Drosophila melanogaster the male specific lethal (MSL) complex is required for upregulation of expression of most X-linked genes in males, thereby achieving X chromosome dosage compensation. The MSL complex is highly enriched across most active X-linked genes with a bias towards the 3′ end. Previous studies have shown that gene transcription facilitates MSL complex binding but the type of promoter did not appear to be important. We have made the surprising observation that genes driven by the glass multiple reporter (GMR) enhancer-promoter are not dosage compensated at X-linked sites. The GMR promoter is active in all cells in, and posterior to, the morphogenetic furrow of the developing eye disc. Using phiC31 integrase-mediated targeted integration, we measured expression of lacZ reporter genes driven by either the GMR or armadillo (arm) promoters at each of three X-linked sites. At all sites, the arm-lacZ reporter gene was dosage compensated but GMR-lacZ was not. We have investigated why GMR-driven genes are not dosage compensated. Earlier or constitutive expression of GMR-lacZ did not affect the level of compensation. Neither did proximity to a strong MSL binding site. However, replacement of the hsp70 minimal promoter with a minimal promoter from the X-linked 6-Phosphogluconate dehydrogenase gene did restore partial dosage compensation. Similarly, insertion of binding sites for the GAGA and DREF factors upstream of the GMR promoter led to significantly higher lacZ expression in males than females. GAGA and DREF have been implicated to play a role in dosage compensation. We conclude that the gene promoter can affect MSL complex-mediated upregulation and dosage compensation. Further, it appears that the nature of the basal promoter and the presence of binding sites for specific factors influence the ability of a gene promoter to respond to the MSL complex.In  Drosophila eye is well suited to genetic investigations, as it is a dispensable tissue with mutant phenotypes that are relatively easy to identify Rh1 (ninaE) promoter, upstream of a minimal promoter from the hsp70 gene hid gene from GMR kills eye cells proportional to the GMR-hid expression level; a smaller eye with higher levels GMR-hid-derived eye size GMR-hid to screen for modifiers of Drosophila dosage compensation, but observed that GMR-hid did not report on dosage compensation. We have thus investigated why GMR-hid failed to respond, and report that the minimal hsp70 promoter appears refractory to the compensation machinery.The Drosophila males (XY) lack a dose of X-linked genes in comparison to (XX) females, but compensate for this by doubling transcription from the single X chromosome msl2 RNA is repressed by the female-specific SXL protein in vitro assay Drosophila cells GMR-hid to respond to dosage compensation, but found that it was unable to fully do so. We argue that an active gene is not sufficient for recognition by the dosage compensation machinery, even if near a HAS, and specifically that the hsp70 minimal promoter appears to hinder dosage compensation.The dosage compensation machinery can also affect genes that are not endogenous to the X chromosome. Translocations of genomic fragments between the X chromosome and autosomes exhibit “spreading” of the MSL complex from the X-linked sequences into the adjacent autosomal regions GMR-lacZ transgene was not linear with respect to gene dose, at least in the environment of the female X chromosome. The activity in females with two copies of the transgene was more than twice the activity in single-copy females, suggesting some synergy in homozygosity. Further, we detected some repression of expression from GMR in the larval imaginal tissue, consistent with that first reported for an earlier Glass-based eye reporter that contained a longer glass response element We also report two further observations of oddities in expression from GMR that will be of interest to those working with this enhancer-promoter. Activity of beta-galactosidase produced by a GMR-hid to screen for mutations that affected the end product of dosage compensation: the equalization of phenotype.Our understanding of the mechanism of X chromosome dosage compensation may need to better appreciate the balance between the MSL complex and the wider network of chromatin regulators. Identification of all factors that play a role in dosage compensation is not trivial. The five MSL proteins  were identified based on mutant phenotypes of male-specific lethality GMR-hid system to report on Drosophila dosage compensation, with the aim of then conducting a mutagenesis screen for modifiers of dosage compensation. To obtain a sex-specific difference in eye size, we created P-element transgenic lines carrying GMR-hid, and screened for those genetically linked to the X chromosome  male eyes were only slightly smaller than heterozygous female eyes; both sexes having a small range of sizes that just overlapped. The line with greatest difference and least overlap between the sexes (line C60) was selected for further analysis.We followed two strategies to adapt the romosome . Transgeted eyes  represeeach sex . Surprismsl1, msl2, and mle. Flies heterozygous for both GMR-hid and each msl deficiency  had similar sized eyes to those without the msl deficiency  in the X-linked roX1 gene roX1 noncoding RNA is a component of the MSL complex. roX1 expression is tightly regulated through a combination of constitutive repression and male-specific hyper-activation by the MSL complex that antagonises the repression roX1 DHS attract significant amounts of MSL complex to the insertion site, clearly visible by immuno-fluorescent labelling of polytene chromosomes, and a nine-copy multimer of the DHS is particularly effective roX1 DHS is also capable of mediating increased activity of a coupled lacZ reporter gene at autosomal insertions In a complementary approach, we sought to directly attract MSL complex to autosomal insertions of roX1 DHS-GMR-hid, and (roX1 DHS)9-GMR-hid, and observed the eye phenotype in flies carrying autosomal insertions of each , GA-rich motifs that are found predominantly in intergenic or intronic sequences  females . It appeGMR-hid to report might have been due to an inability to report two-fold transcriptional changes as an altered eye size. However, we observed a wide range of possible eye sizes (that likely reflected position effects on transgene expression), and the system has displayed great sensitivity previously GMR-hid was not amenable to dosage compensation. An alternative, although unlikely explanation, was the MSL complex was not recruited to the transgenes containing a roX or 18D11 high affinity site. Yet, in (roX1 DHS)9-GMR-hid lines  gene. We have previously shown that X-linked insertions of arm-lacZ generally acquire dosage compensation attP landing sites GMR-hid at each of these locations had eyes similar to those with P-element insertions of the transgene , nearly sufficient to compensate for deficiency against two-copy females (bottom panels), although the response at 6E was slightly lower. However, male activity in lines with GMR-lacZ was only slightly above single-copy female activity, providing effectively no dosage compensation against two-copy females. The apparent discrepancy between the two ratios (slight hyper-activity but no dosage compensation) is explored below. As the lacZ sequences were identical between arm-lacZ and GMR-lacZ, these results imply that the GMR-hsp70 regulatory sequences were responsible for poor dosage compensation.We targeted both activity . We compGMR-lacZ fail to compensate? Perhaps the construct actively blocked the compensatory machinery, although no such elements have yet been reported. Alternatively, the construct may have lacked some feature necessary for transcription to be compensated. We selected the landing site at 2A, which supported strong expression and rates of transformation, and tested the effect of a variety of modifications to GMR-lacZ on the male hyper-activity and dosage compensation. Since the MSL complex binds to the body of active X-linked genes with a bias towards the 3′ end, we first considered replacing the 3′ hsp70 sequences with those from SV40 that were used for arm-lacZ. The GMR-lacZ-SV40 line produced much higher levels of beta-galactosidase than GMR-lacZ, possibly because the lacZ RNA is less stable with hsp70 3′ UTR sequences GMR-lacZ but the increase was not statistically significant. To enhance binding of the MSL complex to the 3′ end of the transgene, we inserted of an MRE (from 18D11) in the SV40 3′ UTR of GMR-lacZ-SV40. However, we found no measureable effect on male hyper-transcription , and is slightly enriched on the X chromosome arm promoter does not contain an MRE. However, an MRE is found within the gene, but this is downstream of the promoter fragment used in arm-lacZ . We isolated a short fragment of the armadillo promoter (arm*) spanning the cluster of DRE-like elements and five GAGA-like elements, and tested it upstream of GMR-lacZ. The fragment did not include the transcription start site. The activity levels and sex ratio from arm*-GMR-lacZ were similar to those in flies without the arm fragment .We next considered that the fragment . We nextDRE/GAGA-GMR-lacZ. We targeted the construct to the attP landing sites and 6E and 20C, and found similar responses of the transgene at all locations. We checked that the observed increase was specific to the X chromosome, by targeting the construct to an autosomal attP (at 86F). Indeed, males and females with DRE/GAGA-GMR-lacZ at 86F had similar activity levels  than those without  from the X-linked dosage compensated Pgd gene GMR-pgd-lacZ was much reduced compared to GMR-lacZ in both sexes  and dosage compensation appeared to be partially restored.Lastly, we considered that the th sexes , which wGMR-lacZ, arm*-GMR-lacZ) had no dosage compensation, and those with high male hyper-transcription  had only modest dosage compensation. Because (hemizygous) males in each measurement were genetically identical, this anomaly can only be explained by differences in female expression. Due to experimental variation, the raw male or female activities cannot be directly compared without first controlling for variation by normalization, as with a male to female ratio. But if we instead calculate the inverse  ratios, then we have comparable activity estimates of females, normalized to single-copy males  is sufficient to dosage compensate. But females with two copies of all GMR-based constructs have more than twice the activity of single-copy females. This explains the discrepancy: a slight male hyper-activity was only sufficient to equal about half the activity of paired females, and full compensation would require male hyper-activity of around 2.5 to 3.0-fold (log2 of 1.3–1.6). The significance of this homozygous effect is discussed below.The two methods for comparing male and female activity of all GMR-based constructs appeared to give slightly different results. Constructs with very low hyper-transcription  is sufficient to lead to cell death through expression of a ala5tetO-hid transgene, but only in the absence of tetracycline arm-tTAWe placed the tetO-GMR-lacZ to lines with arm-tTA, n1-tTA, or s1-tTA activators, and raised the offspring in the absence of tetracycline. We measured the male hyper-activity of beta-galactosidase in adult heads . A separate cross to y w flies , which lack tTA, showed that the tetO sequences alone had little effect on activity from GMR-lacZ 9-GMR-hid lines may have been sufficient to relieve repression on mini-white. However, there was little difference in eye size, suggesting that GMR-hid was not responding to the bound MSL complex. Lack of response to MSL complex is also consistent with our observations that strong MSL binding sites had no effect on GMR-lacZ beta-galactosidase activity. While formally possible that local MSL complex was limiting at each site, the large number of lines screened makes this unlikely. Certainly local MSL complex at the 2A, 6E, and 20C attP sites was sufficient to up-regulate arm-lacZ, yet had no effect on GMR-lacZ. The most likely explanation is that expression from the GMR-hsp70 enhancer-promoter was not responsive to MSL complex.This was most evident in the phenotype of the UAS-hsp70 TATA transgenic promoter are induced with GAL4 mof gene, but only when mof is transcribed mof is transcribed from a tubulin promoter, GAL4 UAS sequences, or even in an antisense direction, Kind and Akhtar conclude that “the type of promoter and direction of transcription are not [important]”. It appears that some feature of transcription itself, or an active chromatin state, may be responsible for recruitment of MSL complex to the many genes on the X chromosome. This may involve the chromo domain of MSL3, which recognizes histone H4 monomethylated at lysine 20 GMR-lacZ failed to acquire compensation, at the same X-linked sites where arm-lacZ did, argues that transcription alone is not sufficient for a gene to be compensated, and that the importance of a gene's promoter in dosage compensation cannot be ignored. Indeed, studies of the X-linked white gene concluded that the gene contains multiple elements that influence dosage compensation, some near the promoter and some in the coding region Drosophila dosage compensation Our observation that the promoter can affect dosage compensation was somewhat surprising. The MSL complex binds the male X chromosome most strongly at 150 to 300 mostly non-coding GA-rich MRE sequences hsp70 basal promoter was replaced with the weaker basal promoter from the X-linked dosage compensated Pgd gene. This suggests that the Pgd basal promoter contains elements that are important for recruitment of the MSL complex to the reporter gene and that the hsp70 basal promoter lacks these elements. The white gene promoter was concluded to be important for dosage compensation because autosomal white transgenes driven by the full heat-inducible hsp70 promoter were expressed equally in males and females white transgenes are typically expressed at higher levels in males than females hsp70-white transgene is not compensated because the hsp70 promoter does not respond to the MSL complex. The Pgd promoter did not contain any obvious binding sites for known transcription factors. Widely expressed genes such as Pgd and arm generally contain similar levels of RNA polymerase II at 5′ and 3′ regions of the transcription units hsp70 enhancer-promoter. The DREF and/or GAGA factors could create a local chromatin environment that facilitates recruitment of the MSL complex or could interact directly with the complex. It may now be beneficial to separate the effects of DRE and GAGA elements, and determine such characteristics as motif strength, placement, and repeat spacing. Such studies may explain why addition of a fragment from the arm promoter that contained predicted GAGA and DREF sites, did not lead to a significant increase in GMR-lacZ expression in males. We found no support for the notion that late or tissue-specific expression was a factor in poor compensation, although it is possible that these factors affect compensation of other genes. Thus we conclude that genes driven by the GMR-hsp70 enhancer-promoter do not respond to the MSL complex because of the nature of the hsp70 basal promoter and because it lacks elements such as GAGA and DREF binding sites.Our efforts to determine why genes driven by the GMR enhancer-promoter do not respond to the MSL complex were complicated by the surprising observation that activity in homozygous females was more than twice that of hemizygous females. We believe this may be because of a complex interplay between the Glass transcription factor and an unknown repressor. As a consequence of the hyperactivity in homozygous females, the comparison of the activity of one dose males with one dose females provides a more accurate view of male hyperactivation. Of the various gene constructs evaluated, two showed a significant increase in activity in males. In one construct, the pgd-GMR-lacZ and DRE/GAGA-GMR-lacZ transgenes. Knowing that MSL complex can act to relieve repression in males, such changes would also register in our tests as an apparent increase in male hyper-activity. However, we cannot distinguish between general repression with male-specific relief, and more simple male enhancement, as our assays were designed to measure the final result of dosage compensation.A curious possibility is that any one of the promoter modifications could have caused or increased repression of the transgene, perhaps via the putative repressor detected in larval staining for beta-galactosidase. This may be reflected in the generally lower beta-galactosidase activity in lines with Lsp1alpha gene has been particularly well studied Lsp1alpha is not enriched for H4K16ac in the tissue in which it is active runt gene, which is dosage compensated by an MSL-independent mechanism lacZ transgenes driven by the Pgd promoter are enriched for H4K16ac Can our results shed any insight into why other X-linked genes are not dosage compensated? The non-compensated X-linked arm-lacZ, and is not a property of the beta-galactosidase assay, which is linear across a 100-fold range of protein extract concentration Diptera pair through all mitotic stages, not just during meiosis GMR-lacZ cassettes at different locations, but found that the differing activity levels at each location precluded conclusions based on mixed genotypes. Whatever the cause, the non-linear effect has consequences for dosage compensation of GMR-lacZ, as male expression needs to be more than doubled to equal the paired female levels.Beyond the promoter, our experiments also revealed that another factor can affect the degree of compensation. Two copies of all GMR cassettes had more than twice the beta-galactosidase activity of one copy, instead increasing three- to four-fold. As we only measured this effect in females with X-chromosomal insertions, it would be interesting to extend the experiments to autosomes and males. The non-linear effect did not occur with msl-2, with median decreases of about 1.3 to 1.7-fold Drosophila males have indeed evolved to co-opt gene buffering as a part solution to X chromosomal haploidy, then dosage compensation should be viewed as a combinatorial process. At the level of an individual X-linked gene, male expression may be affected by any or all of several mechanisms, including aneuploidy-type buffering, dose-dependent regulatory feedback, and MSL-mediated up-regulation. Given that the paired female X-linked genes might also be affected by transvection, dosage compensation could be seen as merely the result of a combination of mechanisms. Care must be taken in the design of experiments to ensure the correct biological process is measured.More generally, a description of X chromosome dosage compensation as a transcriptional doubling by the MSL complex may be too simple. When quantified by microarray, X chromosomal transcripts reduced a variable amount after knock-down of GMR enhancer appears to reflect more than simple activation by the Glass transcription factor. We observed a general lack of GMR-lacZ expression in larval imaginal tissues beyond those that express Glass. This repression pattern is similar to that observed for another Glass reporter, “38-1”, a pentamer of a larger (38 bp) fragment from the Rh1 proximal enhancer spanning the Glass binding site glass overexpression (from a hsp70-glass transgene) in imaginal tissue with the exception of a small number of cells in the leg disc. In contrast, in response to glass overexpression, 38-1 directs lacZ expression in many cells in the brain and central nerve cord. A shorter 29 bp enhancer (GMR) responded more strongly to glass overexpression, directing lacZ expression in many cells in the brain and in all cells of the eye/antennal and leg discs. It was concluded that shortening the enhancer to 29 bp had deleted the binding site for a putative repressor that inhibits Glass activity in non-photoreceptor cells GMR-lacZ used in this study confirmed that we were using the pentamer of the 29 bp element. One explanation for our results is that the repressor binding site was not completely removed when shortening the fragment from 38 to 29 bp, merely weakened to allow dominance of high levels of Glass over the repressor. Alternatively, the 38 bp enhancer contains binding sites for two repressors, one of which is retained in the 29 bp GMR enhancer. Additional studies would be required to distinguish between these two possibilities. In either case, it appears that the tissue-specific expression directed by the 29 bp GMR enhancer is due to combined action of the Glass activator and an unknown repressor.Finally, it should be noted that the tissue-specificity of the Standard molecular cloning techniques hid cDNA hid cDNA, was inserted into the EcoRI site of pGMR-1 GMR-hid cassette used for most GMR-hid lines. A XhoI to NotI fragment of pCaSpeR3 roX1 DHS short or pCaSpeR3 roX1 DHS short multimer roX1 DHS or its multimer, was inserted into the XhoI site of pGMR-1, with the aid of oligonucleotides (5′-TCGACGTTTAAACGGTTGGCC-3′ and 5′-AATTGGCCAACCGTTTAAACG-3′) annealed to link EcoRI and XhoI sites, to create pCL02 or pCL03, respectively. A EcoRI to EcoRI fragment of pBS SK hid cDNA containing hid was inserted into the EcoRI site of pCL02 or pCL03, to create pRGH and pR9GH, which contained the roX1 DHS-GMR-hid or (roX1 DHS)9-GMR-hid cassettes, respectively. pCaSpeR3 18D10-L monomer or pCaSpeR3 18D10-L4mer GMR-hid-18D and GMR-hid-(18D)4 cassettes, respectively. An EcoRI to EcoRI fragment of pTAattB attB, was treated with Klenow fragment and inserted into the EcoRV and SmaI sites of pBluescript II KS+ (Invitrogen) to create pBSattB. pBSattB was partially digested with XhoI and re-ligated to remove the XhoI site, then a HindIII to HindIII fragment of pGMR-1, encoding mini-white and GMR, was inserted into the HindIII site to create pBSw+GMRattB. An EcoRI to EcoRI fragment of pBS SK hid cDNA, encoding hid cDNA, was inserted into the EcoRI site of pBSw+GMRattB to create pGHattB, which contains the GMR-hid cassette used for phiC31-mediated transformations .An EcoRI to EcoRI fragment of pBS SK attB, was inserted into the EcoRI site of pCaSpeR-arm-betagal arm-lacZ cassette. pCaSpeR-arm-betagal was digested with XbaI, treated with Klenow fragment, and digested with SmaI, then the fragment encoding lacZ was inserted into pBC KS + (Invitrogen) to create pBClacZ. A BamHI to EcoRI fragment of pBClacZ, encoding lacZ, was inserted into the BglII and StuI sites of pBSw+GMRattB to create pGLattB, which contains the GMR-lacZ cassette. To make pGMR-pgd-lacZ, pGLattB was digested with XbaI  and EcoRI and the excised hsp70 minimal promoter was replaced with a 70 bp linker that contained the Pgd minimal promoter. The 70 bp linker was made by annealing the oligonucleotides 5′-CTAGAACAGTGCCATATATAGATTGTAACATTAGGAGCTCAAATCATTGTTGGAACACAAACCACAAAG-3′ and 5′-AATTCTTTGTGGTTTGTGTTCCAACAATGATTTGAGCTCCTAATGTTACAATCTATATATGGCACTGTT-3′. pGL-H was made by digestion of pGLattB with HindIII  and re-ligation to remove the site in the pUC backbone. A SmaI to HindIII fragment of pCaSpeR-arm-betagal, encoding lacZ and SV40 3′ UTR, was inserted into the EcoRI (filled) and HindIII sites of pGL-H to create pGLSV40attB, which contains the GMR-lacZ-SV40 cassette. A SmaI to HindIII fragment of pBS2N17merHF12-1x12 lacZ, SV40 3′ UTR and the 18D site, was inserted in the (blunted) EcoRI and (remaining) HindIII sites of pGL-H to create pGLSV4018DattB, which contains the GMR-lacZ-SV40-18D cassette. A XhoI to XhoI fragment of a PCR amplicon (primers 5′-CCGGAATTCTCGAGTGGAATGTAAACAATGCCACAGAC-3′ and 5′-CCGGAATTCTCGAGTAAACGGAACAGAATCACAGATGC-3′) of the armadillo promoter, from pCaSpeR-arm-betagal was inserted in the XhoI site of pGLattB to create pCL12, which contains the arm*-GMR-lacZ cassette. Four oligonucleotides  were annealed to create 3 DRE and 4 GAGA sites, digested with XhoI, and inserted into the XhoI site of pGLattB to create pCL13, which contains the DRE/GAGA-GMR-lacZ cassette. A XhoI-SacI fragment of pW.T.P-2 tetO, was inserted with the aid of oligonucleotides (5′-CTTTAAACGGTTGGCG-3′ and 5′-TCGACGCCAACCGTTTAAAGAGCT-3′), annealed to link SacI to XhoI, in the XhoI site of pGLattB to create pCL21, which contains the tetO-GMR-lacZ cassette. The linkers are 3′ of the tetO.An EcoRI to EcoRI fragment of pTAattB, encoding GMR-hid eye size with msl deficiencies, msl2 msl1 mle/CyO males were mated to virgin GMR-hid C60 females, and Cy and Cy+ offspring compared. To observe GMR-hid eye size with constitutive expression of msl2, virgin GMR-hid C60 females were separately crossed to w; msl1L60/CyO; P{w+  =  hsp83-msl2} males 1 w1; msl1L60/CyO, pr cn2 yy+ males. Curly offspring of each cross were compared. To measure GMR-lacZ activity with constitutive expression of msl2, w; msl1L60+ = hsp83-msl2}/CyO; P{w males were crossed to virgin w1118; P{w+mC =  hs.hid}3, Dr1/TM6C, cu1Sb1 females, then the resulting curly-winged, stubble-bristled males were mated to virgin females carrying the appropriate lacZ cassette at 2A, and Sb and Sb+ daughters compared.To observe (roX1 DHS)9-GMR-hid were captured with a Magnafire camera and software (Optronics), and the RGB colour balance manually altered to best match other images. All other images were captured with an Olympus DP-70 camera, with the provided software or with Soft Imaging System analySIS FiVE 5.0, and balanced RGB on white card to control for exact lighting conditions. The exposure time was automatically determined by the software.Right-hand-side adult eyes that best represented the modal average eye size of each line were illuminated with reflected halogen light. Images of the lines carrying P-element mediated transformation of Drosophila was performed as previously described attP landing site and expressed recombinase in the germ-line y w line, established as homozygous or balanced lines, and confirmed by PCR both within the insert and across the insert/landing site boundary.2 transformation then often applied to allow better comparisons of different ratios. Two sample, two-tailed Student's t-tests were conducted in the R software environment, using (the default) Satterthwaite's approximation. Tissue dissected from climbing third instar larvae was stained with X-gal essentially as described by Glaser et al. Beta-galactosidase assays of hemisected adults or heads were conducted based on the procedure of Simon and Lis Figure S1GMR-hid did not respond to the MSL complex. Eyes of flies that carried a single copy of the GMR-hid transgene (line C60), and were either wild type for the msl genes, heterozygous for msl1, msl2 and mle or constitutively expressed msl2 (hsp83-msl2). If GMR-hid on the X chromosome responded to levels of the MSL complex then the eye size in msl1 msl2 mle heterozygous males should have been larger than control. Similarly, eyes of females that expressed msl2 should have been smaller than control.(TIF)Click here for additional data file.


Drosophila: the Male-Specific Lethal (MSL) and the Polycomb group (PcG) complexes. These two serve as models for understanding targeting, because they have been extensively studied and play vital roles in Drosophila, and their targets have been documented at high resolution. Furthermore, the proteins and biochemical properties of both complexes are largely conserved in multicellular organisms, including humans. While the MSL complex increases gene expression and PcG members repress genes, the two groups share many similarities such as the ability to modify their chromatin environment to create active or repressive domains, respectively. With legacies of in-depth genetic, biochemical and now genomic approaches, the MSL and PcG complexes will continue to provide tractable systems for understanding the recruitment of multiprotein chromatin complexes to their target loci.Chromatin-binding proteins must navigate the complex nuclear milieu to find their sites of action, and a constellation of protein factors and other properties are likely to influence targeting specificity. Despite considerable progress, the precise rules by which binding specificity is achieved have remained elusive. Here, we consider early targeting events for two groups of chromatin-binding complexes in  Genetic information is encoded at the DNA level, but most interpretation of genetic information occurs at the protein level. In addition to histones, numerous non-histone proteins interact with chromatin and DNA to bring about the proper, cell-specific interpretation of the genome.Drosophila: the male-specific lethal (MSL) complex and the Polycomb group (PcG). These two groups can be considered as models for addressing how chromatin factors are targeted, as their sites of action have been documented at high resolution.One major question in chromatin biology is how protein players find their proper sites of action. In genomes ranging in sizes from megabases to gigabases of DNA, transcription factors, which recognize their sites in a DNA sequence-specific manner, are only found localized to a fraction of their numerous consensus sequences throughout the genome. It is no wonder, then, that complete understanding of the potentially more complicated phenomenon of chromatin targeting has remained elusive . Here, wDrosophila. Dosage compensation is the means by which X chromosome gene expression is adjusted to balance gene expression from the autosomes. In flies, this is achieved by hypertranscription of the active genes on the single male X chromosome. The MSL complex is made up of five core proteins (figure 2a and figure 2b). The first step involves initial targeting to several hundred chromatin entry sites  carrying a degenerate sequence motif. Two prominent CESs are the roX RNA genes that produce the ncRNA components of the complex. The second targeting step involves sequence-independent spreading in cis to most active genes. It is for this second step that roX RNAs seem most critical (reviewed in reference . Ho. Hoet alb).In contrast to the MSL complex, the structural and functional analysis of PREs, which were originally characterized in the Bithorax cluster, has shown that there is no strong consensus sequence that can be simply defined as a PRE motif. Instead, PREs often contain diverse combinations of sequence motifs for multiple DNA-binding proteins, including Pho/Phol, SP1/KLF proteins, GAGA factor (GAF)/Pipsqueak (Psq), Dorsal switch protein 1 (Dsp1), Grainyhead (Grh) and Zeste , reflectfigure 3in vitro. Furthermore, double mutants of pho and phol have a synergistic effect leading to Hox gene misexpression, suggesting that Pho and Pho-like act redundantly in Hox gene silencing .F.FDrosopet al. [The modEncode data allowed Alekseyenko et al.  to exploDrosophila species, even within orthologous PREs [en or invected (inv) PREs show PSS at only about 60% and 20–45% of insertion sites, respectively [et al. [en PRE activity. This suggests that, in the context of this reporter assay, there is competition between the PRE and neighbouring regulatory elements, such as enhancers, for control over the transcriptional state of the reporter gene.PREs have different combinations of diverse motifs without any preferential number, order or spacing . Furtherous PREs . It is uectively . This ph [et al.  observedFab-7 PRE transgene showed that spreading of H3K27me3 is blocked by RNA polymerase II bound promoter regions and active chromatin marks rather than by insulator elements [Insulators have been identified as another type of regulatory element that affects PRE activity, potentially by blocking the spreading of PcG proteins or H3K27me3 marks –104. Genelements ,108,109.et al. [bithoraxoid (bxd) PRE are cooperatively required for silencing by the PRE, whereas the Dsp1 binding motif is not essential for the PRE activity. In addition, replacement of the bxd PRE in a reporter construct with the iab-7 or iab-5 PREs from Abd-B revealed that these PREs are interchangeable, indicating equivalent functional capabilities. On the other hand, direct comparison between the Fab-7 PRE and one of the vestigial (vg) PREs at four ΦC31 site-specific integration loci demonstrated that these two PREs exhibit differential silencing traits in a genomic-context-dependent manner, indicating that different PREs can have distinct properties [en PREs supports the idea that different PREs produce different functional outcomes. PRE1 and PRE2 of the engrailed gene not only have a different number, order and spacing of motifs, but also require different numbers of Pho motifs for silencing activity. In addition, an AT-rich region only found in PRE1 is required for full PSS activity. These differences led to distinct expression patterns in embryonic and larval reporter assays in which the reporter construct was integrated at the same genomic site [The sensitivity of PREs to genomic context makes comparison of functional differences between PREs challenging. Therefore, site-specific recombination tools such as gene conversion and ΦC31 integration have been very important to examine the effects of mutation or deletion of sites within a PRE, and for direct comparison between different PREs in a constant genomic context ,110. Usiet al.  showed tmic site .6.An additional avenue that has yet to be fully explored with respect to MSL complex targeting is the three-dimensional structure and folding of the genome . This piin situ hybridization, it has been reported that PcG-targeted regions can contact other PcG-bound regions or promoters over long distances [b) [Much more intensive investigation of higher-order structure has occurred in the PcG field. Through PRE transgenic analyses and techniques such as DNA adenine methyltransferase identification (DamID), chromatin conformation capture assays (3C and associated technologies), and fluorescent istances –116. On nces [b) . In furtMcp and Fab elements, Li et al. [Mcp and Fab-7 elements does not depend on the ability to recruit PcG complexes, but rather on insulator DNA sequences flanking or within the PRE element. In subsequent investigations by the same group, CTCF binding sites were observed to be critical for long-range interactions of reporter constructs; in comparison, the PRE was observed to be needed only for proper and stable sorting to subnuclear structures [Recently, however, the idea that PcG proteins themselves mediate long-range interaction has been challenged. Using transgenes containing the i et al.  showed tructures . Additio7.In review of the topics covered so far—the biochemical properties of the proteins and complexes themselves, the sequence motifs associated with their binding, and the state of the chromatin around targets, both locally and at longer range—it seems clear that each of these factors plays at least an incremental role in determining the initial targeting of chromatin complexes. In this section, we consider evidence for the role of RNAs in the initial targeting of these model complexes.Drosophila, in contrast to reports in mammalian systems, in which numerous lncRNAs have been proposed to function in PcG targeting [The long non-coding roX RNAs are an integral part of the MSL complex . They arargeting .A recent report proposes a role for the small interfering RNA (siRNA) pathway in targeting of the MSL complex to the X chromosome. Menon & Meller  observe Fab-7 region directed 21–23 nt small RNAs in transgenic flies was due solely to the Fab-7 element in the transgene; the endogenous locus itself does not stimulate the production of such siRNAs.Similarly, the direct involvement of RNAi machinery components in PcG-mediated silencing is being debated in the PcG field. The positive correlation of Ago2 ChIP data with PcG ChIP-chip data from modENCODE led Taliaferro and co-workers to suggest that Ago2-mediated transcriptional repression, which is independent of the catalytic activity of Ago2, might be related to PcG-mediated silencing . Previouet al. showed that the binding profile of Ago2 overlaps with TrxG proteins as well as PcG proteins, and Ago2 not only preferentially associates with active promoters, but also acted as a TrxG protein opposing the Pc mutant phenotype in a genetic screen [Therefore, the role of RNAi components in PcG-mediated silencing still seems uncertain, and potentially tied to the structure of the specific transgenes used in transgenic assays. A recent analysis of the possible role of RNAi components in PcG-mediated silencing at the endogenous BX-C locus showed that depletion of RNAi components did not result in any significant change in the repressed chromatin state of the locus, and direct comparison of Ago2-bound small RNAs and PcG-target promoter regions showed no enrichment of small RNAs for such PcG targets . In addic screen .In one additional potential link to small RNAs, analysis of the chromatin environment at transcription start sites proximal to PREs showed that these regions are enriched with H3K4me1 and H3K4me2 marks, and many of them generate short RNAs owing to stalled PolII , consist8.Drosophila miranda become dosage-compensated by the acquisition of CES-like sequences [D. miranda version of CLAMP can similarly explain the selection of CES-like sequences will be an important test. Interestingly, although the mechanisms of dosage compensation are not conserved in nematodes and in mammals, two other model systems that have been intensively studied, the ‘nucleate and spread’ models for targeting to the X chromosome, appear quite similar in the three systems [As can be seen, the constellation of factors and sequence characteristics that may be involved in initial targeting of complexes to chromatin makes investigation of this topic a complicated endeavour. In the case of MSL targeting, primary sequence characteristics of CESs are certainly a major factor, as novel sex chromosomes generated by chromosomal fusions in equences . The rec systems ,131,132.PcG targeting seems to involve multiple proteins, and no single consensus sequence (even the Pho/Phol consensus) can adequately predict PcG binding. Furthermore, the manner by which PREs are typically studied is with transgenic reporter assays in which recruitment of PcG is not directly assayed; rather, functional silencing of the transgene is tested. However, PcG recruitment does not always lead to silencing, but may instead only modulate transcription levels (e.g. at the Psc/Su(z)2 locus) ,133–135.With the advent of higher-throughput genome editing technologies, such as the CRISPR/Cas9 system, it is becoming more feasible to do the types of experiments that may reveal requirements for targeting and function without removing sites from their endogenous locations. It may finally be possible to expand from single site assays to look at more global recruitment: mutation of a majority of MREs or PREs in a chromosomal region. New chromatin immunoprecipitation techniques that enhance the resolution of binding sites may also help to refine known binding patterns or reveal sequence motifs. Hopefully, these technologies will allow investigators to address the many outstanding questions that still exist in the area of PcG and MSL initial targeting.


Living cells are controlled by networks of interacting genes, proteins and biochemicals. Cells use the emergent collective dynamics of these networks to probe their surroundings, perform computations and generate appropriate responses. Here, we consider genetic networks, interacting sets of genes that regulate one another’s expression. It is possible to infer the interaction topology of genetic networks from high-throughput experimental measurements. However, such experiments rarely provide information on the detailed nature of each interaction. We show that topological approaches provide powerful means of dealing with the missing biochemical data. We first discuss the biochemical basis of gene regulation, and describe how genes can be connected into networks. We then show that, given weak constraints on the underlying biochemistry, topology alone determines the emergent properties of certain simple networks. Finally, we apply these approaches to the realistic example of quorum-sensing networks: chemical communication systems that coordinate the responses of bacterial populations. Genes encode proteins, which in turn carry out the processes required for the maintenance of cellular life. During the process of gene expression, the genetic information is first transcribed or copied onto a short-lived messenger RNA (mRNA) molecule. This mRNA is then translated repeatedly into a protein, as specified by the genetic code: a set of three consecutive nucleotides of mRNA uniquely specifies one of twenty possible amino acids, a series of which are strung together to form the protein .a), is not the entire story however. Every cell in the human body carries the same complement of genes, yet a heart cell and a brain cell are made up of very different proteins. Even in a single-celled organism such as a bacterium, different proteins are expressed at different times. The bacterium Escherichia coli is able to assemble flagella when it needs to swim, and pili when it needs to anchor itself to a surface; it will produce a metabolic enzyme only when its substrate is present, and synthesize DNA repair proteins only when subject to shock. In short, genes can be turned on and off.This basic description, the ‘central dogma of molecular biology’ a, is notb).This simple but powerful idea was first proposed by Jacques Monod in the 1940s , ch. 7. figure 1A can activate gene B and gene C, but repress gene D, and so on. There is a compelling case to be made for the existence of such networks in living cells. Consider that a bacterial genome contains about 4000 genes, whereas the human genome contains about 25 000 genes—a surprisingly modest difference at first glance, given that the human body is made up of more than 200 cell types, not to mention higher degrees of organization required to specify a complex tissue such as the brain. A deeper analysis suggests that gene number is not the correct measure of complexity: the properties of a cell are specified by the proteins contained within it; the range of possible cell types is therefore determined by the range of possible combinations of expressed genes, and grows exponentially with gene number. How are all such combinations to be accessed, however? We know that distinct external signals can drive cells to differentiate into distinct types. However, such signals do not directly interact with individual genes, turning them on or off. Once the differentiation process is triggered, various combinations of gene expression must arise through the intrinsic behaviour of the genes themselves. That is, there must be a network of genetic interactions which, based on very few external regulatory cues, is able to produce the correct expression patterns. The manifest complexity of cellular behaviour strongly implies the existence of complex regulatory networks within.These new arrows are loaded with implications: they permit us to assemble complex networks of transcriptional and regulatory interactions. Gene Escherichia coli and the yeast Saccharomyces cerevisiae, there is a growing body of detailed information regarding transcriptional and regulatory interactions . This binding event can be represented ask+ and k− for this clubbed reaction. Indeed, there might be several configurations of the bound state: other combinations in which the DNA can bind these proteins. Let sj represent these various states (including the one in which the DNA is bare). The probability of occurrence of each state in thermodynamic equilibrium is then m1. More generally, the probability per unit time that the reaction (P+) or right to left (P−) isP+=P−, givingK is the equilibrium constant. This result is usually presented as the principle of mass action. The concentration of a given promoter state is the total DNA concentration multiplied by the probability of occurrence of that state. If we agree to measure all free energies as differences from that of the bare configuration, a comparison of . The probability that the DNA is bare is given byX]=1/K (c).We can now use these general results to study a few relevant examples, where we now explicitly treat multi-step reactions. Consider a DNA region DX is εX b. The pr [X]=1/K c.D represents a promoter, and that the protein X is a repressor, which acts to prevent transcription by the polymerase P. Let the free energy of the state DP be εP. If the two proteins X and P bind independently, then the free energy of the doubly bound state DXP will be the sum of the individual binding energies.  The energies of the various bound states in this scenario are indicated in figure 3a. The only state from which transcription can proceed is the state DP. Applying the equilibrium prescription, we find that this state occurs with probabilityX and P bind independently, so the probability that state DP occurs is the probability that P is bound multiplied by the probability that X is not bound (the latter being given by (K1=K4=eεX− and K2=K3=eεP−.)Suppose now that the DNA region given by ). It is A and B, to which X can bind in any order (b). Let the free energies of the two singly bound states be εA and εB, and that of the doubly bound state be εAB=εA+εB+ΔεAB. These assumptions correspond to the most general situation, of which the following are special cases: if the two sites are identical, then εA=εB; if X binds independently to these sites, then ΔεAB=0. The energy term ΔεAB corresponds to some interaction between the two bound copies of X. If the binding of a single molecule makes it more favourable for another to bind, a condition referred to as positive cooperativity, then ΔεAB<0. Conversely, in a situation of negative cooperativity, ΔεAB>0, and the binding of one molecule interferes with the ability of the other to bind. Positive cooperativity is the norm among transcription factors that act multiply. Let us see what effect this will have. Assume, for simplicity, that |εA|∼|εB|≪|ΔεAB|. In the kinetic framework, this corresponds to K1≪K2, and K3≪K4, with the detailed balance condition again as shown in . In the literature, as a first approximation, binding probabilities are often parametrized as Hill equationsn being the Hill coefficient (a measure of cooperativity) and [X0] being the half-saturation concentration. The hyperbola (n=1) and the sigmoid (n=2) can both be parametrized in this way (c). These parameters, among many others, are required to provide a detailed biochemical description of any genetic network.In many instances, transcription factors bind to multiple sites. Suppose the promoter in question contains two sites, ny order b. Let thshown in . We find]=1/K1K2 c. In theyperbola  (with n=this way c. These 3.(a)lac operon, which is regulated by both a repressor and an activator  evolves asThe rate of protein creation per promoter, nP/V can therefore vary by as much as a factor of two over the cell cycle. We will usually ignore this variation, assuming the promoter concentration to be constant, and absorbing it into the quantity αi. We will also assume that cell volume grows exponentially, so V (t)∝eγt. The growth rate γ is related to the cell doubling time TD as γ to give the net decay rate γi. Protein degradation and dilution might themselves depend on the concentrations of some subset of proteins present in the system .,33.32,33ologists –36: negaologists ,38; posiologists ,39–41; aologists ,42–44.F(b)Y and is regulated by an activator X (a). Cooperative interactions can result in a Hill-type dependence of the gene expression level on the activator concentration. Setting x=[X] and y=[Y ],x is measured in units of the half-saturation concentration  and timare with ). The vab). Assume that the protein is a repressor that behaves as shown in (x at which the rate of creation f(x) and the rate of destruction g(x) balance one another. We see from figure 6a that the negative-feedback system settles into a steady state intermediate between 0 and A (something that cannot be captured in a pure binary description). If the expression level of the system is transiently increased above this steady state, the resulting drop in the creation rate quickly restores equilibrium. In fact, the auto-repressed system equilibrates more rapidly than an unregulated system with the same steady state, as shown in figure 6a; this has the effect of suppressing stochastic fluctuations [Rapid equilibration and noise reduction by negative feedback: consider what happens when a gene negatively regulates its own expression b. Assumeshown in :4.3Thetuations .Figure b). This can be achieved by closing the loop in (b). In the continuous model, this would correspond to having multiple values of x at which the rates of creation and destruction balance one another. For hyperbolic activation (n=1), we find just one stable expression state. However, for sigmoidal activation (n>1), the system can have two stable states, separated by an unstable state that forms a threshold (b). Trajectories that begin above this threshold are driven to the high state, whereas those that begin below the threshold are driven to the low state. The behaviour of the system therefore depends on its history, a phenomenon known as hysteresis. Suppose that we begin with a group of cells in the low expression state, then fully induce expression in some of these cells by means of an external signal such as a morphogen. Even once this signal is removed, the induced cells will maintain their high-expression levels. The positive-feedback network thus forms the basis for cellular memory, allowing cells of identical genotype to achieve different phenotypes depending on the external signals received.Memory and bistability by positive feedback: we next allow the gene to positively regulate its own expression b. This c loop in :4.4We ctivated b. In thehreshold b. Trajecc). In the context of electronics, such systems are known as flip–flops. The binary version of this system is capable of maintaining two distinct internal states: if we choose one gene to be active, then the other must be inactive. In terms of concentrations,u=0, along which dx/dt=0, and v=0, along which dy/dt=0. The fixed points or steady states of the system occur where these curves, known as nullclines, intersect. Once again, we must ask of each fixed point whether it is stable or unstable. In this case, a graphical analysis shows that, for n=1, the system has a single stable fixed point along the diagonal x=y (c). For n>1, this symmetric fixed point becomes unstable, and two asymmetric stable fixed points are created, one corresponding to high x-expression, and the other to high y-expression (d). As in the case of the positive feedback network, the flip–flop provides a mechanism for cellular memory.Memory and bistability with a flip–flop: a pair of genes that repress one another is similar to a single gene that activates itself c. In theonal x=y c. For n>pression d. As in d). In a sense, this is an extended version of a negative feedback circuit we saw previously, and the binary model predicts that it should oscillate. Importantly, because the feedback now comes with a delay, oscillations can be shown to occur in the corresponding continuous system as well. Consider the following activator–repressor pair:x is slow to respond to changes in y, this fixed point is stable and any oscillations are damped (a). However, if x responds sufficiently rapidly, the fixed point becomes unstable, and the system enters a sustained limit-cycle oscillation (b). Hysteretic oscillators of this kind are known to form the molecular basis for circadian rhythms and other types of periodic phenomena in living cells [Hysteretic oscillator: we again look at a system of two genes, but now one of them is an activator, while the other is a repressor d. In a se damped a. Howeveillation b. Hysterng cells .Figure e). The binary system is clearly oscillatory. The continuous analogue may be specified asi=0 is identified with i=3. The system has a symmetric fixed point xi=x0. For sufficiently high n, this fixed point can become unstable, forcing the system into a limit-cycle oscillation (c).Ring oscillator: finally, let us consider a system with three genes, each repressing the next in sequence e. The biillation c.5.(a)N distinct regulatable promoters, each of which has binding sites for up to M distinct transcription factors. In addition, we are given Next promoters whose transcriptional outputs can be controlled using extracellular signals. Each promoter can be made to express one or more transcription factors; the same transcription factor might be expressed by multiple promoters, in which case its total level is obtained by summing. We assume that the levels of all transcription factors can be measured. To simplify the discussion, we discretize the system so that all the inputs and outputs can take on any one of the states x∈{0,1,…,Ω−1} with inputs saturating at the maximal level. Reasonable values of these quantities are N, Next approximately 2–10, M∼2–5 [Ω∼10.Suppose we are given 0, M∼2–5 , and Ω∼1ΩM distinct inputs. For each promoter i, let this information be summarized as a function αi. The set {αi|i=1,…,N} represents the biochemical specification of the system. There are ΩΩNM possible biochemistries .A promoter is specified by defining its response to N+Next promoters is driving each of the M inputs of a given promoter. The M×(N+Next) connectivity matrix for promoter i has the formj and k run over inputs and promoters, respectively; and each entry can take on values 0 or 1. The set {Ci|i=1,…,N} represents the topological specification of the system and there are approximately 2NM(N+Next) possible topologies (ignoring degeneracies). Notice that the biochemical space explodes much more rapidly than the topological space.We next turn to topology, which involves specifying which of the N′ext≤Next external inputs, and therefore can be put into ΩN′ext configurations. How completely can we probe the biochemistry of such a system? To get a rough idea, let us make the following simplifying assumptions: for each external configuration, the feedback system achieves a unique steady state; and as we cycle through configurations, a given promoter cycles through a random sample (with repeats) of its ΩM possible states. The probability that a given state is missed over ΩN′ext samples is N′ext<M our sampling is extremely sparse; if ΩM samples our fractional coverage is (1−1/e).Consider a feedback network constructed with some complicated Next≥M, we can choose to construct a synthetic genetic network with the trivial feed-forward architecture :5.2whi et al. . For N′e(b)I; this enzyme generates a molecule known as acyl-homoserine lactone (AHL) that can diffuse freely between cells. Cells ‘receive’ this signal using a transcription factor designated R; when R is bound to AHL it functions as an activator, driving transcription at a promoter henceforth designated pX. The capability of I/R systems to issue and receive signals can have a variety of uses [I/R quorum-sensing systems are interesting because they are invariably found in a particular positive-feedback configuration: the enzyme I is expressed downstream of the R-dependent promoter pX [There are several natural contexts in which bacterial cells in a population stand to benefit by coordinating their actions . Many ba of uses . Because of uses . Topologmoter pX ,52.FiguI/R systems has been reported previously [ρ; the concentration ϕ of AHL in the medium; and the intracellular concentrations YI and YR of the enzyme I and transcription factor R. AHL levels will be proportional both to the enzyme levels and to cell density: ϕ(t)=μρ(t)YI(t). The transcriptional output of promoter pX is a function of instantaneous AHL and R levels. This biochemistry is summarized:pA and pB, the system can be wired into the following topologies:pX. If the proteins I and R have translation rates QI, QR and decay rates γI, γR, respectively, the feedback systems are described by the following differential equations:αI and αR are control parameters: transcription rates that are constant in time but whose values can depend on external inputs; the function αX embodies the frozen biochemical parameters; and the structure of the equations indicates the feedback topology. There are evidently two reasons why the responses of R-feedback and I-feedback systems might differ. The first is biochemical: the promoter logic αX is an asymmetric function of its two inputs YI and YR (a). The second is structural or topological: the input YI is multiplied by the cell density, whereas the input YR is fed in directly  causing these two variables to influence the dynamics in completely distinct ways.A computational and experimental characterization of eviously . We revie format  specify  and YR a. The sedirectly b,c causiYI and YR as functions of ρ. Under positive feedback, two distinct classes of responses can arise (a). For monostable responses , transcription increases smoothly with cell density. For bistable responses , there is a range of cell densities over which two stable transcription levels coexist. For each topology, a bifurcation analysis can be used to obtain regions of parameter space that give rise to the different response types [Figure 10b shows a two-dimensional slice of the parameter space: a biochemical parameter n  is varied along the x-axis; the control parameters αI or αR are varied along the y-axis. We see that the R-feedback topology is constrained: it is restricted to a single response type independent of the regulator level, once biochemical parameters are frozen. However, the I-feedback topology is versatile: it can be tuned between smooth and abrupt density-dependent response types by varying the regulator alone. This versatility might underlie the observed preference for I-feedback systems among diverse bacterial species: an organism that is able to rapidly modify its response in the face of an uncertain and fluctuating environment gains a crucial fitness advantage. Versatility is a purely topological property of the system, made without reference to specific biochemical parameter values.If cell density varies slowly compared with intracellular protein concentrations, equation  can be san arise a. For mose types , support6.a). Control parameters (such as the transcription rates αI or αR) are the software: they can respond directly and dynamically to external inputs, and vary on time scales from minutes to hours. Biochemical parameters (such as the Hill coefficient n) are the firmware: they can be changed incrementally by mutations, infrequent events that might become fixed in a population only over hundreds of generations. Network topology is the hardware: it is possible to switch topology but this requires rare, potentially disruptive, large-scale DNA rearrangements. The topological hardware and biochemical firmware are essentially frozen, leaving only the regulated software to vary freely at short time scales.There are three types of changes that can be used to modulate the response of genetic networks, operating on completely distinct time scales a. Controa priori distribution of parameter values that are likely to occur in nature. It is therefore impossible to estimate or compare the volumes of regions in parameter space that give rise to any set of specified behaviours . Even in this situation, topology provides a useful organizing framework. Consider the region of parameter space of some genetic network associated with some desired behaviour. If this region in the case of topology-II is completely contained within that in the case of topology-I, then we can be certain that the topology-I is more likely to generate the desired behaviour, without knowing anything about the likelihood of occurrence of parameters (figure 11b). The analysis thus generates a partial ordering among topologies independent of the actual biochemistry, and suggests a means to search the space of all possible topologies for interesting networks. Searching through topologies in this manner might be the only approach possible if the very existence of certain interactions is in doubt. For each topology, we would scan over parameter values to identify the range of possible behaviours. It could be the case that several topologies are consistent with some desired outcomes. In that case, it might be necessary to add additional biologically relevant constraints: robustness to parameter variation; adaptation to external changes; power consumption efficiency; and so on. The approach of searching topological space with constraints is emerging as a powerful means to understand the design principles of complex genetic networks in the absence of detailed biochemical data [When studying natural genetic networks, the approach to take depends on the extent of available data. If topology is known and key parameters identified, we can use experimental measurements to constrain as many parameters as feasible. Of the remaining parameters we can try to identify a few that are expected to be critical, and investigate all possible system behaviours as their values are varied. This approach is incomplete, however, because of a further unknown that is often ignored: we rarely, if ever, know the cal data –58.We might soon achieve a nearly complete understanding of certain simple organisms through a systematic analysis of the networks that govern their behaviour. Eventually such techniques might even give us predictive power, allowing us to guess at the inner workings of organisms based solely on the annotated sequences of their genomes. However, on very long time scales, the structure of a network must itself be dynamic: natural selection can be thought of as driving a search through topological space, converging on network architectures that generate biologically useful outcomes ,60. As m


With this in mind, we sought to identify the Bmp3 promoter in mouse (M. musculus) through functional and comparative genomic analyses. We found that the minimal promoter required for expression in resides within 0.8 kb upstream of Bmp3 in a region that is highly conserved with rat (R. norvegicus). We also found that an upstream region abutting the minimal promoter acts as a repressor of the minimal promoter in HEK293T cells and osteoblasts. Strikingly, a portion of this region is conserved among all available eutherian mammal genomes (47/47), but not in any non-eutherian animal (0/136). We also identified multiple conserved transcription factor binding sites in the Bmp3 upstream ECR, suggesting that this region may preserve common cis-regulatory elements that govern Bmp3 expression across eutherian mammals. Since dysregulation of BMP signaling appears to play a role in human health and disease, our findings may have application in the development of novel therapeutics aimed at modulating BMP signaling in humans.The Bone Morphogenetic Protein (BMP) pathway is a multi-member signaling cascade whose basic components are found in all animals. One member, BMP3, which arose more recently in evolution and is found only in deuterostomes, serves a unique role as an antagonist to both the canonical BMP and Activin pathways. However, the mechanisms that control BMP3 expression, and the  Drosophila, SMA in C. elegans). Strikingly, this basic mechanism is highly conserved across all animals The Bone Morphogenetic Protein (BMP) pathway is a signaling cascade that has ancient origins in the evolution of animals, arising 1.2–1.4 billion years ago Although the first observation of BMP activity in mammals was its ability to induce ectopic bone formation Drosophila, respectively) share dramatic identity, BMP3 is highly divergent from other BMP ligands in that it falls into an intermediate phylogenetic clade between TGF-β/Activin and BMP ligands in vivo analyses suggest that BMP3 serves an inhibitory function. For instance, while BMP ligands promote osteogenesis Bmp3 knockout mice have high bone mass, indicating that BMP3 acts as a negative regulator of osteogenesis in vivoXenopusArising more recently in evolution, the BMP ligand BMP3 serves a unique function by antagonizing the canonical BMP and Activin pathways. Homologs of BMP3 have only been identified in deuterostomes, but are present as early as echinoderms  through functional and comparative genomic analyses. Having found that the minimal promoter resides within 0.8 kb upstream of Bmp3 in M. musculus, we also identified a highly conserved element (ECR) upstream of the homologous Bmp3 locus in every available eutherian mammal genome but not in any non-eutherian animal. We determined the minimal ECR that is present in all reference eutherian mammal genomes and identified the transcription factor binding sites conserved between M. musculus, rat (R. norvegicus), and human (H. sapiens). Collectively, our findings suggest that the high level of conservation of the Bmp3 upstream ECR may preserve common cis-regulatory elements that govern Bmp3 expression across eutherian mammals.Despite significant progress in distinguishing BMP3 as a unique inhibitory ligand among BMPs, the mechanisms that regulate BMP3 expression are unclear. For instance, BMP3 exhibits a restricted expression pattern Bmp3 in M. musculus were generated from M. musculus genomic DNA using primer pairs as detailed in Bmp3 minimal promoter, genomic fragments contained the first 63 nt of Bmp3 exon 1 in order to include the annotated Bmp3 transcription start site. For directional cloning into pGL4.14 (Promega), which is a promoter-less plasmid that encodes firefly luciferase, or pGL4.26 (Promega), in which firefly luciferase is under the control of the herpes simplex virus Thymidine Kinase minimal promoter, 5′ XhoI and 3′ HindIII restriction enzyme cut sites were appended to the genomic fragment by PCR. For directional cloning into pJL114, in which firefly luciferase is controlled upstream of the Bmp3 minimal promoter in pJL114, 5′ SacI and 3′ XhoI restriction enzyme cut sites were appended to the genomic fragment by PCR. Ligation was performed using DNA Ligation Kit (Takara) at 16°C for thirty minutes and transformed into OneShot TOP10 E. coli (Invitrogen) using the manufacturers′ protocol.A series of plasmids containing fragments from the region upstream of HEK293T, UMR-106, and C2C12 cells were obtained from ATCC; primary calvarial osteoblasts were isolated from newborn wild type mice as described by Owen & Pan Taq polymerase (NEB) according to the manufacturer's protocol. RT-PCR primers were designed to be complementary to M. musculus, R. norvegicus, and H. sapiens and cross exon boundaries .For RT-PCR analyses, cells were scraped into PBS, centrifuged for 5 min at 500 x g at 4°C, the PBS aspirated, then cells were lysed and RNA collected using the RNeasy Mini Kit (QIAGEN) according to the manufacturer's protocol. cDNA was synthesized using the Transcriptor First Strand cDNA Synthesis Kit (Roche) according to the manufacturer's protocol. Newborn mouse hind limb RNA  served as a positive control. PCR on cDNA was performed using OneLuciferase activity was assayed using the Dual-Glo Luciferase Assay System (Promega). Cells were seeded at 3 k/cells per well in a 96-well plate. The next day, a plasmid encoding Renilla luciferase  and test plasmids driving firefly luciferase were co-transfected into cells using XtremeGENE (Roche). After 48-hours, firefly and Renilla luciferase activity was quantified using a luminometer ; expression of firefly luciferase under the control of the CMV promoter served as a positive control. Experiments were performed in triplicate or greater and are expressed as mean±SEM firefly luciferase/Renilla luciferase ratio. Data were normalized to the promoter-less (pGL4.14) or Thymidine Kinase minimal promoter (pGL4.26) control firefly luciferase plasmids.Bmp3 upstream minECR was constructed using the Los Alamos National Laboratory's Simple Consensus Maker (http://www.hiv.lanl.gov/content/sequence/CONSENSUS/consensus.html) using “Output aligned” parameter. For identification of transcription factor binding sites, DNA sequences were first aligned using zPicture DNA sequences were aligned using BLASTN post hoc Newman-Keuls correction for multiple pairwise comparisons using GraphPad Prism. A p value of <0.05 was considered significant.Statistical significance was determined by One-way ANOVA with M. musculus Bmp3 promoter, we first used ECR Browser Bmp3 transcription start site between M. musculus and the closely related R. norvegicus. This revealed a high level of nucleotide identity (≥75% across sliding 100 nt window) in the approximately 1.9 kb region proximal to the annotated transcription start site of M. musculus Bmp3 .To identify the lus Bmp3 . Within M. musculus and R. norvegicus, we predicted that the M. musculus Bmp3 promoter would reside within the conserved region that abuts the Bmp3 transcription start site instead of farther upstream in the poorly conserved region. We established a reporter system using HEK293T cells wherein firefly luciferase expression is controlled by fragments from the putative M. musculus Bmp3 promoter. We first confirmed that HEK293T cells express Bmp3 basally  identified binding sites for a number of general  and pathway-specific transcription factors , many of which are conserved with R. norvegicus  between the distal and proximal blocks  that regulates the expression of Bmp3. However, the overall level of conservation between M. musculus and R. norvegicus is too high to allow us to make this conclusion. For this reason, we extended our nucleotide conservation analysis by performing pairwise alignments of the Bmp3 upstream regions between M. musculus and more distantly related species. We were unable to use ECR Browser for these analyses as the species that are aligned to M. musculus in this database are limited. Instead, we performed alignments using BLASTN in sequential 500 nt sections from M. musculus against the full-length 5 kb region upstream of Bmp3 from the other species. This approach also allowed for the possibility of genomic insertions or deletions. To ensure the fidelity and accuracy of these analyses, we focused our attention on the thirty-nine NCBI Reference Sequence (RefSeq) animal genomes, at least twenty-eight of which contain an annotated Bmp3 or Bmp3-like gene.The high degree of identity in the distal block/IvS between M. musculus was found among the other two muroideans, R. norvegicus and Chinese hamster (C. griseus)  . Strikinmusculus . This paanatinus , nor in M. musculus and R. norvegicus upstream of Bmp3 is an ECR among eutherian mammals. To test this directly, we first aligned the region upstream of Bmp3 in M. musculus and H. sapiens using ECR Browser M. musculus spanning from position −1642 to −1138 upstream of the Bmp3 transcription start site that is within the distal block conserved between M. musculus and R. norvegicus .These findings suggested that all or a portion of the distal block conserved between rvegicus . The absBmp3 CoreECR, we generated plasmids in which portions of the repressive distal block/IvS region were placed upstream of the Bmp3 minimal promoter. This revealed that the CoreECR is as effective as the complete ∼1 kb region in its ability to repress the Bmp3 minimal promoter  eutherian mammals .We then used BLASTN to align the  mammals ; in eachM. musculus lies upstream of Bmp3 in each eutherian mammal in the RefSeq genome database. However, the total length and identity of the Bmp3 upstream ECR varies (Bmp3 upstream ECR (minECR) with a consensus length of 297 nt , which comprise >73,580,051 sequences. This failed to identify any EST with significant alignment to the consensus Bmp3 upstream minECR- the highest identity was 85% over 59 nt . For comparison, exon 1 of M. musculus Bmp3 readily aligned to multiple ESTs from M. musculus and other species .Additionally, we confirmed that the Bmp3 upstream ECR is a cis-regulatory element unique to eutherian mammals. However, since the number of RefSeq genomes is fairly small at present, we extended our investigation to species for which a RefSeq genome is not available. We used BLASTN to align the consensus Bmp3 upstream minECR to the whole-genome shotgun sequence database, which comprises 191 animal species. This revealed significant conservation of the Bmp3 upstream minECR in thirty-one of thirty-nine eutherian mammals that were not represented by the RefSeq cohort .Our findings suggest that the q cohort ; notablycis-regulatory role(s) played by the Bmp3 upstream ECR. To do so, we first used rVista 2.0 to identify the transcription factor binding sites (TFBSs) in the consensus Bmp3 minECR sequence  are present in the consensus minECR sequence . This revealed that all of the TFBSs within 3.8 kb upstream of Bmp3 that are conserved across the RefSeq rodent genomes (9/9) reside within the limits of the minECR and are present in the consensus minECR sequence . These findings indicate that the consensus minECR could serve as a valid predictor of highly conserved cis-regulatory elements that govern Bmp3 expression.Finally, we sought to validate the evolutionary conservation of the ista 2.0 . This re CoreECR . Moreovesequence . For seccis-regulatory elements controling Bmp3 expression in M. musculus. Functional characterization of various genomic fragments revealed the 0.8 kb region proximal to the annotated M. musculus Bmp3 transcription start site to be the minimal promoter in HEK293T cells, UMR-106 osteosarcoma cells, and primary calvarial osteoblasts. This region corresponds to a highly conserved block (≥80% identity across a sliding 100 nt window) that is shared between M. musculus and R. norvegicus and contains binding sites for a number of both general and pathway-specific transcription factors. We analyzed the 5 kb upstream region and exon 1 of Bmp3 from M. musculus using Neural Network Promoter Scan Bmp3 expression. Our findings are consistent with a previous report that attained promoter activity using the 2 kb region upstream of Bmp3 in R. norvegicusIn the present study, we took a comparative genomics approach to identify potential Bmp3 minimal promoter is poorly conserved between M. musculus and H. sapiens, although this finding is consistent with a previous report comparing the promoter for Bmp3b  between M. musculus and H. sapiensBmp3b likely arose from duplication of the Bmp3 gene, or vice versa Bmp3 and Bmp3b in M. musculus failed to demonstrate any significant nucleotide identity , suggesting that Bmp3 and Bmp3b have evolved unique mechanisms regulating their expression. This idea is supported by the fact that the spatio-temporal expression domains of Bmp3 and Bmp3b differ quite drastically We were surprised to find that the M. musculus and R. norvegicus as the minimal promoter, we turned our attention to the remaining highly conserved region (−1.9 kb to −0.8 kb upstream of Bmp3). Interestingly, appending this region to the minimal promoter repressed promoter activity. Fidelity of the genomic fragment in this reporter plasmid was confirmed by bi-directional sequencing and our finding was consistent in each repetition of our assay, leading us to conclude that this region is capable of repressing basal Bmp3 expression in each cell type tested. This is supported by the fact that, when we examined potential TFBSs in this region by rVista2.0 After identifying the proximal block conserved between M. musculus and each of the other thirty-nine complete RefSeq genomes revealed that a portion of the distal block was conserved with every eutherian mammal (15/15), but not in any non-eutherian species (0/24) even though a Bmp3 or Bmp3-like locus has been annotated for at least thirteen non-eutherian species . We went on to narrow this conserved region to 297 nt that is shared between all eutherian mammals in the RefSeq cohort, and then found this minimal ECR in thirty-one additional eutherian mammals represented in the whole-genome shotgun sequence (WGS) database . As with the RefSeq database, we did not find the minimal ECR in any of the 136 non-eutherian animal genomes in the WGS database. Of note, genomic sequences are presently available from only four non-eutherian mammals . Thus, we conclude from our findings that the Bmp3 upstream ECR is a eutherian mammal-specific cis-element, but are aware that future studies are required to definitively show if this ECR is also found in metatherian or prototherian mammals.BLASTN alignment of the 5 kb regions between Bmp3 upstream minECR is a highly conserved genomic region near the minimal promoter that represses basal promoter activity. This arrangement is similar to the conserved, high GC-content short-range repressive elements that have been described near the Bmp2 promoter Bmp3 upstream minECR bears no alignment to these regions and the GC content is only 45% . To examine possible regulatory mechanisms, we examined the Bmp3 upstream minECR using rVista 2.0, which not only pinpoints consensus TFBSs using the TRANSFAC database but combines this information with sequence conservation analyses of the surrounding 20 nt to identify the most biologically relevant TFBSs M. musculus and H. sapiens upstream of Bmp3 reside within the limits of the minECR, and the majority of these are predicted to be high-affinity binding sites. Moreover, even among the more closely-related rodents M. musculus, R. norvegicus, C. griseus, and C. porcellus we found that all of the TFBSs within 3.8 kb upstream of Bmp3 (9/9) reside within the limits of the minECR .The cis-regulatory elements upstream of Bmp3 in mammals provides a foundation for future studies examining modulation of Bmp3 expression. In support of this, the TFBSs that we identified are highly consistent with what has previously been reported on the regulation of Bmp3. For instance, binding sites for HNF1, VDR, AP1, and NF-kappaB, all of which have been shown to regulate Bmp3 expression Bmp3 upstream minECR.Our identification of a highly conserved block of potential Bmp3 in regulation of bone formation. Similar to many osteogenic BMP ligands, Bmp3 is expressed in osteoblasts Bmp3 knockout mice have high bone mass Bmp3 promoter could determine mechanisms to reduce Bmp3 expression in diseases of low bone mass such as osteopenia and osteoporosis.Of particular interest to us is the role of Bmp3 promoter from M. musculus and determined that this region is highly conserved with R. norvegicus. We also found that a highly conserved upstream region abutting the minimal promoter is able to repress the minimal promoter. A portion of this region is conserved among all available eutherian mammal genomes (47/47), but not in any non-eutherian animal (0/136). We also identified multiple conserved transcription factor binding sites in the Bmp3 upstream ECR. Collectively, these findings suggest that the high level of conservation of the Bmp3 upstream ECR may preserve common cis-regulatory elements that govern Bmp3 expression across eutherian mammals.We identified the minimal Figure S1Bmp3 expression analysis. RT-PCR for Bmp3 in HEK293T cells (A), UMR-106 cells (B), primary mouse calvarial osteoblasts, and C2C12 cells 9D) compared to Hprt housekeeping control. Newborn mouse hind limb cDNA was used as a positive control in all experiments (only shown in A). Intervening lanes from a single gel removed in A (indicated by white bar).(TIFF)Click here for additional data file.Figure S2Bmp3 upstream ECR (minECR) present in all RefSeq mammals.Determination and individual nucleotide conservation of the minimal  BLASTN (Version 2.2.26+) was used to align the M. musculus: H. sapiens CoreECR sequence to all sixteen available eutherian mammal RefSeq genomes in order to identify the Bmp3 upstream ECR shared with M. musculus. Each full-length ECR was then aligned using MUSCLE in MEGA5 Bmp3 upstream ECR (minECR) with a consensus length of 297 nt (319 nt as shown when including insertions found in some species). The consensus sequence was determined using Los Alamos National Laboratory's Simple Consensus Maker. Uppercase letters in the consensus sequence indicate 100% conservation, while lowercase letters indicate the majority nucleotide. “n” indicates no consensus nucleotide could be determined and “.” indicates a gap. For each individual species, a dash indicates a match to the consensus, while “A, T, C, or G” indicates a mismatch to the consensus. The mean identity to the consensus minECR is 90% . A partial match to the SINE2-type repeat MIRb, found primarily in primates, is denoted in red.(TIFF)Click here for additional data file.Table S1Primers used for firefly luciferase reporter plasmid construction.(XLSX)Click here for additional data file.Table S2M. musculus Bmp3.Transcription factor binding sites upstream of (XLSX)Click here for additional data file.Table S3Bmp3 5 kb upstream regions between M. musculus and other mammals.Nucleotide alignments of the (XLS)Click here for additional data file.Table S4Bmp3 upstream Evolutionary Conserved Region (ECR) in RefSeq animal genomes.Conservation analysis of the (XLS)Click here for additional data file.Table S5Bmp3 upstream Evolutionary Conserved Region (minECR) in all available animal genomes.Conservation analysis of the minimal (XLS)Click here for additional data file.Table S6Bmp3 upstream ECR and conservation in Rodents/Primates.Transcription factor binding sites in the consensus minimal (XLSX)Click here for additional data file.Table S7M. musculus and H. sapiens in the region upstream of Bmp3.Transcription factor binding sites conserved between (XLS)Click here for additional data file.