XIST from nine different locations in human HT1080 cells.X-chromosome inactivation is a striking example of epigenetic silencing in which expression of the long non-coding RNA XIST initiates the heterochromatinization and silencing of one of the pair of X chromosomes in mammalian females. To understand how the RNA can establish silencing across millions of basepairs of DNA we have modelled the process by inducing expression of Localization of XIST, depletion of Cot-1 RNA, perinuclear localization, and ubiquitination of H2A occurs at all sites examined, while recruitment of H3K9me3 was not observed. Recruitment of the heterochromatic features SMCHD1, macroH2A, H3K27me3, and H4K20me1 occurs independently of each other in an integration site-dependent manner. Silencing of flanking reporter genes occurs at all sites, but the spread of silencing to flanking endogenous human genes is variable in extent of silencing as well as extent of spread, with silencing able to skip regions. The spread of H3K27me3 and loss of H3K27ac correlates with the pre-existing levels of the modifications, and overall the extent of silencing correlates with the ability to recruit additional heterochromatic features.cis-acting silencer when expressed from nine different locations throughout the genome. A hierarchy among the features of heterochromatin reveals the importance of interaction with the local chromatin neighborhood for optimal spread of silencing, as well as the independent yet cooperative nature of the establishment of heterochromatin by the non-coding XIST RNA.The non-coding RNA XIST functions as a The online version of this article (doi:10.1186/s13059-015-0774-2) contains supplementary material, which is available to authorized users. XIC), which is located at Xq13 in humans, is the region of the X that is necessary for the chromosome to be inactivated, and contains the XIST gene (X-inactive specific transcript) [To avoid a functional gene dosage imbalance between the sexes, one of the two X chromosomes in female placental mammals is transcriptionally silenced . This prnscript) –4. Remarnscript) . The coanscript) , 7, revinscript) ). In addnscript)  with pernscript)  resultinin vitro model for the events of XCI  provides an iewed in ). Early iewed in , althougiewed in . After tilencing , consistng [XIST , 16. SATfunction ; howevertivation . More reic cells , and XISic cells , 20. In H3K27me3 . Loss ofH3K27me3 –24. Chared genes .Smchd1 gene results in loss of DNA methylation and partial to full reactivation of approximately 20 % of the X-linked genes in mouse [There is substantial evidence for cooperativity of multiple silencing pathways in the initial silencing of the chromosome, with XCI able to proceed in the absence of key components of the silencing machinery such as PRC2 –28, PRC1in mouse , 32. Surin mouse , consistin mouse , but ratin mouse .Xist/XIST transgenes into autosomes, and localization of the RNA to the autosome is able to induce many features of the Xi including nucleolar localization [XIST transgene was integrated into chromosome 21 in induced pluripotent stem cells from an individual with Down’s syndrome and corrected gene expression from chromosome 21 to near normal disomic levels [XIST a better understanding of the influence of the chromatin neighborhood is necessary.Spread of silencing to autosomal genes has been observed in unbalanced X/autosome translocations; however, the extent of autosomal silencing is highly variable in both humans (reviewed in ) and miclization , 21, 38.c levels . Togethec levels , and forXIST expression from an inducible transgene [XIST expression from the myriad of changes that occur during differentiation provides an opportunity to dissect the role of XIST in XCI. By using this inducible XIST transgene to examine the influence of XIST expression induced from nine different integration sites we aimed to establish a hierarchy to the features that are recruited by XIST and determine if any were influenced by the genomic context of the XIST integration. We were able to identify features  that are recruited to all integrations and thus are compatible with any of the sites/genomic contexts tested, a feature that is not recruited to any site examined (H3K9me3) while another set of features  are dependent on genomic context, but appear independent of each other.XIST expression is required to induce the cascade of changes that cooperatively silence the X, but relatively little is known about the process by which the non-coding RNA recruits these changes. In human somatic cells we have reported the recruitment of several features of XCI, including gene silencing, following ransgene , 40. TheXIST cDNA has been previously reported to localize in cis to the chromosome from which it is transcribed, resulting in the recruitment of some chromatin modifications and repression of both flanking reporter genes as well as flanking endogenous genes [XIST cDNA into nine different FRT sites in HT1080 cells containing a constitutive Tet-repressor transgene to allow induction of XIST by treatment of the cells with the tetracycline analog doxycycline (DOX). The sites of the FRT integrations were identified by inverse PCR and sequencing to be 1p, 3q, 4q, 7p, 7q, 8p, 12q, 15q, and an X chromosome FRT site at Xq23 was previously reported [In the HT1080 cells an inducible us genes , 40. To reported . The intP ≤0.01). Co-hybridization with fluorescently labelled Cot-1 also showed depletion of Cot-1 hybridization coincident with the XIST cloud at each integration site, observable in the line diagrams of signal intensity across the XIST cloud , a trend shared with the other integration sites ; howevees Table . PerinucEGFP reporter gene at the 3q integration site [XIST into the FRT site results in expression of an upstream Hygromycin (Hyg) gene. Robust silencing of Hyg was observed at all integrations after 5 days of XIST expression . As only the repeat A region of XIST is required for silencing of the flanking reporter genes [XIST repeat A and a DsRed reporter driven by the mouse Pgk1 gene promoter, which is normally X-linked and subject to XCI (p21), i(3)(p10), i(3)(q10), der(4)t, der(5)t, der(11)t see additional details in methods). We generated allele-discriminating pyrosequencing assays to examine silencing of candidate genes flanking the integration sites , all XIST integration sites except 4q showed at least one gene with significant allelic silencing. There was much more variability between integration sites for endogenous gene silencing than was seen for the silencing of Hyg. The 8p-integrated XIST clone displayed the most silencing, with four out of the five genes tested showing 60–80 % silencing. The 4q clone, in contrast, showed no significant silencing for any of the three genes tested. Two different integration sites on chromosome 7 showed quite different results, with only one of seven genes assayed showing over 20 % silencing for the 7p integration site, while five of the seven genes showed over 20 % silencing with XIST expressed from the 7q integration site. There was also discontinuous spread of silencing. For example, in the 1p integration site clone, two genes located approximately 200 kb from the XIST transgene failed to silence (1 % silencing), whereas the RHBDL2 gene located approximately 400 kb from the XIST transgene silenced by approximately 70 %. In addition to variation between the integration sites in the number of genes that were silenced, there were also significant differences in the extent of gene silencing between genes that showed silencing. More than half of the significant changes demonstrated less than 50 % silencing of one allele, and a significant change as small as 6 % for the ZNF710 gene on 15q was observed, indicating that XIST can cause a continuum of silencing.Given the capacity of the XIST RNA to silence in )(p21), i(p10), i(ble XIST . Individ)(p21), i(p10), i. Consistent with our candidate gene analysis, the increase in proportion of genes in this range was not significant for the 1p integration site. Examination of expression changes as an allelic change is more sensitive to the partial reduction of expression of one allele, and should also still detect changes in allelic expression when the total expression level of the gene is regulated in trans although the number of genes that can be examined is reduced by the requirement for an expressed polymorphism. We examined the allelic change in expression on chromosomes 1, 8, and 12 . The biological duplicate for RNA-seq was highly concordant for the percent allelic gene silencing for 8p . The proportion of genes showing allelic silencing for genes flanking the 1p integration site was significant (P = 0.0269); however no significant change was seen for the 12q integration site. We show only the distal end of chromosome 1 as the HT1080 cells carry a translocation of one chromosome 1 to chromosome 4 . We validated several of the observed changes by pyrosequencing or q-PCR, and the assays are included in Additional file r = 0.9341; P <0.0001). Of the 17 genes showing at least 50 % allelic silencing in both 8p RNA-seq replicates, two genes (DLC1 and STC1) did not show a decrease in total expression, suggesting that auto-regulation could be another source of discrepancy between total read and allelic read changes. The high rate of validation of the observed changes by pyrosequencing of biological triplicates substantiated that silencing spread to endogenous genes. Intriguingly, on chromosome 8 there appeared to be domains of silencing separated by areas that were more resistant to the action of XIST. In order to explore what features might lead to differential susceptibility to silencing we performed chromatin immunoprecipitation followed by sequencing (ChIP-seq) for induced and uninduced cells with XIST integrated at 8p.As our candidate genes examined by pyrosequencing had shown significant reductions in gene expression that ranged from as little as 6 % to approximately 80 % silencing of one allele, we did not expect complete silencing of one allele that would reduce expression levels overall by 50 %. We chose a stringent threshold of 30–60 % total reduction to classify genes as silencing, at which level a significantly greater proportion of genes were observed within 30 Mb of the 8p and 12q integration sites compared to the genome . In Fig. r = 0.5904; P = 0.0007). Mouse Xist localizes to sequences that are in contact with the integration site as determined by chromatin conformation capture [r = 0.4996; P = 0.0006), although we did not identify an association of allelic silencing with domains designated as closed nor open [r = 0.4599; P = 0.0003) and loss of acetylation . Given the proposed role for repetitive elements in the XCI process [ChIP-seq for both the archetypical facultative heterochromatic mark H3K27me3 and the active mark H3K27ac showed significant, but opposing changes on chromosome 8 upon DOX induction of XIST from the 8p integration site, and these changes were even more dramatic on the short arm where ted Fig. , b. Surpted Fig. . This un capture , 44, and capture  .Xist transgenes . Thion site .XIST transgene in iPS cells [Spread of silencing to autosomes in these HT1080 cells is not seen to the extent that is observed for X/autosome translocations , 49 or aPS cells , suggestPS cells  we see XPS cells , 25. TheWe observe a Cot-1 hole and increased perinucleolar association at all integration sites, consistent with previous reports that XIST/Xist expression from autosomes increases perinucleolar association , 21. In The other feature that we observed at all integrations examined was H2AK119u1, which is established by the PRC1 complex. We observed H2AK119u1 in the absence of SMCHD1, macroH2A, and H3K27me3 in the 1p integration cells, suggesting that XIST may be able to directly recruit the PRC1 complex, in agreement with the PRC2-independent PRC1 recruitment previously suggested in mice [Xist expression [Xist [XIST-dependent. We observed that macroH2A is not required for XIST-induced silencing, which is consistent with the ability of mice that are knocked-out for both macroH2A1 and macroH2A2 to continue to undergo XCI [r = 0.8; P = 0.01), with the proportion of cells positive for SMCHD1 always equal or lower to the percent seen to be perinucleolar. Thus perinucleolar localization may be required but not sufficient to recruit SMCHD1. The strong ability of the X integration to recruit all features confounded the ability to detect correlations; however, it appears that the majority of the features are independently recruited to the chromosome that expresses XIST. For several of the integration sites two or more independent clones with similar XIST levels behaved similarly supporting that the variation we observe is due to the integration site. In addition, the 4q and 12q clones were derived from a different subclone of HT1080 from the other autosomal integration sites; however, they did not appear to be more similar to each other, arguing against the variation arising during subclone generation.Interestingly, no single factor was seen to associate with more robust silencing Fig. , demonstpression , and is on [Xist ; yet intergo XCI . The recThe differential recruitment of chromatin marks depending on integration site suggests that multiple silencing pathways not only work in parallel to promote silencing, but that their recruitment is favored by different underlying DNA sequences, consistent with previous studies using genes that escape from inactivation to identify 12 features of the DNA sequence of the X chromosome that may be involved in the spread or maintenance of XCI .Overall, we saw variability in the recruitment of chromatin marks between the integration sites, highlighting the importance of the integration site in modulating XIST function. Localization of XIST with a concomitant depletion of Cot-1 RNA, recruitment of H2AK119u1, and a shift to perinucleolar location was seen at all integrations, and thus reflect features established by XIST independent of the local chromatin environment. In contrast, recruitment of SMCHD1, macroH2A, H3K27me3, and H4K20me1 appeared to be strongly influenced by the site of XIST expression. As some silencing was observed at all integrations, this study demonstrates that silencing in human somatic cells can occur in the absence of macroH2A, SMCHD1, and H3K27me3/H4K20me1 recruitment, underscoring the independent but cooperative nature of the X-chromosome inactivation process. The X chromosome demonstrated the most consistent ability to recruit the heterochromatic marks of XCI, consistent with an evolutionary accumulation of DNA features enabling the recruitment of heterochromatic marks to the X chromosome.lacZeo (Life Technologies) at low concentrations. The full-length inducible XIST cDNA construct [2 in DMEM supplemented with penicillin/streptomycin, non-essential amino acids, and 10 % V/V fetal bovine serum. XIST expression was induced with the addition of 1 μg/mL doxycycline to the culture medium.HT1080 HH1 cells were transfected with pcDNA6/TR, and two subclones (HT1080HH1-2-3 or 2–12) expressing high RNA levels of the Tet-repressor were subsequently transfected with pFRT/onstruct  was co-tSouthern blotting identified eight FRT integrations that appeared single copy. Inverse PCR utilizing primers complementary to a sequence within the integrated pFRT plasmid was used to identify the precise integration site of the transgenes in the HT1080 cell lines. The ends of linearized plasmids are subject to exonuclease activity, thus the actual integrated transgene often lacks several hundred of base pairs on each end which was first identified by a series of PCR assays prior to restriction endonuclease digestion with a frequently-cutting restriction endonucleases identified to cut in the remaining plasmid, followed by ligation with T4 DNA ligase (Invitrogen) to create circular DNA molecules. The captured genomic DNA was amplified by nested PCR with primers facing outward from the plasmid fragment for sequencing and the genomic location was identified using the BLAT algorithm . No X-liHyg-resistance and assessed for loss of Zeomycin sensitivity.While cancer derived, the HT1080 cells remain diploid with four structural rearrangements detected by spectral karyotyping (p21), i(3)(p10), i(3)(q10), der(4)t, der(5)t, der(11)t. Using our allelic pyrosequencing assays we observed instability of chromosome 3 in two of nine clones  and homPgk1 promoter , then permeabilized with 0.5 % Triton-X 100 in CSK for 8 min on ice and then fixed in 4 % paraformaldehyde for 8 min at room temperature. Coverslips were stored at 4 °C in 70 % ethanol. Just prior to RNA FISH, the coverslips were immersed in 100 % ethanol for 5 min and left to air dry. FISH was performed with two probes: an XIST probe and a Cot-1 (Invitrogen) probe. Both probes had been directly fluorescently labeled using the Nick Translation Reagent Kit  with Spectrum red-UTP (Vysis) for Cot-1 DNA probes and Spectrum green-UTP (Vysis) for XIST probes. Approximately 150 ng of each probe was mixed together along with 20 μg salmon testes DNA then air dried in a speed vacuum, resuspended in 10 μL deionized formamide, denatured at 80 °C for 10 min, and then mixed with 10 μL hybridization buffer . This was pipetted onto a small square of Parafilm and the coverslip was placed on top of the probe mixture. Another piece of Parafilm was then placed on top and the edges were sealed to prevent the drying out of the coverslip. Hybridization took place overnight in a humidified chamber at 37 °C. The next day the coverslips were rinsed as follows: 20 min in 50 % formamide/50 % 4XSSC at 37 °C, 20 min in 2X SSC at 37 °C, and 20 min in 1X SSC at room temperature. Coverslips were then stained with DAPI and mounted onto microscope slides with Vectashield (Vector Laboratories). Cells were observed on a Leica inverted microscope (DMI 6000B) at 100X magnification and images were obtained using a Retiga 4000R (Q-Imaging) camera with Openlab software (PerkinElmer). Images were processed using Adobe Photoshop CS4 to reduce background and correct for variation in FISH efficiency between different images. A one-way ANOVA test in GraphPad was used to determine significantly different signal sizes. Line scans were generated using Image J software (NIH) by drawing a line through the area of interest and plotting the RGB intensities across the line.Cells were grown on glass coverslips. Upon removal from cell culture the coverslips were first rinsed in ice-cold CSK buffer , ‘perinucleolar only’ , ‘both’ , or ‘neither’. XIST signals scored as ‘both’ are included in the ‘perinuclear’/‘perinucleolar’ percentages in Table For combined RNA FISH and immunofluorescence, coverslips, which had been stored at 4 °C in 70 % ethanol, were first rinsed in PBS then placed onto a small amount of PBT (PBS with 1 % BSA and 0.1 % Tween 20) containing 0.4 U/μL Ribolock RNase inhibitor. Coverslips were sealed between two layers of Parafilm and left in the blocking buffer at room temperature for 20 min, then transferred from blocking buffer to PBT containing 1:100 primary antibody and 0.4 U/μL Ribolock, sealed between two layers of Parafilm and left at room temperature for 4–6 h. Coverslips were then washed three times, for 5 min each time, at room temperature in PBS containing 0.1 % Tween 20, then put onto a small amount of PBT containing 1:250 fluorescently labeled secondary antibody and 0.4 U/μL Ribolock, sealed between two layers of Parafilm and left at room temperature in the dark for 45 min. Coverslips were then washed three times, for 5 min each time, at room temperature in the dark in PBS containing 0.1 % Tween 20. Coverslips were then fixed in 4 % PFA in PBS for 10 min at room temperature in the dark, and washed for 5 min in PBS before continuing on to RNA FISH, making sure that the coverslips remained in the dark throughout the RNA FISH procedure. Antibodies used in immunofluorescence include: anti-H3K27me3 (07–449 from Millipore); anti-macroH2A (07–219 from Millipore); anti-SMCHD1 (ab31865 from Abcam); anti-H2AK119u1 (05–678 from Millipore); anti-H3K9me3 (07–442 from Millipore); anti-H4K20me1 (07–440 from Millipore).ACTB or PGK1. Primer sequences are found in Additional file RNA was isolated from cell pellets stored at −70 °C using TRIZOL (Invitrogen) according to the manufacturer’s instructions and then treated with DNase1. cDNA was generated in the range of 0.5–2.5 μg RNA using M-MLV reverse transcriptase for qPCR on a StepOnePlusTM Real-Time PCR System , using Maxima Hot Start Taq (Thermo Scientific) and EvaGreen dye (Biotium). The following conditions were used: 95° for 5 min, followed by 40 cycles of , and a melt curve stage of . The expression levels of genes of interest were normalized to the expression of Taq DNA polymerase (Invitrogen), 0.5 μM forward and reverse primers, and 50–100 ng of cDNA for  × 35 cycles, and 72 °C for 10 min for final extension. One of the forward and reverse primers was biotinylated for template isolation during pyrosequencing preparation. Universal M13 primer was also used for some assays , 0.2 mM dNTPs, 0.625 U r = 0.9934 (P <0.0001). A second RNA-seq of the 12q and 1p integration sites with No DOX and following 5 days DOX produced 76 bp paired-end reads. Total RNA expression was quantified in FPKM using Cufflinks. ChIP of H3K27me3 (Active Motif antibody 61017) and H3K27ac (Active Motif antibody 39133) was performed on the 8p clone with and without DOX treatment, as previously done [P value = 1 × 10−3) and to quantify the level of H3K27me3 in 2 kb and 1 Mb non-overlapping windows.For both ChIP-seq and RNA-seq, library preparation and sequencing was done according to Illumina protocols, and reads were aligned to hg18 reference genome using Tophat. RNA-seq was performed as previously described , first osly done  using 20cis with the XIST transgene. Variant sites were next combined based on regions of interest, either gene location or genomic regions for RNA-seq and ChIP-seq, respectively. Variant sites that had an allelic ratio 0.15 greater or less than the overall gene allelic ratio for the sample were removed and a new overall gene ratio was calculated. For ChIP-seq, variants with reads >2-fold different from the average number of reads in the region of interest were also excluded. Percent allelic silencing for a given gene or region of interest was defined as /control allelic ratio. The calculated ratio was the frequency of the allele with lower reads in the experimental sample for each gene or region of interest. We also required that there were at least 4 reads and 5 reads with allelic information per gene (RNA-seq) and region of interest (ChIP-seq), respectively, in both the control and experimental samples to be considered for analysis.Allelic information for both RNA-seq and ChIP-seq was obtained with a custom workflow in Galaxy –58. BrieXIST transgene (AGPAT5 for 8p clone).We obtained normalized Hi-C data for human female fibroblast line IMR90  and the http://dx.doi.org/10.6084/m9.figshare.1529822).The sequence data described are available in GSE68109. Additional microscopy images are available in Figshare (


Chlamydomonas reinhardtii zygotes undergo a program of differentiation into a diploid zygospore that is accompanied by transcription of hundreds of zygote-specific genes. We identified a distinct sequence motif we term a zygotic response element (ZYRE) that is highly enriched in promoter regions of C. reinhardtii early zygotic genes. A luciferase reporter assay was used to show that native ZYRE motifs within the promoter of zygotic gene ZYS3 or intron of zygotic gene DMT4 are necessary for zygotic induction. A synthetic luciferase reporter with a minimal promoter was used to show that ZYRE motifs introduced upstream are sufficient to confer zygotic upregulation, and that ZYRE-controlled zygotic transcription is dependent on the homeodomain transcription factor GSP1. We predict that ZYRE motifs will correspond to binding sites for the homeodomain proteins GSP1-GSM1 that heterodimerize and activate zygotic gene expression in early zygotes.Upon fertilization  Changes in ploidy are fundamental transitions for eukaryotic cells undergoing sexual reproduction. Fertilization merges the contents of two gametic cells and initiates a zygotic development program that leads to formation of a new individual in diploid or sporophyte-dominant species or to recombination and meiosis in haploid or gametophyte-dominant species.Chlamydomonas reinhardtii is a haploid unicellular green alga that reproduces mitotically (vegetative reproduction) under nutrient replete conditions. Nitrogen starvation triggers gametic differentiation into one of two mating types (plus and minus) that are governed by a single multigenic locus, mt, on Chromosome VI with two haplotypes, mt+ and mt− , and formation of a thick, environmentally resistant cell wall that protects the dormant zygospore. Initiation of zygotic development is triggered by formation and nuclear translocation of a heterodimeric KNOX/BELL-type homeodomain protein dyad GSM1/GSP1 whose subunits are expressed individually in mt− (GSM1) or mt+ (GSP1) gametes  and its importance for activating zygotic gene expression was tested using luciferase reporter constructs. When the reporter was fused to the promoter of zygotic gene ZYS3 luciferase expression was brought under zygotic control. When ZYRE elements were fused to a tubulin minimal promoter they also conferred zygotic expression. Furthermore, the ZYRE-controlled luciferase expression was not observed when the mt+ parent carried a gsp1 mutation. These experiments show that the ZYRE element is necessary and sufficient to drive zygotic gene expression under the control of a key regulator of zygotic development, GSP1. We propose that ZYRE elements may be directly bound and regulated by the zygotic homeodomain transcription factor GSP1-GSM1 that is formed upon fertilization.Recently, we performed a genome-wide transcriptome study that enabled the comprehensive identification of 627 early zygotic genes  and 6145c mt− (CC-1691) were used for transformation experiments, and mating tester strains R3 mt+ (CC-620) and CJU10 mt− (gsp1 mt+ mutant strain was generated from F1 progeny after crossing 21gr mt+ and gsp1 mt− strain LMJ.SG0182.003395 distributed by the Chlamydomonas Stock Center (www.chlamycollection.org) through the Chlamydomonas Library Project  that were amplified with primers containing XbaI and XhoI sites (or compatible sites for ligation). PCR fragments were cut with appropriate enzymes and ligated to XbaI-XhoI cut pHsp70A/RbcS2-cgLuc. Figure S1 describes sequences of transgenes used in the luciferase expression experiments.Luciferase reporter constructs were made as follows: The Table S2. ZYS3 promoter (ZYS3p(−118 to +97) relative to TSS) was amplified with ZYS3p-XbaI-F and ZYS3UP-XhoI-REV. ZYS3 promoter without ZYRE motifs (ZYS3p(−94 to +97)) was amplified with ZYS3-nomotif-F and ZYS3UP-XhoI-REV. ZYS3 ZYRE motifs conjugated directly to 5′-UTR (ZYS3p(−118 to −95: +1 to +95)) were amplified with ZYS3-motif-5UTR-F and ZYS3UP-XhoI-REV. ZYS3 5′-UTR only (ZYS3p(+1 to +97)) was amplified with ZYS3-5UTR-F and ZYS3UP-XhoI-REV. ZYS3 with a synthetic ZYRE sequence replacing its endogenous ZYREs (Z-ZYS3p(−94 to +97)) was amplified with ZYS3-Z-nomotif-F and ZYS3UP-XhoI-REV. The β-tubulin TUB2 minimal promoter (TUB2p(−53 to +113); upstream region containing the TATA box and 5′-UTR) was amplified with TM-XbaI-NdeI-F and TM-SalI-R. A chimeric promoter of ZYS3 ZYREs and TUB2 minimal promoter (ZYS3p(−118 to −95)::TUB2p(−53 to +113)) was produced by PCR amplification with TM-XbaI-A-F and TM-SalI-R. For DMT4, which has a ZYRE-like motif (CGTGACGTGAC) within its first intron, an in-frame translational fusion was made with its promoter, first exon first intron and a part of the second exon (DMT4) amplified with CMT1UP-XbaI-FWD and CMT1UP-XhoI-REV . The Ѱ-DMT4::cgLuc construct with mutated ZYRE was constructed by the one-step site-directed protocol ; cycles: 98° for 2 min and 30 cycles of 98° for 30 sec, 50° for 30 sec, and 72° for 30 sec, followed by 72° for 7 min. Primers are summarized in loxP site and the hygromycin resistant marker gene aph7”) via Cre/lox-mediated recombination .Resultant promoter-luciferase constructs were recombined with the plasmid pKS-aph7”-lox  were transformed using a modified version of a previously published electroporation protocol (2 with continuous illumination from 150 µE red (625 nm) and 150 µE blue (465 nm) LED lights at 25° until they reached a density of 1 × 106/ml. Cells were harvested by centrifugation at room temperature and resuspended at 3.5 × 108 cells/ml in TAP with 50 mM sorbitol. Around 1 µg DNA and 1−5 × 107 cells in 300 µl TAP with 50 mM sorbitol were placed into 4 mm cuvettes, prechilled on ice for 15 min, and then electroporated using a GenePulser Xcell , with settings of 800 V, 25 µF, no resistance. Transformants were recovered in 10 ml TAP + 50 mM sorbitol in low light for 16 hr and then concentrated and plated on two TAP agar plates supplemented with 30 µg/ml hygromycin for selection. After 5 d single transformants were picked into 200 µl TAP media in clear 96-well plates, grown for 5 d in constant light, and then pinned onto TAP agar plates under 2 × 32 W fluorescent lights. After 5 d a toothpick full of cells from each pin spot was scraped from the plate and resuspended in 200 µl of nitrogen free-HSM (NF-HSM) to complete gametogenesis. A 50 µl aliquot of gametes from each transformant was mixed separately with testers of each mating-type (R3 mt+ or CJU10 mt−) and incubated under light for an additional 2 hr prior to measurement of luciferase activity. Mating activity, scored as agglutination between plus and minus gametes, was confirmed under the dissection microscope. Another 2 hr later, luminescence activity was measured as follows: Plates were processed and read in a FLUOstar Optima  with automated injectors. Just prior to each reading, 100 µl of coelenterazine solution  was injected into a plate well containing 100 µl cell mixture, and luminescence was detected at the maximal gain setting (4095). Strains not expressing luciferase were used to measure background. Transformants that showed greater than fourfold more luminescence when mixed with gametes of the opposite mating-type testers vs. same mating-type testers were scored as positive for zygote-specific expression. In almost every case, when transformants were mixed with testers of the same mating-type the luminescence values were similar to background levels (Table S3).Cells . Out of ten significant MEME motifs eight were present in a small number of genes, but two reached the count limit of 50. One of these abundant motifs was a low-complexity G-rich sequence that is widespread in the C. reinhardtii genome and was not pursued further. The other high-frequency motif was an 11-mer with the sequence complexity characteristic of a bona fidecis-regulatory element, YGACAYGAC, that we named ZYRE (Table S1). ZYRE is similar to but more refined and statistically supported than the CGtGACATGaCC motif previously identified from a limited set of zygotic genes (Table S1 and cis-regulatory element (DMT4 (Table S1) show significant de-enrichment of ZYRE in their promoters  and introns . For both ZYRE-containing reporters we observed a fraction of transformants (∼10%) that had zygotic upregulation of luciferase, while the majority of remaining transformants showed either low or background levels of luciferase activity. The observed fraction of positive transformants in our experiments is consistent with results from other studies where random integration of transgenes often results in silencing (Table S3). Importantly, upregulation of zygotic luciferase activity was rarely observed in transformants that had missing or mutated ZYRE elements (Table S3). Thus, ZYRE elements can function as zygotic transcriptional enhancers both upstream of and within transcription units. Similar results as those described above were obtained when reporters were tested in mt− strains meaning that the parental gamete type in which the ZYRE reporter resides does not influence its activity  in more detail. The ZYS3 promoter contains three ZYRE motifs in a 25 bp region from –94 to –118  to a tubulin minimal promoter that has been employed previously in reporter constructs  and minus (GSM1) gametes respectively, and which heterodimerize and enter the nucleus upon fertilization . It is possible that the motif search criteria we used missed partially degenerate ZYRE-like sequences in these genes, as was the case with DMT4, or that alternative GSP1-GSM1 binding sites exist for those genes. In addition some zygote-specific genes may be regulated indirectly by zygotic transcription factors that act downstream of or independent of GSP1-GSM1. Among our zygote-specific genes (Table S1), there are five that encode putative transcription factors: ZYS1A and ZYS1B (RLS7 (EZY18 (70-like putative transcription factor (Cre03.g193400). An interesting area of future study will be to determine whether motifs that are present in zygotic promoters (Table S4) which do not contain detectable ZYREs are recognized by GSP1 and GSM1 or other zygotic transcription factors.Many (77%) but not all nd ZYS1B , 2004, R


Hox transcription factors (TFs) are essential for vertebrate development, but how these evolutionary conserved proteins function in vivo remains unclear. Because Hox proteins have notoriously low binding specificity, they are believed to bind with cofactors, mainly homeodomain TFs Pbx and Meis, to select their specific targets. We mapped binding of Meis, Pbx, and Hoxa2 in the branchial arches, a series of segments in the developing vertebrate head. Meis occupancy is largely similar in Hox-positive and -negative arches. Hoxa2, which specifies second arch (IIBA) identity, recognizes a subset of Meis prebound sites that contain Hox motifs. Importantly, at these sites Meis binding is strongly increased. This enhanced Meis binding coincides with active enhancers, which are linked to genes highly expressed in the IIBA and regulated by Hoxa2. These findings show that Hoxa2 operates as a tissue-specific cofactor, enhancing Meis binding to specific sites that provide the IIBA with its anatomical identity. •Meis provides a ground state that is common to all the branchial arches•Hoxa2 recognizes Meis prebound sites in the second arch that contain Hox motifs•Hoxa2 enhances Meis binding, which coincides with active enhancers, at these sites•Hoxa2 modulates the ground-state binding of Meis to instruct second arch identity Hox transcription factors instruct specific morphologies in the branchial arches, a series of segments in the developing vertebrate head. Amin et al. found that Hoxa2 operates as a tissue-specific cofactor, enhancing binding of the homeodomain transcription factor Meis to specific sites that provide the second arch with its anatomical identity. The body plan of vertebrates involves the formation of similar repetitive segments, which subsequently diversify to give rise to different body parts. A key discovery has been that Hox genes endow the initially identical segments with their distinct identities .Branchial arches are a useful model of segmental specification. This transient, metameric series of tissue bands appears in the head region of vertebrate embryos. Cranial neural crest (CNC) cells, emerging from areas of the hindbrain that express different Hox genes, colonize the branchial arches . All braIntensive work in the past years has revealed that Hox genes are present in nearly all animals, and the principles of Hox gene organization and function are highly conserved throughout the animal kingdom . DespiteAs DNA-binding transcription factors, Hox display notoriously low binding specificity in vitro, yet they accomplish the task of selecting specific target genes to control segment morphology . Hox proHoxa2 and Meis levels. Our results highlight the importance of genomic profiling TFs in their native, tissue-specific expression domains to understand the mechanisms governing segment-specific identity.To understand how Hox operate in vivo, we mapped Meis, Pbx, and Hoxa2 binding in the native area of competence of Hoxa2, the IIBA. Through this analysis we uncovered a synergistic interaction between Hoxa2 and Meis TFs. Collectively, our findings show that Meis TFs provide a ground state that is common to all segments (arches). Hoxa2 recognizes a subset of Meis prelabeled sites, which contain Hox recognition motifs. By selectively binding to and enhancing a subset of Meis binding, Hoxa2 modifies the branchial arch ground state, established by Meis, to instruct IIBA-specific morphologies. This specific synergy between Hoxa2 and Meis is further reinforced by a positive-feedback loop, which locks IIBA cells in a state of high Meis1 and Meis2 . We extracted chromatin from the IIBA of E11.5 embryos and confirmed that Hoxa2 binds to Meis1 and Meis2 . Therefore, because Meis1 and 2 are highly conserved in vertebrates   , we obseebrafish , and mirebrafish . CollectTo identify the molecular mechanisms controlled by Meis1/2 in the branchial arches, we mapped Meis genomic occupancy in both Hox-negative (IBA) and Hox-positive (IIBA) arches in mouse. Meis bound many regions  in both Hox-positive (II) and -negative (I) branchial arches  and S2, Hoxa2 and Hoxb2) are expressed in arch II, and paralogs of group 3 are expressed in arch III, while the first arch does not express any Hox genes   was contained in a 4.0 kb fragment sufficient to drive gene expression in the hindbrain rhombomere 3 (r3) and r5 and in the CNC migrating from r4 into the IIBA  A. In coner peak) A. In sumtive IBA B. The Methe IIBA . In a cotogether C. The Hotebrates D, and, atibodies E.Hoxa2 proximal promoter is conserved from mouse to zebrafish, we turned to the zebrafish embryo model to examine the effects of Meis knockdown on Hoxa2 expression. After injecting one-to-two-cell-stage embryos with morpholino oligonucleotides targeting translation of meis transcripts expressed in the branchial arches, we observed a downregulation of hoxa2b expression in the hindbrain and IIBA of all embryos (39/39 affected). These embryos still expressed dlx2, which labeled the developing branchial arches  distance from Meis binding, and only a small fraction of Hoxa2-binding events was apparently unrelated to Meis binding A. A simiFunctional annotation of Hoxa2/Meis/Pbx-shared regions identified enrichment of genes in overlapping functional categories with the entire Hoxa2 ChIP-seq C, while Hox proteins form dimers with either a Pbx protein or a Meis protein, as well as trimers with one Pbx protein and one Meis protein  and Hoxa2-negative (IBA) branchial arches. Meis peaks with a higher binding signal in the IIBA (measured by fold enrichment [FE]) were preferentially located close (200 nt) to Hoxa2 binding A, suggesMeis1; 1.3-fold change in Meis2)   and were−3). GREAT showed that top Meis binding in both branchial arches was associated with genes involved in skeletal development, consistent with the control of skeletal development by Meis TFs. Association with middle ear morphogenesis was also detected, which is consistent with the middle ear forming from both IBA and IIBA. We observed the same differential association using the top 1% of Meis ChIP-seq replicates in the IBA and IIBA (data not shown). In sum, the enhanced Meis binding in the IIBA, caused by Hoxa2, appears to target specific biological processes in the IIBA.Finally, we asked whether enhancement of Meis binding in the IIBA reflects biologically meaningful differences in the branchial arches. We focused on the top 1% of Meis peaks for analysis (containing peaks with FE > 60), as only high-FE Meis-bound regions showed a skewed distribution relative to Hoxa2 distance B. Functi−15), which also were enriched in the fraction of top Meis binding in the IIBA. In sum, these observations indicate that the assembly of Hox-Meis complexes is sequence based.We interrogated DNA sequence motifs to identify the mechanisms underlying synergistic binding of Hoxa2 and Meis in the IIBA. We found TGACAG, which corresponds to the canonical Meis recognition motif, as the most highly overrepresented motif in the entire Meis ChIP-seq and in highly enriched Meis peaks unrelated to Hoxa2 binding  A. In conHoxa2 mutants  from genes . Zfp703 and Zfp503 encode for two related zinc-finger proteins that act as transcriptional repressors. In zebrafish, they are required for the formation of r4 and the expression of r4-specific genes, including hoxa2 . In addition, the synergy between Hoxa2 and Meis is reinforced by genetic interaction. A positive-feedback loop expands XMeis3 activates Hox genes in Xenopus  to shape arch-specific morphologies . The widLikewise, more posterior arches (third and fourth arches) share the first arch default state . In contOur work shows that, molecularly, Hoxa2 does not entirely reprogram the epigenomic landscapes provided by Meis TFs in the branchial arches. Rather, Hoxa2 truly acts as a tissue-specific cofactor to specify the identity of the second arch, where it modulates the ground-state program established by Meis proteins. Our results provide a radically new framework to understand how Hox transcription factors control development in vertebrates.Hoxa2 mutant mice were described previously (eviously . CD1 miceviously .HEK293T cells were transfected using Fugene 6 (Promega), with a total of 1 μg DNA, containing 550 ng pGL3-Hoxa2 promoter (Hoxa2 promoter from −220 to +1 cloned in pGL3 [Promega]) and 150 ng of each Hoxa2, Meis1, and Pbx1a in pCDNA3 expression vector or pCDNA3 control (Life Technologies). Cells were harvested 24 hr after transfection for luciferase reporter assays (Promega). IBAs were dissociated into single cells and infected using supernatants from Ecotropic-Phoenix packaging cells, transfected with pMYs-IRES-GFP (Cell Biolabs) or pMYs-Hoxa2-IRES-GFP . The infChIP-seq experiments have been deposited in ArrayExpress . ChIP-seq and ChIP assays were performed as described previously  using thMus musculus) genome using BFAST 0.7.0a (http://liulab.dfci.harvard.edu/MACS/), using the matched-input DNA reads as a control. For peak calling, the “nomodel” parameter was used and the mean fragment size set at 200 bp. The threshold p value was set to p < 1 × 10−4. Binding regions with false discovery rate < 10% were selected . The first replicate of Meis IBA and Meis IIBA underperformed, but 85.4% and 84.5% of the called regions were contained within the second replicates. In view of this, the second replicate was used in downstream analyses. The location of binding regions, defined by their summit region coordinates relative to RefSeq gene structure, was determined using CEAS version 0.9.9.8 (http://liulab.dfci.harvard.edu/CEAS/). The comparison of genome coordinates and the generation of the conservation profile used GALAXY (http://bejerano.stanford.edu/great/) using the “basal plus extension” association rules. Plots and overlay density plots were generated using a python code available on request.For ChIP-seq analysis, 50 bp sequences from Meis IBA ChIP, Meis IIBA ChIP, Pbx IIBA ChIP, Foxc1 ChIP-seq, and matched-input DNA controls were used. The Meis samples were run using two biological duplicates for the ChIP and matched-input DNA controls. Sequence reads were mapped to the NCBI37 (mm9/July 2007) release of the entire mouse (T 0.7.0a . The mapd GALAXY . Motif dd GALAXY . The anaS.A., D.Z., M.L., and F.L. carried out the experiments and analyzed the data. I.J.D, J.H., and M.R. performed computational analysis and interpretation of the data. N.B., C.S., F.S., S.A., and M.L. designed the experiment and analyzed and interpreted the data. N.B. wrote the manuscript. All authors read and edited the manuscript prior to submission.


A well-established cascade of transcription factor (TF) activity orchestrates adipogenesis in response to chemical cues, yet how cell-intrinsic determinants of differentiation such as cell shape and/or seeding density inform this transcriptional program remain enigmatic. Here, we uncover a novel mechanism licensing transcription in human mesenchymal stem cells (hMSCs) adipogenically primed by confluence. Prior to adipogenesis, confluency promotes heterodimer recruitment of the bZip TFs C/EBPβ and ATF4 to a non-canonical C/EBP DNA sequence. ATF4 depletion decreases both cell-density-dependent transcription and adipocyte differentiation. Global profiling in hMSCs and a novel cell-free assay reveals that ATF4 requires C/EBPβ for genomic binding at a motif distinct from that bound by the C/EBPβ homodimer. Our observations demonstrate that C/EBPβ bridges the transcriptional programs in naïve, confluent cells and early differentiating pre-adipocytes. Moreover, they suggest that homo- and heterodimer formation poise C/EBPβ to execute diverse and stage-specific transcriptional programs by exploiting an expanded motif repertoire.DOI:http://dx.doi.org/10.7554/eLife.06821.001 Human body fat consists mostly of fat-storing cells called adipocytes. These cells develop in two main steps. First, mesenchymal stem cells—which can potentially become one of many different types of cell—commit to becoming pre-adipocyte cells. These pre-adipocytes then develop into mature adipocytes. Proteins called transcription factors control both steps of this process by binding to particular sites in the DNA of the cell and activating certain genes that control the cell's identity and activity.Various different transcription factors are known to stimulate the development of mesenchymal stem cells into adipocytes. Experiments performed on cells that have been grown in the laboratory suggest that the cells must also be tightly packed together to become adipocytes.Cohen et al. have now investigated the role a protein called C/EBPβ plays in the development of adipocytes, and have found that it plays different roles at different stages of development. When mesenchymal stem cells become tightly packed, more of another protein called ATF4 is produced. This protein binds to C/EBPβ, and the resulting two-protein complex then binds to sites on the DNA of the mesenchymal stem cell to activate genes that turn the stem cell into a pre-adipocyte. Reducing the amount of ATF4 in mesenchymal stem cells reduces the number of pre-adipocytes that develop.When not bound to ATF4, and in response to certain cell signals, C/EBPβ binds to different DNA sites and helps pre-adipocytes develop into mature adipocytes. Whether transcription factors other than ATF4 partner with C/EBPβ to change DNA-binding-site preferences remains unknown. Future studies will search for these, with the aim of providing a clearer molecular understanding for how C/EBPβ acts as a ‘bridge’ that links together the two stages of adipocyte development.DOI:http://dx.doi.org/10.7554/eLife.06821.002 Post-mitotic adipocytes arise from the differentiation of MSCs in a process termed adipogenesis . The celPPARγ  and activates thousands of putative enhancers with increased sensitivity to DNase I, enrichment for activating histone modifications, and induced binding by TFs . Acting PPARγ , and thePPARγ .While DMI is a potent trigger for adipogenesis in both mouse 3T3-L1 fibroblasts and human mesenchymal stem cells (hMSCs), it is effective only when cells are densely packed . Here, wWe also observe a surprising enrichment for C/EBPβ-binding sites at RNAPII enhancers responding to high-seeding density prior to the addition of DMI cocktail. While lacking a canonical, palindromic C/EBPβ motif, these enhancers exhibit dramatic enrichment for an asymmetric, composite motif that juxtaposes half-sites for canonical C/EBP and AP-1 motifs. We demonstrate that these hybrid motifs recruit C/EBPβ as a heterodimer with another bZIP family member, ATF4. Genome-wide binding by ATF4 demonstrates its exclusive co-localization with C/EBPβ, and depletion of ATF4 decreases adipogenesis. Together, these observations suggest that a program of C/EBPβ-ATF4-dependent gene expression triggered by high-seeding density plays an important role in priming hMSCs for adipogenesis. The observation that C/EBPβ hetero- and homodimeric complexes exhibit different sequence specificities provides novel mechanistic insights into how C/EBPβ can be differentially targeted to control distinct programs of gene expression at distinct phases of adipocyte differentiation, and revises the prevailing view that C/EBPβ is transcriptionally inactive in the absence of exogenous adipogenic stimuli .RNAPII is recruited to active enhancers on a global scale , and we The uncommitted, HD-primed, and DMI-induced RNAPII clusters mapped to distinct gene ontologies (GOs) . CytoskeIntriguingly, the HD-primed, but not the uncommitted and DMI-induced, RNAPII sites associated with a CHOP/ATF4/hybrid motif . It is aTo test whether C/EBPβ drives the activity of HD-primed enhancers via a non-canonical binding site as predicted by the motif analysis of RNAPII sites, we screened C/EBPβ-binding regions at all RNAPII-annotated regions for the presence of a canonical CEBP sequence (TTGCnnAA)  or a hybAKR1C1, ALOX15b, FKBP5, HSD11b1, and ZBTB16) were activated by DMI treatment along with a control adipocyte marker (PDK4). As a whole, these findings suggest that C/EBPβ targets a non-canonical C/EBP motif to activate HD-primed enhancers. Furthermore, although the addition of DMI cocktail increases C/EBPβ occupancy globally, including sites associated with the HD-primed enhancers, it reduces the activity of the HD-primed enhancers while inducing enhancers harboring canonical C/EBP motifs.The HD-primed and DMI-induced enhancers are active during distinct stages of differentiation, and employ different motifs to recruit C/EBPβ, suggesting that distinct classes of C/EBPβ-recognition sequences confer temporal regulation of gene transcription during adipocyte development. To address this idea, we placed several RNAPII-annotated regions that are targeted by C/EBPβ and harbor either a hybrid or canonical motif into luciferase reporters, and assayed their activity in response to DMI cocktail . InteresHybrid motifs recruit C/EBP TFs as heterodimers with bZip proteins from the AP-1 or ATF families . TherefoGenomic occupancy of ATF4 is associated predominantly with genes involved in tRNA aminoacylation for protein synthesis, response to endoplasmic reticulum (ER) stress, and the unfolded protein response . ATF4 ocEssentially no ATF4 peaks were observed that lack co-bound C/EBPβ, suggesting heterodimer binding. To interrogate whether ATF4 and C/EBPβ can simultaneously occupy the same genomic loci, we performed sequential ChIP experiments . After aTo investigate whether ATF4 regulates adipocyte differentiation, we depleted its expression in primary hMSCs, and in the well-characterized mouse pre-adipocyte cell line 3T3-L1 . TransieEscherichia coli to ensure homogenous preparations that lacked other potential bZip binding partners typically present in mammalian cell extracts  has successfully examined TF binding with mponents . Here, wTitration of C/EBPβ over two orders of magnitude produced cistromes with robust peaks  that scaTo investigate the effects of heterodimerization on the global genomic distribution of C/EBPβ, we titrated ATF4 into the IVC. Upon addition of ATF4, C/EBPβ exhibited reduced occupancy of C/EBPβ-homodimer sites and de novo binding at sites not represented in the homodimer cistromes generated with higher C/EBPβ concentrations . These rOur data reveal that C/EBPβ controls distinct gene expression programs in hMSCs at different stages of adipogenesis . Prior tThis study identifies ATF4 as a cell density sensor that links the physical and chemical requirements for adipogenesis. High cell density has long been known to be an important condition for adipocyte differentiation in vitro . It is lMice lacking ATF4 are lean and resist diet-induced obesity, although they have adipose tissue . Our demThe binding profiles of ATF4 and C/EBPβ in hMSCs can be explained by the inherent sequence specificities of the homo- and heterodimeric complexes. First, genomic binding of ATF4 requires heterodimerization with C/EBPβ. While elegant biochemical studies have demonstrated the propensity of ATF4 and C/EBPβ to heterodimerize , early sThe IVC demonstrates that C/EBPβ and ATF4 target distinct DNA half sites, TTRS and TGAT, respectively. The relative plasticity of C/EBPβ homodimer in accommodating multiple DNA sequence variants is borne out in vivo, as the top motif associated with the cellular C/EBPβ cistrome shows considerable degeneracy at the −1 and −2 positions. In contrast, the C/EBPβ-ATF4 heterodimer is restricted to hybrid motifs of two inverted half-sites, TTRSATCA. Thus the biochemical properties of the heterodimer are sufficient to explain the nearly 1:1 correspondence between ATF4 binding and the presence of hybrid motifs in vivo. Interestingly, ATF4 heterodimerizes with the C/EBP related protein, CHOP, in the context of ER stress to bind hybrid motifs , suggestC/EBPβ homodimers bind to hybrid sequences with low affinity relative to the C/EBPβ-ATF4 heterodimer, and this interaction requires elevated protein concentrations. DMI-treated hMSCs show persistent occupancy of the hybrid sites by C/EBPβ despite low ATF4 levels. An interesting possibility is that persistent binding is attributable to the elevated C/EBPβ concentrations in differentiating cells, however the effects of DMI-induced post-translational modification of C/EBPβ on homodimer binding cannot be excluded. Notably, robust RNAPII recruitment at hybrid sites was observed in cells only in the presence of ATF4. Thus, hetero vs homodimeric binding may have important consequences for licensing transcription activity, especially under conditions where the basal activity of C/EBPβ is limiting, possibly due to associated repressor complexes .Our findings may have broad impact on the understanding of bZip TFs that readily form different dimer pairs , as well2) or high  densities. For differentiation studies, DMI medium was added to cells that had been cultured overnight at the desired cell density. DMI formulation was as follows: DMEM with 10% FBS, 2 mM glutamine, 1 μM dexamethasone, 0.5 mM IBMX, and 0.2 mM indomethacin, and 10 μg/ml insulin. 3T3L1s and C3H10T1/2 cells were obtained from ATCC.hMSCs were obtained from Lonza and maintained in low glucose Dulbecco's modified Eagle's medium containing supplemented with 10% fetal bovine serum and 2 mM glutamine. hMSCs were used at passages 4 through 7. For cell density manipulations, hMSCs were re-plated at low (2000 cells/cm2) in duplicate wells. Cells were then treated with DMI medium for an addition 24 hr followed by treatment with passive lysis buffer. Relative luminescence (Firefly to Renilla ratio) was determined on a Glomax luminometer. hg19 genomic coordinates for each reporter are as follows: ASNS, chr7:97501555-97502014; MTHFD2, chr2:74426247-74426355; SLC7A5, chr16:87887210-87887550; Intergenic hybrid, chr1:204476231-204476350; control C/EBPβ (LAMβ1), chr7:107598937-107599756; ASTN2, chr9:119212591-119213520; HSD11b1, chr1:209893618-209894063; PPARγ2, chr3:12391004-12392991. Oligos for subcloning candidate enhancers are listed in the Candidate enhancers were amplified from hMSC genomic DNA by touchdown PCR, using oligos that added 5′ XhoI and 3′ BamHI restriction sites. Amplified DNA was gel isolated, digested, and ligated into pGL4.24. Genomic coordinates for candidate enhancers are reported in the Supplemental Information. C310T1/2 cells were co-transfected with 1.8 mg of luciferase reporter construct and 0.2 mg of CMV-Renilla plasmid using the TransIT LT1 transfection reagent. Following an overnight transfection, cells were re-plated at high cell densities , further stripped of protein by Proteinase K treatment and phenol/chloroform extraction, and sheared by sonication with a Covaris M220 focused ultrasonicator. ATF4 and C/EBPβ were expressed in BL21(DE3) E. coli from pET30a vectors carrying an N-terminal His Tag and full-length cDNAs. Proteins were purified by Co2+ affinity chromatography and quantified by comparison with a BSA standard after SDS-PAGE. EMSA binding reactions were resolved by native 6% PAGE, with 10 mM MgCl2 in the gel to increase the DNA-binding specificity of bZip proteins (Binding reactions were performed in 20 μl (EMSA) or 100 μl (IVC) with 10 mM HEPES pH 7.8, 50 mM NaCl, 10 mM MgClproteins , and werThe following antibodies were used for ChIP: C/EBPβ , GR , ATF4 , RNAPII , H3K27ac , ATF2 , ATF-3 , C/EBPg , GADD153 , FOS , FOSL1 , JUND , and MAFF/G/K . The first IP for sequential ChIP was performed with whole cell extract derived from approximately 5 million formaldehyde-crosslinked hMSCs. After overnight incubation at 4°C with antibody and protein G sepharose in 1 ml ChIP buffer , IPs were washed and then eluted by incubation at 65°C for 15 min in 50 μl ChIP elution buffer  with 10 mM DTT. Beads were pelleted and the supernatant transferred to a new tube. A subsequent wash with 50 μl ChIP elution buffer was performed with the beads, and the supernatants combined. Eluted protein-DNA complexes were added to 1 ml ChIP buffer with 5 mg/ml BSA and 2 μg nonspecific DNA. Samples were split into two equal parts and volumes raised to 1 ml with ChIP buffer. One aliquot received antibody against a particular TF, the other rabbit IgG, and samples were incubated at 4°C for 1–2 hr. Immune complexes were captured by protein G sepharose, washed, eluted, and incubated overnight at 65°C to reverse formaldehyde crosslinks. DNA was subsequently isolated for PCR. Primers are listed in ChIP-Seq libraries were prepared from formaldehyde-crosslinked hMSCs, using approximately 2–8 million cells per condition, depending on the abundance of the factor of interest. Libraries were sequenced as previously described . AnalyseRNAPII data was windowed into 20 bp bins, and a USHP measured for each bin. To eliminate artifacts introduced by regions of low ChIP signals caused by poor mappability, regions of no RNAPII sequence coverage were masked based on their mappability scores from the ENCODE Duke Uniqueness 35 bp track. Masked regions  that were juxtaposed to bins with enriched RNAPII signal were assigned an interpolated USHP score, that is, the average USHP score of adjacent upstream and downstream bins unaffected by mappability. RNAPII data were smoothened by recalculating USHP scores as the moving average of a 60 bp window centered on each 20 bp bin. Genomic regions with high sequencing depth in the input (no ChIP) tracks were subtracted from the RNAPII data to remove false positive peaks. Finally, each bin was assigned a ‘USHP slope’ score computed from the difference of the USHP values for the bin lying 60 bp downstream vs the bin 60 bp upstream divided by the intervening bp length .RNAPII features were defined as the intervals spanning regions of increasing USHP slopes paired to regions of decreasing USHP slopes. Briefly, RNAPII bins were thresholded for USHP slope scores and clustered to detect regions of positive or negative USHP slopes. Each positive sloping region was joined with the nearest downstream negative slope region, with a maximum cutoff distance of 400 bp between the 3′ and 5′ ends of the positively and negatively sloping regions. Adjacent RNAPII features were then evaluated for peak merging based on the peak to trough ratios (rations < 2.5 were merged), such that complex RNAPII features comprised of poorly resolved multiplets were treated as a single feature. To evaluate enrichment of sequence reads within the putative RNAPII features, features within 2 kb were clustered, and these domains expanded to ±1 kb to sample the local RNAPII background. Peak-associated bins within these domains were masked and the average USHP signal per bin in the local neighborhood computed. RNAPII features with USHP maxima that were 3.5-fold greater than the mean of non-peak associated local USHP bins were used in subsequent analyses. In addition, RNAPII features intervals were subject to a signal threshold requiring a tag count of ≥20 per feature or a tag density of at least 14.5 sequence tags per bp. All steps of the RNAPII feature detection algorithm were scripted through the Galaxy server, and will be available as published workflows.2 fold change of the condition-specific tag count relative to the mean tag count observed for all four RNAPII tracks . Heat maps were generated in TreeView . Each peak was assigned attributes to indicate whether RNAPII exhibited a twofold or greater change in occupancy for each pairwise comparison of experimental conditions, with three possible attributes per comparison . Following attributed-based group assignment, the complexity of the clustering results was simplified by grouping together functionally related clusters. Similar classes of RNAPII were detected with non-supervised clustering methods (k-means clustering using Cluster 3.0). For heat map representations of the RNAPII data, the data was transformed to represent the logTreeView .For each peak interval, motif analysis was centered on the bins scoring in the top 25th percentile of USHP values observed within the peak interval. A minimum window size of 400 bp around these top 25th percentile bin was used for motif mining. Windows around the top 25th percentile of bins were limited to a maximum length of 600 bp (peaks that failed this threshold were excluded). Overlapping windows  were merged into single intervals. Motif mining was performed with HOMER using the hg19r genome build and the ‘size given’ peak option. Sequence motifs reported in http://www.ncbi.nlm.nih.gov/geo/) under accession number GSE68864.Supplemental figures and files are available. Genome-wide data sets are available at the NCBI Gene Expression Omnibus . Similarly, the author response typically shows only responses to the major concerns raised by the reviewers.eLife posts the editorial decision letter and author response on a selection of the published articles . An edited version of the letter sent to the authors after peer review is shown, indicating the substantive concerns or comments; minor concerns are not usually shown. Reviewers have the opportunity to discuss the decision before the letter is sent  and 3 reviewers, one of whom, Stephen Farmer, has agreed to share his identity.Thank you for sending your work entitled “ATF4 licenses C/EBPβ activity in human mesenchymal stem cells primed for adipogenesis” for consideration at The editor and the reviewers discussed their comments before we reached this decision, and the editor has assembled the following comments to help you prepare a revised submission.All the reviewers considered your studies to be well executed. However, a major concern is the biological relevance of the reported discovery, in particular the role of cell density or confluence as a “trigger” during adipogenesis in vivo. If the authors would like to make the argument that this mechanism relates specifically to adipogenesis their data are incomplete. ATF4 knockout mice have adipose tissue although they are leaner and resist diet induced obesity, but there is little evidence they have a defect in adipogenesis. It would appear from a number of studies that ATF4 plays a more important role in regulating energy metabolism than in directly affecting adipogenesis. In fact, the authors never directly assess adipogenesis but rather lipid accumulation, which can be affected by altering cellular metabolism. The authors should attempt to distinguish between the two: it would be relatively straightforward to examine the cells metabolically, for instance flux analysis, metabolomics, simple glycolysis assays. Without some attempt to distinguish between a differentiation phenotype and a cellular metabolic phenotype many of the conclusions about adipogenesis are not supported by the data. Furthermore, in addition to measuring oil droplet accumulation a qPCR survey of dynamic adipocyte differentiation marker expression should be conducted to elucidate the block in greater detail.A previous study by Yu et al. (cited in the manuscript) has suggested that ATF4 controls expression of C/EBPβ and PPARγ through promoter binding in a 3T3 model. The current manuscript suggests a model where ATF4 binding at enhancer elements is critical. Can the authors distinguish these two models experimentally, e.g. through rescue of adipogenesis in ATF knockdown cells through C/EBPβ overexpression?The authors describe 1451 ATF4 ChIP-seq peaks  that are All the reviewers considered your studies to be well executed. However, a major concern is the biological relevance of the reported discovery, in particular the role of cell density or confluence as a “trigger” during adipogenesis in vivo. If the authors would like to make the argument that this mechanism relates specifically to adipogenesis their data are incomplete. ATF4 knockout mice have adipose tissue although they are leaner and resist diet induced obesity, but there is little evidence they have a defect in adipogenesis. It would appear from a number of studies that ATF4 plays a more important role in regulating energy metabolism than in directly affecting adipogenesis.We appreciate the reviewers’ concerns. We have made the following modification to the second paragraph of the discussion to provide a reference point for readers: This study identifies ATF4 as a cell density sensor that links the physical and chemical requirements for adipogenesis. High cell density has long been known to be an important condition for adipocyte differentiation in vitro . It is lRegarding adipogenesis in ATF4 knockout mice, we agree and make the following modification to the Discussion’s third paragraph: Mice lacking ATF4 are lean and resist diet-induced obesity, although they have adipose tissue . Our demIn fact, the authors never directly assess adipogenesis but rather lipid accumulation, which can be affected by altering cellular metabolism. The authors should attempt to distinguish between the two: it would be relatively straightforward to examine the cells metabolically, for instance flux analysis, metabolomics, simple glycolysis assays. Without some attempt to distinguish between a differentiation phenotype and a cellular metabolic phenotype many of the conclusions about adipogenesis are not supported by the data. Furthermore, in addition to measuring oil droplet accumulation a qPCR survey of dynamic adipocyte differentiation marker expression should be conducted to elucidate the block in greater detail.PPARγ and C/EBPα, both encoding adipose-determining transcription factors, is significantly decreased in hMSCs depleted for ATF4 in response to adipogenic signals. Several downstream gene targets important for adipocyte differentiation also have reduced expression, suggesting that ATF4 affects lipid accumulation by promoting adipogenesis. These data have been added to We thank the reviewers for raising this point; clarifying how ATF4 affects lipid accumulation in response to adipogenic signals improves the study. To directly address adipogenesis, we measured the expression of key differentiation markers in hMSCs with normal and reduced levels of ATF4. Transcriptional activation of A previous study by Yu et al. (cited in the manuscript) has suggested that ATF4 controls expression of C/EBPβ and PPARγ through promoter binding in a 3T3 model. The current manuscript suggests a model where ATF4 binding at enhancer elements is critical. Can the authors distinguish these two models experimentally, e.g. through rescue of adipogenesis in ATF knockdown cells through C/EBPβ overexpression?C/EBPβ or PPARγ expression. Both mRNA and protein for C/EBPβ appear unchanged in hMSCs depleted of ATF4 (Cebpβ (-152 to 0) and Pparγ (-360 to -58) promoters as evidence for direct regulation of these genes, yet ATF4 displays little or no interaction with the CRE motif in unbiased genome-wide studies from mouse embryonic fibroblasts (Nature Cell Biology 2013. doi:10.1038/ncb2738) and hMSCs (this work).Our data do not indicate that ATF4 directly regulates  of ATF4 . It shouPPARγ locus, 40 kb downstream of the PPARγ2 TSS, yet it lacks enrichment for either RNAPII or H3K27ac in high-density-seeded cells, suggesting little or no enhancer activity under these conditionsFurthermore, we do not observe binding at either region in hMSCs . A singlWe appreciate the suggestion to distinguish between conflicting models. However, rather than restoring function, forced expression of C/EBPβ under ATF4 knockdown conditions will yield abnormally high C/EBPβ levels, limiting the interpretation of any potential effects on adipogenesis. Forced C/EBPβ expression has been shown by the Spiegelman and Farmer labs to trigger adipogenesis in NIH-3T3 cells, likely by overwhelming and/or overriding the system and obviating the need for pre-adipocyte-specific transcription. Thus, we do not believe that this experiment can resolve how ATF4 contributes to adipogenic commitment.The authors describe 1451 ATF4 ChIP-seq peaks  that are found in HD MSC cells. Presumably, the peaks fall mainly within cluster 10-11 fromsince there is an enrichment of the hybrid motif, but it is hard to follow if the ATF4 binding events also exhibit a certain GO enrichment which could clarify the biological context ATF4 binding (or absence thereof) might have. The manuscript might greatly benefit from a clearer analysis and display of ATF4-binding events/hybrid motif usage.We thank the reviewers for these suggestions, as they have led to an improved study. GO results derived from mapping ATF4-binding sites to nearby genes are now included in We have also expanded the third paragraph of the Discussion based on these results as follows: “It is of interest to note that ATF4 plays a central role […] demand for protein synthesis during adipogenesis.”


Siganus canaliculatus was the first marine teleost demonstrated to have the capability of biosynthesizing long-chain polyunsaturated fatty acids (LC-PUFA) from C18 precursors, and to possess a Δ4 fatty acyl desaturase (Δ4 Fad) which was the first report in vertebrates, and is a good model for studying the regulatory mechanisms of LC-PUFA biosynthesis in teleosts. In order to understand regulatory mechanisms of transcription of Δ4 Fad, the gene promoter was cloned and characterized in the present study. An upstream sequence of 1859 bp from the initiation codon ATG was cloned as the promoter candidate. On the basis of bioinformatic analysis, several binding sites of transcription factors (TF) including GATA binding protein 2 (GATA-2), CCAAT enhancer binding protein (C/EBP), nuclear factor 1 (NF-1), nuclear factor Y (NF-Y), hepatocyte nuclear factor 4α (HNF4α) and sterol regulatory element (SRE), were identified in the promoter by site-directed mutation and functional assays. HNF4α and NF-1 were confirmed to interact with the core promoter of Δ4 Fad by gel shift assay and mass spectrometry. Moreover, over-expression of HNF4α increased promoter activity in HEK 293T cells and mRNA level of Δ4 Fad in rabbitfish primary hepatocytes, respectively. The results indicated that HNF4α is a TF of rabbitfish Δ4 Fad. To our knowledge, this is the first report on promoter structure of a Δ4 Fad, and also the first demonstration of HNF4α as a TF of vertebrate Fad gene involved in transcription regulation of LC-PUFA biosynthesis.Rabbitfish  FADS1 gene, while Δ6 activity is encoded by the FADS2 gene  vector (Promega). The distance of insert fragments D4, D3, D2 and D1 to the putative transcription start site (TSS) +1, predicted as the first base of the first non-coding exon, was -1166 bp, -723 bp, -262 bp and +203 bp, respectively (For identifying the core promoter region within the cloned 5’ flanking sequence of the rabbitfish Δ4  493 bp) . The PCRectively . The conFad, online software TRANSFAC® and TF Binding® were used to search the potential binding sites in the 5’ flanking sequence of the Δ4 Fad. Since no genome data or promoter structure of Δ4 Fad is currently available in any vertebrate, and all teleost Fads including Δ6 Fad, Δ6/Δ5 Fad, Δ4 Fad belong to FADS2 gene cluster [Fad and the reported promoter sequences of Δ6 desaturase from Dicentrarchus labrax [Gadus morhua [Salmon salar [Homo sapiens [Danio rerio genome  was performed to search for conserved cis elements .In order to identify possible TF binding sites in the core promoter of the rabbitfish Δ4 s labrax , Gadus ms morhua , Salmon on salar , Homo sa sapiens  and Δ6/ΔFad promoter, the construct of deletion mutant D2 was treated as wild-type and site-directed mutants were produced from this with mutation sites designed in the middle of the primer according to the manufacturer's protocol. The strategy of site directed mutation was shown in TM site-directed mutagenesis kit . PCR with recombinant plasmids pGL 4.10-D2 as template was performed by one cycle (95°C for 30 sec), 15 cycles . After the reaction, the PCR product was incubated on ice for 5 min, and digested with MutazymeTM  for 1 h. Finally, the digested product was transformed into DH5α competent cells for selection, and positive clones subsequently sequenced (Shanghai Sangon Biotech Co.). Site-directed mutation of insert fragment D2 in recombinant plasmids was confirmed by sequence comparison with the wild-type deletion mutant D2. The site-directed mutation plasmids from D2 were named as SD1, SD2, SD3, SD4, SD5, SD6, SD7 referring to the binding sites of the corresponding TFs GATA binding protein 2(GATA-2), CCAAT enhancer binding protein (C/EBP), nuclear factor 1 (NF-1), TATA box binding protein (TBP), nuclear factor Y (NF-Y), sterol regulatory element (SRE) and hepatocyte nuclear factor 4α (HNF4α) in the core promoter region, respectively. These site-directed mutants were isolated with High Pure Plasmid Isolation Kit for further transfection in HEK 293T cells.To determine the potential functions of predicted TF binding sites on the core promoter activity, site-directed mutation of recombinant plasmids was carried out. For the rabbitfish Δ4 Fad promoter were transfected into HEK 293T cells grown in the presence of eicosapentaenoic acid  or absolute ethyl alcohol (AEA) carrier alone. HEK 293T cells were grown for 24 h to 80% confluence, then transfected with 100 ng of each reporter firefly luciferase construct with Lipofectamine® 2000 Reagent , and co-transfected with 0.01 ng of vector pGL4.75 (Promega Corporation), an internal control vector to normalize variations in transfection efficiency, constitutively expressing renilla luciferase by CMV promoter. The empty vector pGL4.10 with no promoter sequence in the 5’ flanking region of the reporter gene (firefly luciferase) was used as a negative control in each transfection assay. The LC-PUFA, EPA, had been demonstrated to been effective suppresser of lipogenesis in HEK293 [Δ4 Fad promoter. Based on these earlier studies on effects of PUFA, 50 μM EPA was an appropriate concentration for detection. Fresh cell culture medium with 50 μM EPA or with the same volume absolute ethyl alcohol (control) was replaced at 24 h after transfection.In order to detect the influence of the reporter vectors above (progressive deletion mutants and site-directed mutants) on promoter activity, the mutants were transfected into the mammalian cell line, HEK 293T cells, which were seeded in 96-well cell culture plates in 100 μl High Glucose Dulbecco's Modified Eagle Medium (DMEM) (Gluta MAX)  with 10% fetal bovine serum per well . To confirm the core promoter regions sufficient to initiate transcription and the area responsive to PUFA, progressive deletion mutants of Δ4 n HEK293  and AS cn HEK293 , and thuTM luciferase assay system (Promega), and chemical luminescence intensity was detected in duplicate readings using a microplate reader . The promoter activity was calculated from the chemical luminescence intensity ratio of firefly: renilla luciferase for each construct, and then compared with the activity of vector pGL4.10 luciferase [The effect of site-directed mutation on the transcriptional activity of the core promoter was determined by co-transfecting HEK 293T cells with site-directed mutants and pGL4.75. The ratio of site-directed mutants and pGL4.75 in the transfection complex was the same as for the progressive deletion mutants above. The cells were incubated in 100 μl DMEM +10% FBS and transfected with plasmid complex. Each plasmid complex was transfected in triplicate in three independent experiments. Cell culture medium was replaced with 75 μl DMEM +10% FBS at 24h after transfection. Luciferase assays were performed at 48 h after transfection with the Dual-Glociferase .Fad, nuclear and cytoplasmic proteins were extracted from rabbitfish liver tissue by Beyotime Nuclear Extract Kit  and quantified by Sangon non-Interference Protein Assay Kit . A 5’ end biotin-labeled probe of 289 bp covering the transcription elements was designed and incubated with the proteins so as to determine whether interaction existed between the TFs and the core promoter of the Δ4 Fad. The 5’ end biotin-labeled probe was obtained from 50 μl PCR reaction system including 20 μl ddH2O, 25 μl 2 × Pfu PCR Master Mix, 2 μl 10 mM 5’ end biotin-labeled forward primer, 2 μl 10 mM 5’ end biotin-labeled reverse primer and 1 μl pGL 4.10-D2 recombinant plasmid (100 ng/μl), while the competitor probe was made from the same system with unlabeled primers. PCRs consisted of an initial step (95°C for 3 min), followed by 35 cycles  and a final extension step (72°C for 5 min). Both the labeled and unlabeled primers for probes in EMSA were supplied by Shanghai Sangon Biotech Co., Ltd. According to the manufacturer’s instructions, EMSA of 20 μl reaction system was performed with Beyotime Chemiluminescent EMSA Kit  in the following groups: negative control group , experimental groups of liver cytoplasmic proteins  and liver nuclear proteins , competitor groups of liver cytoplasmic proteins  and liver nucleus proteins . The binding reaction was subjected to a 4% non-denaturing polyacrylamide gel and transferred to a nylon membrane. The 5’ end biotin-labeled DNA was detected using a streptavidin-horseradish peroxidase conjugate and a chemiluminescent substrate. The signal was then detected by autoradiography with X-OMAT BT X-ray film .To confirm the existence of TF binding sites in the core promoter of rabbitfish Δ4 TM Streptavidin Magnetic Beads  and Amicon®Ultra-0.5 Centrifugal Filter Devices  were used to bind and isolate proteins, respectively. The DNA-protein complex of the gel shift band was obtained from 100 μl EMSA reaction system including 20 μl liver nucleus protein (105.6 μg), 10 μl biotin-labelled probe (360 ng), 20 μl binding buffer and 50 μl H2O. One hundred μl of magnetic bead suspension was put into a 1.5 ml micro centrifuge tube, and a magnetic stand used to collect beads and the storage buffer was removed. After pretreating the magnetic beads by washing using 500 μl Tris-buffered saline with Tween® 20 surfactant (TBST) for 10 s, 100 μl biotinylated protein was added to the magnetic beads and incubated with gentle mixing for 60 min at room temperature. The magnetic stand was applied to collect the beads, and bound protein eluted using TBST buffer. 500 μl sample was added to the Amicon® Ultra filter  and centrifuged at 14,000 × g for 10 min. The isolated protein solution was analyzed by liquid chromatography coupled with tandem LC-MS technology .To identify the binding of TFs to core promoter, Pure ProteomeAfter adjusting the pH to 8.5 with 1 M ammonium bicarbonate, total protein (100 μg) extracted from each sample was chemically reduced for 1 h at 60°C by adding DTT to 10 mM and carboxyamidomethylated in 55 mM iodoacetamide for 45 min at room temperature in the dark. Then Trypsin Gold (Promega) was added to a final substrate/enzyme ratio of 30:1 (w/w). The trypsin digest was incubated at 37°C for 16 h. After digestion, the peptide mixture was acidified by 10 μl of formic acid for further MS analysis. After protein digestion, each peptide sample was desalted using a Strata X column (Phenomenex), vacuum-dried and then resuspended in a 200 μl volume of buffer A . After centrifugation at 20000 × g for 10 min, the supernatant was recovered to obtain a peptide solution with a final concentration of approximately 0.5 μg/μl. 10 μl supernatant was loaded on a LC-20AD nano-HPLC  by the autosampler onto a 2cm C18 trap column. The peptides were eluted onto a 10 cm analytical C18 column (inner diameter 75 μm) packed in-house. The samples were loaded at 8 μL/min for 4 min, then the 44 min gradient was run at 300 nL/min starting from 2 to 35% B , followed by 2 min linear gradient to 80%, and maintenance at 80% B for 4 min, and finally return to 5% in 1 min. The peptides were subjected to nanoelectrospray ionization followed by tandem mass spectrometry (MS/MS) in a QEXACTIVE  coupled online to the HPLC. Intact peptides were detected in the Orbitrap at a resolution of 70 000. Peptides were selected for MS/MS using high-energy collision dissociation (HCD) operating mode with a normalized collision energy setting of 27.0 and ion fragments were detected in the Orbitrap at a resolution of 17500. A data-dependent procedure that alternated between one MS scan followed by 15 MS/MS scans was applied for the 15 most abundant precursor ions above a threshold ion count of 20000 in the MS survey scan with a following Dynamic Exclusion duration of 15 s. The electrospray voltage applied was 1.6 kV. Automatic gain control (AGC) was used to optimize the spectra generated by the Orbitrap. The AGC target for full MS was 3e6 and 1e5 for MS2. For MS scans, the m/z scan range was 350 to 2000 Da. For MS2 scans, the m/z scan range was 100–1800 Da. Functional annotations of the proteins were conducted using Blast2GO program against the non-redundant protein database.Fad promoter activity, over-expression vector pcDNA3.1-HNF4α was constructed with clone vector plasmid pEASY-Blunt-Zero-HNF4α (GenBank: JF502073.1) and expression vector pcDNA3.1 (Invitrogen) double digested by XbaI and EcoRI-HF , then with target HNF4α fragments and empty vector pcDNA3.1 ligated by T4 DNA ligase. Constructed vector pcDNA3.1-HNF4α was sequenced (Shanghai Sangon Biotech Co.) for accuracy. Progressive deletion mutants of Δ4 Fad promoter (100 ng/well) and pcDNA3.1-HNF4α (50 ng/well) were co-transfected into HEK293T cell lines seeded in 96-well cell culture plates, while pGL4.75 (0.02 ng/well) was added as an internal control vector. HEK 293T cell was incubated in 100 μl DMEM + 10% FBS and transfected with plasmid complex. Each plasmid complex was transfected in triplicate in three independent experiments. Cell culture medium was replaced with 75 μl DMEM + 10% FBS at 24 h after transfection. Luciferase assays were performed at 48 h after transfection with the Dual-GloTM luciferase assay system, and chemical luminescence intensity was detected in duplicate readings using a microplate reader.To verify the effect of rabbitfish HNF4α on Δ4 ® T7 Ultra Kit  according to the manufacturer to generate capped mRNA with a poly(A) tail. Plasmid pEASY-Blunt-Zero-HNF4α was constructed with pEASY-Blunt-Zero vector  and HNF4α cDNA, which was used as a template for a linearized DNA template above with T7 promoter primer and antisense primer of HNF4α containing termination codon in a pfu-PCR reaction , and stored in -80°C for further transfection into rabbitfish primary hepatocytes, described previously [mRNA transcription was performed on a linearized DNA template containing T7 promoter and rabbitfish HNF4α cDNA sequence (GenBank: JF502073.1) using mMESSAGE mMACHINEreaction . PCR proeviously TM MessengerMAXTM Reagent (Invitrogen). At 48 h after transfection, the primary cells transfected or not were treated with Trypsin-EDTA (Invitrogen), and centrifuged 1500 × g, 2 min to precipitate the cells. The total RNA was extracted from the cell pellet by RNeasy® Plus Mini kit , detected by electrophoresis and quantified by Nanodrop 2000 Spectrophotometer . Then 0.2 μg total RNA were reverse transcribed into cDNA with oligo-dT primer by the SuperScript® III First-Strand Synthesis System (Invitrogen). Expression of target genes  was measured by Q-PCR. Copy numbers of the genes HNF4α, Δ4 Fad (GenBank: GU594278) were normalized to reference gene 18S rRNA (GenBank: AB276993) calculated by the comparative threshold cycle (Ct) method [2O and 2 μl cDNA template (10 ng/μl). The PCR program was consisted of an initial DNA denaturation of 94°C for 5 min, followed by 45 cycles at 95°C for 10 s, annealing 60°C for 20 s, extension 72°C for 20 s, with a final extension step at 95°C for 5 s, 65°C for 1 min, 40°C for 10 s. The reactions of each sample were performed in triplicate.The rabbitfish primary hepatocytes were seeded in two 6-well plates  in DMEM/F12 with 15% FBS medium. When grown for 48 h to 80% confluence, the cells were then transfected with 5 μg/well HNF4α mRNA by Lipofectamine) method . SpecifiHNF4α and Δ4 Fad gene expression after rabbitfish HNF4α mRNA treatment (n = 6) were analyzed by one-way analysis of variance (ANOVA) followed by Tukey's multiple comparison test or Student's t-test using Origin 7.0. A significance of P < 0.05 was applied to all statistical tests performed.All data were presented as means ± SEM. The data of promoter activity influenced by progressive deletion, site-directed mutations, pcDNA3.1-HNF4α (n = 3), Q-PCR data of Fad promoter. The first base of the first non-coding exon was regarded as the putative TSS, which was defined as the +1 position in the sequence. There was an intron of 561 bp length between two noncoding-exons, the first exon was 106 bp and the second 29 bp. Based on progressive deletion of the 5’flanking sequence of the Δ4 Fad, the deletion of fragments D4 (-1166 bp to -724 bp) and D3 (-723 bp to -263 bp) caused a gradual increase of promoter activity, whereas significantly reduced promoter activity occurred when the region of fragment D2 (-262 bp to +203 bp) was deleted, suggesting that the core promoter region was located at -262 bp to +203 bp . Based on bioinformatic analysis of the core promoter, a series of site-directed mutants of the core promoter was constructed and transfected into HEK 293T cells for detection of transcriptional activity. Compared with the wild type D2, mutation of binding sites for NF-1, NF-Y, HNF4α and SRE caused a highly significant decrease in transcriptional activity, while the effect of GATA-2 and C/EBP mutation was also significant (P < 0.05)  . DeletioFad  was used to analyze the peptide fragments with the method of GO (Gene Ontology) and COG (Cluster of Orthologous Group of proteins)  (roteins) . There wFad, the influence of rabbitfish HNF4α over-expression on Δ4 Fad transcription was determined. The over-expression vector pcDNA3.1-HNF4α was constructed and co-transfected into HEK 293T cells with progressive deletion mutants of Δ4 Fad. Deletion mutants D1, negative control D0 and site-directed mutant of HNF4α in D2 showed no response to HNF4α treatment, while the activity of D4, D3, D2 was increased significantly by HNF4α over-expression (P < 0.05)  . These rFad was further confirmed in rabbitfish primary hepatocytes. After the rabbitfish HNF4α mRNA, which was synthesized in vitro, was transfected into the rabbitfish primary hepatocytes, the mRNA of Δ4 Fad gene was significantly increased , five Sp1 elements and a DR-1 element (PPARα binding element) were predicted in the promoter region [S. canaliculatus Δ4 Fad. Therefore, the S. canaliculatus Δ4 Fad promoter shared the same binding site for C/EBP as in G. morhua Δ6 Fad promoter, but did not contain either the Sp1 element that was reported in S. salar and H. sapiens Δ6 Fads2 promoters, or the DR-1 element of the H.sapiens Δ6 FADS2 promoter. However, results suggested that the structure of S. canaliculatus Δ4 Fad promoter was highly conserved in comparison to promoters of Δ6 Fad or Δ6/Δ5 Fad from other fish. This was consistent with all these genes being Fad orthologues [S. canaliculatus Δ4 Fad core promoter, which was predicted by software TRANSFAC®, did not exist in the corresponding region of promoter sequences of Δ6 Fad from D. labrax [G. morhua [S. salar [H. sapiens [Fad from D. rerio . It is not known at present whether HNF4α was not a common TF involved in Fad regulation as identified herein for rabbitfish, and more Fad promoter structure is yet required, specifically from the fish with Δ4 Fad including Senegalese sole [The position of p to TSS . For thepromoter , a Sp1 er region . The pre. labrax , G. morh. morhua , S. salaS. salar , and H.  sapiens  and Δ6/Δese sole  and striese sole . To dateS. canaliculatus Δ4 Fad transcription, in consideration of the probe length (less than 300 bp), the probe of core promoter for the EMSA did not contain these two elements, and so these factors were not further studied for interaction between nucleus proteins and core promoter. With respect to EMSA, the LC-MS protein analysis did not identify TFs NF-Y and SREBPs in the core promoter, which might be caused by low expression of NF-Y and SREBPs in the S. canaliculatus liver sample as a low content of these TFs would reduce the opportunity to bind to the probe of rabbitfish Δ4 Fad core promoter. Although LC-MS did not identify the existence of GATA-2, C/EBP, NF-Y or SREBPs, according to the site-directed mutation assay, these TFs might still be the potential regulator in the transcription of S. canaliculatus Δ4 Fad.Mutation of the GATA-2 and C/EBP binding sites had significant influences on S. canaliculatus Δ4 Fad. HNF4α is a TF of the nuclear receptor (NR) superfamily that constitutively binds fatty acids [FADS2 (Δ6 desaturase) transcription found that HNF4α did not affect Δ6 desaturase expression when co-transfecting CV1 cells with expression vectors of human FADS2 promoter and HNF4α [Fad promoter. Mutation of this site caused a significant reduction of Δ4 Fad promoter activity. Moreover, the proteins identified by LC-MS confirmed the existence of HNF4α in the core promoter. All these results confirmed the existence of a binding site for HNF4α in the S. canaliculatus Δ4 Fad promoter. Finally, the over-expression of S. canaliculatus HNF4α also showed that this TF could increase the process of Δ4 Fad transcription in HEK 293T cells. Promoter activity of D1, D0 and HNF4α element mutant showed no major difference between treatment with empty pcDNA3.1 vector and pcDNA3.1-HNF4α. While compared with the control group (pcDNA3.1 vector), D4, D3 and D2 promoter activity increased significantly after HNF4α overexpression, suggesting HNF4α could increase Δ4 Fad promoter activity in HEK 293T cells. To further functionally confirm whether HNF4α is a transcription factor of rabbitfish Δ4 Fad gene, the effects of HNF4α overexpression on Δ4 Fad mRNA was detected by Q-PCR in rabbitfish primary hepatocytes. HNF4α mRNA overexpression increased Δ4 Fad gene expression, suggesting that Δ4 Fad could be a target gene of HNF4α. As for NF-1, it activates transcription through direct interaction with basal TFs [Fad promoter activity and it was also identified by LC-MS. A further identification of such TFs involved in Δ4 Fad gene expression is required to perform as the HNF4α assay above.HNF4α and NF-1 were confirmed as regulatory factors of transcription of ty acids , 36, thety acids . Furtherty acids . Up to nty acids , involvity acids , 40. Witty acids , 42, fatty acids . Howevernd HNF4α . The preasal TFs  in the hasal TFs . In presFad promoter of S. canaliculatus was cloned and characterized in the present study, this representing the first report on the promoter structure of a Δ4 Fad, and also the first demonstration of HNF4α as a TF of a vertebrate Fad gene, suggesting a new regulatory mechanism in LC-PUFA biosynthesis.In summary, the Δ4