To this end, we generated Jmjd3 global and conditional knockout mice. Global deletion of Jmjd3 induces perinatal lethality associated with defective lung development. Tissue and stage-specific deletion revealed that Jmjd3 is dispensable in the later stage of embryonic lung development. Jmjd3 ablation downregulates the expression of genes critical for lung development and function, including AQP-5 and SP-B. Jmjd3-mediated alterations in gene expression are associated with locus-specific changes in the methylation status of H3K27 and H3K4. Furthermore, Jmjd3 is recruited to the SP-B promoter through interactions with the transcription factor Nkx2.1 and the epigenetic protein Brg1. Taken together, these findings demonstrate that Jmjd3 plays a stage-dependent and locus-specific role in the mouse lung development. Our study provides molecular insights into the mechanisms by which Jmjd3 regulates target gene expression in the embryonic stages of lung development.Histone demethylases have emerged as important players in developmental processes. Jumonji domain containing-3 (Jmjd3) has been identified as a key histone demethylase that plays a critical role in the regulation of gene expression; however, the  Jmjd3 deletion results in perinatal lethality that is associated with respiratory failure caused by defective lung development. Tissue and stage-specific deletion show that Jmjd3 is dispensable for mouse lung development in the later stages (after E9.5). Jmjd3 deficiency downregulates the expression of genes critical for lung development through interactions with specific transcription factors and epigenetic protein complexes. Our findings provide new insights into the role and molecular mechanism of action of Jmjd3 in embryonic lung development.A chromosome in the eukaryotic nucleus is an organized package of DNA coiled around histone proteins. DNA contains genes and other nucleotide sequences as well as histone proteins including H1, H2A, H2B, H3, and H4. Gene expression is dynamically regulated by DNA and histone modifications, such as methylation and demethylation. The protein Jumonji domain containing-3 (Jmjd3) is a critical demethylase that regulates gene expression. Here, we found that Jmjd3 plays an important role in the regulation of mouse lung development. Global  Histone methylation state is dynamically regulated by histone methyltransferases and demethylases in vivo function. Jmjd3 expression is induced by vitamin D and proinflammatory stimuli in macrophages and is required for the expression of INK4A-ARF, Nodal, and Irf4 in fibroblasts, mouse embryonic stem cells (ESCs), and macrophages, respectively Jmjd3 knockout (KO) mice have demonstrated the importance of Jmjd3 in differentiation and development in vivo. Jmjd3 has been shown to play a crucial role in the regulation of macrophage development and differentiation Jmjd3 deletion in mice indicate the critical requirement for Jmjd3 during development Despite the identification of Jmjd3 as a key H3K27 demethylase, little is known regarding its SP-B in mice and humans results in lethal neonatal RDS, which is characterized by a virtual absence of lung compliance and increased amounts of incompletely processed proprotein SP-C SP-B gene mutation SP-B expression are associated with epigenetic alterations remains largely unknown.Lung development is a complex process that requires the participation of many transcription factors and developmentally regulated genes at several different stages. This complex process begins with the formation of airways from embryonic lung buds that originate from the foregut endoderm and branch into the millions of alveoli required for the first breath after birth in vivo, we generated Jmjd3 global and tissue-specific knockout mice. We found that Jmjd3 ablation induces perinatal lethality associated with respiratory failure caused by defective lung development. Tissue-specific deletion of Jmjd3 and tamoxifen (TM)-induced temporal deletion revealed Jmjd3 is dispensable in the later embryonic development (after E9.5) stages. Gene expression profiling, tissue staining, and chromatin immunoprecipitation-sequencing (ChIP-Seq) analyses showed that Jmjd3 deficiency markedly reduces a set of genes critical for lung development, in particular SP-B. Jmjd3-mediated changes in target gene expression are associated with alterations in H3K27 and H3K4 methylation levels in the proximal promoter region. Jmjd3 regulates SP-B expression in a locus-specific manner through interactions with the transcription factor Nkx2.1 and the epigenetic protein Brg1. Together, our findings show that Jmjd3 plays a stage-dependent and locus-specific role during embryonic lung development. This function of Jmjd3 is associated with the epigenetic regulation of lung surfactant protein gene expression.To investigate the function and mechanisms of Jmjd3 in vivo functions of Jmjd3 during development, Jmjd3 KO mice were generated by homologous recombination technique +/−Jmjd3 mice were fertile and viable. However, homozygous Jmjd3-deficient mice were not recovered at weaning time (P21) from the heterozygous Jmjd3+/− mating pairs  up to postnatal day 0 (P0). However, homozygous Jmjd3-deficient mice died shortly after birth (P0) with kyphosis and severe lordosis (Jmjd3-deficient newborns and their wild-type (WT) littermates had pink skin color at birth; however, Jmjd3-deficient newborns became cyanotic within minutes due to respiratory failure , SP-A, SP-B, SP-D, and Clara cell 10 kDa secretory protein (CC10). Consistent with this observation, qPCR and immunoblot analyses showed that AQP-5, SP-B, SP-D, and CC10 mRNA and protein expression were markedly reduced in Jmjd3-deficient lung tissues compared with WT lung tissues  were markedly increased in Jmjd3-deficient lungs compared with WT lungs  by immunofluorescence staining and found increased H3K27me2 and H3K27me3 in −/−Jmjd3 MEFs  and 292 genes had decreased H3K27me3 in Jmjd3-deficient lungs compared with WT controls  containing AQP-5 on chromosome 15. Our ChIP-Seq data showed that H3K27 and H3K4 methylation of both SP-B and AQP-5 were altered, whereas H3K27 and H3K4 methylation in neighbouring loci were not significantly changed. This data suggests that Jmjd3 targets specific loci for histone demethylation and does not affect histone methylation in adjacent chromosome regions  containing  regions .SP-B gene expression Jmjd3 and FLAG-tagged Nkx2.1  SP-B expression by interacting with Nkx2.1 in the SP-B promoter region.Jmjd3 is a general H3K27 demethylase without specific DNA binding properties and the mechanisms by which it regulates specific gene expression remain unknown. Therefore, we reasoned that Jmjd3 might be recruited to specific gene promoters through interactions with key transcription factors and/or epigenetic proteins. To test this possibility, we first determined whether Jmjd3 regulates target gene expression by interacting with Nkx2.1. Nkx2.1  is a key transcriptional activator of d Nkx2.1 . To deteSP-B expression Jmjd3 and FLAG-tagged Brg1 . WT Jmjd3 significantly enhanced Nkx2.1 and Brg1-mediated SP-B promoter activity, whereas mutant Jmjd3 failed to do so  and −/−Jmjd3 mice were generated by homologous recombination technique Jmjd3 deletion in mice, TM (Sigma T5648) was dissolved in corn oil (Sigma) at a concentration of 10 mg/ml and intraperitoneally (IP) injected into 8-week-old mice at 9 mg TM/40 g of body weight Wnt1-Cre strain was obtained from the Jackson Laboratory (022137). SPC-Cre, and CCSP-Cre and floxedUtx mice were kindly provided by Dr. Brigid Hogan , Dr. Frank DeMayo (Baylor College of Medicine) and Dr. Kai Ge , respectively.Jmjd3 floxed mice (Jmjd3 expression in heterozygous and homozygous WT MEFs and WT and Jmjd3−/− E17.5 lung tissues was determined using the following Jmjd3 primer set: (forward) 5′-AAGTGGGGACAAGGAGACCT and (reverse) 5′-AAGTGGGGACAAGGAGACCT. WT and Jmjd3−/− E17.5 lung tissues were also evaluated for the expression of SP-A (5′-CTCCAGACCTGTGCCCATATG and 5′-ACCTCCAGTCATGGCACAGTAA), SP-B (5′-ACGTCCTCTGGAAGCCTTCA and 5′-TGTCTTCTTGGAGCCACAACAG), SP-C (5′-ACCCTGTGTGGAGAGCTACCA and 5′-TTTGCGGAGGGTCTTTCCT), SP-D (5′-CTCTGAGGCAGCAGATGGA and 5′-ATCAGGGAACAATGCAGCTT), CC10 (5′-TCCTAACAAGTCCTCTGTGTAAGA and 5′-AGGAGACACAGGGCAGTGACA), AQP-5 (5′-ATGAACCCAGCCCGATCTTT and 5′-ACGATCGGTCCTACCCAGAAG), ABCA3 (5′-TTGCCCTCATTGGAGAGCCTG and 5′-TCCGGCCATCCTCAGTGGTGGG), Nkx2.1 (5′-TCCAGCCTATCCCATCTGAACT and 5′-CAAGCGCATCTCACGTCTCA), UTX (5′-ATCCCAGCTCAGCAGAAGTT and 5′-GGAGGAAAGAAAGCATCACG), Ezh2 (5′-GCCAGACTGGGAAGAAATCTG and 5′-TGTGCTGGAAAATCCAAGTCA), Hoxa5 (5′-TCTCGTTGCCCTAATTCATCTTT and 5′-CATTCAGGACAAAGAGATGAACAGAA), Hoxb1 (5′-TTGCCCTGGAAACTGTAAAG and 5′-AATTTGCCAACAACCCATC), Esrrg (5′- TGCACCGGGCTCTGTCAAGGAA and 5′-AATCCATGTGCGCCCGACAACC), Reln (5′-TCCAGGCTCAGCACCAAGCCAA and 5′- TGGATCTTGCCTTCTGACGCCCTT), Kirrel3 (5′-AGAAAGTCACAGCTCCGCTCGGT and 5′-ACGGGAGGGTTGCAGAAAGGCT), March4 (5′-GCCCCTCCCCTTGGTTCCATCAAA and 5′-CGAGGAGGAGAAAGCGAAGCCACT), Fog2 (5′-CGCCTTTGTGGTGGACTTTGACT and 5′-GCTTCTCGTTGCCTCCCACTACA), COUP-TFII (5′-AGTACTGCCGCCTCAAAAAG and 5′-CGTTGGTCAGGGCAAACT), Wt1 (5′-AATGCGCCCTACCTGCCCA and 5′- CCGTCGAAAGTGACCGTGCTGTAT) and β-Actin (5′-GTGGGCCGCTCTAGGCACCA and 5′-TGGCCTTAGGGTTCAGGG).Total RNAs were isolated from E17.5 lung tissues and primary MEFs using Trizol reagent (Invitrogen). A total of 1 µg of RNA was converted to cDNA using Superscript III Reverse Transcriptase (Qiagen) with random hexamer primers. qPCR was carried out using SYBR Green mix on the ABI 7000 PCR machine. +/+Jmjd3 and −/−Jmjd3 lungs using the Affymetrix Genechip microarray system. RNA quality assessment and microarray analysis were performed by the Microarray Core Facility of Baylor College of Medicine. RNA quality was checked using the NanoDrop ND-1000 spectrophotometer and Agilent 2100 Bioanalyzer. Total RNA samples of 250 ng were labeled using the 3′ IVT Express Kit with the new standard Affymetrix linear amplification protocol and then reverse transcribed to produce double-stranded cDNA. The cDNA product was used as a template for the in vitro transcription of biotin-labeled cRNA. The labeled cRNA was quantified using the NanoDrop ND-1000 spectrophotometer. A total of 15.0 µg of the labeled cRNA was fragmented and the concentration rechecked. A hybridization cocktail containing Affymetrix spike-in controls and fragmented labeled cRNA was loaded onto a GeneChip array. The array was hybridized for 16 h at 45°C with rotation at 60 rpm and then washed and stained with a strepavidin, R-phycoerythrin conjugate stain. Signal amplification was done using biotinylated anti-streptavidin. The stained array was scanned on the Affymetrix GeneChip Scanner 3000. The images were then analyzed and quality control metrics were recorded using Affymetrix GCOS software version 1.1.2. Pairwise comparisons were made between WT and Jmjd3-deficient lungs. Raw P values were adjusted by the Benjamini-Hochberg method for 5% false discovery rate to yield adjusted p values. The criteria for significance of differentially regulated genes were established as ≥1.3-fold change with an adjusted P value<0.05. Pathways were analyzed (Ingenuity Systems) to determine the ratios of known genes within each pathway that were significantly changed between WT and KO lungs.Microarray analysis was performed on RNA samples prepared from E17.5 Lung tissues were collected, ground into fine powders in liquid nitrogen, and transferred to lysis buffer  supplemented with protease inhibitor cocktail  and 2 µM phenylmethylsulphonyl fluoride (PMSF). Cells were washed in phosphate-buffered saline (PBS), harvested, and lysed in RIPA buffer supplemented with protease inhibitor cocktail and 2 µM PMSF. After sonication and centrifugation, protein supernatants were collected, and protein concentrations were determined using a Bradford Protein Assay Kit (Sigma). Protein extracts were boiled in SDS sample buffer for 5 min, loaded directly onto a 4–12% SDS gel, transferred onto nitrocellulose membranes (Bio-Rad), blocked with 5% milk, and incubated with primary and corresponding secondary antibodies. The following antibodies were used: anti-H3K4me1 (07-436), anti-H3K4me2 (07-030), anti-H3K4me3 (07-473), anti-H3K9me1 (07-450), anti-H3K9me2 (07-441), anti-H3K9me3 (07-442), anti-H3K27me1 (07-448), anti-H3K27me2 (07-452), and anti-H3K27me3 (07-449) from Millipore; anti-SP-A (sc-7700), anti-AQP-5 (sc-9891); anti-SP-B (sc-13978), anti-SP-C (sc-13979), anti-SP-D (sc-13980), and anti-CC10 (sc-25555) from Santa Cruz Biotechnology; anti-H3(9715) and Ezh2 (4905) from Cell Signaling; anti-CD11b (17-0113-81), anti-F4/80 (15-4801-82), and anti-Gr1 (17-5931-81) from eBioscience; mouse anti-Flag, anti-HRP-flag, and anti-β-actin from Sigma; rabbit anti-UTX (ab36938) from (Abcam); and anti-Jmjd3 (ab1022a) from Abgent.Jmjd3 KO embryos at E11.5 Tissues and embryos were fixed in 10% neutral formalin. The fixation time was dependent on embryonic stage: E<12.5 were fixed for 2 h, E12.5-E17 for 4 h, and E>17.5 for 24–48 h as previously described AQP-5, SP-A, SP-B, SP-C, SP-D, and CC10 were evaluated by ChIP-PCR and ChIP-qPCR using the following specific primers: AQP-5-AF, 5′-AACCTGCGGAGGGGGAAGGT; AQP-5-AR, 5′-CGTCCCCACCCCCACTCCAT; AQP-5-BF, 5′-CACCACCCCAGGGTCCCCAA; AQP-5-BR, 5′-CCTGCTCTGCGCTCGGCAAT; AQP-5-CF, 5′-CAGGAGAGCCCAGCACGCAC; AQP-5-CR, 5′-CAGTGTAGACTGGCCCGGCG, SP-A-AF, 5′-GCACACGTACGGAAGCCGGA; SP-A-AR, 5′-CCTGCGGTGCTCAGTGGCAA; SP-A-BF, 5′-CCTTTCTGCTTCTTTCCTATGGCCG; SP-A-BR, 5′-AGCAAAACATCAGAACAACCAAAACTA; SP-A-CF, 5′-ATGGCTGCTTCCTGTCCGGC; SP-A-CR, 5′-CCCGCACACAGAGCCTGCAA, SP-B-AF, 5′-AGGGCCCGGACACATAGAGG; SP-B-AR, 5′-CTGAGGCCCAGGGCAGAGGT; SP-B-BF, 5′-GGGTGTGAGGTGACACGCCG; SP-B-BR, 5′-CTGTGGTGGGGGTGACCACG; SP-B-CF, 5′-TGTCACCAGTGGCACAGTGGAA; SP-B-CR, 5′-AGGTGAGCACTGCCATACCAGG; SP-B-DF: 5′-GTAGAGGATTGAGAAGCCT; SP-B-DR, 5′-CAGCCTGACTTTGTTCAC; SP-B-EF, 5′-CGTGACTCTCTGAAGAAGGC; SP-B-ER: 5′-AAACGACACCCTGGAAGTG; SP-B-FF, 5′-CCCTTGTTTGACGGTGAA; SP-B-FR: 5′-TGGCTGCCTACTGCTTAGT; SP-C-AF, 5′-ACAGGCAATCCCAGATCGCTGA; SP-C-AR: 5′-GTCCCAGCCATCTCTGCCCCT; SP-C-BF, 5′-AGAGGGGCAGAGATGGCTGGG; SP-C-BR, 5′-GCTTGGGACAGCACCTGGGG; SP-C-CF, 5′-AGGCCCAGTCCTTCACCCCTG; SP-C-CR, 5′-GCCTACTGGAGGATGGACAGTCA; SP-D-AF, 5′-TCAGAGGACTAATGACAGCCTT; SP-D-AR, 5′-CAGCAGGGACAGACATACCA; SP-D-BF, 5′-GGAAGATGGAAGAACAAGGG; SP-D-BR, 5′-CTACAAAGGCAGCAACCTG; SP-D-CF, 5′-TGTGTGTGTGTGTGTGTGTGA; SP-D-CR, 5′-CACCTCTGTTTGTCAGGCTC; CC10-AF, 5′-TCCCACCAGCACCATAGTA; CC10-AR, 5′-CACCTTCTCCATTTCCACA; CC10-BF, 5′-CTAACAATGCCCAAGAATCG ; CC10-BR, 5′-GGAGACCCTTCAGGAATCA; CC10-CF, 5′-CTCCACTGCCTGAATACTCC; CC10-CR, 5′-ACTTGGTCATCTTCTCCGTG.Chromatin was prepared from lung tissues of E17.5 embryos. ChIP-PCR and ChIP-qPCR were performed using Jmjd3 antibody (Abgent) and selected histone marks, respectively. Immunoprecipitated chromatin DNA and input were used for ChIP-PCR and ChIP-qPCR with specific primers. Briefly, whole lungs of E17.5 embryos were quickly chopped into tiny pieces with a scalpel and fixed in 1% formaldehyde in 1X PBS at room temperature for 15 min. Glycine was added to a final concentration of 0.125 M. Samples were rotated at room temperature for another 5 min, centrifuged at low speed for 5 min, and the supernatants decanted. The pellets were washed once with ice-cold 1X PBS and disaggregated mechanically through a 0.5-µm cell strainer in 1 ml of ice-cold 1X PBS. Immunoprecipitation was performed on single cell suspensions using the ChIP Assay Kit according to the manufacturer's instructions . Antibodies used included anti-Jmjd3; anti-mono-, di-, and trimethylated H3K4 antibodies; anti-mono-, di-, and trimethylated H3K9 antibodies; and anti-mono-, di-, and trimethylated H3K27 antibodies. DNA binding of Jmjd3 and methylation of lung marker genes including +/+Jmjd3 and −/−Jmjd3 embryos were collected and prepared for ChIP-PCR as previously described Jmjd3 knockout were assessed using the fold changes of background-subtracted read densities around 5 kb and 2 kb of the TSSs. The statistical significance of the fold change was assessed using the MA-plot-based method. The fold change and the false discovery rate cutoffs were set to 1.5 and 10%, respectively.Lungs of E17.5 t test with GraphPad Prism 4.0 software. A P value<0.05 was considered significant.Data are represented as mean ± standard deviation (SD) where indicated. Statistical analysis was performed using the Student's Figure S1A) H&E staining of Jmjd3-deficient lung tissues at P0. Type I and type II cells and capillary beds were altered in Jmjd3-deficient lungs. Bar = 50 µm. (B) All lobes were smaller in Jmjd3-deficient lungs at E17.5 compared with WT lungs. L, left lobe; S, superior lobe; P, post-caval lobe; M, middle lobe; I, inferior lobe. (C) H&E staining of Jmjd3-deficient lung tissues at E17.5.Jmjd3 deficiency induces developmental defects in the lung. ((TIF)Click here for additional data file.Figure S2f/fJmjd3:f/fUtx:SPC-Cre, f/f:SPC-CreJmjd3, and f/f:SPC-CreUtx pups at P0 appeared to be normal and were able to grow to adulthood.UTX does not compensate for Jmjd3 deficiency. All (TIF)Click here for additional data file.Figure S3Jmjd3 KO tissues. (A) Pre-Bötzinger complex-specific gene expression in the brainstem of Jmjd3 KO and WT mice at E17.5. and P0. (B) Pre-Bötzinger complex-specific gene expression in Jmjd3 KO and WT E17.5 lung tissues. (C) Respiratory muscle gene expression in diaphragm and intercostal muscle at P0. Data are presented as the mean ± SD from three independent experiments. *P<0.05, **P<0.01 (Student's t test).Gene expression in (TIF)Click here for additional data file.Figure S4A) Total methylation of H3K27, H3K4, and H3K9 in Jmjd3-deficient lungs. (B) Immunostaining of WT and Jmjd3−/− primary MEFs with anti-mono-, anti-di-, and anti-trimethylated H3K4, H3K9, and H3K27 antibodies. DAPI nuclear staining is shown in blue. Texas-red (TR) staining indicates histone methylation. (C) Ectopic expression of Jmjd3 rescues total H3K27me3 in Jmjd3−/− MEFs. Lane 1, wild-type; lane 2, Jmjd3−/− MEFs; lane 3, Jmjd3−/− MEFs infected with control retrovirus; lane 4, Jmjd3−/− MEFs infected with retroviruses expressing Flag-Jmjd3. (D) Immunostaining of H3K27 methylation in Jmjd3−/− MEFs infected with Flag-Jmjd3 (F-Jmjd3) retrovirus. White arrowheads indicate infected cells.Jmjd3 deletion affects global H3 histone methylation and lung marker gene promoter methylation. ((TIF)Click here for additional data file.Figure S5A) ChIP-Seq analysis of H3K4me3 and H3K27me3 methylation in the regulatory and gene body regions of genes related to lung development. Scale = 2 kb. (B) ChIP-Seq analysis of H3K27me3 and H3K4me3 levels in several RRG-related genes in Jmjd3 KO and WT E17.5 lung tissues. Open boxes indicate 5 kb regions around TSS sites. Red arrows indicate the transcription direction. (C) ChIP-PCR analysis of Jmjd3 binding to AQP-5, SP-A, SP-C, SP-D, and CC10 proximal promoter regions. The lung tissues of Jmjd3+/+ and Jmjd3−/− embryos at E17.5 were formalin fixed, homogenized, and sonicated before immunoprecipitation with anti-Jmjd3 antibody. The binding regions were analyzed by PCR with specific primers. The designed primers and their location on the gene promoters (right panel). IgG was used as negative control. The red ovals represent Jmjd3 binding to the indicated regions. (D) ChIP-qPCR analysis of H3K27 and H3K4 mono-, di-, and trimethylation levels in the regions upstream of the TSS sites of AQP-5, SP-D, and CC10 in Jmjd3−/− and WT lungs. The location of the designed primers A, B, and C is around −1000 bp, −500 bp, and +1 bp, respectively (top panel). Data are presented as the mean ± SD of methylation enrichment (fold) from three independent experiments. *P<0.05 (Student's t test).ChIP-Seq and ChIP-PCR analysis of lung marker and RRG genes. ((TIF)Click here for additional data file.Figure S6Utx and FLAG-tagged Nkx2.1 do not show an interaction between UTX and Nkx2.1.UTX does not interact with Nkx2.1. Immunoprecipitation and immunoblot analysis of 293T cells expressing HA-tagged (TIF)Click here for additional data file.Figure S7Catalytic domain of Jmjd3 is required for enhancing SP-B-promoter-driven luciferase activity. Up, the measurement of luciferase activity; bottom, protein expression level of Jmjd3 fragments, FLAG-Brg1, FLAG-Nkx2.1, and β-actin. NS, non-specific bands.(TIF)Click here for additional data file.Table S1+/−Jmjd3 breeding pairs.Genotyping analysis of embryos and newborns from (JPG)Click here for additional data file.Table S2−/−Jmjd3 and WT embryonic lungs at E17.5.Microarray analysis of gene expression in (XLSX)Click here for additional data file.Table S3ChIP-Seq analysis of H3K4 and H3K27 methylation around +/− 5 kb of TSS.(XLSX)Click here for additional data file.Table S4ChIP-Seq analysis of H3K4 and H3K27 methylation around −2 kb of TSS.(XLSX)Click here for additional data file.


The trophic peptide hormone adrenocorticotropic (ACTH) stimulates steroid hormone biosynthesis evoking both a rapid, acute response and a long-term, chronic response, via the activation of cAMP/protein kinase A (PKA) signaling. The acute response is initiated by the mobilization of cholesterol from lipid stores and its delivery to the inner mitochondrial membrane, a process that is mediated by the steroidogenic acute regulatory protein. The chronic response results in the increased coordinated transcription of genes encoding steroidogenic enzymes. ACTH binding to its cognate receptor, melanocortin 2 receptor (MC2R), stimulates adenylyl cyclase, thus inducing cAMP production, PKA activation, and phosphorylation of specific nuclear factors, which bind to target promoters and facilitate coactivator protein recruitment to direct steroidogenic gene transcription. This review provides a general view of the transcriptional control exerted by the ACTH/cAMP system on the expression of genes encoding for steroidogenic enzymes in the adrenal cortex. Special emphasis will be given to the transcription factors required to mediate ACTH-dependent transcription of steroidogenic genes. The adrenal gland is a key component of the hypothalamus–pituitary–adrenal (HPA) axis, thus playing a crucial role in the adaptation of organism to stressors. The adrenocorticotropic hormone (ACTH) belongs to this regulatory circuitry, being one of the most potent physiological modulators of adrenal cortex steroidogenesis and trophicity , 2. It ede novo from acetate in the adrenal  to the inner mitochondrial membrane (IMM) is known to be promoted by StAR (see below), the molecular mechanism underlying cholesterol transport and loading into the OMM is still under investigation . The enzCYP11A1 gene) catalyzes breakage of the bond between positions 20 and 22 in the cholesterol side chain . P450c11β  and P450c11AS  catalyze 11β-hydroxylase, 18-hydroxylase, and 18-methyl oxidase activities. At the level of the endoplasmic reticulum, we distinguish: P450c17 (encoded by the CYP17A1 gene) that catalyzes both 17α-hydroxylase and 17,20-lyase activities; P450c21 (encoded by the CYP21A1 gene) that catalyzes 21-hydroxylation in the synthesis of both glucocorticoids and mineralocorticoids; and P450-Aro (encoded by the CYP19A1 gene) that catalyzes the aromatization of androgens to estrogens.Cytochrome P450s are a group of oxidative enzymes. In the human genome, genes for 57 CYPs enzymes have been described. Seven of them are called “type 1” enzymes. They are targeted to mitochondria and receive electrons from NADPH via a flavoprotein (ferrodoxin reductase) and a small iron-sulfur protein (ferredoxin). “Type 2” enzymes are localized in the endoplasmic reticulum and received electrons form NADPH via a single 2-flavin protein called P450 oxidoreductase . Six P45+ or NADPH/NADP+) as cofactors to either reduce or oxidize a steroid through a hydride transfer mechanism . Interestingly, patients displaying mutations in P450c17 are not able to produce cortisol, but show increased corticosterone production  and relatively little 3βHSD2.The pattern of steroid hormones secreted by each adrenal zone is determined by the enzymes expressed in each zone . Specifixpressed . The zonoduction , like inStar gene causes defects in steroidogenesis, with consequent male pseudohermaphroditism and lethality within 1 week after birth  is an orphan member of the nuclear receptor superfamily, which acts as a key regulator of adrenogonadal development and tissue-specific gene expression in steroidogenic cells. Parker and Morohashi groups identified SF-1 by its capacity to bind to and activate transcription from multiple P450 steroidogenic enzyme promoters, which display one or more SF-1-binding sequences in close proximity to the TATA box , 73. FurStAR gene transcription is induced by both angiotensin II and cAMP via increased SF-1-binding to a cAMP-responsive region within the first 350 bp upstream of the transcription initiation site  on the gene promoter -like binding site, which are essential for cAMP-driven transcription  to synergistically promote cription . Interescription , 86.CYP11B2 gene in the zona glomerulosa, where it is responsible for mineralocorticoid production via an SF-1-binding site and a CRE  and the 17,20-lyase reaction of 17α-hydroxylated steroids (leading to androgen production). Studies carried out on H295R cells revealed that CREs are located within the first 63 bp upstream of the transcriptional initiation site and that a second basal transcription element lies between −184 and −206 bp (nrb/NonO and polypyrimidine tract-binding protein-associated splicing factor (PSF), which binds within those first 60-bp upstream of the transcriptional start site, stimulating CYP17 expression in response to ACTH/cAMP signaling , exhibit somatotroph hypoplasia and dwarfism  (see below) with subsequent stimulation of their transcriptional activity , 103–105dwarfism . Moreovedwarfism , 7, 101.CYP11A1 gene transcription in rodent placenta and ovary  . At leasStar gene (281/272 bp), which is also bound by CREB/ATF (see above) and Fos/Jun (see below). Further, two putative C/EBP-binding sites have been identified within the human StAR promoter TCA]. AP-1 is composed by a mixture of homo- and hetero-dimers formed between Jun  and Fos  family members , which shows homology with the AP-1/TRE sequence and overlaps also with the CRE2 sequence  associated with hypogonadotropic hypogonadism (HHG)  , 142. DAfactors [–145; revfactors . In addfactors [ and FSH factors [ downreguTranscriptional coregulators are crucially implicated in nuclear receptor-mediated transcriptional activation –151 and StAR has been shown by several studies  and the silencing mediator of retinoic acid and thyroid hormone receptor (SMRT) increase the repressive activity of chicken ovalbumin upstream-transcription factor 1(COUP-TF-1)  .CYP17 gene transcription by the recruitment of a histone deacetylase to the SF-1/NonO/PSF complex that binds to the CYP17 promoter (CYP17 transcription rate in H295R cells (ASAH1) gene transcription via the binding of CREB to multiple region of the ASAH1 promoter. This event triggers the recruitment of CBP/p300 with a related increase in the trimethylation of Lys4 on histone H3 (H3K4) on the ASAH1 promoter in H295R cells , reached a peak after 15 min of ACTH administration (around twofold) and returned to basal levels after 30 min , which have been implicated in ogenesis –172. Indr 30 min . They shr 30 min . Moreover 30 min . Those sr 30 min . Those er 30 min . Altogetr 30 min . The ACTr 30 min , 173. Ser 30 min , 175. Thr 30 min , 177. Fu5R cells . They alis event . Those eis event , 179. This event . Moreovetivation .The studies outlined here have given an important contribution to the understanding of the impact of ACTH on the regulation of steroidogenic gene expression in the adrenal cortex. Given the pivotal role played by ACTH/cAMP signaling in the acute and chronic regulation of steroid secretion and the implication of those hormones in diverse arrays of fundamental physiological processes, an in-depth investigation is needed to completely elucidate the ACTH-dependent transcriptional regulatory mechanisms that control steroid secretion. Indeed, some aspects addressed in this review still represent a challenge for future studies, which might provide the scientific community with a broader picture of the complex signaling pathways and the intricate transcriptional circuitries that coordinately ensure optimal hormonal output.All authors listed, have made substantial, direct, and intellectual contribution to the work, and approved it for publication.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


S. mansoni lay 300 eggs daily. A large fraction of the total mRNA in the mature female worm encodes one eggshell protein, Smp14. We report that the nuclear receptors SmRXR1 and SmNR1 regulate Smp14 transcription through the recruitment of two histone acetyltransferases (HATs), SmGCN5 and SmCBP1. The treatment of HEK293 cells with histone deacetylase (HDAC) inhibitors (NaB or TSA) produced an 8-fold activation of the SmRXR1/SmNR1-mediated Smp14 promoter activity. Incubation with synthetic HAT inhibitors, including PU139, significantly impaired the Smp14 promoter activity in these cells. Worm pairs cultivated in the presence of PU139 exhibited limited expression of Smp14 mRNA and protein. ChIP analysis demonstrated chromatin condensation at the Smp14 promoter site in worms treated with PU139. ChIP also revealed the presence of H3K27me3 and the absence of RNA Pol II at the Smp14 promoter region in the PU139-treated worms. Most significantly, the PU139-mediated inhibition of Smp14 expression resulted in a significant number of abnormal eggs as well as defective eggs within the ootype. In addition, scanning electron microscopy revealed structural defects and unformed eggshells, and vitelline cell leakage was apparent. The dsRNAi-targeting of SmGCN5 or SmCBP1 significantly decreased Smp14 transcription and protein synthesis, which compromised the reproductive system of mature female worms, egg-laying and egg morphology. Our data strongly suggest that the inhibition of Smp14 expression targeting SmGCN5 and/or SmCBP1 represents a novel and effective strategy to control S. mansoni egg development.Treatment and control of schistosomiasis relies on the only available drug, praziquantel, and the search for alternative chemotherapeutic agents is therefore urgent. Egg production is required for the transmission and immunopathology of schistosomiasis and females of  Schistosoma mansoni is a parasitic worm that causes schistosomiasis, a debilitating disease in Africa and South America. Female worms mated with males produce hundreds of eggs that can reach the environment to propagate the biological cycle, or become trapped in host tissues, triggering inflammation and pathology. Because eggshell formation is a key step in egg development and viability, we have studied the molecular mechanisms of S. mansoni eggshell development, focusing on a major eggshell gene, Smp14. Using a variety of technical and biological approaches, we obtained strong evidence that eggshell formation depends on nuclear receptors and coactivators with chromatin modifying activities, mainly histone acetylation. Inhibition or partial deletion of S. mansoni histone acetyltransferases impaired the expression of Smp14, culminating in a severe negative effect on eggshell formation. Our findings will contribute not only to a better understanding of sex and tissue-specific gene regulation in S. mansoni but also provide an alternative strategy for interfering with the egg production, which might be targeted in novel therapeutics directed against this parasite. S. mansoni lay a large number of eggs daily, which are responsible for both the transmission and pathogenesis of schistosomiasis S. mansoni eggshell development. The major S. mansoni eggshell protein is Smp14 Smp14 gene is exclusively expressed in mature vitelline cells in the vitelline duct and within the egg, enclosed by the eggshell, inside the ootype Smp14 is the most abundant mRNA transcript in sexually mature females and accounts for 10% of the mRNA of the entire organism Smp14 transcription is regulated by the nuclear receptor heterodimer SmRXR1/SmNR1, which recruits coactivators with histone modifying (acetylation and methylation) activities in vitro assembly of the SmRXR1/SmNR1 heterodimer and the two major histone acetyltransferases (HATs) in S. mansoni, SmGCN5 and SmCBP1, on a DNA response element within the Smp14 promoter region in vivo, the role of SmGCN5 and SmCBP1 in the transcriptional regulation of Smp14. We demonstrated that histone acetylation and chromatin remodeling by both schistosome HATs are essential for the activation of Smp14. More significantly, the inhibition of the histone acetyltransferase activity of SmCBP1 or SmGCN5 by a HAT inhibitor or RNA interference decreased Smp14 transcription and was associated with a severe negative effect on eggshell formation. Taken together, our data strongly suggest that histone acetyltransferase activity can represent a possible target for limiting parasite transmission, i.e., by decreasing the number of viable eggs deposited in the environment and reducing the production of the soluble egg and eggshell antigens involved in the immunopathogenesis of schistosomiasis.Schistosomes are large metazoan pathogens that parasitize over 200 million people worldwide, resulting in up to 300,000 deaths per year Smp14 promoter. Because schistosome cell lines are not available, we used HEK293 cells as a surrogate system to investigate Smp14 transcriptional regulation by schistosome nuclear receptors. We observed a SmRXR1/SmNR1-dependent transcriptional activation of the Smp14 promoter . As seen in Smp14 promoter was immunoprecipitated along with the nuclear receptors and HAT coactivators. These data strongly suggest that in vivo, SmGCN5 and SmCBP1 are recruited to the Smp14 promoter through the stable interaction with SmRXR1/SmNR1, and acetylate histone H3.First, we investigated whether the SmRXR1/SmNR1 heterodimer activated the transcription of a reporter gene under the control of the promoter . BecauseR1/SmNR1 . To furtin vitro using an immobilized histone peptide substrate and a heterogeneous assay format. The quantification of acetylation was measured using anti-acetyl lysine antibodies and a europium-labeled secondary antibody. Time-resolved fluorescence was used to determine the europium concentration, which is an indication of the degree of histone acetylation. Whereas the N-aryl derivatives PU139 and SF7 acted as broad-spectrum HAT inhibitors, the N-benzyl congener PU141 was selective for the closely related HAT subtypes CBP and p300  demonstrated the greatest inhibitory activity , the mRNA levels were decreased by the same amount as observed following the two-day incubation . The Smp14 promoter was co-immunoprecipitated by the anti-RNA polymerase II antibody  and an effective decrease in the Smp14 protein level . Surprisingly, the S. mansoni genome does not present genes encoding members of the p160 HAT coactivators Smp14 promoter.The data presented in this study clearly demonstrate that the tivities . DespiteSmp14 expression, then the inhibition of these enzymes should have a direct effect on egg development in vivo. Indeed, this study demonstrated that the targeted inhibition of the catalytic domains of the schistosome HATs by PU139 treatment or the disruption of HAT transcription/translation by dsRNAi significantly decreased the Smp14 mRNA and protein levels, culminating in gross morphological eggshell defects and hindering egg development  and were approved by the committee for ethics in animal experimentation of the Nord-Pas de Calais region (Authorization No. AF/2009) and the Pasteur Institute of Lille (Agreement No. A59-35009).S. mansoni was maintained in Biomphalaria glabrata as the intermediate host and in Syrian hamsters (Mesocricetus auratus) as the definitive host A Puerto-Rican isolate of 2. The cells were plated one day before transfection in 24-well plates at a density of 80,000 cells per well. The transfections were performed using 150 ng of each plasmid . The cells were lysed 24 h after transfection, and the analysis of luminescence as a measure of luciferase activity was performed . The sodium butyrate (NaB), trichostatin A (TSA) or HAT inhibitors were added to the plated cells at 6 hours after transfection. MTT and LDH assays were used to measure the cell viability (data not shown). Statistical significance was determined using Student's t-test.The full-length cDNA of SmGCN5 The adult worms were obtained by whole-body perfusion of hamsters following a 6-week infection, and the worms were cultivated as previously reported 4 and 0.8% K3Fe (CN)6; washed in 0.1 M cacodylate buffer, pH 7.2; dehydrated in a graded ethanol series (20°–100° GL) for one hour each step; critical-point dried in CO2; mounted on metallic stubs and coated with gold (20–25 nm deposited). The samples were examined under FEI Quanta 250 scanning electron microscope, operating at 15–20 kV and a pressure of 2–8×10−4 Pa. Transmission electron microscopy was performed on a Tecnai G2 microscope . Eggs  were washed in 0.1 M cacodylate buffer, pH 7.2; post-fixed in 1% OsO4 and 0.8% K3Fe (CN)6; washed in 0.1 M cacodylate buffer, pH 7.2; dehydrated in a graded acetone series (20°–100° GL) for one hour each step and embedded in Polybed 812 epoxide resin. Thin-sections (60 nm) were collected on copper grids, stained for 30 minutes in 5% aqueous uranyl acetate, for 5 minutes in lead citrate.The eggs were counted and photographed every 24 h, and the worm motility and morphology were monitored daily. Approximately 300 eggs from three independent experiments were measured (length and width) as previously reported ChIP analysis was performed following an adapted protocol for schistosomes, as previously reported In preparation for the double-stranded RNA (dsRNA) synthesis, 500-bp fragments of the firefly luciferase , SmCBP1 or SmGCN5 cDNAs were amplified by PCR using the gene-specific primers listed in Figure S1Smp14 promoter occupancy by schistosome nuclear receptors and HAT coactivators. ChIP analysis of fifty adult worms freshly perfused. Chromatin was extracted and immunoprecipitated with antibodies directed against acetylated H3, SmRXR1, SmNR1, SmGCN5 and SmCBP1. ChIP DNA (Smp14 promoter) was analysed by agarose gel and stained with EtBr.(TIF)Click here for additional data file.Figure S2PU139 treatment does not interfere with the mRNA levels of the target genes. The same parasites that were cultivated in the presence of PU139 or vehicle (DMSO) and evaluated for Smp14 expression Click here for additional data file.Figure S3Inhibition or partial depletion of histone acetyltransferases do not compromise the S. mansoni male reproductive system. (A) Adult worm pairs were cultivated for two days in the presence of PU139 or vehicle, fixed and analyzed by confocal laser scanning microscopy. Note that even after treatment, the male and female worms remained paired . Scale bars: 10 µm. (B) Adult worm pairs that received dsRNAs for LUC, SmCBP1 or SmGCN5 were cultivated for seven days and analyzed by confocal laser scanning microscopy. Details of the testicular lobes (tl) are shown in all panels. Scale bars: 20 µm.(TIF)Click here for additional data file.Table S1Five small synthetic pyridoisothiazolones were selected to test against members of the histone acetyltransferase (HAT) family  using histone H3 (amino acid residues 1–21) as a substrate. The data represent the IC50 value [µM] ± standard error. * Numbers in parentheses refer to compound numbering in the reference that describes the structure and synthesis.(DOCX)Click here for additional data file.Table S2List of oligonucleotides used in the study. For the ChIP analysis, three regions of the Smp14 promoter were amplified using the referenced primers. For ChIP, the Smp14 gene ATP region comprised the sequence from −28 to the ATG initiation codon (+70); the Smp14 gene DR17 region comprised the sequence from −480 to −398 and contained a hormone response element; and Smp14 gene DR5 region comprised the sequence from −210 to −113 and contained a second hormone response element. For the RNAi experiments, the oligonucleotides contained the T7 promoter sequence (underlined). The bolded text indicates the restriction enzyme sites in the SmGCN5 primers.(DOCX)Click here for additional data file.


To elucidate the molecular mechanisms involved in bovine ACSL1 regulation, we cloned and characterized the promoter region of ACSL1. Applying 5′-rapid amplification of cDNA end analysis (RACE), we identified multiple transcriptional start sites (TSSs) in its promoter region. Using a series of 5′ deletion promoter plasmids in luciferase reporter assays, we found that the proximal minimal promoter of ACSL1 was located within the region −325/−141 relative to the TSS and it was also located in the predicted CpG island. Mutational analysis and electrophoretic mobility shift assays demonstrated that E2F1, Sp1, KLF15 and E2F4 binding to the promoter region drives ACSL1 transcription. Together these interactions integrate and frame a key functional role for ACSL1 in mediating the lipid composition of beef.The nutritional value and eating qualities of beef are enhanced when the unsaturated fatty acid content of fat is increased. Long-chain acyl-CoA synthetase 1 (ACSL1) plays key roles in fatty acid transport and degradation, as well as lipid synthesis. It has been identified as a plausible functional and positional candidate gene for manipulations of fatty acid composition in bovine skeletal muscle. In the present study, we determined that bovine ACSL1was highly expressed in subcutaneous adipose tissue and  The nutritional value and eating qualities of beef are enhanced when the unsaturated fatty acid content of fat is increased. Longer chain n-3) polyunsaturated fatty acids such as docosahexaenoic acid DHA; 22:6, n-3) and eicosapentaenoic acid EPA, 20:5, n-3) also have well-recognized beneficial effects in terms of reducing the risk of cardiovascular disease, cancer, and type-2 diabetes, as well as critical roles in brain function, visual development in the fetus, and the lifelong maintenance of neural and visual tissues12:6, n-3  polyunsa, n-3 als14C-DHA and18209Intramuscular fat deposition and the fatty acid profiles of beef are determined mainly by lipid metabolism, which dictates the balance between fat deposition and fat oxidation in skeletal muscle. ACSL1 belongs to the class of acyl-CoA synthetases, which are essential for the activation, transport, and degradation of fatty acids, as well as lipid synthesis1112longissimus thoracis. Furthermore, we analyzed molecular mechanisms involved in ACSL1 regulation and found that transcriptional activity of ACSL1 gene was dependent on E2F1, Sp1, KLF15, and E2F4 transcription factors. Understanding the transcriptional regulation of ACSL1 may provide further information on regulatory roles of ACSL1 gene in mediating the lipid composition of beef.The expression of ACSL1 is important for regulating the fatty acid composition of bovine skeletal muscle, but the transcription factors that contribute to the control and regulation of its expression have not been characterized. In this study, we first determined the tissue distribution of ACSL1 mRNA and found that bovine ACSL1was highly expressed in subcutaneous adipose tissue and longissimus thoracis, liver, omasum, small intestine, abomasum, kidney, spleen, cecum, large intestine, rumen, reticulum, and lung tissue. As shown in longissimus thoracis, but low in the rumen, reticulum, and lung tissue.To detect the tissue distribution of bovine ACSL1 mRNA, qPCR was performed using cDNA from 14 bovine tissues and organs:, subcutaneous adipose, heart, In order to analyze the molecular mechanisms involved in ACSL1 regulation, we performed 5′-RACE to identify the TSS of ACSL1 gene. Antisense primer R1 and nested primer R2 , complemTo measure the activity of potential cis-acting elements and determine the minimum sequence required for activity, a series of nine reporter constructs with progressively larger deletions from the 5′ end of the promoter were generated. The effects of these modifications were evaluated upon transfection of the corresponding luciferase reporter plasmids into 3T3L1 and C2C12 cells, and the results of these analyses are shown in http://www.genomatrix.com) with a cutoff value over 90%. We identified four consensus motifs as the transcription factor recognition sites for E2F1, Sp1, KLF15, and E2F4 (http://www.urogene.org/methprimer/) revealed one CpG island located within the region −335/−57 relative to TSS-1, which had a GC content of 78.5%  to confirm whether E2F1, Sp1, KLF15, and/or E2F4 bind to the ACSL1 promoter longissimus thoracis . E2F transcription factors regulate both proliferative and metabolic genes31353637Sp1 is known to be act as both a negative and a positive regulator of gene transcription4041394041KLF15 is a member of the Spl-like/KLF family, which are closely related zinc-finger DNA-binding proteins, important regulators of glucose, amino acid metabolism, and lipid utilization454648longissimus thoracis and the expression of the ACSL1 is regulated by multiple transcription factors, including E2F1, Sp1, KLF15 and E2F4. Epigenetic modification in the ACSL1 promoter with an effect on transcription of the gene remains to be investigated. These results may lead to enhanced understanding of the regulation of ACSL1expression in other models, as well as providing new insights into the regulatory mechanisms and biological functions of the ACSL1 gene in mediating the lipid composition of beef.In conclusion, our study revealed that bovine ACSL1 is highly expressed in subcutaneous adipose tissue and −ΔΔCt methodFourteen tissues were obtained from three adult Qinchuan cattle. Total RNA was extracted from the tissues using a Total RNA kit  and then reverse-transcribed using a PrimeScript™ RT reagent Kit with gDNA Eraser  . cDNA from reverse transcription of individual animal samples for each tissue was pooled for qPCR which was performed using a SYBR Green PCR Master Mix kit  and 7500 System SDS V 1.4.0 . All of the primers used in the real-time PCR experiment are listed in longissimus thoracis was reverse-transcribed with PowerScript RT . PCR was performed using a Universal Primer A Mix (UPM)  and gene-specific primers (The transcription initiation site (TSS) of the bovine ACSL1 gene was determined using a BD SMARTTM RACE cDNA amplification kit  according to the manufacturer’s instructions. Briefly, 1 μg of total RNA isolated from the  primers  located http://www.genomatix.de software was used to analyze putative transcription factor binding sites on the positive and negative chain of the ACSL1 promoter, and to ensure the site-directed mutagenesis would not create new binding sites of other transcription factors. All the constructs were sequenced in both directions .In order to clone the bovine ACSL1 promoter region, we designed gene-specific primers to amplify a 2.0-kb genomic region upstream of the bovine ACSL1 gene TSS. The PCR product of 1954 bp was isolated from agarose gel using a Gel Extraction Kit (omega), and it was cloned into T-Vector pMD19 (simple) and submitted (GenBank No. KT232078). For the generation of the luciferase reporter construct, the 2.0-kb bovine ACSL1 promoter fragment was excised from the T-Vector pMD19 (simple) by digestion with Sac1 and Xho1  and ligated into the pGL3-basic vector digested with the same restriction enzymes. This plasmid was named pGL3-1933. Plasmids pGL3-1328, -1034, -730, -325, -251, -207, -140 and -80, which contained′ unidirectional deletions of the promoter, were generated by PCR using specific primers with the sequence of the Sac1 and Xho1 restriction sites incorporated and pGL3-1933 as template. Substitution mutation constructs were generated by a QuickChange Site-Directed Mutagenesis Kit . The MatInspector programme available online at 2 and passaged using standard cell culture techniques.Two mouse cell lines (C2C12 myoblasts) and 3T3L1 cells were maintained in Dulbecco’s modified Eagle’s medium  with 4500 mg/L glucose, where the medium was supplemented with 10% fetal bovine serum (FBS) , 100 units/mL penicillin , and 100 mg/mL streptomycin. The cells were incubated at 37 °C with 100% humidity under 5% CO5 cells/well in 24-well dishes and 24 h later, they were transfected with the plasmids described above using X-tremeGENE HP DNA transfection reagent  according to the manufacturer’s instructions. Briefly, cells were plated at 80–90% confluence and cultured overnight at 37 °C with DMEM containing 10% FBS, but without antibiotics. The transfection reagent was mixed with opti-MEM (6 μ/150 μL medium) (GIBCO-Invitrogen), incubated for 5 min at room temperature, combined with different plasmids (2.4 μg/150 μL for pGL3 and 0.03 μg/150 μL for pRL-TK), and then incubated for 20 min at room temperature. In total, 100 μL of the DNA-transfection reagent mixture was added to each well (in triplicate). At 48 h after transfection, the cells were washed with PBS and total lysates prepared using passive lysis buffer (Promega Corp). Luciferase activity was measured using the Dual Reporter assay system (Promega Corp) and NanoQuant Plate™ . The level of firefly luciferase activity was normalized to Renilla luciferase activity and expressed as arbitrary units.Cells were plated at a density of 1.2 × 10Nuclear extracts from C2C12 cells were prepared using the Nuclear Extract Kit  according to the manufacturer’s protocol. The protein concentration in the nuclear fraction was determined using the Bradford dye assay . All of the DNA probes used in the EMSA assays .


Drosophila intestine is orchestrated by Intestinal Stem Cells (ISCs). Following damage or stress the intestinal epithelium produces ligands that activate the epidermal growth factor receptor (EGFR) in ISCs. This promotes their growth and division and, thereby, epithelial regeneration. Here we demonstrate that the HMG-box transcriptional repressor, Capicua (Cic), mediates these functions of EGFR signaling. Depleting Cic in ISCs activated them for division, whereas overexpressed Cic inhibited ISC proliferation and midgut regeneration. Epistasis tests showed that Cic acted as an essential downstream effector of EGFR/Ras signaling, and immunofluorescence showed that Cic’s nuclear localization was regulated by EGFR signaling. ISC-specific mRNA expression profiling and DNA binding mapping using DamID indicated that Cic represses cell proliferation via direct targets including string (Cdc25), Cyclin E, and the ETS domain transcription factors Ets21C and Pointed (pnt). pnt was required for ISC over-proliferation following Cic depletion, and ectopic pnt restored ISC proliferation even in the presence of overexpressed dominant-active Cic. These studies identify Cic, Pnt, and Ets21C as critical downstream effectors of EGFR signaling in Drosophila ISCs.Epithelial renewal in the  Drosophila intestine is orchestrated by intestinal stem cells (ISCs). EGFR signaling also plays an important role in regulating ISC proliferation in flies. However, the mechanism by which EGFR/Ras/MAPK signaling promotes ISC proliferation is poorly understood. Here we demonstrate that the transcriptional repressor, Capicua (Cic), mediates these functions of EGFR signaling. We found that the critical role of Cic as a negative regulator of cell proliferation in the fly midgut is consistent with its tumor suppressor function in mammalian cancer development. The direct target genes of Cic were identified by ISC-specific mRNA expression profiling and DNA binding mapping (DamID) method. Cic represses cell proliferation via regulating string (stg), Cyclin E (CycE), and the ETS domain transcription factors Ets21C and pointed (pnt). Using genetic tests we show that these interactions are meaningful for regulating stem cell proliferation. Combining our knowledge of Cic with what was previously known about CIC in tumor development, we propose that human CIC may regulate Ets transcription factors and cell cycle genes in Ras/MAKP-activated tumors.Studies suggest that epidermal growth factor receptor (EGFR) signaling activation is a causal driver of many stem cell-derived epithelial cancers, including colorectal cancer. As in the human intestine, epithelial renewal in  EGFR/Ras/MAPK signaling has diverse functions in regulating cell proliferation, growth, differentiation and survival in most animal cells . AbundanDrosophila midgut is an outstanding model system to study the basis of epithelial homeostasis due to its simple structure, similarity to the mammalian intestine, and powerful genetics. As in the mammalian intestine, epithelial turnover in the fly midgut is carried out through a dynamic process mediated by intestinal stem cells (ISCs). ISCs undergo cell division to renew themselves and give rise to transient cells called enteroblasts (EBs), which can further differentiate into either absorptive enterocytes (ECs) or secretory enteroendocrine (EE) cells. When damaged or aged cells are lost from the fly’s gut epithelium, ISCs respond by dividing to replenish the epithelium .,33.DrosoDrosophila’s intestinal stem cells (ISCs) activates these cells for rampant proliferation , indicating that Cic regulates ISC proliferation cell autonomously. When we used another ISC-specific driver Dlts to knock down cic in ISCs specifically, we not only detected the same overporoliferation phenotype  was used to express UAS-cic-RNAi in ISCs and their undifferentiated daughters, the EBs ts was used to knock down cic in EBs. Increased mitoses were observed after depleting cic in EBs  for 12 hours to generate an enteric infection. ISCs from control midguts, which expressed GFP only, showed regeneration-associated proliferation  had Cic 90% CI) . We nextells see . Cic bin dataset . Hence,  dataset . In adding sites . To furtnfection . The sigcic-RNAi .stg and CycE because they are transcriptionally induced in proliferating ISCs A” was searched in the peaks. The total number of occurrences was quantified, as well as the number of peaks that contained the searched pattern. In order to estimate the significance of the pattern, the background was generated by randomly moving the peaks in the genome for 1000 times. The occurrence of the pattern in the random sequences was then fitted to a Negative Binomial distribution by using the fitdistr function in R . Binding reactions were carried out in a total volume of 20 ml containing 60 mM Hepes pH 7.9, 20 mM Tris-HCl pH 7.9, 300 mM KCl, 5 mM EDTA, 5 mM DTT, 12% glycerol, ~1 ng probe, 1 mg poly (dI-dC), 1 mg BSA and 1 ml of programmed or non-programmed (control) TNT lysate. After incubation for 20 min on ice, complexes were resolved on 5% non-denaturing polyacrylamide gels run in 0.5X TBE at 4°C, and visualized by autoradiography.EMSA experiments were conducted using derivatives of Cice embryo . Wild-tyRp49 –Forward: TCGATATGCTAAGCTGTCRp49 –Reverse: GGCATCAGATACTGTCCCTTGβ-tubulin-Forward: ACATCCCGCCCCGTGGTCβ-tubulin-Reverse: AGAAAGCCTTGCGCCTGAACATAGpnt-Forward: ACGCCCTATGATGCTCAATCpnt-Reverse: TATCCAGACCCAAGGTGCTCpntP1-Forward: CGACTGCGAACAATCTGGTpntP1-Reverse: TTGCTGGTGTTGTAGCCTGTpntP2-Forward: TTAGCCAATTGAACGGCATCpntP2-Reverse: GCACAGATCCTTGCATCCATEts21C-Forward: CCGGGCACTCAGGTACTACTEts21C-Reverse: CATACTGGAGGCCGGATCTaos-Forward: AGAACCCATGGCTTACATGCaos-Reverse: CGTCGCGGGTGTTAAGTTACyan-Forward: CTGCTGGTCATCGTGCTTAGyan-Reverse: GACCTCAGTGTGAGCAGCAAstg-Forward: CAGCATGGATTGCAATATCAGTAstg-Reverse: CAACGTCGTCGTCGTAGAACCycE-Forward: ACAAATTTGGCCTGGGACTACycE-Reverse: GGCCATAAGCACTTCGTCS1 FigThe figure was reproduced from the cBioPortal for Cancer Genomics web page and modified to show only cancers with >3.3% alteration frequency. Asterisks mark colorectal cancer data from Genentech .(TIF)Click here for additional data file.S2 Figesgts system. esg+ progenitor cells (green), PH3+ (red) nuclear DNA (blue). (A) Control adult midgut (B) Cic knock down midgut after 4 days induction 29°C. Scale bars represent 50μm. (C-H) Cic mutant clones were analyzed using the MARCM system. ISC clones (green), DNA stained with DAPI (blue). Control  and mutant  ISC clones were induced with the MARCM system and examined 10 days, 20 days and 30 days later. Mutant ISCs divided faster and generated bigger clones. Scale bars represent 100μm. RNAi-mediated depletion of Cic in ISCs and EBs using the (TIF)Click here for additional data file.S3 FigDlts system. ISCs are marked by GFP (green). Sample was also stained with anti-PH3 to detect mitoeses (red) and DAPI to detect nuclear DNA (blue). (A) Control adult midgut, (B) Cic depleted midgut after 4 days induction at 29°C. Dramatic increases in the number of GFP positive cells were observed in cic depleted midguts, as was large increase in ISC mitoses.  RNAi-mediated depletion of Cic in EBs using the Su(H)ts system. EB cells are marked by GFP (green). Samples were also stained with anti-PH3 (red) and DAPI (blue). (C) Control adult midgut (D) Cic depleted midgut after 4 days induction 29°C. Increases in the number of GFP positive cells and mitoses were observed in cic knockdown midguts. (E) Midguts as in A-C were scored for PH3+ cells after 4 days of induction of cic-RNAi in ISCs or EBs. (F) After 4 days induction of cic-RNAi in ISCs or EBs, midguts were scored for GFP+ or GFP- mitotic cells. Most mitotic cells were GFP+ when cic-RNAi was induced in ISCs using the Dlts system, whereas in midguts in which cic was depleted in EBs, most of the mitotic cells were GFP- and likely ISCs. This indicates a non-cell autonomous effect. (G) Midguts were scored for PH3+ cells after 4 days of induction of cic-RNAi using the esgts system, which targets gene expression to ISCs and EBs. Dramatic increases in the number of GFP positive cells were observed in cic knockdown midguts as was a large increase in ISC mitoses. Statistical significance was determined by Student’s t test . Error bars in each graph represent standard deviation. Scale bars represent 20μm. RNAi-mediated depletion of Cic in ISCs using the (TIF)Click here for additional data file.S4 FigP.e. infected Cic-DamID samples. (B) Cic binding sites in the aos locus from Cic-DamID-Seq using midgut ISCs. The black peaks are from control animals, and the grey peaks are from P.e. infected animals. Plot represents the log2 ratio between the Dam-fusion signal and the Dam-only signal. Red arrows point out TGAATG(G/A)A motifs. The aos transcription unit is shown below the graph. Yellow boxed regions indicate the ORF. (C) mRNA expression ratio change of aos was analyzed by qRT-PCR and normalized to β-Tub and Rp49 with non-amplified mRNA from FACS-sorted progenitor cells.(A) Graph showing fold change of peaks from Cic-DamID and (TIF)Click here for additional data file.S5 FigP.e. infection. (B) Cic binding sites in the Ets21C locus from Cic-DamID-Seq from esg+ cells. Black peaks are from control samples and grey peaks are from P.e. infected midguts. The Y-axis represents the log2 ratio of the Cic-Dam fusion signal to the Dam-only signal. Red arrows point out TGAATG(G/A)A motifs. (C) Normalized mRNA expression fold change of pnt, pntP1, pntP2 and Ets21C in cic transheterozygous mutant midguts.(A) mRNA expression heatmap of Ets transcription factors, showing fold change inductions from RNA-Seq data from whole midguts upon 6 hours (TIF)Click here for additional data file.S6 FigpntP2 overexpression on ISC proliferation. Transgene expression was induced using the esgts system at 29°C for 4 days. Samples were stained with anti-GFP (green), anti-PH3(red) and DAPI (blue) to mark DNA. (A) Control adult midgut. (B) pntP2 overexpressing midgut. The pntP2 over expressing midgut had more GFP+ ISCs and EBs (green). (C) pntP2 and cicΔC2 over expressing midgut. GFP positive progenitor cells were still able to proliferate in the pnt, cicΔC2 over-expressing midgut. (D) pnt mutant clones analyzed by the MARCM system. The size of the clones was quantified by counting cell numbers per clone. pntΔ33 is a pntP1 specific mutant allele, pntΔ78 is pntP2 specific mutant allele and pntΔ88 is pnt null mutant allele that affect both isoforms. Only the pntΔ88 detectably suppressed clone expansion. (E) Mitotic ratio of the pnt mutant clones was scored by calculating the average number of mitoses in each clone. (F) Quantification of ISC mitoses (PH3 positive cells) in pnt and cic depleted midguts or yan depleted midguts, using esgts system. Fewer mitotic ISCs were observed in the pnt and cic double knock down midgut than in the cic knockdown midguts, showing that pnt is required downstream of cic. Yan depletion had no effect on ISC proliferation. (G) pnt mutant clones were generated in a cic depleted background using the MARCM system. The size of the clones was quantified by counting cell numbers per clone. Only the pntΔ88 null allele suppressed the growth of cic-depleted ISC cell clones. (H) yan expression ratio as measured by qRT-PCR in yan-depleted midguts, using two different yan-RNAi lines. Statistical significance was determined by Student’s t test . Error bars in each graph represent standard deviation. Scale bars represent 50μm.(A–C) Effect of (TIF)Click here for additional data file.S7 FigDrosophila Ets21C). (B) Cross-cancer alteration summary for EGR (the human orthologs of Drosophila Pnt).The figure was reproduced from the cBioPortal for Cancer Genomics web page and modified to show only cancers with >3.3% alteration frequency. (A) Cross-cancer alteration summary for EGR (the human orthologs of (TIF)Click here for additional data file.S1 TableP.e. infection.Two lists of peaks from Cic DamID-seq and Cic DamID-Seq upon infection are included in this table. Each data sheet presents the specific genomic location of the peaks with detailed information for the Cic binding peaks such as, chromosome, Peak starting sites, Peak ending sites, Log2 fold change of CicDam/Dam-only and Summit of the peaks. (S1-1) Cic binding peaks from Cic DamID-Seq. (S1-2) Cic binding peaks from Cic DamID-Seq upon (XLSX)Click here for additional data file.S2 TableesgtsUAS-GFP cells expressing cic-RNAi for 4 days. Listed data include Flybase gene ID, gene symbol, Log2-fold-change for each gene as well as significance .The table included the full list of genes whose mRNA expression was significantly changed  in sorted (XLSX)Click here for additional data file.S3 Tablecic-RNAi, and also associated with one or more Cic binding peak from the Cic DamID-Seq analysis. In addition, the normalized Log2 fold change of the peaks following P.e. infection, associated with the genes was also shown in the table. Some of the peaks disappeared after P.e. infection, so they were marked as #N/D (not detected). Derived from the data in List of Flybase gene IDs for genes that were both upregulated upon (XLSX)Click here for additional data file.S4 Tablep-value with Benjamini-Hochberg correction) and numbers of Cic Dam-ID peaks, found in their introns or within 5kb range of the transcription start site. Genes that have Cic binding sites are shaded grey.Information of well-known growth promoters was listed in the table with their log2 Fold change in RNA-Seq and significance ((PDF)Click here for additional data file.S5 TableThree Lists of the non-protein coding RNAs such as tRNA, snRNA & snoRNA and non-protein coding genes that have Cic binding sites in their loci within the 5 kb range in the transcription start site were included in this table. Each data sheet shows the specific genomic location of each peak with detailed information for the Cic binding peaks such as, chromosome, Peak starting sites, Peak ending sites, Log2 fold change of CicDam/Dam-only, FlyBase ID and Gene symbol. (S5-1) Cic binding tRNAs. (S5-2) Cic binding snRNAs & snoRNAs. (S5-3) Cic binding non-protein coding genes.(XLSX)Click here for additional data file.


SESN3 has been implicated in multiple biological processes including protection against oxidative stress, regulation of glucose and lipid metabolism. However, little is known about the factors and mechanisms controlling its gene expression at the transcriptional level. We performed in silico phylogenetic footprinting analysis of 5 kb upstream regions of a diverse set of human SESN3 orthologs for the identification of high confidence conserved binding motifs (BMo). We further analyzed the predicted BMo by a motif comparison tool to identify the TFs likely to bind these discovered motifs. Predicted TFs were then integrated with experimentally known protein-protein interactions and experimentally validated to delineate the important transcriptional regulators of SESN3. Our study revealed high confidence set of BMos (integrated with DNase I hypersensitivity sites) in the upstream regulatory regions of SESN3 that could be bound by transcription factors from multiple families including FOXOs, SMADs, SOXs, TCFs and HNF4A. TF-TF network analysis established hubs of interaction that include SMAD3, TCF3, SMAD2, HDAC2, SOX2, TAL1 and TCF12 as well as the likely protein complexes formed between them. We show using ChIP-PCR as well as over-expression and knock out studies that FOXO3 and SOX2 transcriptionally regulate the expression of SESN3 gene. Our findings provide an important roadmap to further our understanding on the regulation of SESN3. SESN1/2/3), while most invertebrates contain only a single sestrin gene[NRF2 (nuclear factor erythroid derived 2 like 2)-regulated pathway [AMPK (AMP-activated protein kinase) and mTORC1 (mechanistic target of rapamycin complex 1)[mTORC1 [SESN gene in fruit fly leads to triglyceride accumulation in its body [SESN3 gene expression and function in hepatocytes and mouse livers. Over expression of SESN3 dramatically reduces the ethanol-induced hepatic steatosis [SESN2 and SESN3 have also been shown to regulate insulin sensitivity and glucose homeostasis [SESN3 expression are not well studied. Understanding the complex regulatory mechanisms that regulate the SESN3 is of importance, as new therapeutic targets for metabolic diseases might be discovered.Sestrins belong to a small family of evolutionally conserved proteins. They are distinct from any other characterized eukaryotic protein families because they do not have any previously identified domain structures. Mammals pathway ,5. Seconteatosis . In addieostasis ,8. Howevin vitro [in vivo [in silico [Transcription factors (TFs) are known to bind specifically to gene’s promoters at the regulatory positions (binding motifs) and thus contribute to its transcriptional regulation and cellular function. Various in vitro , in vivo[in vivo  and in sn silico  approachn silico .SESN3 orthologs from a diverse set of primates and rodents (with at least 85% sequence homology with human) to perform phylogenetic footprinting [SESN3 upstream region. Predicted binding motifs were further analyzed by Tomtom (a motif comparison tool from MEME-SUITE) to identify motif specific potential transcription factors. Predicted TFs were integrated with documented protein-protein interaction in BioGRID [SESN3 gene.In this study, we used the upstream regulatory regions of human printing . We emplprinting ,15 which BioGRID  to decipSESN3 orthologs and their upstream regulatory regions were extracted (FASTA sequences) from ENSEMBL. These SESN3 sequences from human and its 10 orthologs (Primates and Rodents) were taken and executed using MEME-SUITE, an open source hub of bioinformatics tools. Prediction of novel regulatory motifs was performed by using phylogenetic footprinting, an in silico method coupled with downstream computational analysis. Based on this, consensus sequences in upstream region were discovered by MEME analysis. These consensus sequences were further analyzed using the Tomtom tool which enables the comparison of predicted motifs with Position Weight Matrices (PWM) of TFs for overlap. Further, protein-protein interaction network was constructed between the potential TFs by utilizing the available physical interactions in BioGRID to delineate the important regulators and the network of interactors controlling the expression of SESN3 gene.Human-SESN3 gene is located on chromosome 11. We obtained DNA sequences for the human SESN3 gene (Ensembl ID ENSG00000149212) from the ENSEMBL database. There are 5 transcripts reported for the human SESN3 gene . Here we limit the width of discovered binding motifs in MEME analysis to reflect the widths of most established PWMs—which typically vary in length between 4bp to 30bp [DNA binding motif discovery using the of tools , 15. MEMof tools . These m to 30bp –28.Transcription Factors (TFs) are thought to bind specifically to their corresponding binding motif (BMo) and reguSESN3 upstream regions.We used a set of 2201 DNA motifs ranging between 4bp and 30bp in length (average length 12.7) from TRANSFAC, 843 DNA motifs ranging between 7bp and 23bp in length (average length 12.7) in Jolma et al and 979 DNA motifs ranging between 5bp and 30bp in length (average length 13.0) in JASPAR CORE and UniPROBE Mouse. Hence, we rationalized that a motif length between 4bp to 30bp for the discovered motifs, would be able to capture most of these recognition sequences in the SESN3 upstream regions. Only the TF associations which are identified at p ≤ 1e-03 with E-value < 10 were considered as statistically significant for the 5kb upstream regions.PWMs of various discovered motifs were used as input file for Tomtom and compared with already reported PWMs of TFs in the above described databases to identify the potential TFs binding to the SESN3 genes in UCSC genome browser (http://genome.ucsc.edu/cgi-bin/hgFileUi?db=mm9&g=wgEncodeUwDnase). The images generated from the browser were positioned according to the coordinate of the SESN3 upstream region of block diagram and studied for active BMo.DNase I hypersensitive sites (DHS) are open chromatin region of DNA, sensitive to DNase I cleavage. It is believed that, the occurrence of DHS, notably in the promoter region  is an inFOXO3 and SOX2 genes. The constructs also contained FLAG tag sequence on the N-terminus. After 48 hours of transfection, cells were processed for chromatin immunoprecipitation (ChIP) analysis for the predicted TF binding sequences as previously described [FOXO3 ChIP forward primer 5’-ACAAATCCTGGTACGCTGGA-3’, reverse primer 5’–CAGGACTGTGCATTATGACATCA– 3’; SOX2 ChIP forward primer 5’–CCAGTAGGCGATGCAAGTTA– 3’, and reverse primer 5’–CTAGACGCCCGCAACCTG– 3’.Human HEK293 cells were transfected with plasmid DNAs carrying coding sequences for control GFP (green fluorescent protein), human escribed . The seqFOXO3 and SOX2 CRISPR/Cas9 single guide RNA (sgRNA) sequences were designed using an online program at crispr.mit.edu (Dr. Feng Zhang lab) for gene knockout. The selected two sgRNA sequences for the human FOXO3 and SOX2 genes are: 5’-CACTTCGAGCGGAGAGAGCG-3’ (FOXO3 sgRNA1), 5’-TCCACTTCGAGCGGAGAGAG-3’ (FOXO3 sgRNA2), 5’-TGGGCCGCTTGACGCGGTCC-3’ (SOX2 sgRNA1), and 5’-ATGGGCCGCTTGACGCGGTC-3’ (SOX2 sgRNA2). The DNA oligonucleotides were cloned into a lentiCRISPRv2 vector  as described previously [GFP, FOXO3, or SOX2 sgRNA plasmids. The transfected cells were selected using puromycin (1 μg/ml) for 7 days, and then maintained in the culture medium containing 0.5 μg/ml puromycin.Human eviously ,34. To gGFP, human FOXO3, and SOX2 genes were cloned into a pcDNA3 vector using PCR amplification and restriction digestion.The coding sequences for Human HEK293T and HepG2 cells were cultured in DMEM/high glucose medium containing 10% FBS. HEK293T cells were transfected with plasmid DNA using polyethylenimine and HepG2 cells were transfected using TurboFect reagent (Thermo Fisher Scientific).PPIA) was chosen as an internal control gene. Primer sequences are listed as follows: human PPIA forward primer: 5’- AGGTCCCAAAGACAGCAGAA-3’, human PPIA reverse primer: 5’-GAAGTCACCACCCTGACACA-3’, human SESN3 forward primer: 5’-GTACCAACTGCCGGAAAGTG-3’, and human SESN3 reverse primer: 5’- CCACTGTGTTTGCTTGGACA.Total RNAs were isolated from cultured cells using TRI Reagent (Sigma). mRNA levels for selected genes were analyzed by real-time PCR. Peptidylprolyl isomerase A  across most of the body fluids like blood, liver secretome, and multiple tissue types  are DNase I enzyme sensitive regions of chromatin, where chromatin has less condensed structure due to chromatin remodeling for facilitating transcriptional activation and other downstream events . We used-03 and E-value <10). All possible TFs predicted to bind to the discovered motifs were catalogued and shown in FOXOs, SMADs, SOXs, HNF4A, and TCFs (see SOX2 and FOXO3 using ChIP-PCR approach in HEK293 cells (See SOX2 and FOXO3 transcription factors were found to exhibit significantly enriched binding to the predicted location in the human SESN3 promoter region compared to a negative control GFP (Green Fluorescent Protein)   . Thus, tma cells , but thell line) , suggestpression .SESN3 as shown in -44) among which, motifs overlapping with the DHS signals  from Tomtom analysis and their corresponding motifs overlapping with DHS signals thereby resulting in a subset of TF-TF interactions which are likely to control SESN3 promoter. The resulting network of 30 nodes with 60 interactions is shown in SMAD3, TCF3, SMAD2, HDAC2, SOX2, TAL1 and TCF12. FOXOs, which have been documented to regulate the SESN3 gene transcription[SMAD3, suggesting their interplay to combinatorially control SESN3.Further, in order to prioritize these predicted TFs  and to kions see . This reciations . Such phfunctions,51. It iscription were alsSOX2 contains highly conserved DNA binding domains known as HMG (High-mobility group) box domains which facilitate the binding with DNA for transcriptional control [-05, E-value = 0.07) to motif 4. This TF was found physically interacting with TCF3, POU5F1 (OCT-4), SOX6, HDAC2 and in addition to that it is also interacting with TCF12, thus indirectly bridging with SMAD3- another major hub of TF-TF interaction network ( control . Our stu network .HNF4A) belongs to the HNF4 family. It is known to bind to DNA either as a homodimer or as a heterodimer with other transcription factors such as SMAD3 [, [-03, E-Value = 6.67) Protein-protein interaction network data suggests its assistive role in regulating the SESN3 gene along with SMADs.Hepatocytes nuclear factor 4 alpha SMADs are signaling cascade associated proteins that act as transcriptional mediators of multiple signaling pathways. For instance–they modulate the transcriptional activity of target genes by transforming growth factor-beta-1[SP1[FOXOs [HNF4A [-04, E-value = 3.76) to be bound by SMAD3 further supporting the binding specificity of this TF to SESN3 regulatory regions. SMAD3 works as a master regulator consistent with our observation that it forms a hub with most other high confident TFs as is evident from our interaction network analysis.or-beta-1. They aror-beta-1,58 and ior-beta-1 in the ueta-1[SP1, FOXOs [P1[FOXOs ,61, HNF4s [HNF4A  etc. MotSESN3 gene promoter using an unbiased computational approach. We found high confidence set of TFs correspond to these identified novel BMos and obtained hubs of TF-TF interaction network that include SMADs, SOXs and TCFs. FOXOs, which have been documented to regulate the SESN3 gene transcription[SMAD3, suggesting their interplay to combinatorially control SESN3. Some of them including FOXO3 and SOX2 have also been experimentally validated for their binding affinity in identified BMos using ChIP-PCR technique. Our findings can form a roadmap to further our understanding on the regulation of the SESN3 gene.This work is among the first efforts to identify transcription factor binding sites in the scription were alsS1 Fig(PDF)Click here for additional data file.S2 Fig(PDF)Click here for additional data file.S3 Fig(PDF)Click here for additional data file.S4 FigSESN3 in (A) human cell lines and (B) mouse liver (8 week adult and 14.5 days embryo) using ENCODE project, represented by UCSC browser visualization tool. An overlap of DHS signal was found and shown as dark band over respective motifs in block diagram. The combined best matches of a sequence to a group of motifs were shown by combined p value. Sequence strand specified as “+” (input sequence was read from left to right) and “-” (input sequence was read on its complementary strand from right to left) with respect to the occurrence of motifs. The two coordinates on x-axis represents the 3 kb upstream and 2 kb instream regions as base distance (in blue) and genic distance  of SESN3 gene.DNase I hypersensitive region was shown in 3 kb upstream and 2 kb instream sequences of (PDF)Click here for additional data file.S5 Fig(PDF)Click here for additional data file.S6 FigProtein interaction network between TFs constructed for all possible predicted transcription factors using BioGRID database with TFs belongs to DHS signaled BMo were shown in asterisk “*”.(PDF)Click here for additional data file.S1 Table(XLSX)Click here for additional data file.S2 TableFor each motif, significance, location from start (0 in block diagram) and sequence at location for each species was documented.(XLSX)Click here for additional data file.S3 Table(XLSX)Click here for additional data file.S4 Table(XLSX)Click here for additional data file.S5 Table(XLSX)Click here for additional data file.


Although the locations of promoters and enhancers have been identified in several cell types, we still have limited information on their connectivity. We developed HiCap, which combines a 4-cutter restriction enzyme Hi-C with sequence capture of promoter regions. Applying the method to mouse embryonic stem cells, we identified promoter-anchored interactions involving 15,905 promoters and 71,984 distal regions. The distal regions were enriched for enhancer marks and transcription, and had a mean fragment size of only 699 bp — close to single-enhancer resolution. High-resolution maps of promoter-anchored interactions with HiCap will be important for detailed characterizations of chromatin interaction landscapes.The online version of this article (doi:10.1186/s13059-015-0727-9) contains supplementary material, which is available to authorized users. The mix was placed in a 95 °C heat block for 10 min to denature the DNA, and then centrifuged for 10 s at maximum speed. The mixture was then transferred to a 0.2 ml PCR tube containing 100 ng of the appropriate probe set (4.5 μl volume). The mixture was vortexed for 3 s and centrifuged for 10 s and placed in a thermocycler set at 47 °C for incubation for 64–72 h. The thermocycler’s heated lid was set to 57 °C.Hybridization of the probes to the Hi-C material was done exactly according to the manufacturer’s instructions (Roche Nimblegen Inc). Briefly, 1 μg of Hi-C material was mixed with 5 mg COT DNA, 1 μl of 1000 μM Universal Oligo, and 1 μl of 1000 μM Index Oligo and dried down in a vacuum concentrator on high heat  heated to 47 °C was added to the beads, pipetted up and down 10 times and incubated for 5 min at 47 °C. The mix was then placed in the magnet and liquid was removed once clear. The wash with 1× Stringent wash buffer was repeated once more. Then, 200 μl of 1× wash buffer I was added to the mixture and mixed by vortexing for 2 min; the beads were collected using the magnet and liquid was discarded once it was clear. The same steps were then repeated using 300 μl wash buffer II (except this time vortexing for 1 min) and 200 μl wash buffer III (except this time vortexing for 30 s). To elute the captured material from the beads, 50 μl of PCR-grade water was added to the beads and they were stored at −20 °C until further use.After the incubation, the mixture was washed to eliminate unhybridized probes. Wash buffers  and 100 μl of streptavidin beads were prepared for each hybridization according to the manufacturer’s instructions. The hybridization mix was mixed with 100 μl of streptavidin beads, further mixed by pipetting up and down 10 times and placed back in the thermocycler at 47 The resulting DNA libraries were sequenced 100 bp from both ends (paired-end sequencing) on a HiSeq 2000 (Illumina Inc.). This is long enough to map to ~90 % of the genome , includiPaired-end sequences were aligned to the mouse genome (build mm9) through HiCUP  which usWe designed sequence capture probes against mouse promoters compiled from multiple sources. RefSeq and Ensembl annotations were used together with transcription start sites from DBTSS (from 25 May 2010) and MPromDb (from 28 May 2010). There were in total 53,501 target sequences (targeting closest upstream and downstream MboI sites of each promoter and negative controls) and the probes covered 93.5 % of the target bases . DBTSS is based on full-length mRNAs, and mostly corresponds to RefSeq and Ensembl. MPromDb is based on RNA polymerase II and H3K4me3 ChIP-seq data for different cell types, including ESCs. From annotated transcript start sites, we searched for the closest restriction cut sites (GATC) on each side, and chose the last 150 bp before the cut site as the captured regions. When restriction sites were <300 bp apart we chose the whole region between them. From these regions, Nimblegen designed the actual probe sequences. We also selected exonic and intergenic control regions which were included in the same probe selection pipeline.2 = 0.065, Pearson correlation coefficient). G+C content of HiCap distal regions was slightly higher than the genome average (47 ± 6.7); 40–70 % for 99 % of HiCap distal regions. Promoter–promoter interactions were called similarly, but required that both ends of the paired reads aligned with probes belonging to promoters. We also mined the raw read pairs for interactions involving only distal regions. For this purpose we collected all distal regions from significant promoter–distal interactions and performed similar analyses for read pairs with both ends originating from a HiCap distal region. Following is a breakdown of called interactions and how they distribute over expressed and non-expressed genes. We detected at least one interaction for 73 % of the expressed genes  and for 48 % of genes with no detectable expression . Unsaturated sequencing could account for the fact that we did not detect any interaction for 27 % of the expressed genes. It is not surprising, however, that we did not detect any interaction for 52 % of genes with no expression as they might not be involved in distal interactions. There are also cases where only one of the alternative promoters of the same gene is involved in a distal interaction; therefore, it is fairer to assess the number of genes with interactions rather than the number of promoters.HiCUP software available at Babraham Bioinformatics  was usedWe downloaded enhancer regions inferred from different ChIP-seq experiments carried out in mESCs  and [GEO:GSM1156609] (for mESCs).We performed Capture-C (3C coupled with sequence capture) using our custom promoter probes. We downloaded Capture-C raw reads from the Gene Expression Omnibys (GEO) database with sample IDs [GEO:GSM1156607] and [GEO:GSM1156608] . Reads were aligned to the mouse genome (mm9 assembly) and a comprehensive collection of splice junctions  using BoP value using the χ2 test with Uh and Uc as observed and Nh/Nc as their expected ratio. For the compound test including all TFs, we summed all values of Nc, Nh, Uc, and Uh and performed the same tests.To assess the power of HiCap interactions to predict differentially expressed genes after TF perturbation, we constructed the following test   to deterP values in Fig. P values by Stouffer's z-score method, to compute two-tailed P values. The P values were also significant (P < 10−300) without this consideration for distance and network degree.We mined the HiCap interactions between promoters and enhancers to enumerate the occurrences of motifs Fig. –j. As a P values as the number of randomizations with at least as many pairs as the non-randomized, or one less . Due to the 1000 randomizations, the minimum possible P value was 0.001. P values were then adjusted to false discovery rates using the Benjamini–Hochberg method.We tested if gene pairs connected through promoter–promoter, promoter–enhancer–promoter interactions more often shared annotated gene function. To this end, we used the gene ontology service DAVID . First wRaw sequence reads have been submitted to the NCBI Sequence Read Archive  under S under ac


Nap1 can bind H2A–H2B dimer in the cytoplasm and shuttles the dimer into the nucleus. Moreover, Nap1 functions in nucleosome assembly by competitively interacting with non-nucleosomal histone–DNA. However, the exact roles of these chaperones in assembling Htz1-containing nucleosome remain largely unknown. In this paper, we revealed that Chz1 does not show a physical interaction with chromatin. In contrast, Nap1 binds exactly at the genomic DNA that contains Htz1. Nap1 and Htz1 show a preferential interaction with AG-rich DNA sequences. Deletion of chz1 results in a significantly decreased binding of Htz1 in chromatin, whereas deletion of nap1 dramatically increases the association of Htz1 with chromatin. Furthermore, genome-wide nucleosome-mapping analysis revealed that nucleosome occupancy for Htz1p-bound genes decreases upon deleting htz1 or chz1, suggesting that Htz1 is required for nucleosome structure at the specific genome loci. All together, these results define the distinct roles for histone chaperones Chz1 and Nap1 to regulate Htz1 incorporation into chromatin.Histone variant Htz1 substitution for H2A plays important roles in diverse DNA transactions. Histone chaperones Chz1 and Nap1 (nucleosome assembly protein 1) are important for the deposition Htz1 into nucleosomes. In literatures, it was suggested that Chz1 is a Htz1–H2B-specific chaperone, and it is relatively unstructured in solution but it becomes structured in complex with the Htz1–H2B histone dimer. Nap1 (nucleosome assembly protein 1) can bind (H3–H4) Saccharomyces cerevisiae, replaces H2A in nucleosomes and has diverse roles in eukaryotic cells  DNA samples, and DNA was then amplified by PCR. Amplified DNA from the IP and input samples were labelled using a ULS aRNA Fluorescent Labelling DNA Kit (Kreatech). Labelled DNA from the ChIP and input samples were hybridized to htz1Δ, chz1Δ and nap1Δ cells were grown in the YPD medium to an OD600 of 1.0. All cells were then treated with 1% (v/v) formaldehyde for 20 min and incubated with 125 mM glycine for 5 min. Cell permeabilization, micrococcal nuclease digestion, protein degradation and DNA purification steps were performed as previously described  of more than 40000 probes that covered the entire yeast genome and constructed binding profiles for three proteins . Our results indicated that Chz1 did not bind chromatin (log2 binding ratio<1) , which was consistent with the chromatin fractionation test shown in We also performed genome-wide ChIP-chip analysis of Htz1, Chz1 and Nap1. For each of the three proteins, we obtained the intensity values [binding ratio   (The binding profiles of Htz1 and Nap1 were strongly correlated (<10−200) C, sugges4×10−38) D, suggesTo understand the organizations of Htz1 and Nap1 at promoters, we calculated the average binding ratio profiles for Htz1 and Nap1 by aligning the binding profiles for 5419 yeast genes at TSSs E. The prP value=1.4×10−20)  nucleosome upstream of the TSS, Htz1 is ~+24 bp upstream of the dyad position; in the first (i.e. +1) nucleosome downstream of the TSS, Htz1 is ~−37 bp downstream of the dyad position F. At the4×10−20) F. This w4×10−20) F.r<−0.93, P value<0.01) between the mRNA frequency and the enrichment of Nap1 and Htz1 surrounding TSSs was observed (r≤−0.60) . This suggests that Nap1 falls off the chromatin with the loss of Htz1-containing dimers in regions of highly transcribed genes. It is notable that we observed a negative relationship between Htz1 and transcription frequency, as Htz1 incorporation into nucleosomes is a preliminary step in opening chromatin for transcription when the Htz1-containing nucleosome is unstable [The enrichment of Htz1 near TSSs is closely correlated with gene expression levels ,4,41. Weobserved G. The hiunstable ,12. In yunstable ,43.chz1Δ, nap1Δ and chz1Δnap1Δ, deletion strains. We also determined the genome-wide occupancy loci of Htz1 in the deletion strains.To systemically uncover the effects of the Chz1 and Nap1 chaperones on Htz1 deposition in chromatin, we performed protein expression test for Htz1, Htz1 acetylated on K14, and H2A in wild-type, chz1 mutant. Interestingly, the nap1 deletion strain had higher Htz1 levels in the chromatin pellet than the wild-type strain. Furthermore, the chz1–nap1 double mutant also had increased levels of Htz1 on chromatin like the nap1 single mutant, which indicated that Nap1 may have a dominant role regulating Htz1 incorporation. In contrast, the levels of H2A in chromatin were unaffected in mutant strains.We first carried out a chromatin fractionation assay to compare the levels of Htz1 on chromatin in wild-type and mutant strains A. Surprinap1Δ and chz1Δ nap1Δ strains   B. In thee strain C. Additin strain C, indicanap1-deletion strain was almost three times than that of in the wild-type strain (chz1 and nap1 (chz1Δ nap1Δ)  C. More thtz1 deletion strains. Based on our ChIP-chip data, we chose eight loci with significantly binding with Htz1 and Nap1. We performed the ChIP and RT–PCR to analyse the binding of Nap1 in wild-type and HTZ1 mutant . Interestingly, we discovered that binding of Nap1 at these eight loci is dramatically decreased upon deletion of HTZ1, which clearly prove the notion that Nap1-binding is Htz1-dependent.To understand whether the association of Nap1 with genomic DNA is dependent on Htz1, we constructed Nap1-myc chz1Δ<wild-type<nap1Δ<chz1Δ nap1Δ, corresponding with the effects of Chz1 and Nap1 on the binding of Htz1  between the −1 and +1 nucleosomes , gene body (TSS ~ TTS) and other region. Deletions of the two chaperones caused an ~9% decrease in number of Htz1-bound probes at promoters  providing further evidence that both chaperones play roles in guiding Htz1p to the proper binding sites.http://www.yeastgenome.org/cgi-bin/GO/goSlimMapper.pl). The average Htz1-binding ratio profile of each cluster was calculated around TSSs. The PCC was calculated using binding profiles for the wild-type and mutant strains, showing the effects of gene deletions on the Htz1-bound genes for the 40 gene clusters.To analyse the regulatory dynamics of Htz1p binding in the deletion mutant strains, 5419 yeast genes were grouped into 40 gene clusters according to their GO annotation  DNA sequences [chz1 mutants suggesting that Chz1 partly contributes to the Htz1-binding specificity.We used GOMO to track the target genes of the motifs. Results indicated that the target genes of the AG-rich motif associates with transcription factor activity and transmembrane transport process [Htz1 favours TATA-less promoters . Our reshtz1Δ, nap1Δ, chz1Δ nap1Δ, chz1Δ htz1Δ and nap1Δ htz1Δ strains. The correlation analysis showed that the transcriptome profile of htz1 was poorly correlated with that of both chz1 and nap1 . Around the TSSs, the average binding profile of ac-Htz1 in the chz1Δ mutant was obviously higher than that of the wild-type strain –(D). The average read intensity near TSSs is shown in supplementary Figure S9(E). The typical distribution pattern was repeated in both the wild-type and mutant strains . An NFR is shown around TSSs and REG sites . The NFR was flanked by two well-positioned nucleosomes and a phased organization of nucleosomes (nucleosome array) was observed downstream of the TSS.htz1 deletion, especially for the −2, −1, +1 and +2 nucleosomes near TSSs , the nucleosome occupancy of Htz1-bound genes dramatically decreased with ear TSSs A. In conear TSSs A did notcleosome A. Moreove strain B. These chz1Δ or htz1Δ mutants, the nucleosome occupancy in the nap1Δ mutant was not different from that in the wild-type strain . This suggests that Htz1 deposition is specific for those Htz1-bound genes. That is, H2A cannot completely compensate for Htz1 deficiency at Htz1-bound genes. Htz1-bound DNA sequences are adenine- and guanine-enriched. Finally, we observed that the association between the transcription frequency and nucleosome occupancy near TSSs was not changed in the deletion mutants.First, according to previous reports ,30 and oractions ,48, and ractions ,48–50. Nmachines . We founDeposition of Htz1 on promoters has been showed to be typical yeast genes whose transcription is frequently reprogrammed . Two HtzAccording to literatures and our present work, we proposed a model to illustrate the mechanisms of Nap1 and Chz1 in depositing Htz1 in yeast . In this model, Nap1 and Chz1 have different roles in Htz1 deposition. Nap1 binds with Htz1–H2B dimer, forming a Nap1–Htz1–H2B complex, in cytoplasm and then the complex is imported into the nucleus. In nucleus, Htz1–H2B–Nap1 interacts with SWR1 and completes the deposition of Htz1. Chz1 does not associate with Htz1 in cytoplasm. Htz1 deposition mediated by Chz1 is occurs in nucleus. During the nucleosome disassembly under conditions of transcription, DNA repair and other process, Htz1–H2B is released from chromatin into the nucleus. For the deciduous Htz1–H2B, their deposition need help of Chz1. Chz1 catches the released Htz1–H2B and delivers it to SWRI and realize the deposition. However, there is another possibility to explain the increased level of Htz1 incorporation in the absence of Nap1. Rather than Nap1 and Htz1 competing for binding to the same genomic loci, it may be likely that the absence of Nap1 may also decrease the incorporation of conical H2A, which would allow Htz1/H2B dimmers to assemble in inappropriate places.