Drosophila are monosomic for the X chromosome, but survive due to dosage compensation. They use the Male Specific Lethal (MSL) complex composed of noncoding roX RNA and histone modifying enzymes to hypertranscribe most genes along the X ∼1.6–1.8 fold relative to each female allele. It is not known how the MSL complex achieves this precise adjustment to a large and diverse set of target genes. We carried out a genetic screen searching for novel factors that regulate dosage compensation in flies. This strategy generated thirty alleles in a previously uncharacterized gene, over compensating males (ocm) that antagonizes some aspect of MSL activity. The mutations were initially recovered because they derepressed an MSL-dependent eye color reporter. Null ocm mutations are lethal to both sexes early in development revealing an essential function. Combinations of hypomorphic ocm alleles display a male specific lethality similar to mutations in the classic msl genes, but ocm males die due to excessive, rather than lack of dosage compensation. Males that die due to very low MSL activity can be partially rescued by ocm mutations. Likewise, males that would die from ocm mutations can be rescued by reducing the dose of various msl and roX genes. ocm encodes a large nuclear protein that shares a novel cysteine rich motif with known transcription factors.Male  Drosophila utilize a genetic mechanism to determine sex. Embryos with two X chromosomes develop into females while those with only one adopt a male phenotype Drosophila seems to have evolved from a general chromatin remodeling machine that in other species is responsible for most histone H4K16 acetylation roX RNA partner binds all chromosomes indiscriminately The dosage compensation complex in Drosophila. A new targeting mechanism was needed to limit the MSL complex to the X. This involved the large noncoding roX RNAs (RNA on X chromosome) and the MLE  helicase thought to play some role in folding roX RNA or assembling MSL proteins onto roX RNA msl2 mRNA under the negative control of the female-specific master regulator of sex determination, SXL  DrosophilaDrosophila females survive without the ancestral functions of the MSL proteins. The same is likely true for male autosomal genes. Each of the msl genes is single copy and as the name suggests, null mutations appear to have no impact on females. The exception is mof where null mutant females are viable, but recovered at only half the expected numbers msl genes were freed to shift functions At least three major innovations were necessary to recruit the MSL proteins to the new function of hypertranscribing the male X in roX2 have identified many promising new candidates in the dosage compensation pathway including a Zn finger protein, CG1832, that may provide the sequence specificity for targeting MSL complex to chromatin entry sites ocm  gene cause elevated dosage compensation of the eye color reporter. Certain combinations of hypomorphic missense alleles display a male-specific lethality as seen with the classic msl genes. Our analysis demonstrates that ocm is a negative regulator of the MSL complex limiting dosage compensation to two-fold. Reduction of OCM selectively kills males due to excessive dosage compensation. Recovering a wide allelic series of ocm mutations allowed us to identify roles in oogenesis, spermatogenesis, and early development in addition to its role regulating the MSL complex.We are interested in finding any additional factors that may be needed to help the MSL proteins carry out their new dosage compensation function. Genetic screens looking for the male-specific lethal phenotype have probably reached saturation. Biochemical purification of the MSL complex requires stable protein interactions that survive extraction and has had limited success in finding new partners miniwhite eye color gene occasionally land in regions of repressive chromatin so that little or no eye pigment is made. If the transgene carries roX1, one of the genes producing the large noncoding RNA component of the MSL complex, the MSL complex is targeted to the transgene and acetylates surrounding chromatin so that the miniwhite gene is expressed msl1 and mle in this screen 166 complementation group, initially represented by three alleles recovered in a small pilot screen using the GMroX1-75 mosaic eye reporter . We repeated a modified version of the screen on a larger scale to recover a total of 30 mutations in ocm w mutation that has been used extensively to assay for position effect variegation ocm had no dominant effect on the speckled pigmentation in males or females . Changing the mosaic pattern to solid red suggested elevated dosage compensation locally around the roX1 autosomal reporter when OCM levels were reduced  when the X chromosome carried wild type alleles of roX1 and roX2 regardless of whether ocm was wild type or heterozygous  that use a haploid-diploid strategy of sex determination. This failure is unlikely due to poor sequence assemblies because homologs for all the msl genes were easily found in these same species. Alignments between OCM containing insect species revealed a number of conserved motifs separated by highly diverged regions  from salivary glands with anti-OCM antibodies did not detect any reproducible signal in either sex (data not shown).Previous high throughput surveys showed that l clones . These rocm has additional phenotypes that might provide clues to its biochemical function. Because we could detect low activity in a few of our mutants with early stop codons, we selected the original P1048Zocm allele for lethal analysis because it is the only frameshift allele recovered (11 base pair deletion). Crossing P1048Zocm to Df(3R) BSC155 produced hemizygous progeny that did not develop past second instar larvae. Survival to this stage might depend upon maternally provided OCM, but we were unable to make ocm null eggs because the gene is needed for oogenesis (see below). Mutant larvae were lethargic but continued to move for several days before dying with grossly normal morphology.Besides its role in dosage compensation, ocm null animals die at second instar, ocm null cells are able to form clones in imaginal discs .msl gene die (50% too little X products). We used a genetic strategy to search for new genes that might regulate the dosage compensation pathway to achieve this precise set point. The miniwhite eye color marker adjacent to roX1 provided an easily scored phenotypic reporter for changes in MSL complex activity that are too small to affect male viability. Rather than look for subtle changes in the color of individual pigment cells, our assay relies on large changes in the fraction of eye tissue where the MSL complex successfully overcomes silencing of miniwhite.The MSL complex has been intensively studied as a model to understand how an RNA-dependent chromatin remodeling machine can produce a subtle, but essential two-fold change in global transcription across the male X chromosome. The MSL complex must act on hundreds of unrelated genes and successfully adjust their expression within a very narrow range. XXX:AA females die (50% too much X products) and XY:AA males mutant for any ocm gene (CG3363) that all produced similar increases in eye pigmentation from the MSL-dependent reporter exclusively in males. Most mutants were strong loss of function or complete nulls such that homozygotes of both sexes died by second instar larvae. However, the few hypomorphic missense alleles recovered were especially informative because they revealed a second dosage compensation phenotype, recessive male-specific lethality. Male lethality could potentially occur at any number of points in the dosage compensation pathway from the initial sex determination decisions, through production of MSL proteins subunits or roX RNAs, assembly of complex, targeting to active X-linked genes, or biochemical activity of the complex. For instance, ectopic MSL2 expression kills most females and renders the few escapers sterile, similar to ocmocm females do not ectopically express MSL complex and msl mutations do not rescue ocm female toxicity disprove this idea. Excluding subtle defects in subunit production is more difficult, but several observations argue against this idea. First, separately reducing production of MSL1, MSL2, MLE or roX1 RNA each partially rescues ocm males arguing that it is the full complex rather than one individual component that is affected. Similarly, ocm mutations do not affect cis spreading behavior of MSL complex from ectopic autosomal sites of roX1 transcription. Spreading behavior is highly sensitive to the ratio of MSL proteins subunits to nascent roX1 RNA suggesting individual components are produced in normal amounts and efficiently assembled into complexes ocm activity is reduced. One way this might occur is if OCM removed chromatin marks deposited by the MSL complex. However, the predicted OCM sequence shares no obvious similarity to known histone deacetylases, phosphatases, or deubiquitylases.We recovered 30 independent EMS alleles of the ocm and wildtype cells are juxtaposed, any miniscule differences in staining can be attributed confidently to the presence of OCM , S2 cells (data not shown) and developing egg chambers was observed either. One potential difficulty in working with OCM is its apparently poor expression levels across all developmental stages e of OCM . For thiocm males, although we could only recover adult males from the weakest ocm alleles. By contrast, the defect in oogenesis is so early that no recognizable egg chambers are formed. Null mutants complete embryonic development but die as young second instar larvae. These female phenotypes clearly demonstrate that OCM is also required for pathways unrelated to the MSL complex. We looked to see if the modular pattern of amino acid sequence conservation might correspond to functionally distinct regions of the protein by determining the codons changed in many alleles. We did notice that the five missense alleles that allow female development but cause excessive MSL activity all cluster in two small, highly conserved segments. While these must be necessary for proper dosage compensation, they cannot be exclusively dedicated to that function because adult females also suffer early blocks to oogenesis. Several of the MSL subunits have recently acquired novel domains that are necessary for the specialized function of dosage compensation of the male X Drosophila species that use the MSL complex for dosage compensation of the male X compared to more distantly related insects that use the MSL proteins for other purposes. No obvious new adaptation was found in fly OCM. One explanation for the wider conservation of OCM motifs implicated in Drosophila MSL activity might be that OCM also modulates MSL activity in insects that use it for other pathways.OCM plays multiple roles in development judging from the pleiotropic phenotypes seen in the different alleles. The defect in spermatogenesis is most likely late because motile sperm are present in escaper roX2 expression in cultured cells identified CG3363 as the strongest repressor of MSL activity CG3363 appeared in two independent genetic screens using different strategies provides confidence that it is a newly recognized negative regulator of the dosage compensation pathway.A recent RNAi screen for regulators of roX1 mutation is ex6roX1 and the roX2 allele is 52Df (1) roX2+4[wΔ4.3] transgene supplies essential adjacent genes lost in the roX2 deletion.Larvae and flies were raised on standard cornmeal-yeast-agar-molasses medium containing propanoic acid at 25°C. In all experiments the The full genotypes of the flies in y w; [GmroX1-75C].A–B) L714X/+y w; ocm; [GmroX1-75C].C–D) L714X; [GmroX1-75C].y w;msl1+/+ ocmE–F) y w; [GmroX1-58D].G–H) Q1673X/+y w; ocm; [GmroX1-58C].I–J) m4y w.K–L) m4; ocmG1646E/+.y wM–N) y w. The ocm allele used for viability assay is indicated beside the graph.O) The wildtype flies are w+GMroX1] reporter at all times because pilot experiments showed the modifiers produced a more conspicuous phenotype when the reporter was paired. The [GMroX1-75C] and [GmroX1-58D] transgenic stocks used were also as previously described The mutagenesis was carried out as described in CyO, y+ balanced males from each of the modifier mutants were mated to comparable females and the presence of nonbalanced progeny (yellow body and flat wings) was scored.2A.y w; ocmG1646E/CyO, y+ virgins crossed to y w/Y; msl ocmL1658N/CyO, y+ males where msl is either msl1L60, msl21, or 1mle. The interesting class of progeny had flat wings and yellow body.The crosses used for 2B. The control classes were produced by w; msl1hypo/CyO, Roi sib crosses to produce homozygotes or crosses to y w; msl1L60/CyO to produce hemizygotes. The double mutants were made by mating y w/Y; msl1hypoocmL1658N/CyO, y+ males to y w; msl1L60ocmL1658N/CyO, y+ virgins.2C.y w roX1 roX2 [w+ cos4Δ]; [w+GMroX1-75C] virgins crossed to y w/Y; msl1L60ocm/CyO, y+ males where the different ocm alleles are showed for each column.ocm chromosomes by back crossing to the starting chromosome until BAC 117N13 could rescue ocm homozygotes to Mendelian frequencies.Standard P element transformation was used for BAC rescue experiments The following primer pairs were used to amplify genomic DNA from adult flies followed by Sanger sequencing separately from both strands. Several background polymorphisms relative to the reference sequence were found during sequencing, but not shown. Likewise, a few nonsense mutations carried nearby missense mutations as well, but only the nonsense mutation is shown.ocm alleles were first recombined to the [FRT42D] transgene as described +]++ ocm/[FRT42D] [y+] [GMR-hid] [w+ cl]+y w; [FRT42D] [UAS-FLP] as described y w; Q1297X[FRT42D] ocm was crossed to y w [ey-flp]; [FRT42D], [Ubi-GFP]/Cyo or y w [Ubx-flp]; [FRT42D], [Ubi-GFP]/Cyo flies to generate mitotic clones in the eyes and wings discs respectively. The flies stocks were gifts from the Bellen’s lab.Six Immunostaining on imaginal discs and embryos were performed as described A segment of the gene spanning codon 1287–1773 was subcloned into the pMAL-C2X vector . The fusion protein was expressed and run on a SDS page acrylamide gel. The protein was excised from the gel and injected into guinea pigs . The animals were sacrificed and the anti-serum collected after 91 days.Adult flies and embryos were first dounced in 50 mM Tris-HCl (pH 8.5), 300 mM NaCl and 1% NP-40 containing protease inhibitor  as described in Figure S1Ocm do not affect the morphology of the X chromosomes or spreading. To check for gross morphology of the X chromosome, an attempt was made to retrieve third instar larvae from L1658N/S1590F (weakest combination), L1658N/G1646E, L1658N/W1401X and S1590F/Q1297X (strongest combination) crosses for MSL1 immunostaining. The morphology of the X in L1658N/W1401X larvae is normal in both A) males and B) females. We did not see altered X morphology in L1658N/S1590F and L1658N/G1646E as well (data not shown). No male larvae could be retrieved from S1590F/Q1297X and L1658N/W1401X is the most severe ocm heteroallelic combination where male third instar larvae can be recovered (See Table 1 for comparison). We also assayed the ability of ocm to affect spreading of the MSL complex around the GmroX1 transgene inserted at 75C. C) The MSL complex binds to the roX1 transgene and appears as a single sharp band (red arrow). D) Reducing 50% ocm activity does not affect the ability of the transgene to recruit MSL complex (red arrow). E) The MSL complex spreads megabases from the same GmroX1 transgene when the fly is mutant for roX1 and roX2 (yellow line). F) Spreading seems to be unaffected after reducing 50% ocm activity (yellow line). The genotype for the larvae is C) y w; [GmroX1-75C]/+, D) y w; ocmL714X/+; [GmroX1-75C]/+, E) y w roX1 roX2; [GmroX1-75C]/+, F) y w roX1 roX2; ocmL714X/+; [GmroX1-75C]/+.(DOCX)Click here for additional data file.Figure S2Hypomorphic mutations in ocm. The region corresponding to the black box in (DOCX)Click here for additional data file.Figure S3Sequence alignment of Cys431. Upper half shows the sequences of insect OCM. The central two proteins from lepidoptera contain only one copy of the cys repeat. The lower half shows MGA proteins from chordates. The last four sequences are from Daphnia proteins. Daphnia 222106 has the best match to the consensus sequence, a second cys-rich region shared with OCM (not shown) and a THAP DNA binding motif.(DOCX)Click here for additional data file.Figure S4ocm is needed for oogenesis. All three samples are shown at the same magnification. A. Paired ovaries from L1658N mutant are bundles of empty ovarioles . B. Same ovaries stained with anti-OCM. C. Individual dissected wild type ovariole containing a germarium through stage 8 egg chamber stained with anti-OCM. We could not detect any discernible OCM staining in any part of the tissues.(DOCX)Click here for additional data file.Table S1ocm were isolated in three separate EMS screens. The first three alleles were isolated with [w+GMroX1-75C] as were the next 16 alleles. The last eleven alleles were isolated using [w+GMroX1-56D]. The alleles that were sequenced are indicated with the first letter indicating the wild type codon, the codon number, and the replaced amino acid with X = stop codon. All of the missense alleles retained some activity as did several of the stop codons when placed in combination with missense alleles. B. Hypomorphic nonsense mutations. Female viability relative to the balancer sisters recovered in the same cross when mated to two weak hypomorphic alleles (805 and 127). Five of the six nonsense alleles have a cytosine base (underlined) followed by the stop codon. This allows low levels of translational readthrough producing low OCM activity making them hypomorphs. The bottom nonsense allele is followed by a guanine, produced no escapers and hence, classified as a null allele.A. Thirty alleles of (DOCX)Click here for additional data file.Table S2ocm alleles. y w; ocma/CyO y+ mothers were crossed to y w/Y; ocmb/CyO y+ fathers. Any viable progeny were recognized by yellow body color and flat wings. For each cross the actual number of CyO y+ sons (B) and daughters (D) are given along with the % viability of ocma/ocmb sons (A) and daughters (C) where the expected ratio is 1∶2, ocm: CyO. Most heteroallelic combinations were lethal to both sexes, but in some combinations (gray fill) daughters were viable but all sons died. In all cases the recovered females failed to produce any eggs. Recombination was used to remove secondary lethal mutations from chromosomes carrying missense mutations. Success was measured by the ability to recover homozygous ocm progeny in Medelian ratios that carried the 117N13 ocm+ transgene. That allowed the viability of homozygotes lacking the transgene to be measured (dark boxes).Complementation table of a subset of (DOCX)Click here for additional data file.


Introduction: Autoimmune diseases such as systemic lupus erythematosus, rheumatoid arthritis, and multiple sclerosis affect millions of people worldwide. Interferon regulatory factor 5 (IRF5) contains polymorphisms associated with these autoimmune diseases. Two of these functional polymorphisms are found upstream of the IRF5 gene. rs2004640, which is a single nucleotide polymorphism and the CGGGG insertion/deletion (indel) were studied. IRF5 uses four different promoters for its four first exons: 1A, 1B, 1C, and 1D. Each promoter was analyzed, including functional differences due to the autoimmune-risk polymorphisms.Results: IRF5 promoters were analyzed using ChIP-Seq data (ENCODE database) and the FactorBook database to define transcription factor binding sites. To verify promoter activity, the promoters were cloned into luciferase plasmids. Each construct exhibited luciferase activity. Exons 1A and 1D contain putative PU.1 and NFkB binding sites. Imiquimod, a Toll-like receptor 7 (TLR7) ligand, was used to activate these transcription factors. IRF5 levels were doubled after imiquimod treatment (p < 0.001), with specific increases in the 1A promoter  and 1D promoter . A putative binding site for p53, which affects apoptosis, was found in the promoter for exon 1B. However, site-directed mutagenesis of the p53 site showed no effect in a reporter assay.Conclusion: The IRF5 exon 1B promoter has been characterized, and the responses of each IRF5 promoter to TLR7 stimulation have been determined. Changes in promoter activity and gene expression are likely due to specific and distinct transcription factors that bind to each promoter. Since high expression of IRF5 contributes to the development of autoimmune disease, understanding the source of increased IRF5 levels is key to understanding autoimmune etiology. Alternative splicing is a method of making different transcripts from one genomic region. One type of alternative splicing involves the use of multiple first exons. This is termed alternative promoter splicing, since each first exon must have its own promoter. Alternative promoter splicing occurs in around half of human genes .The gene interferon regulatory factor 5 (IRF5) is a transcription factor which controls immune signaling, cytokine expression, the cell cycle, and apoptosis –5. It exThe IRF5 gene contains several GWAS-identified polymorphisms associated with autoimmune diseases. Among them, most do not have an assumed effect. Although IRF5 contains several polymorphisms associated with autoimmunity, only four have been identified as functional polymorphisms . Two of The rs2004640 SNP is a G or T polymorphism near the 3′ end of exon 1B. The SNP is within the splice junction, such that when the G allele is present, the splice junction is not recognized and exon 1B cannot be spliced onto exon 2 . UnsplicThe CGGGG indel rs77571059) is found 64 bp upstream of the transcription start site for exon 1A. Each allele has either three (3×) or four copies (4×) of the CGGGG repeat sequence. The 4× copy-number variant allows binding of additional SP1 transcription factors . This 4×59 is fouInterferon regulatory factor 5 exons 1A, 1B, 1C, and 1D each have a distinct transcriptional start site, and as is the case with every first exon, each exon 1 of IRF5 has its own promoter. IRF5’s four promoters have not been thoroughly characterized, although previous studies on the 1A and 1C exons’ promoters revealed that they are controlled in part by an IRF element (IRFE) and an interferon stimulatory response element (ISRE), respectively . Herein,Autoimmune diseases are caused by environmental triggers in those with a genetic propensity. Increases in IRF5 expression due to these promoter polymorphisms could lead to an autoimmune-risk state. A hallmark of lupus and those at genetic risk for lupus is the presence of heightened levels of interferon and interferon-response genes; the interferon signature . IRF5 isFor SLE, an environmental trigger is likely to be Epstein–Barr virus (EBV) infection , 30. EBVInterferon regulatory factor 5 uses one of four first exons for each molecule of mRNA – 1A, 1B, 1C, or 1D. Whether or not one of the four first exons of IRF5 would be actively transcribed depends on the cellular transcription factors that are able to bind it. A putative IRF5 exon 1B promoter sequence was identified by using the encyclopedia of DNA elements chromatin immunoprecipitation sequencing (ENCODE ChIP-Seq) data set . This ande novo for each ENCODE transcription factor tested, and compiled in the online database FactorBook , PU.1, SP1, and TCF12 which binds to enhancer boxes (E boxes). An extra SP1 binding site appears in those with the CGGGG 4× indel. Exon 1B’s promoter was the only IRF5 promoter with a p53 binding site. This is discussed in more detail below. 1B also has SP1, TCF12, IRF4, and early B cell factor (EBF) sites. The 1C promoter was the only promoter with STAT2, activator protein 1 (AP1), and Myc binding sites; it also has SP1 and IRF4 sites. The 1D promoter evaluation showed potential binding sites for only four transcription factors: SP1, CCCTC binding factor (CTCF), IRF4, and NFκB.The promoters for the four first exons of risk), and the other has the 3× variant (1Aprotective). The 1B promoter was cloned using nested PCR to avoid an inverted repeat sequence located ∼2 kbp upstream. The inverted repeat is 1.8 kbp in length, and the two copies have 82.8% identity , was cotransfected as a transfection control for each construct , EBV-transformed human B cells that were generated from three healthy volunteers; U937 cells, a commercially available human monocyte cell line; and Jurkat cells, a commercially available human T cell line. Jurkat cells were used as the negative control, since T cells do not express high levels of onstruct . Values risk and 1Aprotective) demonstrated significantly higher transcriptional activity than the other three promoters in LCL and Jurkat cells   in Jurkat cells when compared to U937 or LCL cells. When comparing LCL to U937 cells, there was no significant difference in IRF5 promoter activity (p = 0.38).Luciferase assay results showed that the 1A promoters (1A) Figure . As experisk promoter activity was significantly higher than 1Aprotective promoter activity (p = 0.019). The 1B promoter, which is only relevant when the risk allele rs2004640 is present, showed activity in LCLs and U937 cells, indicating that it is an active promoter in the same cell types as the other promoters.The autoimmune-risk polymorphisms affected the activity of the promoters. In LCLs the 1AThe levels of IRF5 expression increase due to several signaling pathways, one of which is the Toll-like receptor 7 (TLR7) pathway. Endosomal TLRs such as TLR7 require the ligand to first be endocytosed into the cells, and then merged with the endosome that contains TLR7. Most endosomal TLRs bind to nucleic acids.Toll-like receptor 7 ligation is an important method of activation for pDCs , 41. pDCp = 0.028). IL-6 expression is a common readout for stimulation through TLR7 (p = 0.038) after treatment with imiquimod  . CCR7 did not increase as much after imiquimod stimulation in the risk cells compared to the protective . However, the risk cells demonstrated a small increase in NOXA expression after imiquimod stimulation, and the Calreticulin levels decreased less  in the risk cells than in the protective . A close match to this sequence was found in the 1B promoter Figure B. This iTo test whether activated p53 protein can indeed bind to the p53 binding site, the plasmid which contains the 1B promoter was mutated using site-directed mutagenesis. Mutations were made to the wild-type sequence such that p53 should not be able to bind. The consensus binding site contains four conserved C or G bases which were mutated to A or T on the luciferase plasmid Figure B. The wiThe CGGGG indel polymorphism within the 1A promoter has previously been shown to alter transcription factor binding. When cells have the 4× variant, an additional SP1 binding site is created. This has been shown to increase IRF5 in PBMCs , but decThe 1A promoter has a PAX5 binding site, a gene that activates B cells at early, but not late stages of development . There i(N)GAA motif to IRFs. Further work is necessary to determine in which cell types or with which stimuli the 1A promoter is most active, and in what instances the CGGGG 4× variant alters this activity.The 1A promoter showed increased activity when cells were stimulated with the TLR7 agonist imiquimod. This may be through the PU.1 site through IRF7. IRF7 is known to be activated by TLR7 , and PU.A previous report by Mancl et al. evaluated the 1A and 1C promoters . The 1A The ability of a cell to use and splice the 1B exon is independent of its promoter usage. The 1B promoter is active in persons with both the risk and protective polymorphisms at rs2004640, yet the protective sequence would result in a non-sense transcript, as splicing would not be possible. The risk T allele allows for the exon 1B transcript to be spliced onto exon 2 and this is evidenced by the correlation between the risk T allele and increased levels of both IRF5 and exon 1B usage. The effects of the ability to use the 1B promoter can also be seen in the increased responsiveness of the cells containing the risk allele to imiquimod.Interferon regulatory factor 5’s 1B promoter was predicted to contain a p53 binding site. The only promoter tested which increased in activity after inducing DNA damage was the 1B promoter. The others showed a reduction in luciferase activity (data not shown). The mutated version of the 1B promoter, which contained an altered p53 binding site, showed a slight increase in luciferase activity instead of a decrease, likely suggesting that any p53 binding to this promoter region is inhibitory. The 1B promoter contains SP1, IRF4, TCF12, and EBF binding sites. EBF is a B cell-specific transcription factor . FurtherDuring cloning experiments dealing with exon 1B and its promoter, several sequencing reactions showed <100% sequence identity to the target. It was soon discovered that the primers were annealing to an upstream inverted repeat sequence. This repeat necessitated nested PCR for cloning the 1B promoter. The repeat length is 1.8 kbp, and the two copies have 82.8% identity . The funUsage of exon 1C is lower in cells with the rs2004640 autoimmune-risk factor. The 1C promoter contains putative SP1, IRF4, and EBF sites. It was the only promoter with AP1, Myc, and STAT2 binding sites. AP1 is a heterodimer of Fos and Jun proteins, among others, which are common in immune signal transduction . Myc is Usage of exon 1D is lower in cells with the rs2004640 T allele and in cells with the CGGGG 4× allele. The 1D promoter evaluation showed only four transcription factors’ binding sites: CTCF, IRF4, NFκB, and SP1. NFκB is a target of TLR7 , and thuInterferon regulatory factor 5 is proapoptotic in a p53-independent manner , 65. ThiEpstein–Barr virus infection is a necessary complicating factor when using LCLs as B cell lines. This is especially important since EBV has been shown to affect IRF5 function , 68. TheAutoimmune diseases are complex, multifactorial disorders with both genetic and environmental influences. The promoter variations examined in these experiments are strongly linked to risk for autoimmune diseases, including lupus, multiple sclerosis, and rheumatoid arthritis. Despite much effort, there has not been a dramatic effect associated with these polymorphisms, or really, most of the polymorphisms associated with autoimmune disease. Rather than diminishing their importance, however, the somewhat small effects observed here speak to the fine balance of the immune system. It is likely that even relatively small changes in gene regulation can lead to an imbalance in tolerance or activation of immune cells. Also, these genes are intertwined with other pathways and systems to provide a complex fabric controlling the level of immune responsiveness.2, 15 mM HEPES, 140 mM Na2HPO4, pH 7.2. Transfected cells were lysed and assayed for fluorescence levels before assaying luciferase activity using the Luciferase Assay System (Promega) on a Fusion αHT plate reader (Packard). Luciferase activity was evaluated in proportion to the transfection efficiency.All vectors were sequenced to confirm the proper sequence. The plasmid pMax-GFP (Clontech) expresses the eGFP fluorescent protein, and it was used to measure transfection efficiency. Electroporations were performed using a Nucleofector device (Lonza). The electroporation buffer was 5 mM KCl, 15 mM MgCl2 and passaged at least weekly.Peripheral blood samples were obtained from healthy volunteers after informed consent following a protocol approved by the IRB at Brigham Young University. Peripheral blood mononuclear cells were isolated using lymphocyte separation medium (Mediatech). These cells were induced to form LCLs by incubation with EBV (B95-8 strain) and 2 ng/ml cyclosporin A (Tocris Biosciences). U937 and Jurkat cells were a kind gift from Dr. Kim O’Neill. Cell lines were maintained in RPMI (Sigma) with 10% fetal bovine serum (PerBio) with penicillin/streptomycin/amphotericin  at 5% COGenomic DNA was extracted (Qiagen) from peripheral blood mononuclear cells and genotyped using TaqMan reagents Applied Biosystems (ABI) on a StepOnePlus real-time PCR machine (ABI) at the rs2004640 SNP (ABI SNP Assay C9491614). Homozygous risk or protective individuals were matched by gender and ethnicity. Heterozygotes were not included in the study. The primers and PCR conditions are in Table 6 cells per milliliter.The TLR7 ligand imiquimod (R-837) was used to stimulate cells for some experiments. Cells were treated for 24 h with 25 μg/ml imiquimod (InvivoGen). cDNA preparation, quantitative PCR, primers, probes, and conditions are described elsewhere in the Section “6 cells were used for each condition in each experiment. cDNA preparations were made by extracting RNA using the RNaqueous system (Ambion), followed by DNase treatment (Promega). One thousand nanograms RNA was per condition was then reverse transcribed using SuperScript III reverse transcriptase (Invitrogen Life Technologies). One hundred nanograms cDNA preparations were used as template for quantitative PCR using TaqMan reagents (ABI), or SYBR green reagents For gene expression studies, input RNA levels were normalized using primers to the housekeeping gene GAPDH for SYBR green experiments and β-GUS for TaqMan experiments. For cloning of 5′UTRs and promoters the template genomic DNA from Section “Genotyping of Volunteers and Formation of Paired Samples” was used, with the NEB High GC PCR kit. Primers were purchased from Integrated DNA Technologies. Sequences and PCR conditions are available in Table About 8 × 10Plasmid sequencing used purified plasmid DNA and a primer upstream of the insertion site. Sequencing reactions used Big Dye terminator reagents and the 3730xl DNA analyzer (ABI). See Table t-test was used to compare means for mRNA expression. Paired t-test was used for luciferase levels. An alpha value of 0.05 and two-tailed p values were used in all cases. For experiments using more than two comparisons, ANOVA was used to determine if statistically significant differences were present. Statistical analysis was performed using Data Analysis Plus software (Keller Statistics). ANOVA was performed using the CSBJU online calculator (http://www.physics.csbsju.edu/stats/).A paired An analysis of the promoters for each of the four first exons of IRF5 was performed using the ENCODE ChIP-Seq data set  for deteThe authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Bioinformatic analyses of expression control sequences in promoters of co-expressed or functionally related genes enable the discovery of common regulatory sequence motifs that might be involved in co-ordinated gene expression. By studying promoter sequences of the human ribosomal protein genes we recently identified a novel highly specific Localized Tandem Sequence Motif (LTSM). In this work we sought to identify additional genes and LTSM-binding proteins to elucidate potential regulatory mechanisms.Genome-wide analyses allowed finding a considerable number of additional LTSM-positive genes, the products of which are involved in translation, among them, translation initiation and elongation factors, and 5S rRNA. Electromobility shift assays then showed specific signals demonstrating the binding of protein complexes to LTSM in ribosomal protein gene promoters. Pull-down assays with LTSM-containing oligonucleotides and subsequent mass spectrometric analysis identified the related multifunctional nucleotide binding proteins NonO and SFPQ in the binding complex. Functional characterization then revealed that LTSM enhances the transcriptional activity of the promoters in dependency of the distance from the transcription start site.Our data demonstrate the power of bioinformatic analyses for the identification of biologically relevant sequence motifs. LTSM and the here found LTSM-binding proteins NonO and SFPQ were discovered through a synergistic combination of bioinformatic and biochemical methods and are regulators of the expression of a set of genes of the translational apparatus in a distance-dependent manner. During the human genome project it was recognized that the mere number of protein-coding genes is much smaller than expected. In contrast, entirely new molecular mechanisms have been revealed that add to the complexity of gene regulation. In general, gene expression is regulated by protein complexes that assemble on the DNA at transcription factor binding sites, which then interact with the transcriptional apparatus. With the sequencing of the human and other mammalian genomes, diverse projects were started to identify and characterize the transcribed genomic regions including their transcription start sites (TSS) and proximal regulatory sequence regions (promoter).Bioinformatic analyses of promoter DNA sequences of groups of coexpressed or functionally related genes enable the discovery of gene regulatory mechanisms. In a prior study we investigated the well-characterized promoter sequence set of the essential and highly expressed human ribosomal protein (RP) genes and succeeded in the identification of a novel specific sequence motif . The majThe human ribosome consists of four rRNAs and 79 proteins, which are encoded by 80 genes -4. RP geThe first main goal of the present study was the identification of further genes that might be regulated by a LTSM. By studying mouse and zebrafish RP promoters bioinformatically we first refined the definition of the LTSM sequence motif. Finally, we performed a genome-wide search for genes with LTSM-bearing promoters. Interestingly, the additional LTSM-positive genes mostly encode for products, which are involved in translation.The second main goal of the study was the identification of proteins that bind LTSMs and the functional characterization of the motif. Here we show that the two related nucleotide binding proteins NonO  and SFPQ (splicing factor proline/glutamine-rich)  bind dir1. ATC-ATC tandem motif with a spacer of seven bp, 2. located between +52 and +82 bp relative to the TSS, 3. strictly oriented. The base distribution of the middle part is not considered functionally constrained but rather governed by the genomic background of the actual promoter. Searching the human RP promoters (-500 to +500) with the refined motif definition revealed 50 hits in total, 35 at the preferred location and in the right orientation, two at different locations but in the right orientation and 13 on the reverse-complement strand at no preferred location  because it contains detailed information about transcripts including TSS and exon-intron structure . The proWe decided to focus our search on genes where LTSMs appeared in a similar location relative to the TSS. For each transcript in EnsEMBL55 we scanned the promoter region from +21 to +100 bp relative to the TSS. This resulted in 1460 genes associated to 2078 transcripts that carry LTSM elements  enrichment analysis of the LTSM-positive protein-coding genes using the Ontologizer software and the Parent-Child method revealed that only categories related to translation were enriched among LTSM-positive genes, except for three of six genes in the Purkinje cell GO category  with nuclear extracts of SHP77, COS7 and HEK293 cells to show that LTSM are bound sequence-specifically by nuclear proteins. Radiolabelled double stranded DNA oligonucleotides harbouring the LTSM (LTSM-positive probes) derived from several RP promoters and unlabeled dsDNA competitor probes were incubated with nuclear extracts. The sequences of LTSM-negative competitor probes were chosen from the typical LTSM location of the promoters of RPS6 and RPL13a that are devoid of LTSMs. In several experiments, we tested LTSM-positive probes from the eight RP genes RPL36, RPL32, RPL18, RPL12, RPL11, RPL7a, RPS15 and RPS4X in the presence of LTSM-negative competitor probes. We found specific signals for the five genes RPL36, RPL32, RPL18, RPS15 and RPS4X using HEK293 cell nuclear extract . It should be noted that the LTSM-positive probes of all tested RP genes, including RPL12 and RPL18 that never showed specific signals in EMSA experiments led to the pull-down of NonO and SFPQ Figure . The antThe surprising finding of the tripartite tandem motif (ATC-8-ATC-6-ATC) with the spacers of +1 and -1 in respect to the most common spacer of 7 bp motivated us to test their affinities for NonO and SFPQ binding. Pull-down assays with nuclear extracts from HEK293 cells confirmed that NonO and SFPQ bind to all three tested sequences RPS15a, RPL17 and RPS24 , whereas the insertion of four bp and 53 bp produced similar or even stronger signals  was inserted into the gene trap vector pT1β-geo Figure . The ints Figure . The pats Figure . FurtherThe comparison of human, mouse and zebrafish RP promoters affirmed the functional importance of the LTSM by the rationale of phylogenetical foot printing . This anThe genome-wide analysis did not support a global preference for location or orientation of the LTSM relative to the TSS. However, in the RP promoters of humans, mice and zebrafish LTSM elements were strictly oriented and located. And the insertion of intronic sequences between TSS and LTSM into the pT1β-geo reporter construct led to decreased expression levels. The most likely explanation for our results was that the LTSM binding complex functions as transcriptional activator or enhancer with varying strength depending on its positioning relative to the transcription initiation complex or the TSS. The distance between TSS and LTSM could be one way to fine-tune the transcription levels of RP genes. This concept would also fit our finding that the relatively small insert of four bp led to an enhanced activity of the RP promoter construct.With regard to possible biological functions of the motif it is intriguing that although RP genes are highly and co-ordinately expressed and the sequence motif is evolutionarily conserved, less than half (35 out of 81) of the RP genes are LTSM-positive. Even a search for degenerate motifs in the RP promoters, allowing one of the six ATC-ATC base pairs to deviate, did not reveal instances that cover the entire RP gene set at the preferred position, but led to a high number of questionable matches with no preferred orientation or location instead (data not shown). In summary, gene annotation, scanning the literature, and sequence analysis provided no hint for what separates LTSM-positive and LTSM-negative RP genes . For exaThe central discovery during this work was the identification of the related nucleotide binding proteins NonO and SFPQ as specific LTSM element binders. Although the EMSA experiments showed specific signals of widely varying strength and even no specific signal for three LTSM-positive probes, the pull-down of NonO and SFPQ with each of the tested probes establishes the specificity of our discovery. NonO is highly homologous with the C-terminal part of SFPQ, and NonO has been shown to bind RNA and single stranded DNA through its N-terminus, and double stranded DNA through its C-terminus . The twoIn terms of transcriptional regulation, NonO and SFPQ have been studied intensively as cofactors of hormone receptors, especially androgen receptor, progesterone receptor, and steroidogenic factor 1 ,24-26. TThe results of our reporter assays favour an activating or enhancing function of NonO and SFPQ on RP gene transcription by binding LTSM elements. However, the co-transfection of NonO and SFPQ expressing constructs and the RPL12 reporter construct did not lead to an increased reporter signal. A likely explanation of this result is that the extraordinarily active RPL12 promoter and the transcription factor levels are already optimized such that it is hardly possible to achieve a further increase with manipulations of any nature. Another explanation could be that the LTSM helps to recruit and orient the RNA polymerase complex, but is not a rate-limiting step in transcription. This explanation is consistent with the strong position-dependent requirements of LTSM to the TSS  . Apart fApart from EnsEMBL we examined two other databases CAGE and DBTSS. The Fantom 3 CAGE dataset  containeGene Ontology information and annotation created by the consortium at 1/9/2009 was downloaded from the Gene Ontology website . We usedHEK293, COS7, and SHP77 cells were cultivated as described previously ,32.2) and incubated on ice for 1 h. The sample was transferred to ice cold douncer and homogenized with 25 strokes. The homogenized cells were centrifuged at 2000 rpm for 5 min at 4°C and the resulting pellet was washed once in 1 ml of NE buffer A. The pellet was resuspended in 2 times its volume of NE buffer B  glycerol; 420 mM NaCl; 1.5 mM MgCl2; 0.2 mM EDTA) and incubated on ice for 30 min. Afterwards the sample was centrifuged at 13000 rpm for 20 min at 4°C. The supernatant was transferred to a fresh 1.7 ml Eppendorf tube and an equal volume of NE buffer C  glycerol; 1.5 mM MgCl2; 0.2 mM EDTA) was added. Nuclear extracts were aliquoted into small volumes (100-200 μl), snap freezed in dry ice and stored at -80°C.Collected cells were washed once in cold PBS. The cell pellet was resuspended in 4 times the volume of the pellet in NE buffer A , 2.75 pmol [32P]-dCTP and 2 μmol of each remaining dNTP per 100 ng DNA for 15 min at 25°C. The labelling reaction was stopped using STE buffer  in an amount equivalent to half of the reaction volume. 8 ng of labelled dsDNA oligonucleotide were mixed with 4 × binding buffer  NP40, 20% (v/v) glycerol, 10 mM MgCl2), 40 mM KCl, 5-10 μg of total protein of HEK293 nuclear extracts and, if applicable, 700 fold molar excess of cold competitor probe and 2.5-20 fold molar excess of specific antibody (for antibodies see fishing experiment) in relation to the labelled oligonucleotide were added. The binding reaction took place at 37°C for 60 min. The probes were loaded into a 4% native acrylamide gel (4% (v/v) AA/0.1% (v/v) BA; 5% (v/v) glycerol; 1 x TBE) using 6 x glycerol loading dye and run with 1 × TBE at 250 V. The gel was exposed to a phosphor imager plate (Molecular Dynamics) overnight. The sequences of all DNA probes that were deployed in the EMSA experiments are presented in Additional file EMSAs were made using [eviously . Anneali2), 40 mM KCl, 100 μg of total protein of HEK293/SHP77 nuclear extracts and, if applicable, 1000 fold molar excess of non-labelled competitor probe in relation to the labelled oligonucleotide in a final volume of 1 ml. The binding reaction took place at 37°C for 60 min. After binding the probes were given to 25 μl of streptavidin beads  and inverted at 4°C overnight. The beads were washed three times with binding buffer, whereas a vial exchange took place at every washing step, mixed with Laemmli loading dye and incubated for 5 min at 95°C. The samples were loaded into a 10-14% SDS gel. Subsequently the gel was stained with Coomassie Blue (Fermentas), the bands of interest were cut out and analyzed through MALDI-TOF. Alternatively the proteins were blotted and analyzed using specific antibodies , sc-28730, Santa Cruz). The sequences of all DNA probes that were deployed in pull-down experiments are presented in Additional file 200 ng of double stranded, biotinylated DNA oligonucleotides were mixed with 4 x binding buffer  NP40; 20% (v/v) glycerol; 10 mM MgClFor the luciferase assay the dual-luciferase reporter psiCHECK-2 (Promega) was used. The SV 40 promotor was replaced with the promotor sequence of RPL12 including part of exon 1 that contained the LTSM element but not the ATG start codon  resulting in a relocation of the LTSM to position +4, +29, +53 and +117 related to the original position of +68 of the RPL18 LTSM. The constructs were checked by sequencing  separated from the β-geo  fusion gene, a splicing acceptor site (enSA) and a polyadenylation signal (pA). The promoter region of RPL18 was inserted, including the TSS, the ATG, the splicing donor site and the LTSM of the gene, in detail: - 1000 bp to +90 bp  and a GFP control (pGFP-C1). After 24 h medium was exchanged and harvesting took place after 48 h. The expression of β-galactosidase was assessed using three different readouts, namely X-Gal staining, Western and Northern blot analysis. The pT1β-geo constructs comprised one vector containing the unmodified RPL18 promoter just containing the 3Fe(CN)6; 20 mM K4Fe(CN)6; 2 mM MgCl2; 1 mg/ml X-Gal in PBS) was added to the cells. The samples were incubated at 37°C until colour reaction occurred (1-4 hours).The growth medium was removed; the cells were washed with cold PBS and fixed on ice for 5 min with fixing solution (2% (v/v) formaldehyde; 0.8% (v/v) gluteraldehyde in PBS; pH 7.3). Afterwards the cells were washed three times with PBS and staining solution  NP-40; 0.5% (v/v) deoxycholic acid; 0.1% (w/v) SDS and 1 x protease inhibitor . Proteins were blotted for 75 min at 16 V and analyzed using a specific antibody against β-Galactosidase .et al. [Aat II and EcoR I. The DNA probe matched the 5'-end of the β-geo sequence. Labeling of the probe took place with the Prime-It RmT Random Primer Labeling Kit (STRATAGENE) with subsequent purification from unincorporated nucleotides using MicroSpin G-50 Columns (Amersham Biosciences). The membrane was exposed to a phosphor imager plate overnight and scanned using a phosphorimager storm 820 (molecular dynamics).Cells were harvested using cold PBS. RNA was isolated using TRIzol Reagent (Invitrogen). Northern blotting was carried out following steps 13 to 32 of the protocol of Streit S et al. . The 4905'-TOP, 5': terminal oligo-pyrimidine tract; EMSA: electromobility shift assay; GO: Gene Ontology; LTSM: localized tandem sequence motif; NonO: non-POU domain-containing, octamer-binding protein; SFPQ: splicing factor proline/glutamine-rich; TSS: transcription start siteThe authors declare that they have no competing interests.SR, SS, LT, SG, and DJW carried out the molecular genetic studies, participated in the sequence alignment and drafted the manuscript. HK and MHC participated in the sequence analyses. SR, MV, and DJW participated in the design of the study and performed the statistical analysis. SR, MV, and DJW conceived of the study, and participated in its design and coordination. All authors read and approved the final manuscript.Supplementary Figure S1. Proximal promoters of 51 zebrafish ribosomal protein genes Annotations of the promoter sequences (range: -80 to +100 around the TSS) with sequence motifs: LTSM (cyan underlined), CT-stretch (red box), YY1 (blue box), and TATA (orange box). Exons are marked by a grey background and the ATG translation start codon by an orange background. All sequences are aligned to their predicted TSS. Pink, yellow and green boxes mark G/T-rich, A/G-rich and A/T-rich sequence motifs of low specificity, respectively .Click here for fileSupplementary Table S1. LTSM hits in human RP gene promoters The sequence set was generated by extraction of the genomic sequence from -500 to +500 relative to the TSS of each RP gene. 1 in the strand column indicates that the sequence element has the same orientation as the transcription start, -1 is the reversecomplement. The positions are given relative to the scanned sequence. The TSS is annotated at position 500. Bona fide LTSMs are marked light blue.Click here for fileSupplementary Table S2. Comparison of TSS annotations in various databases To exclude the possibility that inaccuracies in the TSS or the transcript annotations led to the blurring of the positional enrichment of the motif we utilized our well-characterized RP promoter set. We found that for DBTSS and CAGE databases only about half of the RP genes could be correctly assigned. The EnsEMBL database contains gene annotations and transcript annotations. The gene annotations defined the 5-end of a gene as the most 5'-TSS of all its annotated transcripts. For many genes this led to the annotation of a TSS far upstream from the TSS of the most abundant transcript. For the RP gene set this resulted in an average distance between the EnsEMBL annotated TSS and the experimentally verified TSS of 18 or 21 bp depending on the version of the database. EnsEMBL55 contained on average 2.34 transcripts per RP gene. Choosing the transcripts with the closest TSS from EnsEMBL for each RP gene resulted in an average distance of seven bp relative to the TSSs of our set.Click here for fileSupplementary Table S3. LTSM-positive genes from a genome-wide searchClick here for fileSupplementary Table S4. Tripartite LTSM-positive genes from a genome-wide searchClick here for fileSupplementary Table S5. Result of the Gene Ontology (GO) enrichment analysis The table shows the results of the Gene Ontology (GO) enrichment analysis using Otologizer. The first two columns denote the GO ID and GO name. The third column denotes the absolute number of GO terms as annotated in GO. The fourth and fifth column denote the enrichment p-value, without and with adjustments for multiple testing, respectively.Click here for fileSupplementary Figure S2. EMSA experiment with LTSM-positive probes of three genes - complete picture The first lane of each probe contains the labelled doublestranded DNA, in the second lane nuclear extract (NE) was added. Unlabelled specific competitors (SC) were added to block specific protein binding in the third lane. In the last three lanes unspecific competitor (UC) sequence of LTSM-negative RPS6 was added. Specific binding is indicated by black arrows.Click here for fileSupplementary Figure S3. Identification of LTSM-specific binding proteins Pull-down experiment using biotinylated LTSM-positive probes of RPL36, nuclear extract (NE) of SHP77 cells and 1000-fold molar excess of unspecific competitor (UC) sequence of LTSM-negative RPS6. The three dominant bands detected by Coomassie staining (indicated by black arrows) were analyzed using mass spectrometry (n = 2). For identified proteins compare Additional file Click here for fileSupplementary Table S6. List of proteins in LTSM pull-down samples Mass spectrometry (MALDI-TOF) analysis of proteins pulled by biotinylated LTSM-positive probes of RPL36  sequence of LTSM-negative derived from RPS6 was applied in 1000-fold molecular excess. Non-biotinylated LTSM-positive RPL36 probes were used as control. Specific protein signals are indicated by black arrowheads. Proteins were detected using both specific antibodies in conjunction.Click here for fileSupplementary Figure S4. Influence of NonO and SFPQ on RPL12 promotor activity using luciferase assays (A) Schematic view of a part of the human RPL12 gene indicating the amplified region.  Dual Luciferase vector psiCHECK2 with the original SV40 promotor and vector psiRPL12 with the original promotor replaced by the proximal promoter region and part of exon 1 of RPL12. (D) Cotransfection of psiRPL12 and eukaryotic expression vectors containing the genes of NonO and SFPQ (pTarget-NonO and pTarget-SFPQ) did not result in a significantly enhanced promoter activity. GFP expression vector alone served as a control.Click here for fileSupplementary Figure S5. Distance of LTSM to TSS determines the efficiency of transcription Complete picture of Northern blotting experiment detecting β- Galactosidase transcripts via β-Galactosidase specific DNA probe at 5'-end of the transcript .Click here for fileSupplementary Table S7. Sequences of single stranded DNA oligonucleotides used in EMSA experiments The LTSMs are highlighted in grey on the forward strand.Click here for fileSupplementary Table S8. Sequences of biotinylated DNA oligonucleotides used in pull down experiments The LTSMs are highlighted in grey on the forward strand.Click here for fileSupplementary Table S9. Sequences of employed PCR primers and DNA linker oligonucleotidesClick here for fileSupplementary Figure S7. Sequence verification of inserted linkers Sequences obtained by sequencing the described RPL18 region in pGEM-T Easy vector using primer M13 rev (-49). Please, refer to Figure 5 in the main text. Translation start (ATG) is shown in bold letters, inserted Xho I restriction site with insertion of linkers of different lengths (bold letters) is highlighted in dark grey and the LTSM is highlighted in light grey.Click here for file


Here we show that HS4 recruits the E3 ligase RNF20/BRE1A to mediate H2B mono-ubiquitination (H2Bub1) at this insulator. Knockdown experiments show that RNF20 is required for H2Bub1 and processive H3K4 methylation. Depletion of RNF20 results in a collapse of the active histone modification signature at the HS4 chromatin boundary, where H2Bub1, H3K4 methylation, and hyperacetylation of H3, H4, and H2A.Z are rapidly lost. A remarkably similar set of events occurs at the HSA/HSB regulatory elements of the FOLR1 gene, which mark the 5′ boundary of the same heterochromatin region. We find that persistent H2Bub1 at the HSA/HSB and HS4 elements is required for chromatin boundary integrity. The loss of boundary function leads to the sequential spreading of H3K9me2, H3K9me3, and H4K20me3 over the entire 50 kb FOLR1 and β-globin region and silencing of FOLR1 expression. These findings show that the HSA/HSB and HS4 boundary elements direct a cascade of active histone modifications that defend the FOLR1 and β-globin gene loci from the pervasive encroachment of an adjacent heterochromatin domain. We propose that many gene loci employ H2Bub1-dependent boundaries to prevent heterochromatin spreading.Genomic maps of chromatin modifications have provided evidence for the partitioning of genomes into domains of distinct chromatin states, which assist coordinated gene regulation. The maintenance of chromatin domain integrity can require the setting of boundaries. The HS4 insulator element marks the 3′ boundary of a heterochromatin region located upstream of the chicken  FOLR1 and β-globin genes, respectively. These elements recruit enzymes that mediate the ubiquitination of histone H2B. Histone H2B ubiquitination directs a cascade of so-called “active” histone modification events that favor chromatin accessibility. We observe a striking collapse of the active histone modification signature at both chromatin boundaries following the depletion of ubiquitinated H2B. This loss of boundary function leads to the comprehensive spreading of repressive chromatin over the entire FOLR1 and β-globin gene region, resulting in gene silencing. We propose that chromatin boundaries at many gene loci employ H2B ubiquitination to restrict the encroachment of repressive chromatin.The transcription of genes in eukaryotes occurs within the context of chromatin, a complex of DNA, histone proteins, and regulatory factors. Whole-genome profiling of chromatin proteins and histones that are post-translationally modified has revealed that genomes are organized into domains of distinct chromatin states that coordinate gene regulation. The integrity of chromatin domains can require the setting of their boundaries. DNA sequences known as chromatin insulator or boundary elements can establish boundaries between transcriptionally permissive and repressive chromatin domains. We have studied two chromatin boundary elements that flank a condensed chromatin region located between the chicken  There is growing consensus that the non-random chromosomal arrangement of genes in higher eukaryotes enables the sharing of specific chromatin environments that facilitate co-regulation.Drosophila and mammalian cells have revealed prevalent organization of genes into domains, or neighborhoods, of common chromatin state Recent genomic profiling of histone modifications, chromatin factors and nuclear proximity in β-globin gene cluster and upstream region of condensed chromatin that is enriched in the epigenetic hallmarks of heterochromatin The maintenance of chromatin domain integrity can require the setting of boundaries. Boundaries not only allow the partitioning of gene regulation, but also may also maintain the concentration of factors required for heterochromatin structures and normal genome homeostasis HS4 manipulates histone modification signatures to counteract gene silencing S. cerevisiae), which is required for the tri-methylation of H3K4 β-globin locus. We also extend our analysis to look at the 5′ chromatin boundary of the same condensed chromatin and its role in shielding the FOLR1 gene locus.Given that chromosomal silencing has been shown to be processive and stable, we reasoned that the HS4 element needs to act as a constitutive barrier if it is to effectively shield the locus. In this study, we address a hypothesis that HS4 might recruit histone modifications that act as master controllers of the active chromatin state to facilitate barrier stability. Intense study in recent years has begun to unravel the complex language of crosstalk between histone modifications during the establishment of different chromatin states FOLR1 and β-globin gene loci. Firstly, we mapped the presence of ubiquitinated nucleosomes across 50 kb encompassing the chicken β-globin locus  locus is silent −6). Perhaps surprisingly, no enrichment of histone ubiquitination was observed at the promoters or enhancers of the highly active β-globin genes in RBCs. We also mapped histone ubiquitination in 10 day embryo whole brain tissue (FOLR1 and β-globin genes are reported to be silent −5). We also observe significant levels of histone ubiquitination at the FOLR1 gene regulatory elements HSA and HSB in both 10 day embryo RBCs (−5). These elements are situated between the FOLR1 gene and the condensed region and may harbor chromatin boundary activity.N-ChIP analysis of histone ubiquitination was performed on primary red blood cells (RBC) from 10 day chick embryos, in which the n tissue , where bWe sought to determine which of the well characterized activities of the 275 bp core HS4 element are required for the recruitment of histone ubiquitination. We performed N-ChIP analyses of histone ubiquitination at HS4 insulators present on single copy transgenes stably integrated into the early erythroid CFU-E stage cell line 6C2 β-globin sequences (not shown).The histone ubiquitination that is enriched at the HS4 element may occur on any of the core histones. We anticipated that histone H2B is subject to this modification as HS4 is constantly enriched in methylated H3K4 We sought to identify the E3 ligase responsible for H2Bub1 at the HS4 element so that the effects of depleting H2Bub1 could be studied. We used crosslinking ChIP analysis to show that RNF20 interacts with the core HS4 insulator element in erythroid cells . Chickenβ-globin locus and insulated transgenes. This was achieved in 6C2 cells using a lentiviral vector system for doxycycline-regulated expression of miRNA-shRNA . We observe similar profiles of RNF20-dependent H2Bub1 in 6C2 cells . In summary, short term depletion of H2Bub1 is sufficient to disrupt H3K4me3 at the HSA/HSB and HS4 elements, which results in a rapid loss of multiple histone acetylation and chromatin boundary integrity. H3K9me2 appears as the first repressive mark to spread beyond the defective boundaries of the condensed heterochromatin region.We investigated whether the depletion of H2Bub1 and the resulting loss of the active histone signatures at the HSA/HSB and HS4 elements affected the containment of the intervening condensed region. We determined that H3K9me2 and H3K9me3 are restricted to the condensed region upstream of HS4 in wild type 6C2 cells  complexes that target H3-lysine 4. Existing models used to explain atin see . We perfnockdown . The losIL-2R transgene flanked by paired 275 bp core HS4 insulators. It was previously determined that transgene expression from these cells remains constant beyond 80 days of culture, the transgenic HS4 insulators are bound by CTCF, USF1 and VEZF1 The ability of the HS4 element to shield genes from chromosomal position effect silencing in a wide variety of systems is well established IL-2R transgenic cells with lentiviruses that express RNF20 shRNA. The lentiviral miRNA-shRNA system we employed allowed the stable knockdown of RNF20 for at least sixty days . We observed no change in 6C2 cell morphology and only a minimal reduction in cell doubling during this period (not shown). We found that four days of RNF20 knockdown had no effect on transgene expression  are required for heterochromatin boundary integrity in budding yeast FOLR1 and β-globin loci.A number of budding yeast boundary elements are also associated with regions of nucleosome depletion and elevated histone turnover trans histone modification pathway from H2Bub1 to H3K4me3 reported in yeast and man is also conserved in chicken. How the mono-ubiquitination of H2B facilitates H3K4me3 has been subject to intense study over the last few years. Three models have arisen to explain this pathway. The ‘wedge’ model postulated that the bulky ubiquitin moiety would increase the access of H3K4 methyltransferases by non-specifically disrupting chromatin fiber packing in some way trans tail crosstalk in S. cerevisiaeS. cerevisiae that H2Bub1 promotes nucleosome reassembly following RNA polymerase II transcription and enhances global nucleosome stability We have shown that the Recent studies provide compelling evidence that H2Bub1 acts as ‘bridge’ to facilitate H3K4me3. The core SET1 complex can interact with chromatin and mediate H3K4 mono-methylation in the absence of H2B ubiquitination The mono-ubiquitination of H2B is broadly recognized as a mark of transcriptional activity In addition to the recruitment of the E3 ubiquitin ligase RNF20, the HSA/HSB and HS4 boundary elements also require sufficient activity levels of the E2 conjugase RAD6 to enable sufficient levels of H2Bub1 for chromatin boundary stability. There are two broad mechanisms that could result in the persistent H2B ubiquitination of the HS4 insulator. Firstly, the HSA/HSB and HS4 elements might not be subject to the rapid turnover of H2Bub1 associated with promoter clearance and transcription elongation FOLR1 and β-globin loci either side of the condensed region. We find that the heterochromatin-associated marks H3K9me2, H3K9me3 and H4K20me3 are propagated in a continuous manner from the upstream condensed region into the FOLR1 and β-globin loci. The heterochromatin domain expands from a ∼10 kb domain of the condensed region to cover the entire ∼50 kb FOLR1 and β-globin region given time , H3K4me3 (05-745R), H3K9me3 (07-523), H3K27me3 (07-449), H3 (07-690), H3K9acK14ac (06-599), H4K5acK8acK12acK16ac (06-598), H2AK119ub (05-678), H2A.Z (07-594) and CTCF (06-917) were obtained from Millipore. Antibodies against H3K9me2 (ab1220), H3K79me2 (ab3594), H3K79me3 (ab2621), H2A.ZK4acK7acK11ac (ab18262), PAF1 (ab20662), RPB1 (ab5408) and TBP (ab51841) were obtained from Abcam. Antibodies against H4K20me3 were a kind gift from Judd Rice et al, 2001]. Briefly, 6C2 cells (2×107 cells/ml) were crosslinked in fresh growth medium with 1% formaldehyde at room temperature for 20 minutes , 10 minutes  or 2 minutes (H2Bub). Reactions were quenched by adding glycine to a final concentration 0.125 M. The crosslinked cells were washed by PBS twice and then lysed . Cell nuclei collected by centrifugation were washed  followed by chromatin solubilization . Chromatin was fragmented by sonication (Misonix) for a total time of 10 minutes in regular 10 second pulses. Insoluble material was removed by centrifugation at 15,000 g for 10 minutes at 4°C. Sizes of chromatin fragments were ∼500 bp on average.Chicken 6C2 erythroleukaemia cells were grown in αMEM supplemented with 10% FCS, 2% chicken serum, 1 mM HEPES, 25 µM β-mercaptoethanol and 1% Penicillin/Streptomycin solution. Crosslinking chromatin immunoprecipitation was performed as described previously [Litt 7 cells per ml. 1 ml of chromatin was pre-cleared with 5 µg of non-immune IgG and 100 µl (50% slurry in X-ChIP) of protein A/G agarose at 4°C for 3 hours. 10 µg of specific antibody was incubated with pre-cleared chromatin at 4°C with agitation overnight. Binding of protein A/G agarose was carried out at 4°C for 2 hours. The agarose was washed extensively with buffer 1 , buffer 2 , buffer 3  and twice with TE buffer . The bound chromatin was eluted into elution buffer , crosslinks reversed and protein digested. DNA was extracted by phenol/chloroform and ethanol precipitated in the presence of 10 µg of glycogen for quantitative PCR (qPCR) analysis.Soluble chromatin was diluted by X-ChIP buffer  to obtain chromatin from 1×102, 0.4% NP-40 and 10 mM Tris pH 7.5) for MNase (Sigma) digestion in the presence of 1 mM CaCl2. The MNase concentration (X) required to yield mostly di- and tri-nucleosomes was firstly determined. For ChIP experiments, three equal aliquots of nuclei were incubated with ½X, 1X and 2X MNase at 37°C for 17 minutes to obtain representative di- and tri-nucleosomes Circulating red blood cells and whole brain tissue (without major vasculature) were collected from 10 day fertilized chick embryos kindly provided by Aviagen, Ltd. Native nucleosomes were prepared in low salt conditions to ensure retention of all nucleosomes, as described 50 µg of nucleosomes were pre-cleared with 5 µg of non-immune IgG and 100 µl (50% slurry in N-ChIP buffer) of protein A/G agarose at 4°C for 3 hours. 10 µg of specific antibody was incubated with pre-cleared chromatin at 4°C with agitation overnight. Binding of protein A/G agarose was carried out at 4°C for 2 hours. Immunoprecipitated chromatin was collected and washed 5 times with 1 ml N-ChIP wash buffer . Chromatin was eluted with N-ChIP buffer supplemented with 1% SDS followed by 0.5% SDS. Eluates were digested with Proteinase K at 45°C for 2 hours and DNA extracted by phenol/chloroform and precipitated for qPCR analysis.Relative DNA enrichments were quantified in triplicate by TaqMan real-time PCR on a Roche 480 Lightcycler. The primers used in this study were described previously http://hannonlab.cshl.edu). These were scored and four targets were cloned into pEN_hUmiRc2, packaged into lentivirus particles and tested for performance as previously described CAGAGTAACTAGAGAGAAA, was the most potent miR-shRNA and was used throughout this study. Wild type or 8103 . Cell nuclei were collected by centrifugation at 2,500 g for 5 minutes. Nuclear proteins were extracted by incubation in high salt buffer  at 4°C for 30 minutes. Insoluble debris was removed by centrifugation at 16,000 g for 10 minutes at 4°C. Soluble nuclear extract was quantified by Bradford assay (Bio-Rad). 25 µg of nuclear extract or 7 µg of native S1–S2 nucleosome preparations were used for separation by SDS-PAGE. Proteins were then transferred to a PVDF membrane and imaged with HRP-conjugated secondary antibodies on a LAS-3000 imager (Fujifilm). Band intensities corrected for background were quantified using AIDA software.6 cells were harvested by centrifugation at 1,000 g for 5 minutes. Cells were washed twice in 1 ml of Hank's buffered saline solution (Sigma) supplemented with 0.1% BSA and 0.1% NaN3 (HBSS+). Cells were resuspended in 100 µl of HBSS+ and incubated with 10 µl of anti-CD25-PE (Dako) antibody in the dark at 4°C for 30 minutes. Excess antibody was removed by washing twice with 1 ml of HBSS+. After the last wash, cells were resuspended in 500 µl of HBSS+ for FACS analysis. Flow cytometry was performed on a FACSCalibur flow cytometer (BD Biosciences) using CELLQuest software. RNF20 knockdown cells express GFP upon induction with doxycycline. Color compensation was used to correct for GFP fluorescence in the FL2 (585 nm) filter; it was set as FL1 – 1% FL2 and FL2–20% FL1. Data was acquired for 10,000 viable or GFP-expressing cells (FL1  =  530 nm). Histograms were generated using FlowJo software . Mean IL-2R fluorescence intensities of RNF20 knockdown cells were determined and normalized to those of parental reporter transgene cells with wild type RNF20 levels.10Total RNA was isolated from 6C2 cells using TRI reagent (Sigma) and cDNA prepared using SuperScript III (Invitrogen) and random hexamers. The following primers were used SYBR green real time PCR assays:TGCTGCGCTCGTTGTTGA5′ CATCGTCCCCGGCGA5′ ATGCGTCATCTCATCAGCAG5′ TTGGGAAGAAGGGTCATCAG5′ GATTCTGCATGTGCCACTGT5′ AAGACCTGGGTGAAGGGTCT5′ Figure S1http://multalin.toulouse.inra.fr/multalin) of human (NP_062538.5) and chicken (NP_001026605.1) RNF20/BRE1A. The conserved RING domain is boxed. The RNF20 antibodies used in this study were raised against a conserved epitope between residues 125 and 175. B) RT-PCR analysis of RNF20 knockdown following doxycycline induction of shRNA expression. C) Western blotting of 6C2 whole cell extracts before and after doxycycline-induced RNF20 RNAi. Blots probed with anti-RNF20 (expected size of 120 kDa) or anti-TBP (expected size of 38 kDa) are shown with the positions of molecular weight markers (kDa).Knockdown of chicken RNF20. A) Pairwise alignment ((TIF)Click here for additional data file.Figure S2β-globin gene neighborhood in wild type (red bars) and RNF20 knockdown (blue bars) 6C2 cells. The enrichment of each sequence is normalized to the background observed downstream of the inactive ρ-globin gene. Significant changes in ChIP enrichments following RNF20 depletion are represented by asterisks .The ubiquitin ligase RNF20 mediates H2B ubiquitination at the HS4 insulator. Native ChIP of H2BK120 monoubiquitination at sites across the chicken (TIF)Click here for additional data file.Figure S3β-globin gene neighborhood in wild type (red bars) and RNF20 knockdown (blue bars) cells. The enrichment of each sequence is normalized to the background observed at the condensed region (15.850). The location of the core HS4 insulator (21.540) is highlighted in red. Significant changes in ChIP enrichments following RNF20 depletion are represented by asterisks . The location of the core HS4 insulator (site 21.540) is highlighted in red.Loss of H2B ubiquitination does not initially affect H4K20me3 containment. Native ChIP analyses of early erythroid 6C2 cells following four days of doxycycline-induced knockdown of RNF20 expression. Enrichments of H4K20me3 at sites across the chicken (TIF)Click here for additional data file.Figure S4FOLR1 gene in 6C2 cells Crosslinking ChIP analysis of RNA polymerase II  occupancy at the chicken FOLR1/β-globin loci in 6C2 cells. Significant ChIP enrichments are represented by asterisks .RNA polymerase II binding at the transcriptionally active (TIF)Click here for additional data file.


In vertebrates, rod photoreceptor-specific gene expression is regulated by the large Maf and Pax-like transcription factors, Nrl/LNrl and Crx/Otx5. The ubiquitous occurrence of their target DNA binding sites throughout rod-specific gene promoters suggests that multiple transcription factor interactions within the promoter are functionally important. Cooperative action by these transcription factors activates rod-specific genes such as rhodopsin. However, a quantitative mechanistic explanation of transcriptional rate determinants is lacking.Xenopus rhodopsin promoter (XOP) has a bipartite structure, with ~200 bp proximal to the start site (RPP) coordinating cooperative activation by Nrl/LNrl-Crx/Otx5 and the adjacent 5300 bp upstream sequence increasing the overall expression level. The synergistic activation by Nrl/LNrl-Crx/Otx5 also occurred when XOP was stably integrated into the genome. We determined that Crx/Otx5 synergistically activated transcription independently and additively through the two Pax-like cis-elements, BAT1 and Ret4, but not through Ret1. Other Pax-like family members, Rax1 and Rax2, do not synergistically activate XOP transcription with Nrl/LNrl and/or Crx/Otx5; rather they act as co-activators via the Ret1 cis-element.We investigated the contributions of various paired-like transcription factors and their cognate cis-elements to rhodopsin gene activation using cultured cells to quantify activity. The We have provided a quantitative model of cooperative transcriptional activation of the rhodopsin promoter through interaction of Crx/Otx5 with Nrl/LNrl at two paired-like cis-elements proximal to the NRE and TATA binding site. Further, we have shown that Rax genes act in cooperation with Crx/Otx5 with Nrl/LNrl as co-activators of rhodopsin transcription. Xenopus rods harboring NRE mutant promoters, which have drastically reduced expression of a GFP reporter gene [Rhodopsin is an abundantly expressed gene specifically found in rods, and its expression is primarily regulated at the level of transcription (for a recent review see ). The prter gene . By contIn vitro studies have largely supported those in transgenic animals, namely that Nrl and Crx are the key regulators of rhodopsin transcription [in vitro. Crx binds to Nrl through its homeodomain [in vitro is its cooperative (or synergistic) activation by Crx and Nrl [cription . Nrl sticription ,16. Furtcription ,18. Simicription . Althougcription ,19. Rax,cription ,21 and acription . Crx andeodomain . The bineodomain ,18. Botheodomain , Fiz1 [2eodomain  and Qrx eodomain . However and Nrl ,23,26,27in vitro system coupled with mutational analysis of the RPP, we characterized the specific role of the paired-like cis-elements in activation of the Xenopus rhodopsin promoter by Crx and its Xenopus homolog, Otx5, Rax and a novel Xenopus Rax family member, Rax2b [Current models for transcriptional activation describe the complex interplay between distal and proximal promoter regulatory elements, transcription factors and the basal transcription machinery (for example -30). Ther, Rax2b ,33.Xenopus rhodopsin promoter  containing plasmid was 1.5-fold higher in HEK293 cells compared to HEK293T cells ; therefore this difference in activity between the two cell lines reflects higher basal transcriptional activity independent of the specific promoter upstream of the luciferase gene.HEK cells (human embryonic kidney cells transformed by adenovirus 5) are a versatile and rapid system in which to characterize rhodopsin promoter activation, since these cells do not express Nrl, Crx or the endogenous (human) rhodopsin gene. We compared two common lines, HEK293 (ATCC CRL-1573) and HEK293T ,35, the Xenopus homologs of Crx (Otx5) and Nrl (LNrl)  and Nrl (hNrl) or Surprising, the activity in the presence of hNrl-hCrx was 2-fold higher in HEK293T cells compared to HEK293 cells, and contrary to the corresponding basal promoter activities . However, there are many core homeobox sites  is similar in transgenic Xenopus . However, position effects and copy number variation prevent a direct comparison between transgenics and in vitro experiments.The only region of extended conservation in tetrapod rhodopsin promoters is the RPP Figure . Compariions > ~ 0 bp. Hows Figure B. Taken Xenopus -luciferase or XOP(−5361/+41)-luciferase. The activity in the absence of transcription factors between the four clones tested differed widely and by as much as 13 fold, with higher basal activities observed in the shorter promoter . Rax2b is a member of the Rax paired-like homeodomain transcription factor family and in sequence comparisons is the paralog of Rax2a [Using a yeast one hybrid screen with the Ret1 cis-element (GCCAATTAA) as bait and a of Rax2a   co-expressed with individual members of the Rax family alone or in combination with LNrl and/or Otx5 in transfected HEK293T cells. Individually, each Rax transcription factor decreased basal activity of XOP(−503/+41). In combination with Otx5 or LNrl, Rax did not significantly increase activity  utilized by Rax, we measured Otx5-LNrl-Rax2b stimulated promoter activity in the deletion and the XOP promoter mutants in transfected HEK293T cells. Rax2b increased Otx5-LNrl stimulated activity by 30% in the BAT1Δ mutant but had no effect on the level of activity of the Ret1Δ or Ret1/BAT1Δ mutants  and a XOP fragment truncated just before the Ret1 site −128/+41)  is solely responsible for regulating the synergistic activation by Nrl-Crx/Otx5. These results build on previous published studies  which iand Ret4 . It has and Ret4 ,7,15,23.and Ret4 ,23,38, tand Ret4 . Interesand Ret4 -27,39. TA recent study quantifying bovine rhodopsin promoter activity in mouse retinal explants reported that the BAT1 site was not only essential (required) for promoter activation but was the most efficient site in driving activity . The Retin vitro binding assays and to Ret1 by ChIP [We identified a novel retinal homeobox transcription factor, Rax2b, which is a paralog of Rax1a . Rax2 be by ChIP ,20,21,41 by ChIP ,20,41. T by ChIP ,41,42. RXenopus rhodopsin promoter provide additional insights into promoter activation. While the RPP was responsible for the cooperative synergistic activation by Nrl, Crx and Rax, the 5′ upstream promoter sequence (5′ US) distal to the RPP contributed to the activation as well. The level of activity was dependent on the length of the promoter as well as the species of TFs used to activate. This observation was reinforced by the promoter activity assay data in which all three paired-like cis-elements were mutated (m6), resulting in a dramatic but not complete loss of activation. These results suggest that there are regulatory elements upstream of the Xenopus RPP that enhance transcription and are species-specific. This seems plausible since a distal regulatory element, the rhodopsin enhancer element (RER), which activates transcription, has been described for the mammalian promoter and is required for maximal activation of expression in vivo[The transcriptional activity assays performed on different lengths of the n in vivo,5. AlthoXenopus rhodopsin promoter and their contribution to transcriptional activation. The RPP is solely responsible for synergistic activation, however, the 5′US contributes to overall transcriptional activity. We defined quantitatively the individual contributions of Xenopus rhodopsin promoter cis-elements to synergistic activation of transcription by the paired like transcription factors, Rax and Crx/Otx5. We showed specificity of the interaction between the transcription factors and cis-elements. Rax preferentially binds to the Ret1 cis-element and augments synergy, while Otx5/Crx binds to both the BAT1 and Ret4 cis-elements, contributing equally to synergistic activation. These studies characterizing the rhodopsin promoter architecture will advance our understanding of the role of transcriptional network machinery in rod photoreceptor development and homeostasis.In the present study, we characterized regulatory regions of the Approximately 200 bp around the NRE were aligned using CLUSTALW and then manually adjusted to optimize Pax-like cis-elements. Logos were created using WEBLogo3.0. The accession numbers for the sequences used in the alignment are described in Additional file Xenopus laevis adult retina total RNA and subcloned into pCS2. pCS2-hNrl and pCS2-hCrx were generated by PCR from cDNA clones obtained from A. Swaroop and S. Chen, respectively. hNrl was subcloned into the BamHI and Xba I sites, and hCrx into the BamHI and Not I sites in the pCS2 vector. All constructs were verified by sequencing. See Additional file pCS2-Otx5 was obtained from A. Viczian. pCS2-LNrl was subcloned from pMT-LNrl . The pCSXenopus rhodopsin promoter luciferase reporter constructs, pGL2-XOP , were described previously [Xenopus rhodopsin exons and 3′ region was amplified using the -503DWN plasmid as a template and cloned into the Xma I and Mlu I sites of the pGL2 basic plasmid. The XOP(−503/+41) sequence was amplified by PCR and inserted into the Nhe I and Bgl II sites just downstream of the 5.8 kb rhodopsin sequence. Mutations in the cis-elements (ATTA to AGGA) were introduced in both XOP(−5361/+41) and XOP(−503/+41) following the QuikChange XL Site Directed Mutagenesis protocol (Stratagene). The promoter constructs were verified by sequencing. The primers used in cloning are found in Additional file Four WT A DNA fragment that included the FRT sites and hygromycin gene was PCR amplified using the pcDNA5 FRT TOPO vector as template (Invitrogen). The purified PCR fragment was cloned into the Sal I and Bam HI sites of the pGL2-basic vector (Promega), which we named pGL2-FRT. The XOP(−503/+41) and XOP(−5361/+41) promoter fragments were then excised from their pGL2 reporter constructs with Xma I and Bam HI and cloned into the same restriction sites in the modified pGL2/FRT vector. Flp-In 293 T-REx cells (Invitrogen), containing the stably integrated FRT target sites upstream of the Zeocin gene, were co-transfected with the pOG44 plasmid, containing the Flp recombinase gene (Invitrogen), and the pGL2-FRT-XOP(−503/+41) or pGL2-FRT-XOP(−5361/+41) plasmids using Fugene 6 following the manufacturer’s protocol (Roche Diagnostics). Cells were passaged 48 h after transfection. Hygromycin B (150 μg/ml) was added to the culture medium the next day. Twelve individual stable clones expressing XOP(−503/+41)-Luciferase or XOP(−5361/+41)-Luciferase were picked 14 days after selection and expanded. Cell lines were also tested for loss of Zeocin (100 μg/ml) resistence. Only the dually selected clones were used for reporter assays. The promoter constructs were verified by sequencing. The primers used in cloning are found in Additional file 5 and 1.6 × 105 per well, respectively. The stable Flp-In 293 T-REx cell lines were seeded at 2.4 × 105. Cells were co-transfected the next day with Fugene 6 and various combinations of reporter gene and/or expression constructs at a total DNA concentration of 1 μg/well following the manufacturer’s protocol (Roche Diagnostics). After 48 h cells were harvested and lysed with 100 μl/well of Passive Lysis Buffer following manufacturer’s instructions for the Luciferase Reporter Assay System (Promega). Luciferase activity was measured in 10 μl of cell lysate using a Biotek Synergy II plate reader . Luciferase activity from mock transfected.HEK293 or HEK293T cells were seeded into 24 wells at 2.4 × 10Xenopus embryo heads  or adult retinas using an RNAeasy kit (Qiagen) and analyzed using a 2100 Bioanalyzer (Agilent Technologies). Complementary DNA was synthesized from 1 μg total RNA with Superscript II RT polymerase and random hexamers (Invitrogen). cDNA was amplified over 33 cycles using Advantage 2 DNA polymerase (Clontech) and the appropriate primers for histone H4 and Rax family members  and analyzed using a 2100 Bioanalyzer (Agilent Technologies). Complementary DNA was synthesized from 1 μg total RNA using the Quantitect Reverse Transcription kit (Qiagen). Real time PCR reactions were comprised of 1 μl of cDNA (diluted 1:2), 5 μl of SYBR green mix , 0.5 μl each of sense and antisense primers (10 pmol) to target  primers according to MATCHMAKER protocol except that the denaturation time during PCR was increased to 25 s. A fusion library in pGADT7-Rec(2) vector was constructed by homologous recombination in yeast. Yeast were sequentially transformed first with the bait plasmid and then the library. Transformants (6 × 106) were screened on SD/-His/-Ura/-Leu supplemented with 10 mM 3-amino-1,2,4-triazole (3-AT). The screen yielded 30 primary transformants. The plasmid DNA containing the activation domain and cDNA inserts was isolated using standard techniques. The Y190 strain was transformed with these rescued library plasmids and used for mating the secondary screen. Of the 30 transformants, seven survived after mating with the original ROP2-containing Y187 strain on selective plates containing 40 mM 3-AT, but not in plates containing Y187 strain containing scrambled ROP2 sequence bait. The library cDNA inserts were rescued following standard protocols and sequenced.The MATCHMAKER one-hybrid system (Clontech) was used to isolate -element . The baiXenopus opsin promoter; 5′ US: 5′ upstream sequence; LNrl: Xenopus laevis neural retinal leucine zipper; Crx: Cone rod homeobox; Otx5: Orthodenticle-related homeobox; hNrl: Human neural retinal leucine zipper; hCrx: Human cone rod homeobox; Rax: Retina and anterior neural fold homeobox; RCOP: Red cone opsin promoter; RLU: Relative light unit; BAT1: Ret1, NRE, cis-elements in the XOP promoter.TF: Transcription factor; RPP: Rhodopsin proximal promoter; TA: TATA box; XOP: The authors declare that they have no competing interests.SER carried out the molecular biology and cell transfection studies, VM performed the one-hybrid analysis and cloned Rax2b cDNA, XZ prepared the stable cell lines and contributed to their characterization, BEK carried out the sequence analysis. BEK conceived of the study. All authors participated in various aspects of the study design. SER and BEK performed the statistical analysis. The manuscript was drafted by SER and BEK, and edited by all authors. All authors read and approved the final manuscript.Xenopus promoter sequences. Nucleotide composition and number of ATTA sites of the different Xenopus rhodopsin promoter lengths used in this study: XOP(−5361/+41), XOP(−503/+41) and XOP(−145/+41).Characterization of Click here for filecis-element deletions on Xenopus rhodopsin promoter activity. Comparison of luciferase activities in lysates from cells transfected with WT XOP(−503/+41), Ret1∆(−503/+41), BAT1∆(−503/+41) or Ret1/BAT1∆ (−503/+41) alone or with LNrl-Otx5. Activities are presented as mean RLU ± S.E.M. (n = 6).Effect of Click here for filePax-like cis-elements in the RPP with 5′ upstream sequences: Effect on Otx5-LNrl activation. Cells were transiently transfected with a plasmid containing a wild type promoter (XOP(−5361/+41)) or a mutation in cis-element (s)  in the absence (No TF) or presence of Otx5-LNrl. Activities in cell lysates are presented as mean ± S.E.M (n = 6-8). Promoter basal activity (RLU) is shown in parentheses.Mutational analysis of OP−5361/+1) or a mClick here for fileXenopus laevis. (A) RT-PCR was performed using total RNA (1 μg) from embryo heads (stages 35/36 and 39/40) or adult retinas and primers to amplify histone H4 and the three Rx paralogs . (B) Quantitative analysis of Rax expression levels in adult retinas by qRT-PCR.Expression of Rax transcription factors in Click here for filePax-like cis-elements in the RPP: Effect on Rax2b co-activation. Comparison of luciferase activities in lysates from cells transfected with WT XOP(−503/+41), Ret1∆(−503/+41), BAT1∆(−503/+41) or Ret1/BAT1∆ (−503/+41) alone or with LNrl-Otx5-Rax2b. Activities are presented as mean RLU ± S.E.M. (n = 6).Deletion analysis of Click here for filePax-like cis-elements in the RPP with 5′ upstream sequences: Effect on Rax2b co-activation. Comparison of luciferase activity in lysates from cells transfected with XOP(−5361/+41) or XOP(−5361/+41) containing RPP mutants  alone with Otx5-LNrl or with Otx5-LNrl-Rax2b. Activities are presented as mean RLU ± S.E.M. (n = 6-8). Promoter basal activity (RLU) is shown in parentheses.Mutational analysis of OP−5361/+1 or XOP(Click here for fileAccession numbers of tetrapod rhodopsin proximal promoters.Click here for fileAlignment of tetrapod rhodopsin proximal promoters.Click here for fileList of primers used in this study.Click here for file