechanisms, including the dynamic regulation of chromatin structure by ATP-dependent chromatin-remodeling complexes . These rBptf in mice leads to lethality on embryonic day 8.5, demonstrating its requirement for early development  is a core and largest component of the conserved, multi-subunit nucleosome remodeling factor (NURF) complex . NURF loelopment , clinicanomalies . Furthernomalies , melanocnomalies , and T cnomalies . BPTF conomalies . Depositnomalies . While pnomalies , the speBptf-null HSCs exhibited the decreased repopulating capacity, causing severe hematopoietic defects. Our genomic profiling shows that ablation of BPTF in hematopoietic stem/progenitor cells (HSPCs) leads to decreased expression of an HSC-specific gene-expression program, which includes a master transcription factor (TF) regulatory node  known to be crucial for HSC self-renewal and function. We also find that BPTF potentiates the chromatin accessibility of these HSC TF genes. Collectively, our results support a vital requirement of the BPTF chromatin remodeler for the maintenance of adult HSPCs and for the activation of a gene transcription program essential for HSC functions.Using knockout mice, we here show BPTF as a crucial chromatin regulator of hematopoietic stem cells (HSCs). Reconstitution assays demonstrate that Bptf preferentially expressed in the primitive HSPC compartment  designed to ablate Bptf from the bone marrow (BM) upon activation of Mx1-cre by polyinosinic-polycytidylic acid (pIpC). We verified efficient deletion (>95%) of Bptf in the BM  via genotyping and RT-PCR to confirm our model  and 4 weeks after cre induction, we observed a significantly reduced total number of lineage−/SCA-1+/c-KIT+ (LSK) cells and long-term (LT)-HSCs . BptfcKO and Bptff/f LSK cells expressed comparable levels of cKit, an LSK marker (cKit), and comparison of their RNA-seq profiles identified 407 downregulated and 230 upregulated transcripts due to Bptf ablation  showed decreased expression in BptfcKO LSK cells  to profile transcriptomes of the LSK cells sorted from nduction A. As expdeletion A. This p control B, Bptf. K marker B, cKit, and Lmo2 B. Previoand Lmo2 . Consistre genes  and thosre genes  . We used HPC-7 cells because ChIP requires large cell numbers, which prevents the use of primary HSPCs, and HPC-7 cells were previously used as an HSPC mimic to map the genomic binding of HSC regulators . ChIP-seand Lmo2 D. Becausnduction B. Upon B signals C but obs signals E such asand Lmo2 A. We alsand Lmo2 A. TogethBptfcKO HSCs, we predicted that lineage-committed populations in BptfcKO mice would be affected. Four weeks after Bptf deletion, the bones from BptfcKO mice appeared pale and showed significant decrease in the total BM cell number when compared with control , the AP1 complex, and the MLL/KMT2A signature genes. BPTF also sustains an open chromatin state at target “stemness” genes. Thus, BPTF acts as a safeguard of adult hematopoiesis, ensuring HSCs' reconstitution function. In support, BPTF loss caused BM failure phenotypes that are reminiscent of what was observed for KMT2A-null HSCs  or Mx1-cre were purchased from the Jackson Laboratory and crossed to produce Bptff/f;Mx1-cre mice and control littermates. To induce Bptf knockout in the BM, we injected 2- to 3-month-old mice with pIpC (Sigma) three times every other day. UNC-Chapel Hill Institutional Animal Care and Use Committee approved all animal experiments.CD45.2RNA was extracted from sorted LSK cells with the picoRNA Kit (Applied Biosystems) and the RNA-seq library was prepared with Illumina kits according to the manufacturer’s protocol, followed by deep sequencing.G.G.W. designed the project. B.X., L.C., J.M.B., X.L., D.F.A., R.L., S.R., and G.G.W. performed experiments and interpreted data. J.S.P., D.Z., and D.C. analyzed genomic data. G.G.W. wrote the manuscript, and D.F.A. and B.X. critically read the paper.


Aqp2 gene transcription or whether it triggers a broadly targeted transcriptional network. ChIP-Seq quantification of binding sites for RNA polymerase II was combined with RNA-Seq quantification of transcript abundances to identify genes whose transcription is regulated by vasopressin. (View curated dataset at https://helixweb.nih.gov/ESBL/Database/Vasopressin/). The analysis revealed only 35 vasopressin-regulated genes (of 3659) including Aqp2. Increases in RNA polymerase II binding and mRNA abundances for Aqp2 far outstripped corresponding measurements for all other genes, consistent with the conclusion that vasopressin-mediated transcriptional regulation is highly selective for Aqp2. Despite the overall selectivity of the net transcriptional response, vasopressin treatment was associated with increased RNA polymerase II binding to the promoter proximal region of a majority of expressed genes, suggesting a nearly global positive regulation of transcriptional initiation with transcriptional pausing. Thus, the overall net selectivity appears to be a result of selective control of transcriptional elongation.Vasopressin-mediated regulation of renal water excretion is defective in a variety of water balance disorders in humans. It occurs in part through long-term mechanisms that regulate the abundance of the aquaporin-2 water channel in renal collecting duct cells. Here, we use deep DNA sequencing in mouse collecting duct cells to ask whether vasopressin signaling selectively increases  The basic idea of systems biology is to investigate a biological process by studying all relevant components together in parallel to discover mechanismAqp2 gene regulation in the context of data regarding every other expressed gene.In mammals, the peptide hormone vasopressin controls renal water excretion, largely through its actions in the renal collecting duct to regulate the molecular water channel aquaporin-2. Two modes of physiological regulation have been identified: 1) short-term regulation by membrane trafficking134Next-generation DNA sequencing is a recently developed methodology that enables relatively inexpensive large-scale DNA sequencing, and is practical for individual small laboratories pursuing targeted questions like the one in this paper7RNA-seq is an approach, based on deep sequencing of DNA, that allows complete transcriptomes to be identified for a given cell type and permits quantitative analysis of experimental effects on every transcript. ChIP-seq is an approach that comprehensively identifies DNA binding sites for particular proteins over the entire genome. It combines the method of chromatin immuno-precipitation using antibodies specific to a particular protein  with deep sequencing.Cultured mpkCCD cells have been a useful model for understanding regulatory processes in principal cells of the mammalian collecting duct4per se. Although vasopressin increases aquaporin-2 mRNA levels41314Aqp2 gene or is due to a decrease in the degradation rate of the aquaporin-2 mRNA. Data from prior studies in cultured mpkCCD cells suggest that vasopressin does not alter aquaporin-2 mRNA stability16Aqp2 gene in response to vasopressin. To address this, we used ChIP-seq to identify and quantify RNA polymerase II binding throughout the genome. The comprehensive nature of this method provides information about the selectivity of vasopressin’s effect on Aqp2 gene transcription. To provide additional data on the transcriptional effects of vasopressin signaling in collecting duct cells using an independent methodology, we also carried out RNA-seq to identify and measure transcriptome-wide mRNA abundance changes in response to vasopressin.Prior studies have demonstrated that vasopressin increases the steady-state half-life of the aquaporin-2 protein11Aqp2 gene body associated with a large increase in aquaporin-2 mRNA. Of only 35 genes with coincident changes in RNA polymerase II binding and mRNA levels in response to vasopressin, the increases for Aqp2 were by far the greatest, indicating a highly selective effect of vasopressin signaling on Aqp2 gene transcription. Interpreting the results in terms of Shannon information contentOverall, the results show a highly significant increase in RNA polymerase II occupancy across the These studies were done in cultured mouse collecting duct cells (mpkCCD) re-cloned in our laboratory to maximize the response to vasopressinAqp2) for one pair of samples, revealing a marked increase with dDAVP. This figure uses a logarithmic scale for the vertical axis to view the pattern of mapped reads both in the presence and absence of dDAVP.  value for all replicates was 4.52 ± 0.85 , consistent with the prior conclusion that vasopressin markedly increases aquaporin-2 mRNA levels in mpkCCD cells4https://helixweb.nih.gov/ESBL/Database/AVP_Transcr/.To quantify changes in aquaporin-2 mRNA in response to dDAVP and compare the responses to those of all other genes, we used RNA-seq. Nine replicates were analyzed in both dDAVP- and vehicle-treated cells to maximize the ability to detect small changes. 2(dDAVP/vehicle) values for all transcripts. The vertical axis of the plot shows P values from t-tests comparing all dDAVP-treated samples to all vehicle-treated samples. The dashed red lines indicate threshold values  > 2 × SEcc for horizontal axis, where SEcc is the median standard deviation for 3 separate control:control pairs). For this study, both thresholds must be exceeded to consider the transcript significantly changed in abundance, providing a relatively stringent criterion favoring false negatives over false positives. Aqp2 responses to those of all other genes, we used ChIP-seq. This method maps the polymerase as it traverses the gene from 5′ to 3′ to produce mRNA. Thus, changes in transcription can be expected to be associated with coordinate changes in RNA polymerase II binding. The cells are harvested after exposure to dDAVP (0.1 nM) or its vehicle for 24 hours. We profiled 3 experimental pairs (vehicle vs dDAVP). Aqp2 gene for one vehicle:dDAVP experimental pair done at the same time as the RNA-seq measurements shown in 2(dDAVP/vehicle) over all three experiments was 2.78 ± 0.46 (P < 0.05). This result therefore supports the conclusion that the vasopressin-induced increase in aquaporin-2 mRNA abundance is, at least in part, due to increased transcription.To quantify changes in RNA polymerase II binding to each annotated gene in response to dDAVP and compare the 2(dDAVP/vehicle) values . This finding suggests that, in most genes, vasopressin-induced increases in RNA polymerase II binding at the PPR is not associated with increased production of full length transcripts. Figure 2E shows a histogram of log2(dDAVP/vehicle) values for RNA polymerase II binding beyond the PPR (i.e. reads beyond the first 400 bp of the gene bodies) for all genes. As seen, the strong rightward shift was eliminated, leaving relatively few genes that show changes in response to vasopressin. Some examples of the RNA polymerase II binding profiles for selected genes are shown in https://helixweb.nih.gov/ESBL/Database/Vasopressin/ .) values . The ext) values . This fiAqp2, B3gnt7, Arl4d and Nr4a1) show increased RNA polymerase II binding beyond the PPR in response to dDAVP  at multiple serines corresponding to position 5 in the 52 heptad repeats at the COOH-terminal tail of Polr2aAqp2 gene. A summary of all data is provided as a publically accessible webpage at https://helixweb.nih.gov/ESBL/Database/Vasopressin/. The data have also been deposited in Gene Expression Omnibus (GEO) (http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE79584). (Curated data are available at https://helixweb.nih.gov/ESBL/Database/AVP_Transcr/.)To provide a stringently vetted listing of genes undergoing net regulation of transcription in response to dDAVP, we compared the RNA-seq and ChIP-seq responses . The genhttp://brp.kfshrc.edu.sa/AredOrg/), we found putative AREs in 21 of 149 (14.1%) upregulated transcripts from 16Note that in Aqp2, Arl4d, Atp1b1, B3gnt7, Bhlhe40, Cdk18, Gadd45b, H6pd, Id3, Jun, Krt19, Lypd3, Nfkbiz, Nr4a1, Rasl11b, Sat1, Selm, Sik1, Sptbn2, Srxn1, and Tspan1. The comparison shows that, in general, the dDAVP:vehicle ratios were greater with RNA-seq analysis, suggesting that there is a significant degree of ratio compression with the microarrays . In a cDNA array study in which whole inner medullary transcripts were measured in miceVamp8, Zfp36l1, and Tsc22d4. Zpf36l1 is a member of a family of proteins that binds to AU-rich regions (AREs) at the 3′-ends of transcripts and regulates their stabilitiesVasopressin-mediated effects on transcript abundances in collecting duct cells were previously investigated using SAGE analysisroarrays . The SAGBhlhe40, Id3, Nr4a1, and Zfp750, as well as one that was decreased, viz. Jun. All of these have potential roles in regulation of Aqp2 gene transcription. There are two protein kinases, viz., Cdk18 and Sik1, which have potential roles in vasopressin signaling. There are two small GTP-binding protein transcripts that were found to be regulated by vasopressin: Arl4d (increased) and Rasl11b (decreased) with potential roles in vesicle trafficking and regulation of the actin cytoskeleton. Consistent with the role of vasopressin in regulation of apoptosis in collecting duct cellsNfkbiz, Osgin1, Sik1, and Tob1) or decreased (Gadd45b) by vasopressin. The regulated genes listed in Creb1). This transcription factor has been proposed to mediate the effects of vasopressin on gene transcription in collecting duct cells303132Creb1 is very low (TPM = 0.42) compared to a number of other cAMP-regulated b-ZIP transcription factors, suggesting the possibility that more abundant Creb-like transcription factors, such as Atf4 or Creb3, could be involved in the transcriptional effects of vasopressin. Among the b-ZIP transcription factors reported in 35Aqp2 geneIn many cell types, the effects of cyclic AMP on transcription are due to activation of the transcription factor CREB  to be regulated by vasopressin out of 24000 protein coding genes is log2(24000/1) = 14.6 bits. In contrast, if we consider that 35 genes out of 24000 are regulated (2(24000/35) = 9.4 bits. Or if the regulation were less selective, e.g. if one-third of genes were regulated, the required information content would be only log2(24000/8000) = 1.6 bits. If we assume that the information necessary for the regulation is conveyed is via transcription factor binding, more transcription factor binding sites would evidently be needed for selective regulation than for widespread regulation. Specifically, according to O’Neill et al.2(G/M) where G is the number of protein coding genes and M is the number of high affinity binding sites. For most transcription factors, M is typically at least 1000.  or 4.5 bits. Thus, the binding of at least four independent transcription factors would be required to selectively regulate the Aqp2 gene alone (14.6 bits of information), and at least two independent transcription factors would be required to selectively regulate 35 out of 24000 genes (9.4 bits). We conclude then, that the selective transcriptional regulation demonstrated in this paper, implies that transcriptional regulation of Aqp2 must involve multiple transcription factors that bind at independent sites.The foregoing argument can be formalized in terms of information theoryegulated  and 2, tAqp2 gene transcriptionAqp2 gene from several species identified several conserved binding motifs that play putative roles in transcriptional regulation104041Aqp2 geneAqp2 gene, respectively, and are therefore candidates for roles in vasopressin-mediated transcriptional regulation.There are multiple putative transcription factor binding sites in the promotor region that could play roles in regulation of promotor proximal pausing (Drosophila heat shock gene (HSP70) transcriptionPrevious studies in other cell types have demonstrated that most active genes manifest a predominance of RNA polymerase II binding to the PPR pausing , a halt initiation among expressed genes. Recent investigations have shown that certain transcription factors, most notably Myc, have actions that are not selective for specific genes, but rather act as general amplifiers that accelerate the transcription of virtually all expressed geneshttps://helixweb.nih.gov/ESBL/Database/Vasopressin/]). Identification of genomic binding sites for Myc in collecting duct cells has, however, not yet been reported.An interesting aspect of this study is the demonstration that vasopressin increases RNA polymerase II binding to the promotor-proximal region of a majority of expressed genes  and 4B, All experiments were performed in mpkCCD11 cells as previously described Briefly, cells were expanded to ~80% confluence on 25-cm2 plastic flasks (Corning), trypsinized  and resuspended in 10 ml DMEM/F12, and then seeded on permeable supports  at a ratio of 1:10 and grown in 1:1 DMEM/F12 (Invitrogen) containing 2% fetal bovine serum, insulin, dexamethasone, triiodothyronine, epidermal growth factor, selenium, and transferrin as previously describedet al.Samples were diluted in Laemmli buffer  and subjected to SDS-PAGE. Immunoblot analysis using nitrocellulose membranes was performed as described previouslyet al.Samples were diluted in Laemmli buffer and subjected to SDS-PAGE as previously describedImmunofluorescence labeling was done as describedet al.mpkCCD cells were grown in the presence or absence of dDAVP (0.1 nM for 24 hrs) for RNA-seq or ChIP-seq experiments. Procedures for RNA-seq are summarized in BWA). Mapped reads were visualized on the UCSC Genome Browser and the Integrative Genomics Viewer (Broad Institute).Procedures for ChIP-seq are summarized in How to cite this article: Sandoval, P. C. et al. Systems-level analysis reveals selective regulation of Aqp2 gene expression by vasopressin. Sci. Rep. 6, 34863; doi: 10.1038/srep34863 (2016).


The three-dimensional structure of chromatin organized by genomic loops facilitates RNA polymerase II access to distal promoters. The Kaposi’s sarcoma-associated herpesvirus (KSHV) lytic transcriptional program is initiated by a single viral transactivator, K-Rta. Here we report the KSHV genomic structure and its relationship with K-Rta recruitment sites using Capture Hi–C analyses. High-resolution 3D viral genomic maps identify a number of direct physical, long-range, and dynamic genomic interactions. Mutant KSHV chromosomes harboring point mutations in the K-Rta responsive elements (RE) significantly attenuate not only the directly proximate downstream gene, but also distal gene expression in a domain-specific manner. Genomic loops increase in the presence of K-Rta, while abrogation of K-Rta binding impairs the formation of inducible genomic loops, decreases the expression of genes networked through the looping, and diminishes KSHV replication. Our study demonstrates that genomic architectural dynamics plays an essential role in herpesvirus gene expression. Genomic loops and their temporal dynamics play an important role in gene expression of DNA viruses, but are incompletely understood. Here, the authors use capture Hi-C analyses and identify genomic architectural dynamics of KSHV that are regulated by the viral transactivator K-Rta. As reviewed by Palstra et al.2, the concept of ACHs originated, in part, from the fact that the protein concentration of many nuclear factors is below the dissociation constant of protein-protein or protein-DNA interactions. Accordingly, it is necessary to have mechanisms to increase the local concentration of nuclear factors at a given chromatin site. Transcription factors pinpoint their binding sites by three-dimensional scrutiny of nuclear space, and the formation of productive transcription complexes on DNA is intrinsically dynamic4. A higher concentration of factors favors efficient binding to DNA templates by facilitating rapid re-association of dissociating factors at the same or abutting sequences. Thus, the concentration of transcription factors and co-factors near transcription initiation sites is a sensitive limiting component determining the number of transcripts produced. Therefore, spatial and temporal clustering of cognate binding sites is proposed to be an important means to boost the local concentration of factors and thus is indispensable for the regulation of the transcriptional rate of genes5.Tissue-specific cellular gene expression is regulated by the formation of active chromatin hubs (ACHs) at enhancer regions of the genome, where many tissue specific-gene promoters are brought into proximity8. It is important to note that core promoters typically only support low-level basal transcription; cis-regulatory modules or enhancers actually carry most of the regulatory information for gene expression9. These elements can be located near the core promoter or distal to a target gene which is typically found within accessible chromatin11. The combined input of activating and repressing transcriptional factors localized to an ACH then determines its overall regulatory output, which dictates tissue-specific gene expression patterns14. Transcriptional factors, communication proteins, as well as non-coding RNA are suggested to be involved in the assembly of distant regulatory elements18.Development of chromosome conformation capture (3C) techniques has permitted the examination of ACH formation, and numerous studies have indeed demonstrated widespread occurrence of stimulus-responsive enhancer-promoter and promoter-promoter interactions between co-regulated genes21. Such histone modifications correlate with transcriptionally “active”, “poised”, and “repressed” domains of viral chromatin in a manner equivalent to cellular chromosomes22. In addition, viral genomic loops were previously identified in KSHV-infected cells, in which CCCTC-binding factor (CTCF)-cohesin complexes establish physical looping of the KSHV genome18. Disruption of such genetic interactions by ablation of components of the cohesin complex deregulated the KSHV latency-lytic switch leading to viral reactivation23. These results highlight the vital significance of higher-order viral genomic structure to the viral life cycle.Gamma-herpesvirus replication takes place in specific tissues and viral gene expression occurs robustly in a highly-organized fashion. More than eighty viral genes are encoded by a relatively small viral genome, thus gene regulatory information such as the formation of ACHs is likely to be needed and embedded within the viral lifecycle as an accompaniment to DNA sequences required for efficient viral replication. Furthermore, such small viral episomes provide a unique experimental model to study 3D genetic interactions and their association with gene expression. Kaposi’s sarcoma-associated herpesvirus (KSHV) episomes furnish a superb system for defining these mechanisms due to the combination of benefits derived from prior knowledge of defined viral transcriptional factors necessary to initiate viral gene expression, smaller genomic size, simpler genome organization, and a tractable genomic sequence using a bacterial artificial chromosome (BAC) system. Similar to cellular chromosomes, the viral genome is wrapped upon post-translationally modified histones and interacts with multiple histone modifying enzymes24. KSHV lytic replication is initiated by expression of a single viral protein, K-Rta. K-Rta is both essential and sufficient to induce lytic reactivation of the latent KSHV genome in the BCBL-1 cell line model, as well as in the de novo infection model25.Like all herpesviruses, KSHV can exhibit two alternative lifecycles, known as latency and lytic replication. In latency, the viral genome persists in the host as low copy-number nuclear episomes, and its expression is largely silenced with the exception of several genes26. We questioned if the KSHV genome is structured to maximize effects of K-Rta recruitment to achieve robust viral gene expression and also took advantage of an established reactivation system27 to analyze spatiotemporal gene regulation in high resolution. We identified several KSHV genomic loops that encompassed K-Rta direct binding sites, and induction of K-Rta expression further induced genomic loop formation. These studies complement our recent report utilizing immuno-FISH approaches to study KSHV transcription in situ and examine KSHV gene expression in the 3D nuclear space of an infected cell28. The study showed that episomes responding to stimulation aggregated and recruited molecules that formed large “transcription factories”. With the results reported here, we show that KSHV has evolved spatiotemporal gene regulatory mechanisms to assemble viral genomic domains and recruit RNA polymerase II, which results in effective viral gene expression from uniquely structured viral episomes.In this study, we performed an unbiased chromosome conformation analysis by Capture Hi–C analysis, which is 3C combined with next-generation sequencing (NGS)30. We speculated that robust gene activation by K-Rta associates with a unique structure formed by the KSHV genome, which is prefabricated with an architecture designed for K-Rta recruitment. To examine this hypothesis, the K-Rta binding sites in the KSHV genome were precisely mapped with ChIP-seq analysis in the context of the BCBL-1 cell line model. BCBL-1 is derived from a KSHV-infected human primary effusion (body cavity-based) lymphoma (PEL) and contains latent KSHV genomes. The TREx-(F3H3)-K-Rta BCBL-1  subline containing a Tet-inducible Flagx3 and HAx3-tagged K-Rta expression cassette was generated from TREx-BCBL-1 cells27. Doxycycline (Dox)-induced K-Rta expression  in the PAN and K12 promoters have been studied in detail33, which allowed us to prepare mutant KSHV genomic episomes (described below).The KSHV immediate early gene, K-Rta, is both necessary and sufficient to switch latent KSHV into the lytic infection cycleion Fig. . The res26. The capture step was achieved using a custom library of biotinylated oligonucleotide baits  that target the entire KSHV genome, including terminal repeats. This step was incorporated in order to enhance the sensitivity of Hi–C analysis to detect chromatin interactions of the KSHV genome by enabling substantial enrichment of KSHV-derived sequences  were then visualized with Circos34. The number of sequence reads and identified genomic interactions are summarized in Table p < 0.001) (Table BamHI position #14). On the other hand, the number of genomic loops associated with the late and latent gene cluster regions  and the K-Rta promoter locus (BamHI position #14) , which is another major K-Rta recruitment site , as well as to the PAN promoter region (BamHI position #5) were also increased   approach coupled with target sequence capture and NGS1) Table . When reite Fig. , to the 1) Table .Table 1CBamHI digestions, we further generated a higher resolution genomic interaction map by performing Capture Hi–C analyses using the DpnII restriction enzyme. The enzyme digests KSHV genomic DNA at 350 different sites and the frequent digestion allowed us to generate a linear genomic map by quantifying the Capture Hi–C sequencing reads encompassing the region at 3-kb intervals  , the K12 RE (K12 Mu), or both REs (C/T)AG(A/G)(G/T)GGC core sequence37. Using the mutant episomes described above, K-Rta mediated viral gene expression was examined by RT-qPCR to define the importance of a single K-Rta direct binding site to the entire KSHV gene expression program. KSHV RT-qPCR arrays provided an expression read-out for every KSHV ORF over 72 h of reactivation  and KSHV PCR array results  indicated loop formation between regions of the KSHV genome containing the PAN RE and K12 RE, we asked whether binding of K-Rta at one RE would influence binding at the other, presumably proximal RE. K-Rta ChIP analysis was performed using chromatin from dox-induced (0 and 8 h) iSLK cells were first transfected with a PAN RNA expression vector 48 h prior to dox-induced reactivation. This raised the level of PAN RNA 3000-fold above the level present in reactivated PAN Mu cells transfected with empty vector  were detected in the presence of K-Rta  grown in trans-well culture with serum free media  prepare the latent genome for direct K-Rta binding events and (b) facilitate the formation of inducible genomic loops that are associated with distal promoter activation . The underlying causes of this variation are still not entirely clear. Although our 3C results were obtained from cell populations, individual episomes may vary in their looping architecture, as is the case with mammalian genomes47, and loop diversity may contribute to variable reactivation scenarios described above. In this regard, we hypothesize that correct genomic KSHV loop formation and subsequent direct K-Rta binding events are additional factors to spur a subset of K-Rta-expressing cells and/or episomes towards full reactivation. Although the regulation of loop formation is unclear, examples in support of cells containing either preformed (hardwired) loops48 or induced loop formation50 have been reported. The loops we detected in KSHV-positive cells during latency suggest one class of KSHV genomic loops are hardwired. The observed induction of genomic links upon K-Rta expression also indicates the presence of inducible loop formation. Our study with mutant KSHV further suggests that activation of specific promoters, and presumably recruitment of RNA polymerase II complex, is important for inducible genomic loop formation . The results showed there were little changes in the contact frequency of several loci examined  in BCBL-1 cells , 50 μg/ml G418, and 100 μg/ml hygromycin B in the presence of 5% CO2. iSLK cells containing BAC16 WT or BAC16 mutants were cultured in complete DMEM containing 250 μg/ml G418, and 1000 μg/ml hygromycin and 1 μg/ml puromycin. TREx-(F3H3)-K-Rta BCBL-1 cells that expresses Flag × 3 and HA × 3 tags at the N-terminal region of K-Rta were generated and cultured in complete RPMI 1640 containing 50 μg/ml blasticidin and 100 μg/ml hygromycin B. Vero (ATCC CCL-81) and iVero (dox-inducible K-Rta expression) cells were cultured in the presence of 1 µg/ml puromycin. Human gingival epithelial cells (HGEP), obtained from pooled donors, were purchased from CellnTec (#HGEPp). The cells were cultured in CnT-prime epithelial cell medium (CellnTec #CnT-PR).iSLK cell lines containing each recombinant BAC16 were cultured in selection media in 150 mm culture dishes until 80% confluent. For reactivation, selection media was removed and cultures were re-fed with complete DMEM (w/o selection drugs) containing 1 mM sodium butyrate and 1 µg/ml doxycycline. The cultures were reactivated for 4 days. The cells were removed by centrifugation  and the supernatants were passed through a 0.8-µm filter, and virus particles were concentrated by ultra-centrifugation at 25,000 rpm , for 2 h at 4 °C (Beckman SW28 rotor). Viruses were re-suspended in 500 µl of DMEM along with the residual ~500 µl media in the centrifuge tube and stored at –80 °C until use. Viral genomic copy number in the medium was measured by qPCR, as described previously55.56. The PCR utilized pCIneo-3xFLAG-ORF50/RTA as template and the primer pair sSalI-RTA(pENTR), CAT GTC GAC ATG GCG CAA GAT GAC AAG GG and asRTA-EcoRI(pENTR), CTG AGA ATT CTC AGT CTC GGA AGT AAT TAC GCC ATT G. To construct pCW57.1-KSHV K-Rta/ORF50, the KSHV K-Rta/ORF50 fragment was transferred from pENTR4-KSHV K-Rta/ORF50 to the Tet-ON lentiviral vector pCW57.1 (Addgene plasmid # 41393), by using the GATEWAY system .To construct pENTR4-KSHV K-Rta/ORF50, K-Rta/ORF50 fragments were obtained by PCR and inserted into pENTR4 no ccDB (686-1) (Addgene plasmid # 17424)36. Point mutations were introduced at the K-Rta direct binding sites within the response elements of the PAN and K12 promoters33.The KSHV bacmid BAC16 was kindly provided by Dr. Jae U. Jung  and mutagenesis performed according to previously described methodsThe primer sequences for mutagenesis were as followsTctaCccccGacataagcagtttgtcctacTAGGGATAACAGGGTAATCGATTT,. Whole genome sequences of each BAC clone were confirmed by next-generation DNA sequencing as described below.57 with default settings. Using the BAM alignment files as input, SAMtools mpileup58 was then used to perform variant detection. Ultra-high coverage was obtained for most samples (~1200×) and allowed for comparative whole genome sequence analysis. The point mutations in each BAC16 were validated with unbiased variant calling . The results also demonstrated a 24,710 C > T variant originating from the parental BAC16, and an additional mutation within the K12 promoter region of BAC16 K12Mu  (Supplementary Table BAC16 DNA constructs were isolated from 2 l of LB culture with the Qiagen MAXI kit (Qiagen). Genomic DNA preparations were quantified using the Qubit Fluorometer (Life Technologies) and sheared to ~ 180 bp using the Covaris E220 Focused-ultrasonicator . Sequencing libraries were prepared using the NEBNext DNA Library Prep Kit  and multiplexed sequenced  on the Illumina MiSeq platform  per manufacturer’s standard protocols. Raw sequence reads (FASTQ format) were aligned back to the BAC16 KSHV reference  using BWA-mem60. The packaging plasmids  were a kind gift from and Dr. Hiroyuki Miyoshi . To establish Vero cells with inducible expression of K-Rta/ORF50 (iVero cell), Vero cells were infected with the lentiviral particles, and transduced cells were selected and maintained by culture in growth medium containing 2.5 µg/mL of puromycin (InvivoGen). KSHV BAC16 wild-type and mutants were transfected into iVero cells by the calcium-phosphate method, and stably transfected cells selected by culture in complete medium in the presence of 1,000 μg/mL of hygromycin B and 2.5 μg/mL of puromycin.The lentivirus vector was produced by tripartite-transfection of pCMV-VSV-G-RSV-Rev, pCAG-HIVgp and pCW57.1-KSHVK-Rta/ORF50 into 293 T cells (ATCC CRL-3216)iSLK BAC16 lines were prepared by infection with viral supernatants produced in iVero cell lines described above. Each iVero BAC16 line was induced with 1 μg/ml doxycycline + 1 mM sodium butyrate for 4 days. Supernatants were collected, concentrated, and used to infect iSLK cells. For infection, iSLK cells were seeded into a 6-well plate (2 × 105 cells/well), and viral supernatants were added to the cells on the following day. Two days later, the transduced cells were trypsinized and transferred to 10-cm dishes and cultured in complete DMEM containing 1 µg/ml puromycin, 250 µg/ml Geneticin and 1,200 µg/ml hygromycin B.The iSLK cells. Latent or dox-induced iSLK cells were fixed with formaldehyde, digested with restriction enzyme (BamHI), diluted, and re-ligated. The primer design and protocol used has been described previously by Kang et al.18. A diagrammatic representation of the 3C PAN BamHI anchor primers are shown in Fig. The chromosome conformation capture (3C) procedure and analysis was carried out using chromatin derived from BAC16-WT or BAC-mutant infected A custom KSHV genomic capture probe library using xGen Lockdown Probe technology  was designed to target the entire KSHV genome sequence  and manufactured by IDT. The library consists of a pool of 1,152 individually synthesized, 5′-biotinylated, 120mer oligonucleotide baits. A stock solution (0.75 µM) of the pool was prepared in TE (pH 8.0).26. The chromatin prepared from KSHV-infected cells for the 3C procedure described above was used as input for Capture Hi–C. Briefly, infected TREx-K-Rta BCBL-1 or iSLK/r.219 cells (1 × 108) were fixed with formaldehyde, and the chromatin was digested with either BamHI or DpnII restriction enzyme, and then re-ligated under ultra-dilute conditions. DNA-protein crosslinks were reversed by incubation at 65 °C overnight with 0.2% SDS and the DNA purified with the QIAquick PCR Purification Kit (Qiagen). DNA samples were submitted to the UC Davis Comprehensive Cancer Center Genomics Shared Resource for library preparation, target enrichment, and NGS. The DNA preparations were sheared to a mean fragment length of 250 bp using a Covaris E220 Focused-ultrasonicator  and sequencing libraries were then prepared from the DNA chimerae using the NEBNext DNA Library Prep Kit . Target enrichment for KSHV genomic content was performed using a custom-designed KSHV genomic capture probe library (described above) and according to the manufacturer’s standard protocols . Briefly, in-solution hybrid capture was performed by hybridizing libraries (500 ng) with the KSHV genomic capture probe pool (3 pmol) in a mixture containing xGen 1X Hybridization Buffer, Cot-1 (5 µg), and xGen Universal Blocking Oligos at a temperature of 65 °C for 4 h. Hybridized targets were then bound to streptavidin-coupled magnetic beads  by incubation at 65 °C for 45 min, and unbound DNA was removed by a series of high-stringency (65 °C) and low-stringency (room temperature) washes. KSHV genome-enriched Capture Hi–C library DNA was eluted and post-capture PCR enrichment performed with high-fidelity KAPA HiFi HotStart DNA Polymerase  and purified with Agencourt AMPure XP magnetic beads. KSHV-enriched libraries were then validated with an Agilent 2100 Bioanalyzer, and quantitated with a Qubit fluorometer (Invitrogen) and by qPCR-based quantification (KAPA Library Quantification Kit). Libraries were submitted for sequencing (100-bp paired-end) on an Illumina HiSeq 2500 sequencing system. Junction sites (BamHI or DpnII sites) were mapped and visualized with Circos. Because of the relatively small size of the KSHV genome, exclusion of non-digested fragments with an additional capture step by the incorporation of biotin labeled nucleotide at the ends of successfully digested fragments was not performed. Consequently, we have had a relatively larger fraction of non-digested KSHV genomes that might be inaccessible for digestion by restriction enzyme, BamHI. In addition, there were a number of BamHI containing fragments that were unable to be mapped to BamHI sites within the short illumina sequence reads.Long-range chromatin interactions within the KSHV genome were determined in an unbiased, comprehensive manner with Capture Hi–C, by utilizing 3C in conjunction with KSHV genome enrichment and next-generation sequencing (NGS)http://www.bioinformatics.babraham.ac.uk/projects/hicup/). By executing a series of Perl scripts, this performed identification of the Hi–C ligation junctions within the reads , read alignment, and removal of PCR duplicates and common Capture Hi–C artifacts. Subsequently, read pairs derived from the interacting fragments were output in SAM format. Within HiCUP, Bowtie 261 was used to align FASTQ data to the reference KSHV genome sequence  and an in silico BamHI-digested reference genome. Hi–C output was then analyzed with HOMER  to generate a Capture Hi–C tag directory and for identification of significant interactions. Junction sites (BamHI or DpnII sites) were mapped and visualized with Circos plotting (described below). Frequencies of chromatin looping sites are presented as the number of reads obtained with 3C of KSHV-infected chromatin.Paired-end sequence data (FASTQ format) was processed to identify putative Capture Hi–C interaction products (di-tags) using the Capture Hi–C User Pipeline (HiCUP)  Sequence reads were mapped to the KSHV reference genome (NC_009333.1). The number of sequence tags that mapped to a given BamHI or DpnII fragment is indicated in the Circos display and represent the number of unique interactions involving the fragment.3C sequence reads and genomic features of TREx-K-Rta BCBL-1 and iSLK-Wt and PAN-Mu cells presented in Fig. http://www.R-project.org).The R package ver. 3.2.4 was used to visualize genomic links and gene expression. Heatmaps of genomic links were plotted with R package ggplot2 ver. 2.1.0. The gene expression profiles of Human gingival epithelial precursor cells obtained from pooled donors were purchased from CellnTec. The cells were cultured in epithelial medium (CellnTec CnT-PR) as submerged cultures on polystyrene lifts in 12-well plates. Cells were bathed with 800 µl media in the upper chamber of the lift and with 1 ml medium in the lower well. When the cells were confluent, viral stocks were added to the upper chamber and the infection was allowed to proceed for 24 h. Cells were monitored for eGFP expression and RNA was purified using RNeasy columns (Qiagen).62. The antibodies used were rabbit anti-K-Rta IgG63, mouse anti-K-Rta monoclonal antibody , anti-Flag M2 (Sigma F1804), and rabbit and mouse control IgGs . Immunoprecipitated chromatin DNA was analyzed by SYBR Green-based quantitative PCR (qPCR) (Bio-Rad) with primers for each RE as follows: PAN RE-S (agcttgaaggatgatgttaatg), PAN RE-As (gaagcggcagccaaggtgactg), K12 RE-S (ccaagagatccgtcctccgtgcc), and K12 RE-As (agcgggatgctaggtccacgc).Chromatin immunoprecipitation assay (ChIP) assays were performed, as described previously8 TREx- K-Rta BCBL-1 cells were used per assay and immunoprecipitated with 20 µg mouse anti-FLAG M2  or mouse control IgG . 1 ng ChIP-enriched or input DNA was used to generate Illumina-compatible libraries with the KAPA LTP Library Preparation Kit  according to the manufacturer’s recommendations. Libraries were submitted for sequencing (50-bp single read) on an Illumina HiSeq 2500 sequencing system. The ChIP-Seq data was aligned to the human hg19 reference genome assembly and reference KSHV genome sequence  with Bowtie 261. Peak finding was performed with the MACS2  program64 according to the standard parameters described in the developer’s manual. We used the default settings with a minimum FDR  cutoff of 0.05. The peaks and reads alignments were visualized using the Integrative Genomics Viewer (IGV) genome browser from the Broad Institute.Briefly, chromatin DNA from 1 × 1063, anti-LANA  and anti-GAPDH . The uncropped western blotting images are shown in Supplementary Fig. Cells were collected in RIPA buffer  supplemented with 1 mM PMSF and 1× protease inhibitor cocktail (Roche). Antibodies used for immunoblotting were anti-K-RtaiSLK BAC16 WT and BAC16 mutant cells were reactivated with doxycycline (1 μg/ml) and RNA was isolated from these cells 0, 24, 48, and 72 h post-induction using RNeasy columns (Qiagen). cDNAs were synthesized using SuperScript III first-strand synthesis reagents (Invitrogen). Gene expression was analyzed by qPCR using specific primers designed by Fakhari and Dittmer65. KSHV gene expression was normalized to the cellular ACTB signal. RNA harvested from PAN RNA transfected cells included a DNase I treatment step.The data discussed in this publication have been deposited in NCBI’s GEO Database under accession number GSE99950. The authors declare that all other data supporting the findings of this study are available within the article and its Supplementary InformationPeer Review File


The increasing financial burden of orthopaedic implants on our health care system has prompted cost-control measures, such as implant reprocessing. The purpose of this study was to describe the current usage by orthopaedic trauma surgeons of reprocessed external fixators (EFs) for treatment of complex fractures.A 16-question survey about use and perceptions of reprocessed EFs was distributed to 894 Orthopaedic Trauma Association members between August 2016 and June 2017 using a web-based survey system.P = .0004). Eighty-seven percent of respondents believed reprocessing could be cost-effective. The most common reason (32%) for not using reprocessed EFs was coordination/logistics of reprocessing. Concern about litigation was also reported as a main reason for not using (20%) or having recently stopped using (21%) reprocessed EFs.The authors received 243 responses (27%). Thirty-seven percent of respondents reported using reprocessed EFs. Nonprofit hospitals used reprocessed EFs more commonly than did for-profit hospitals (41% vs 15%, Many orthopaedic traumatologists are interested in the reprocessing of EF components but few have reprocessing systems in place at their institutions. A major barrier to implementation is concern about litigation, which is likely unwarranted on the basis of Food and Drug Administration approval and a lack of previous litigation. Reprocessing by the original device manufacturers has yielded substantial savings at our institution and is an example of the cost savings that can be expected when implementing an EF reprocessing system. External fixation is an important and expensive orthopaedic procedure. The increased use of external fixation during the last decade coincides with the acceptance of staged treatment of complex periarticular fractures and injuries amenable to damage-control approaches –4. HowevReprocessing can be performed by the original implant manufacturer, a third-party company, or the hospital , 6. HistIn response to market demand, orthopaedic device manufacturers have begun their own reprocessing programs for some EF components after receiving FDA 510(k) approval. This clearance for retesting, re-evaluation, and recertification [The purpose of this study was to determine the current practices of orthopaedic trauma surgeons with regard to reprocessed EFs. We hypothesized that there would be heterogeneity in practices between institutions because of a lack of knowledge of the reprocessing options and concerns about safety, liability, FDA approval, and questionable cost savings. We reviewed the legal literature associated with reprocessing external devices to assess historical and projected risks of using reprocessed devices. Finally, we performed a cost analysis of the EF reprocessing program used at our institution.Survey questions were formulated by an orthopaedic resident, orthopaedic trauma fellow, and orthopaedic trauma surgeon. After receiving approval from The Johns Hopkins Medicine institutional review board (#IRB00134437) and the Orthopaedic Trauma Association research committee, we distributed the survey to all Orthopaedic Trauma Association members between August 2016 and June 2017 using a web-based survey system . Privacy was maintained by collecting only de-identified data and using recruitment procedures that did not allow tracing of respondents to answers. Survey participation was voluntary and no incentives were provided.The survey consisted of 16 questions. Questions 1–7 asked about characteristics of the respondent’s hospital/health care center environment. Questions 8–10 inquired about the respondent’s current practice of using EFs and experience with reprocessed EFs. Questions 11–16 assessed the respondent’s knowledge and perceptions of reprocessed EFs. Respondents were able to provide open-ended answers, which were reported as the multiple choice option that was closest in meaning. If the open-ended answer did not correspond with any multiple choice option, it was treated as a new answer.P values < .05 were considered significant.Survey data were analyzed using Excel, version 2016, software . Statistical analyses were performed using MedCalc, version 15.0, statistical software . Categorical data were analyzed using Fisher exact tests. The literature on reprocessed medical devices was reviewed to address concerns regarding litigation. We performed searches using LexisNexis, Westlaw, and PubMed for legal cases pertaining to all reprocessed devices in orthopaedic and non-orthopaedic surgical specialties without restrictions on publication date. The following medical subject headings and terms were used in the search: “external fixator,” “reprocessing,” “reprocessed,” “reuse,” “reusable,” and “complication.” To confirm search results, the first author and our senior librarian performed the searches independently using the same criteria. The results were compared, and there were no differences between them.An EF reprocessing system was initiated at our institution in November 2014 with one implant manufacturer . FDA-approved components  that are eligible for reprocessing and recertification by the vendor were included. To evaluate savings, we calculated the cost of all EF components used during 9 months of reprocessing (January to September 2017). Savings were calculated as the difference between the cost of new components and reprocessed components. We also compared the total number of components used with the number that were recertified through the reprocessing process during the same period to obtain the percentage of total costs saved.We sent survey invitations to all 894 Orthopaedic Trauma Association members and received 243 responses (27%). Most respondents worked at nonprofit institutions (84%) and at urban hospitals (56%). Forty-six percent of respondents worked at university-based centers Table .Table 1COnly 37% of respondents reported current use of reprocessed EFs at their institutions. Reasons for not using reprocessed EFs included coordination and logistics of reprocessing 32%), litigation concerns (20%), a gap in knowledge about the process (12%), and doubts about actual cost savings (12%). Recent cessation of reprocessing was reported by 29 respondents. Respondents reported that reprocessing was performed by the original vendor (37%), a third party (34%), or the hospital (12%). Seventeen percent of respondents said they did not know who performed the reprocessing for their institutions . The rates of using reprocessed EFs were also significantly different according to hospital affiliation (P = .001)  than did for-profit hospitals (15%) (1) Table .Table 3EWhen asked what they thought about using reprocessed EFs, most respondents said they believed that reprocessing could be cost-effective (87%) and beneficial for hospital revenue 64%). However, when asked what they perceived to be the most important obstacle to using reprocessed EFs, they cited vendor coordination/logistical challenges of reprocessing (25%), and litigation concerns (20%) . Some of these were legal cases related to reuse of medical devices. However, none of the cases was related directly to the reuse of EFs or EF reprocessing.At our institution, FDA-approved EF components were reprocessed and recertified by the original manufacturer. The overall rate of component recertification (“pass rate”) was 80%. We used reprocessed EFs whenever they were available. However, in every case in which EFs were used, we also needed new components for the EF that are unable to be reprocessed or that failed recertification. The total cost of all EF parts (475) used  during the 9-month study period would have been $389,251 if purchased new, whereas the total cost of purchasing the same number of reprocessed EF parts was $97,999. Therefore, the total 9-month cost savings achieved through this system was $291,252.In the interest of reducing medical costs, one strategy is the use of reprocessed EF devices. Given the novelty of device reprocessing, we sought to assess current orthopaedic traumatologists’ understanding of this system. Our results showed an overall lack of knowledge of the potential value of EF reprocessing. Although most respondents believed that EF reprocessing could be cost-effective (87%) and beneficial to hospital revenue (64%), most respondents (63%) were not using reprocessed components. The most common reasons reported for not reprocessing were vendor coordination (32%) and litigation concerns (20%). A small proportion of respondents (12%) acknowledged their deficient understanding of reprocessing. Increasing surgeon knowledge of reprocessing is a crucial first step in broadening implementation.A cost analysis of EFs reprocessing at our institution showed a 9-month cost savings of $291,252 (75% savings compared with list price). This would equal nearly $400,000 for 1 year of reprocessing. The result is similar to those of previous reports showing an approximate savings of 25–32% of list prices over time –10. The Litigation concerns were reported as a main reason for not using (20%) or having recently stopped using (21%) reprocessed EFs. Possible legal issues raised by the hospital related to informed consent of patients about reprocessed instrumentation could complicate the implementation of this process. However, our review of the legal literature found no reports of litigation pertaining to the use of reprocessed EFs. Likewise, the senior author (EAH) has encountered no legal issues at his institution related to the use of reprocessed EFs during the past 2 years. Although the legal process to implement the use of reprocessed EFs was time-consuming to complete, mainly because of the need to establish billing codes and fees for reprocessed components, the reprocessing system has produced substantial savings with no related litigation.At our institution, EF reprocessing was performed by the original manufacturer of the devices according to their guidelines . Used EFLegal and ethical considerations for the healthcare system pertain to how reprocessed EFs are billed. The Current Procedural Terminology code (20690) for the application of an EF does not distinguish between the use of a newly manufactured device versus a reprocessed device. However, for appropriate billing, a code different from the one used for new parts is required for the reprocessed components. Implementation of an already FDA-approved reprocessing system at other institutions therefore requires the establishment of unique billing codes for new versus reprocessed components to be used by the institution and the original manufacturer.Ethical implications for patients regarding billing and familiarity with a reprocessing system must be considered, as well. It would be unethical to charge patients full price for reprocessed components . SimilarThis study has limitations. Because it is based partially on a survey, there are risks of non-responder bias and self-reporting bias that may alter the findings. We did not assess cost saving data from other institutions or vendors; therefore, results may differ in other practices. Further, our savings analysis might not be generalizable to other institutions because final negotiated vendor prices vary among institutions. Finally, our cost analysis may not correlate with those in other countries, where parts are reprocessed by the institutions themselves, or where resources such as water and electricity used for reprocessing and transportation of reprocessed items may be more expensive, thus making single-use newly manufactured components potentially cheaper than reprocessed parts. However, Mahapatra and Rengarajan  showed rThis study meanwhile has several strengths. Respondents were able to provide open-ended answers to some questions, indicating their actual practices as opposed to being limited by designated responses. All respondents were members of the Orthopaedic Trauma Association who have knowledge of and experience with using EFs. Finally, we obtained a response rate of 27%, which is higher than the typical 10–25% response rates for other Orthopaedic Trauma Association member surveys –16.Many orthopaedic traumatologists are interested in the reprocessing of EF components but few currently have reprocessing systems in place at their institutions. A major barrier to implementation is concern about litigation, which is likely unwarranted on the basis of FDA approval and a lack of previous litigation. Reprocessing by the original device manufacturers has yielded substantial savings at our institution and is an example of the cost savings that can be expected when implementing an EF reprocessing system.


Escherichia coli. By calibrating measurements against the signal from a single RNA polymerase bound at a single promoter, we can calculate both promoter occupancy levels and the flux of transcribing RNA polymerase through transcription units. Here, we have applied the methodology to the E. coli lactose operon promoter. We confirm that promoter occupancy is limited by recruitment and that the supply of RNA polymerase to the lactose operon promoter depends on its location in the E. coli chromosome. Measurements of RNA polymerase binding to DNA segments within the lactose operon show that flux of RNA polymerase through the operon is low, with, on average, over 18 s elapsing between the passage of transcribing polymerases. Similar low levels of flux were found when semi-synthetic promoters were used to drive transcript initiation, even when the promoter elements were changed to ensure full occupancy of the promoter by RNA polymerase.Chromatin immunoprecipitation, followed by quantification of immunoprecipitated DNA, can be used to measure RNA polymerase binding to any DNA segment in This article is part of the themed issue ‘The new bacteriology’. Recall that ChIP, in combination with analysis of immunoprecipitated DNA, permits us to detect protein binding at any chromosomal locus [Many bacteria rely on transcription regulation in order to adapt to fluctuating environments. This often involves the interaction of regulatory activator proteins at or near promoters, which results in recruitment of the DNA-dependent RNA polymerase (RNAP) and subsequent transcript initiation and gene expression. In contrast, when the regulatory proteins are repressors, access to the promoter is blocked and, hence, expression of the corresponding transcription unit is silenced –3. Most al locus , indepenal locus –11. Hereal locus ,12–14, t2.(a)E. coli efficiently cross-links RNAP to bound DNA targets [E. coli K-12 lactose (lac) operon to study RNA polymerase flux. Recall that the lac operon is expressed in a transcription unit from a promoter whose activity is repressed by the Lac repressor protein, and that induction requires a chemical inducer such as isopropyl β-d-1-thiogalactopyranoside (IPTG) [lac operon, we used the lac0 pair of probes that samples the lac promoter, and the lac1–5 pairs of probes that sample approximately 300 base pair DNA sequences that are 518, 1421, 2308, 3691 and 4654 base pairs, respectively, downstream from the transcript start. Because each probe pair creates an amplicon that is a similar size, we can directly compare signal intensity between the different probes.Formaldehyde treatment of cultures of  targets ,9. Commee (IPTG) ,16. To aEscherichia coli K-12 strain MG1655, growing exponentially in medium, either with or without IPTG, was subject to our ChIP protocol , and lac operon (probed with the lac0–5 probes). The data show that the inclusion of IPTG in the bacterial growth media triggers a more than a 100-fold increase in levels of imunoprecipitated DNA, confirming that RNAP association with the lac operon is regulated by the Lac repressor. Accepting that the level of immunoprecipitated DNA corresponding to each probe reflects the amount of transcribing RNAP associated with the chromosomal DNA corresponding to each probe, the data argue that, at least for the first 2000 base pairs of the operon, RNAP levels remain constant, while they decline towards the end of the operon, presumably contributing to polarity effects [ effects . Some qu effects ,19.Figulac operon from the data in lac operon loci, probed with the lac0–5 probes. As expected, rifampicin causes a sharp decrease in the levels of immunoprecipitated DNA corresponding to the lac1–5 probes, but an increase with the lac0 probes. If we take the measured signal with the lac0 probe as indicative of a single promoter-bound RNAP, then we can deduce that, during induction in our growth conditions, the lac promoter is approximately 50% occupied, which is consistent with experimental data showing that the activity of the E. coli lac promoter is limited by the recruitment of RNAP [lac operon. Messenger elongation by RNAP in bacteria is known to proceed, on average, at 20–50 bases per second [In order to calculate the absolute numbers of RNA polymerase molecules associated with the  of RNAP ,21. Furtr second –24. Becar second –27.(b)lac promoter in E. coli strain MG1655 was dependent on its chromosomal location [lac promoter::green fluorescent protein (gfp) cassette that we inserted at different chromosomal locations. We found that expression varied by up to 200-fold according to location, and, using ChIP, we showed that the measured differences were due to different levels of RNAP associated with the gfp gene. Because lac promoter activity is limited by RNAP recruitment [lac promoter with the bacteriophage λ major leftward promoter (LλP), whose activity is known to be limited by RNAP escape rather than recruitment [gfp expression from either our lac promoter::gfp or LλP::gfp fusions inserted into the MG1655 chromosome at different specific locations. The data show that observed differences in expression are much smaller with the LλP::gfp fusions than with the lac promoter::gfp fusions, and this is consistent with the suggestion that the effective concentration of RNAP differs according to location along the E. coli chromosome.In a previous study, we found that the measured activity of the location . To showruitment . Figure (c)E. coli lac operon promoter is often adopted as a paradigm, we wanted to compare results with an unrelated promoter, and so we used a previously constructed set of promoter::lac fusions [galP1 transcript start region. In an initial experiment, we selected the KAB-TTTG promoter that carries the −35 element TAGACA (consensus is TTGACA), an extended −10 element of TTTG (consensus is TGTG) and a −10 hexamer of TATGGT (consensus is TATAAT). lac promoter. Hence, promoter occupation by RNAP, as judged by the ratio of signal without rifampicin to with rifampicin, is approximately 40%, whereas occupancy of the downstream DNA segments corresponding to the Lac1357 and Lac2720 probes, ranges from 10% to 20%, which would correspond to an RNAP flux of one every 30 s.Although the  fusions , where tBecause it is well established that promoter −35 and extended −10 elements contribute to the recruitment of RNAP at bacterial promoters ,33, we r3.E. coli, for quantifying the binding of RNAP in vivo to any specific segment of DNA. For promoter regions, we can directly measure occupancy by RNAP, whereas for regions within transcription units, we can deduce the flux of RNAP. The method exploits rifampicin that specifically targets RNAP and blocks it in open complexes at promoters. Here, we make the assumption that formaldehyde equally efficiently cross-links rifampicin-blocked RNAP, RNAP that is bound and paused at promoters, and elongating RNAP, to cognate DNA targets. This appears reasonable as RNAP has a large molecular mass, and makes intimate contacts with the DNA template in all three situations.We have developed a simple method, based on ChIP with lac operon promoter is limited by RNAP recruitment [lac promoter::gfp fusion differs according to its location on the E. coli chromosome [E. coli chromosome, the promoter will sample different locations, including locations where the effective concentration of RNAP is higher or lower. Consistent with this, a recent live-cell super-resolution microscopy study of RNAP in E. coli showed that the vast majority of non-transcribing RNAP molecules that were ‘searching’ for promoters were DNA-bound [lac operon promoter is not dependent on location [E. coli cell, whereas diffusion of larger RNAP molecules is constrained, and this is consistent with calculations of macromolecular mobility in bacteria [Our results are consistent with previous observations that transcript initiation at the ruitment ,21. We bromosome , becauseNA-bound . Intereslocation . From thbacteria .lac operon is consistent with previous observations, from both in vitro [in vivo [lac promoter or by genetically engineered promoters, even when the promoter is fully occupied. This underscores that promoters, as well as being drivers of transcription, are also bottlenecks, and delays to RNAP result in reduced flux through downstream-transcribed sequences [The low measured flux of RNAP through the in vitro ,27 and i[in vivo  studies,equences ,37. Contequences –40, pausequences ,33,41 anequences ,43. Addiin vivo rates of RNA chain elongation [in vitro [lac operon, contradict the simple view that promoters are simply devices that ‘feed’ RNAP into transcription units [lac promoter have many uses in synthetic biology, their full exploitation will require considerable extension of our current knowledge base.We are aware that the measured rates of RNAP flow that we report here are surprisingly low, and depend critically on estimates of RNA chain growth rates. Hence, it is worth underscoring that measured ongation –24 are cin vitro , and thain vitro ,45. The on units . Hence, 4.(a)lac operon were completed using E. coli K-12 strain MG1655 [crp derivative of strain M182 [lac fusion carried on the low copy number broad host range lac expression vector, pRW50 [The experiments analysing the flux of RNAP through the chromosomal n MG1655 , whereasain M182 , with thr, pRW50 . Fragmenr, pRW50 ,48. The −1 tetracycline, where appropriate, and incubated for 16 h at 37°C with aeration. Cells were then subcultured into fresh media to a final OD650 of 0.03, then incubated at 37°C with aeration until an OD650 of 0.4 was reached. The growth conditions used for fluorescence assays were the same, except M9 minimal media supplemented with 2 mM MgSO4, 0.1 mM CaCl2, 0.1% casamino acids, 0.3% fructose and 100 µM IPTG was used [For the ChIP assays, triplicate single colonies were used to inoculate Luria Bertani (LB) media, supplemented with 35 µg mlwas used .(b)LλP::gfp fusion into the genome of E. coli MG1655, an oligodeoxynucleotide was synthesized to encode an EcoRI restriction target site and the LλP G-12 T up-mutant λ leftward promoter fused to the ribosome binding site of the lacZ gene. This ribosome binding site was used in order to make the fusion comparable to previously used lac promoter::gfp fusion [HindIII site in the pJB plasmids to create an EcoRI–HindIII LλP promoter fragment, which was subsequently cloned into EcoRI–HindIII digested pJB plasmids containing the appropriate homology regions for insertion into the chromosomal targets used previously [LλP promoter::gfp fusion into the target chromosomal loci was achieved exactly as described previously by the gene doctoring chromosome recombineering method [To construct the gene doctoring donor plasmids required to insert the p fusion . This oleviously . Insertig method ,49, and (c)650 of 0.025, and incubated at 37°C, with aeration, until an OD650 of 0.4 was reached. When appropriate, rifampicin was added to the culture to a final concentration of 50 µM, and incubated for 15 min, which is sufficient to trap RNAP molecules at promoters [lac operon . Control primers were used to amplify transcriptionally silent control regions. Enrichment of ChIP samples for RNAP binding was calculated relative to the transcriptionally silent control regions, as previously described [Chromatin immunoprecipitation (ChIP) and quantitative real-time PCR (qPCR) were performed as before, using commercial mouse monoclonal antibody for the RNAP β subunit (Neoclone no. W0002) to immunoprecipitate DNA cross-linked to RNAP ,28,50. Oromoters . Cells w


NH2‐terminal variant Sgk1 isoform, Sgk1_i3 that, like Sgk1, is expressed in the distal convoluted tubule, connecting tubule and collecting duct and can stimulate epithelial Na+ transport . We now demonstrate that, similar to Sgk1, aldosterone and glucocorticoids stimulate Sgk1_v3 expression in cell lines from the collecting duct and airway epithelia. In mice, short term aldosterone infusion and maneuvers that increase endogenous aldosterone secretion including dietary Na+ deprivation and K+ loading increases distal nephron Sgk1_v3 expression in vivo. Although Sgk1_v3 has a different 5ʹ proximal regulatory region from Sgk1, the transcription start sites are less than 1000 bp apart. We cloned the 5ʹ regulatory region for Sgk1 and Sgk_v3 upstream of a luciferase gene and by deletion and reporter gene analysis we localized the corticosteroid regulatory region for Sgk1_v3 to a glucocorticoid response element (GRE) that had previously been identified for Sgk1 . We tested this element with MR in an MR‐null cell line and demonstrate that aldosterone stimulates Sgk1 and Sgk1_v3 via this GRE. We conclude that corticosteroids stimulate Sgk1 and Sgk1_v3 expression in epithelial cells via activation of a common conserved GRE in the 5ʹ flanking region of Sgk1.We previously identified a 5ʹ variant alternate transcript of Sgk1 (Sgk1_v3) encoding an  Sgk1_v2 is widely expressed but, in contrast to the originally reported principal Sgk1 transcript, is not regulated by glucocorticoids . Rat tail collagen was obtained from BD Biosciences . All cell culture media were obtained from Life Technologies . The reporter vectors, pGL3basic, pGL4.1, and pRL‐SV40 were obtained from Promega  and GIPZ shRNA Lentiviral particles were purchased from GE Dharmacon .c14 cells were cultured in defined media containing 2% FBS, antibiotics and other hormones, as previously described  or with non‐silencing shRNAmir control lentivirus according to manufacturer's instructions. Cells were maintained under puromycin selection for isolation of single cell clones. Individual clones were expanded to generate stable cell lines expressing shRNAs. Wild type mpkCCDc14 cells or stable mpkCCDc14 GIPZ lentiviral clones were seeded on collagen‐coated transwell filters  and short‐circuit measurements (Isc) were measured in culture media at 37°C in Ussing chambers as described previously . All animals were fed regular rodent chow until start of experiments. Animals were then fed 0.1% NaCl (TD 92238) or 8% NaCl  for 1 week or a 10% K diet (high K) or 0.1% K diet  for 1 week before sacrifice. In other experiments, aldosterone (1.5 mg/kg body weight) or vehicle diluted in PBS was injected intraperitoneally 2 h prior to sacrifice. All experiments were performed following protocols that were approved by the VAMC Institutional Animal Care and Use Committee.Spe1‐HindIII piece of this 5ʹ sequence that encompassed 1558 bp of sequence 5ʹ to exon 1d and the remaining sequence downstream including 117 bp of exon 1 was subcloned upstream of a modified firefly  luciferase in pGL4.1 to give Sgk1(1 + 3)luciferase. Then ~1000 bp downstream of exon 1d was removed by NcoI digestion to give Sgk1(3)luciferase which included 1558 bp of sequence 5ʹ and flanking exon 1d and 58 bp of exon 1d. The 5ʹ flanking sequence upstream of exon 1d including a previously mutated GRE was similarly subcloned into pGL4.1 to give Sgk1(3)mutGRE. Separately, the initial ~1900 bp of 5ʹ sequence in Sgk1(1 + 3)luciferase was removed by StuI and Acc65I digestion to give Sgk1(1)luciferase which included 532 bp 5ʹ and flanking exon 1.The 5ʹ flanking region of Sgk1 upstream of exon 1 has been cloned previously into the reporter vector, pG3basic , a plasmid vector where the SV40 viral promoter drives sea pansy luciferase (Renilla reniformis). In HT‐29 experiments, 0.5 μg of a rat MR expression vector (gift from David Pearce) or an empty plasmid, pcDNA3 , was cotransfected with the luciferase plasmids. In some experiments, the αENaC promoter‐luciferase construct (‐141 + 55) that does not contain a functional GRE was used as a negative control and compared to a construct containing two copies of the Sgk1 GRE at ‐142nt, αENaC_Sgk1_2xGREluciferase , with 1 ® VILO™ (Life Technologies) to generate cDNAs with the following conditions: 25°C for 10 min followed by 42°C for 60 min for cDNA synthesis, and 85°C for 5 min for termination.Total RNA from cultured cells was prepared with AbsolutelyRNA miniprep kit while total RNA from microdissected distal nephron segments was extracted with Absolutely RNA nanoprep kit . Equal amounts of RNA were subjected to reverse transcription with SuperScriptPrimers used in real time PCR for human and mouse Sgk1, Sgk1_v3 and 18S rRNA have been published , Mann–Whitney  or one‐way ANOVA where applicable, using SigmaPlot® 12 . In all figures statistical significance is indicated where P values <0.05 were considered statistically significant.Data are provided as arithmetic means ± standard error. All data were tested for significance with a Studentʹs c14 cells. We also reported that its encoded protein sgk1_i3 is more stable compared to sgk1 and that it stimulates apical epithelial Na+ transport when expressed in FRT and in mpkCCDc14 cells , chlorocebus sabaeus (green monkey) and microcebus murinus (gray mouse lemur). The primate HRE differs from rat and mouse sgk1 HRE, ACCACAnnnTGTTCT by 2 nucleotides. We have demonstrated that sgk1_v3 is conserved in rodents and like sgk1 that it is regulated by glucocorticoids in human airway epithelial cells (A549) and mouse collecting duct epithelial cells (mpkCCDc14). It is not uncommon for a 5ʹ regulatory element to regulate multiple transcripts of the same gene even when the transcription start sites and proximal promoters are different. While exon 1d and exon 1, the initiating exons for Sgk1_v3 and Sgk1 are only ~700 bp apart in homo sapiens, the transcription start site for exon 1a, the initiating for Sgk1_v2 is a ~135 kb away. Since Sgk1_v2 is not corticosteroid regulated, a transcriptional insulator element is likely to be present upstream of the Sgk1 HRE shielding Sgk1_v2 and possibly other upstream genes from the influence of this HRE.The HRE in the Sgk1 gene is an imperfect palindromic bipartite sequence within the 5ʹ regulatory region that is identical to that seen in several non‐human primates. Interestingly, the transcription start site is upstream of and incorporates this GRE within the 5ʹ transcribed but untranslated region for several other primates including the + diet and high K+ diet regulate Sgk1_v3 in vivo. We identified a shared 5ʹ flanking HRE as the transcriptional basis for corticosteroid‐stimulated Sgk1_v3 and sgkl expression.In conclusion, we demonstrate that glucocorticoids and aldosterone regulate an abundant 5ʹ variant alternate transcript of Sgk1, Sgk1_v3 in multiple epithelial cell lines and physiologically relevant stimuli such as aldosterone, low NaNone declared.