The small non-histone protein Heterochromatin protein 1a (HP1a) plays a vital role in packaging chromatin, most notably in forming constitutive heterochromatin at the centromeres and telomeres. A second major chromatin regulating system is that of the Polycomb/trithorax groups of genes which, respectively, maintain the repressed/activated state of euchromatin. Recent analyses suggest they affect the expression of a multitude of genes, beyond the homeotics whose alteration in expression lead to their initial discovery.Drosophila development, HP1a collaborates with the Polycomb/trithorax groups of proteins to regulate gene expression and that the two chromatin systems do not act separately as convention describes. HP1a affects the levels of both the Polycomb complexes and RNA polymerase II at promoters, as assayed by chromatin immunoprecipitation analysis. Deposition of both the repressive (H3K27me3) and activating (H3K4me3) marks promoted by the Polycomb/trithorax group genes at gene promoters is affected. Additionally, depending on which parent contributes the null mutation of the HP1a gene, the levels of the H3K27me3 and H3K9me3 silencing marks at both promoters and heterochromatin are different. Changes in levels of the H3K27me3 and H3K9me3 repressive marks show a mostly reciprocal nature. The time around the mid-blastula transition, when the zygotic genome begins to be actively transcribed, appears to be a transition/decision point for setting the levels.Our data suggest that early in We find that HP1a, which is normally critical for the formation of constitutive heterochromatin, also affects the generation of the epigenetic marks of the Polycomb/trithorax groups of proteins, chromatin modifiers which are key to maintaining gene expression in euchromatin. At gene promoters, deposition of both the repressive H3K27me3 and activating H3K4me3 marks of histone modifications shows a dependence on HP1a. Around the mid-blastula transition, when the zygotic genome begins to be actively transcribed, a pivotal decision for the level of silencing appears to take place. This is also when the embryo organizes its genome into heterochromatin and euchromatin. A balance between the HP1a and Polycomb group silencing systems appears to be set for the chromatin types that each system will primarily regulate.The online version of this article (doi:10.1186/s13072-015-0010-z) contains supplementary material, which is available to authorized users. Nucleosomes package DNA into the basic unit of eukaryotic chromatin. They are repositioned or evicted by ATP-dependent chromatin remodeling enzymes or altered by covalent modification of their histone tails to mark the chromatin for activation or repression. Against this backdrop of chromatin states, gene expression is regulated and development and differentiation proceed.While nucleosomes define the basic unit, at the genome level chromatin is frequently viewed as being organized into two major forms: euchromatin which is generally associated with gene-rich regions and the chromatin is less condensed, whereas heterochromatin is highly condensed, late replicating, and mostly transcriptionally silent throughout the cell cycle. It is generally gene poor and is associated with telomeres, centromeres, and pericentric regions.Drosophila primarily Su(var)3-9 and Setdb1) to promote H3K9 methylation and facilitate the deposition and spreading of the mark (reviewed in [Drosophila). It is well known for its role in gene silencing and modifies position effect variegation in a dose-sensitive manner (position effect variegation (PEV); the process of gene silencing through the spread of heterochromatin, usually detected in rearranged chromosomes or transgene insertions). More recent studies, however, suggest HP1 also affects the transcription of euchromatin genes and can act positively [A key component and contributor to the constitutive heterochromatin at telomeres and centromeres is heterochromatin protein 1 (HP1). HP1 is a small non-histone protein with two conserved domains, an N-terminal chromodomain which binds chromatin by recognizing methylated lysine 9 on histone H3 (H3K9) and a chromoshadow domain which enables HP1 to dimerize and bind to a variety of proteins through a degenerate pentapeptide motif, PxVxL [iewed in ). HP1 issitively ).Drosophila melanogaster as necessary for maintaining either the repressed/activated state, respectively, of the homeotics, genes which give segments their identity so patterning the body axis. Since their initial characterization, further analyses suggest that their function and regulation is widely conserved across metazoans. Additionally, high-throughput genome-wide techniques suggest that besides the homeotics, several hundreds, perhaps thousands of genes may be regulated by the PcG/trxG genes. Many of their targets appear to be essential developmental regulators [A second major chromatin regulating system is that of the Polycomb/trithorax groups (PcG/trxG) of genes. PcG/trxG genes were first identified in gulators ,6.Drosophila; Eed in mammals), and p55 (CAF1), which modulate the activity of the E(Z) methylase [Drosophila and the homologous mixed lineage leukemia group in vertebrates, which maintains the active state through tri-methylation of H3K4 (H3K4me3) at the promoters of active genes . PC itsiewed in ). ImplicDrosophila embryo, HP1a and the PcG proteins work together to regulate silencing and transcription. This interaction appears to be more acute during the early stages as later in development (larvae), HP1a appears to play less of a role in maintaining the levels of the PcG/trxG marks. Our data additionally suggest that the early Drosophila embryo has promoters which are bivalent with repressive H3K27me3 (PcG) and activating H3K4me3 (trxG) marks, both of which are dependent on HP1a. This dependence is also less clear cut in later stages. We speculate this effect of HP1a on the PcG and H3K27me3 levels may also occur in mammalian embryonic stem cells (ESC). We also find that as the early embryo organizes its genome into heterochromatin and euchromatin, the HP1/H3K9me and PcG/H3K27me3 systems appear to work together to both sense and partition the genome, sorting the chromatin type each will regulate. In the event of reduced maternal HP1a levels, the PcG system serves as a back-up resulting in an increase in H3K27me3 levels at heterochromatin, presumably to decrease the transcription of repetitive sequences which are derepressed by the reduction in HP1a. When H3K27me3 levels are low, there generally appears to be an increase in H3K9me3 levels, suggesting the two systems compensate for each other to achieve silencing. Collectively, these observations suggest the two chromatin systems work at multiple steps/stages to organize the genome.The HP1 and PcG/trxG pathways are thought to have diverged and acquired different functions in maintaining genome stability in multicellular organisms. We find, however, that the two chromatin systems do not act separately. In the early Drosophila sex determination, we found that HP1a plays both a repressive and activating role on the master switch gene, Sex-lethal .In studying al . The poUbx suppresses wing development to form halteres on the third thoracic segment of the fly. It is haploinsufficient and loss of one gene copy increases the haltere size, providing a sensitive readout of Ubx levels. Introducing a copy of the Su(var)2-505 null mutation (HP1a gene name) in heterozygotes of the Ubx130 loss of function allele worsened the phenotype making the haltere larger . This indicates a decrease in Ubx levels and that HP1a has a positive effect on Ubx expression, similar to what we observed at Sxl. It also demonstrates that a canonical PcG/trxG target is affected by HP1a. To confirm that the interaction was not from a background effect, a second allele (Su(var)2-502) was examined and a similar though slightly weaker effect was observed (not shown). Our previous analyses with additional Su(var)2-5 alleles  at Sxl also demonstrated Su(var)2-502 had a weaker effect.Su(var)2-505and Ubx130 . Introduction of absent small and homeotic disks 1 (ash1), a member of the trxG of chromatin modifiers, improved the haltere phenotype suggesting an antagonistic relationship. This was somewhat surprising, as ash1 normally promotes Ubx expression so its reduction should worsen the Ubx130 phenotype, which in the absence of the Su(var)2-505 mutation was the case . E(z)731 improved the haltere phenotype slightly, consistent with its repressive role. By contrast, Su(z)124, slightly enhanced the effect of Su(var)2-505 suggesting the two mutations promote each other’s effect, indicating a mild positive role of Su(z)12 on Ubx. The canonical role of SU(Z)12 in PRC2 is to promote the methylase activity of E(Z) so it should behave like E(Z), which was the case in the absence of the Su(var)2-505 mutation. This unexpected positive effect of Su(z)12 suggested by the Su(var)2-505Ubx130 phenotype could be from an additional role with HP1a as, unlike E(Z), SU(Z)12 also regulates heterochromatin and affects PEV [Ubx expression. The interactions are complex, uncovering unexpected changes in expression levels.To further corroborate this interaction between the two different chromatin silencing systems, we decreased the dose of other PcG/trxG genes simultaneously with ects PEV . Taken tSxl, Ubx, hairy (h), bottleneck (bnk), hunchback (hb) and twinstar (tsr)) were quantified in 2–3 h embryos from wild-type and Su(var)2-505 heterozygous parents , which is in keeping with the canonical role of HP1a as a repressor. However, prior in situ hybridization data had demonstrated that HP1a has a positive role on Sxl, with is reduction severely inhibiting Sxl expression [Sxl mRNA must arise from some of the embryos having higher than normal expression, to compensate for the embryos with poor to low expression. Indeed, the prior in situ hybridizations had suggested slightly higher than normal expression in close to 50% of the older embryos, primarily in cycle 14 but not cycle 13 embryos, suggesting a zygotic effect in the embryos which have a wild-type chromosome.The genetic interactions described above prompted us to examine whether HP1a regulates the transcription of other genes. The mRNA levels of six genes  2-505 parents were not equally affected. Three classes could be observed: one in which the expression is weaker, a second with near normal levels, and a third in which the expression is slightly stronger 2-505 embryos, the stronger class are Su(var)2-505 heterozygotes, and the normal looking embryos are homozygous for the balancer with two normal copies of Su(var)2-5 . Quantifying the embryos from the bnk in situ hybridizations was challenging as its expression changes within the cell cycle; however, comparisons of embryos at the same stage in the cell cycle showed some embryos with either weaker or stronger than wild-type signal  suggesting effects similar to those seen at Ubx, h, and Sxl. Together, these data suggest that (i) HP1a heterozygotes show an increase in expression, presumably from a decrease in repression while (ii) severely reducing/eliminating HP1a adversely affects expression. This dual effect emphasizes that HP1a has more than one role in the transcriptional regulation of euchromatin genes and is consistent with our previous observations of an activating and repressing role at Sxl [As ) Figure B. Unliker Figure B. We suse at Sxl .Ubx expression and it appears to repress several functionally different genes, we wondered if the PcG/trxG of chromatin modifiers were working together with HP1a. To answer this question, chromatin immunoprecipitation using antibodies specific for H3K27me3, followed by quantitative PCR (ChIP-qPCR), was performed on 1–3 h embryos from wild-type and Su(var)2-505 heterozygous parents , segmental patterning , sex determination, cellular membrane organization genes , and genes residing in facultative heterochromatin (concertina (cta) and light (lt)). sgs8, the salivary gland specific gene, represents non-expressed genes. All showed a significant reduction in their H3K27me3 levels in the Su(var)2-505 background 2-505 is very comparable to reducing the H3K27 methylase itself, E(Z), or its binding partner, SU(Z)12 2-505, indicating it is real.Given that HP1a is necessary for proper dentical -20. SecoSu(var)2-505 embryos show a decrease in H3K27me3, we tested whether the opposite mark promoted by the trxG, H3K4me3, was also affected. ChIP-qPCR was performed on 1–3 h embryos from wild-type and Su(var)2-505 heterozygous parents. Of the promoters tested, loss of HP1a significantly reduced H3K4me3 levels at nine genes 124 or E(z)731 parents did not show this consistent decrease in H3K4me3 , suggesting that HP1a is unique in this respect.As the Su(var)2-505embryos than wild-type . Second, qRT-PCR analysis to measure the levels of E(z) mRNA (and the two major H3K9 methylases) shows that there is more message in the Su(var)2-505embryos . Western blots of E(Z) protein show its levels are comparable to wild-type, however . This suggests that it is not the methylase but another activity which is limiting the deposition of H3K27me3. Third, when HP1a levels are reduced using RNA interference, we also find a decrease in H3K27me3 . Finally, introducing a copy of an RFP-HP1a transgene into the Su(var)2-505/CyO background improves or rescues the reduction in both H3K27me3 and H3K4me3 at almost all the promoters . The main exceptions were: for H3K27me3, hb and the heterochromatin genes (lt and cta); for H3K4me3, most indicated a rescue with some genes showing a slight overshoot, except for lt which decreased. The RFP-HP1 transgene was also effective at correcting the elevated levels of Ubx, h, and bnk mRNA in 2–3 h embryos . This suggests that the transcriptional effect is also due to a reduction in HP1a. .)That these effects are from the reduction of HP1a and not from the H3 methylases or a background effect is suggested by several controls . First, Western blot analysis on 1–2 h embryos shows that the levels of maternal HP1a protein are less in the Su(var)2-505genomic sequences to drive expression and is maternally expressed . It is unable to fully rescue the Su(var)2-505mutation to viability however, perhaps because the RFP, which is as large as HP1a itself, blocks the amino terminus of HP1a which may alter its normal transcriptional function. Nevertheless, the significant improvements we observe in the different effects of HP1a, including the repetitive elements with low H3K27me3 levels in the 1–3 h window , support the idea that the changes in the Su(var)2-505embryos are from a reduction in HP1a and not from changes in the histone methylases or some unknown background effect.The RFP-HP1a transgene uses Su(var)2-505 embryos , suggesting that while the promoter regions show a consistent reduction, the effect of HP1a may be local or that other regions in the genome have higher levels of H3K27me3 to offset the drop. To differentiate between these scenarios, the 3′ UTR regions of nine of the ten genes were scored. For Ubx and Sxl, the two largest genes, a region 1 kb downstream from the promoter was also scored. Different heterochromatic sequences were also examined - the 1360 repetitive element, TART and HeT-A telomeric elements, and F-element which is abundant in pericentric heterochromatin as well as other heterochromatic regions across the genome. As seen in Figure Sxl and Ubx, 1 kb downstream of the promoter. Three of the nine  showed an increase, while bnk did not show a change, indicating that these gene regions are not equally affected by the reduction in HP1a. For the repeated elements, 1360 and Het-A were reduced, while TART and F-element did not show a change , this balance is deciphered by the Sxl establishment promoter, SxlPe, to result in its transcription in only females . This afe cycle ,24 (revife cycle ).E(z), Su(z)12, and ash1 mutants were tested against a sensitized background of reduced female promoting X-linked genes (sisterless-a (sis-a), sisterless-b (sis-b)). In this setup, an additional mutation can either worsen or improve female viability depending on whether the gene acts, respectively, positively or negatively in the process . A reduction of ash1 strongly compromised female viability suggesting it facilitates SxlPe expression. This lethality was completely reversed by introducing a constitutive allele of Sxl, supporting the idea that ash1 is needed for Sxl activation and not its maintenance . Decreasing the levels of E(z) and Su(z)12 improved female viability suggesting they act negatively . This is consistent with the opposing activities of ash1 and Su(z)12/E(z) and the idea that PcG/trxG proteins regulate the sex determination decision.Sxl indicates the two chromatin modifying systems can strongly synergize. Mutation of Su(var)3-9 alone weakly enhances female viability suggesting it acts negatively, which is in stark contrast to the poor viability of females with reduced HP1a. However, for all genes, the presence of either a Su(var)3-9 or Su(z)12 mutation improved female viability . This cooperation between the two chromatin systems is most compellingly highlighted by the improvement in female viability when the dose of both Su(z)12 and Su(var)2-5 is halved, an improvement that is more effective than when the dose of Su(var)2-5 and Su(var)3-9 are halved. The interaction results were similar with different alleles of PcG genes , and the trends tended to match the allele strength, suggesting the results are not from background interactions but are related to the mutations themselves.Performing additional genetic interaction tests with Sxl were further analyzed by qRT-PCR for mRNA levels and by in situ hybridization. Reducing the PcG/trxG genes affected both the strength and timing of the SxlPe promoter . We also determined the levels of H3K27me3 and H3K4me3 at SxlPe. H3K27me3 was generally lower in ash1MB03235/TM6, Su(z)124/TM6, and E(z)731/TM6 embryos, compared to wild-type . Not unexpectedly, as they make up PRC2, Su(z)124 and E(z)731 had the stronger effect.The effects of the PcG/trxG genes on Sxl is a bona fide target of the PcG/trxG proteins despite the relatively low levels of their marks compared to more heavily modified genes such as Ubx and hb. We also note that cta, which has relatively low levels of H3K27me3 in embryos, shows significant levels of the mark in larvae as do bnk and nullo, supporting the notion that these genes are regulated by PRC2. The low and high levels of H3K27me3 at promoters additionally suggest at least two classes of targets, an idea consistent with the demonstration that genes such as Polycomb-like, which do not affect all PC targets [Ubx which are highly modified. This potentially explains the two classes we observe.These various analyses demonstrate that  targets , functioDrosophila [Sxl, the apparent bivalency would be from males having the repressive H3K27me3 and females the activating H3K4me3 mark. To determine whether the promoter is indeed bivalent, we analyzed an attached-X chromosome (X^X) stock  where the free X chromosome has a deletion of Sxl (SxlfP7BO). In this stock, only females have Sxl sequences so the signal is representative of only females. H3K27me3 levels at Sxl in this stock were relatively low, so most of the signal in wild-type embryos comes from males. However, both sexes have comparable levels of H3K4me3 . As SxlPe is never expressed in wild-type males [Pe is transcribed.The gene promoters we analyzed had detectable levels of both H3K27me3 and H3K4me3  were scored 12 have been shown to interact both in vitro and in vivo [Drosophila embryos. Indeed, immunoprecipitation (IP) experiments on extracts from 1–3.5 h embryos 2-505 heterozygous parents. With the exception of the lt promoter which was unchanged, all the promoters examined show a decrease in E(Z) levels suggesting HP1a facilitates the recruitment of PRC2 to promoters . PRC2 recruitment to the PRE element in bxd  was also negatively affected by the reduction in HP1a.As HP1a is in complex with PRC2 and its reduction decreases H3K27me3 levels, we tested whether it also affected PRC2 recruitment. ChIPs for E(Z) were performed on 1–3 h embryos from wild-type and Su(var)2-505 and Su(var)2-504, null and strong loss of function alleles, respectively. These larvae survive primarily on maternal HP1a until the third instar stage after which they die without pupating. ChIP-qPCR on wild-type 2-5 alleles are in this background) and Su(var)2-505/04 larvae was performed. Validating our protocol and results, the H3K27me3 signal for the Ubx promoter in wild-type was similar to that in other studies [hb, nullo, and tsr promoters were slightly lower but not significantly different from wild-type while Ubx was actually a little higher in the Su(var)2-505/04 larvae. The heterochromatic genes lt and cta showed a proportionately larger decrease in H3K4me3 than seen in embryos, consistent with the observation that HP1a is required for the robust expression of genes which naturally reside in heterochromatin (reviewed in [Su(var)2-505 so the marks in the Su(var)2-505/04 larvae should at first be comparable to embryos since both start development with approximately 50% maternal HP1a). The H3K27me3 levels in Su(var)2-505/02 larvae by contrast showed even smaller differences from wild-type , suggesting that the chromodomain defective protein from the Su(var)2-502 allele must provide enough activity to almost normalize this mark by the larval stage.As HP1a appears to be required for the deposition of both H3K27me3 and H3K4me3 at gene promoters in early embryos, we wished to determine whether this dependency persists later in development, where it is possible to further reduce the levels of HP1a using two defective alleles -  studies . Surprisiewed in ). The geSu(var)2-505, Pc15 or Su(z)124 where each mutation was inherited from a different parent. Additionally, trans-heterozygous mutants of Su(var)2-505 with either Pc15 or Su(z)124, where the parental origin of each mutation was reversed, were also generated. The three mutations are all strong loss of function, so they essentially reduce gene dose by half. H3K27me3 levels were measured at the promoters of three silenced  and two active genes 2-505 null allele affects the level of H3K27me3 detected in larvae. When the mother provides the mutation, H3K27me3 levels are actually higher than wild-type; however, when it is paternally inherited, they are lower. PC resembled HP1, where the maternal reduction showed lesser effects than the paternal ; however, unlike HP1, the levels did not exceed wild-type at any of the genes. In the case of Su(z)12, reducing its dose significantly reduced the H3K27me3 levels at silent genes regardless of the parent of origin, indicating that maintaining the silenced state is sensitive to the levels of PRC2 12 had a rather modest effect and for Ubx, which normally contains high levels of H3K27me3, there appeared to be no effect. This suggests that unlike silenced genes, genes which are being transcribed can compensate for the reduction in SU(Z)12 or are less sensitive to its reduction.The first point of note is that the parent which contributes the Su(var)2-505 with Pc15. The levels of H3K27me3 were generally higher than when PC alone is reduced, and for all the genes the parent-of-origin effect on H3K27me3 recapitulated the Su(var)2-505 trend: higher than wild-type if maternally inherited, lower if paternally inherited 2-505, the Pc15 mutation is almost invisible, demonstrating that the HP1a dose is epistatic to that of PC, and once again places HP1a upstream of PC. A significant parent-of-origin effect was not observed when Su(z)124 was combined with Su(var)2-505; the only exception was hb where the trend was the reverse of Su(var)2-505 alone, and the levels did not exceed wild-type.The second interesting observation came from combining We note the larvae being compared are genetically identical and only the parental source of the mutation is different. This implies that the amount of HP1a the mother deposits into the egg influences the perceived ‘set point’ for H3K27me3 levels at promoters. To have a parental effect, defining this set point must be a relatively early decision, as ultimately the zygote must transcribe its own genes and the parental source of a mutation should no longer come into play.Su(var)2-505mutation came from the mother, but were unaltered or lower (HeT-A) if the mutation came from the father, indicating a new H3K27me3 set point is also established for heterochromatic regions. Surprisingly, the Pc15 mutation tended toward slightly increasing H3K27me3 but, with the exception of the F-element, had no significant parent-of-origin effect 2-505and Pc15 showed the Su(var)2-505 parent-of-origin effect was maintained for 1360 and TART but erased for HeT-A and F-element, indicating that the various heterochromatic regions are less uniform in their response than euchromatin.A key event which occurs early in development is the formation of well-organized constitutive heterochromatin, beginning shortly before and extending into cellular blastoderm . This prSu(var)2-505/CyO parents over sliding time windows . In 1–3 h embryos, a window which precedes heterochromatin formation, promoters in embryos from Su(var)2-505 heterozygous parents have lower than wild-type H3K27me3 levels 2-505 heterozygous parents and heterozygous Su(var)2-505 larvae with different parents of origin. H3K9me3 was scored as it has been reported to track similarly to H3K9me2 [The histone modification normally associated with HP1a is methylation of H3K9. This prompted us to determine whether the levels of H3K9 methylation also change when H3K27me3 levels are altered. The same regions were analyzed in embryos and larvae; the heterochromatin transition windows of 2–4 and 3–5 h in embryos from  H3K9me2 .SetDB1 and Su(var)3-9, shows them to be slightly higher than wild-type in the 2–3 h window but close to wild-type in the 3–5 h window , so their expression level is unlikely to be responsible for the lower H3K9me3 levels in the 3–5 h window.Repetitive regions which are clearly transcriptionally derepressed Figure C, surprinullo promoter which remained above wild-type but proportionately less so than in the earlier time window).The reciprocity between the levels of H3K27me3 and H3K9me3 is better upheld at euchromatin gene promoters 2-505 is inherited from the mother, H3K9me3 levels are significantly lower than wild-type at the heterochromatic repeat regions 2-505 mutation is inherited from the father, there is no significant change in H3K9me3 levels compared to wild-type, as for H3K27me3 (HeT-A was the only exception with lower H3K27me3). Regardless of the parent of origin, inheriting one copy of Su(var)2-505 derepresses the repetitive elements 2-505), suggesting that, as seen in embryos, these chromatin marks cannot fully compensate for the reduction in HP1a. For the euchromatin gene promoters, we generally also find that when H3K27me3 levels are higher 2-505), H3K9me3 levels are lower and, conversely, when H3K27me3 levels are lower 2-505), H3K9me3 levels are higher .Drosophila embryos, HP1a and PcG/trxG proteins regulate gene expression in a collaborative manner. HP1a is needed for the normal deposition of both H3K27me3 and H3K4me3 at several promoters 2-505/CyO background restored both H3K27me3 and H3K4me3 at most of the promoters we examined, supporting the conclusion that HP1a impacts the deposition of both marks .Our data suggest that in early Drosophila embryos have bivalent promoters . These data indicate the promoter has both H3K27me3 and H3K4me3 in males, where it is never transcribed. Additional support for bivalency comes from the observation that both marks show a dependence on HP1a. HP1a is found at all the promoters and interacts with the members of PRC1 and PRC2. This interaction, with both the PcG and trxG complexes, presumably leads to the methylation of histone H3 at lysines 4 and 27. Consistent with this proposal, PRC1 and TRX-C proteins have been shown to co-occupy many gene promoters in Drosophila [The presence of both H3K27me3 and H3K4me3 at several promoters in 0–2 h embryos when very few zygotic genes are transcribed  suggestss Figure . This arosophila ,42. The Given that HP1a affects both activating and repressive marks, it is perhaps not surprising that we find the same gene can show both a negative and positive response to changes in HP1a. The response depends on the degree of the reduction in HP1a Figure . As seveDrosophila chromosome 4 and pericentromeric regions), and this binding is independent of H3K9 methylation [The binding of HP1a to promoters has been shown to be necessary to maintain an open chromatin structure . If its hylation . It is ahylation .Drosophila HP1a, but not HP1β or HP1γ, directly interacts with SU(Z)12 in vitro through its chromoshadow domain and requires the PxVxL motif [Drosophila HP1a co-IPs with PRC2 components, as well as PC , could control PRC2 recruitment and H3K27 methylation at a subset (21.6% of 958) of common target genes . Their ‘Pe found that the telomeric protein HOAP made a substantial contribution to HP1a recruitment. Presumably, proteins such as HOAP work in combination with general transcription factors to delimit promoter regions as appropriate binding sites for HP1a. In mammalian ESC, many of the PcG target genes have been shown to be bound by three main pluripotency transcription factors, suggesting that at a subset of genes PcG proteins can collaborate with developmental transcription factors to repress transcription [In heterochromatic regions, HP1a is recruited to promoters independently of methylation . Our anacription .Drosophila embryo, nuclei divide very rapidly and the genome is packaged with maternally provided histones which are primarily unmodified. We propose HP1a works with the PcG/trxG genes to maintain a silenced genome, while promoters are bivalently marked poised for expression. In this capacity HP1a is upstream of the PcG/trxG genes. Once PRC2 introduces the initial marks for repression, the dependency on HP1a becomes diminished as evidenced by the Su(var)2-505/04 larvae, which fail to enhance the deficiency in H3K27me3 despite their highly reduced levels of HP1a [The dual roles of HP1a as a repressor and activator are fairly well described, and we demonstrate additional evidence to that effect. At least some of this duality must arise from HP1a impacting the activities of the PcG/trxG genes. This role changes depending on the developmental stage. In the early  of HP1a . There i of HP1a ) may proWe find that over development the H3K27me3 and H3K9me3 repressive systems are dynamic, coordinating their levels with each other at euchromatic regions as well as at repetitive elements where they appear to promote silencing when the regions are derepressed from a reduction in HP1a. Altogether, the picture which emerges is that the time around constitutive heterochromatin formation is a key decision point for setting the levels of the silencing marks. Although HP1a is upstream of the PcG/trxG initially, the silencing marks of the two systems appear to have reciprocity in their levels as the primary domains of their action, heterochromatin and euchromatin, respectively, are established.Drosophila, constitutive heterochromatin formation appears to complete around cellular blastoderm (approximately 3.5 h [Su(var)2-505mutation is reversed. Animals heterozygous for Su(var)2-505 show different H3K27me3 and H3K9me3 amounts on their promoters, depending on which parent contributed the mutation. This parent-of-origin effect is generally reciprocal for both marks: whereas receiving Su(var)2-505 maternally leads to higher H3K27me3 and lower H3K9me3 levels, receiving the mutation paternally leads to lower H3K27me3 levels and H3K9me3 levels that are frequently unchanged relative to wild-type, or higher than the maternally inherited Su(var)2-505mutation.In ly 3.5 h ). AcrossWe speculate that when HP1a silencing is compromised, PRC2 becomes the back-up repressive mechanism. Indeed, the transcripts of repetitive regions show a decrease as the levels of H3K27me3 increase as embryos get older Figure C. In earSu(var)2-505 allele, there is no reduction in the maternal supply of HP1a so the embryo can form constitutive heterochromatin normally. This does not trigger PRC2 to become the heterochromatin back-up system and the H3K27me3 set point is unchanged. The normal dependence for HP1a at promoters for the deposition of H3K27me3 is still present, however, and after formation of constitutive heterochromatin in the heterozygotes the reduction in HP1a is sensed . This results in a decrease in H3K27me3, much like early embryos with reduced HP1a before the formation of constitutive heterochromatin 2-5 parent-of-origin effect dominates the effect of reducing the dose of PC, supporting the idea that it is HP1a which establishes the normal set point, and that its levels are more critical than those of PC.This model also provides an explanation for the differences in levels of the silencing chromatin marks in the two parent-of-origin conditions. The foregoing explains the maternal inheritance. When the father introduces the n Figure . This SuUse of H3K27me3 as a back-up at constitutive heterochromatin is also seen during early mouse embryogenesis. For the first four divisions, the maternal chromosomes use H3K9me3 while the paternal chromosomes utilize PRC1 and H3K27me3. In the absence of maternal H3K9me3, PRC1 becomes the back-up repressive mechanism for the maternal chromosomes which then also use H3K27me3 to form pericentric heterochromatin .Tetrahymena thermophile, the E(Z) homologue EZL1 is responsible for both H3K27 and H3K9 methylation in RNA interference-mediated heterochromatin methylation. Both marks are recognized by the chromodomain protein Pdd1p [Arabidopsis, the HP1 homologue, like heterochromatin protein 1 (LHP1), binds to H3K27me3 in addition to H3K9me2/me3 and may regulate gene expression as part of the PcG silencing complex [Drosophila embryo organizes its genome into euchromatin and heterochromatin, to assign the primary roles of the two silencing systems between the two chromatin types.The cooperation between HP1 and the PcG chromatin systems in regulating expression as well as partitioning the genome may be the offshoot of their having shared the same biological function. The chromodomains of HP1 and PC are very similar . The mouin Pdd1p . In Arab complex . These sOur results demonstrate that HP1a and the PcG proteins work together to regulate both silencing and transcription. This interaction begins early in embryogenesis where HP1a appears to act upstream of the PcG/trxG marks. As the early embryo organizes its genome into heterochromatin and euchromatin, the HP1/H3K9me3 and PcG/H3K27me3 systems work together to both ‘sense’ and partition the genome, sorting the chromatin type each will regulate. In the event of reduced maternal HP1a levels, the PcG system serves as a back-up with an increase in H3K27me3 levels at heterochromatin, presumably to decrease the transcription of repetitive sequences which are derepressed by the reduction in HP1a. When H3K27me3 levels are low, particularly in euchromatin which is the primary domain of the PcG, there generally appears to be an increase in H3K9me3 levels. Our results suggest that at multiple steps/stages the two systems compensate for each other to collectively achieve the required silencing across the genome.http://www.flybase.org/). All crosses were done at 25°C; Ore R and w1118 were the wild-type controls. Embryos from Ore R, w1118; Su(var)2-505/CyO, w; E(z)731, P{FRT(whs)}2A TM6C, Sb1, Tb1 or w; Su(z)124,e P{FRT(whs)}2A/TM6C, Sb1 parents were collected for 1 or 2 h on apple juice plates, aged appropriately for the RNA or ChIP collections, and processed as described. Larvae carrying the mutations of interest were sorted from the balancer class using the tubby marker on the third TM6B chromosome balancer for Pc15}2A/TM6B, Tb1) and Su(z)124, CyO-Kr-GFP was used for the two Su(var)2-5 alleles (Su(var)2-505/04) on the second chromosome. The RFP-HP1a transgene on the third chromosome (BL # 30562) was crossed into w1118; Su(var)2-505/CyO-GFP background for the Su(var)2-505 chromatin modification rescue; reduction of HP1 by RNAi used the transgenic TRiP line GL00531 (BL # 36792), the shRNA was driven by mothers with the maternal GAL4 nos promoter line 40 (BL # 4442). Genetic crosses testing female viability with mutant gene combinations had the progeny counted 8–9 days out from the first day of eclosion. All crosses had a minimum of 232 males, the reference class.Flies were reared under uncrowded conditions on standard cornmeal medium. Description of genes can be found in Flybase 2-505/04 where approximately 65 larvae were used. w1118 was the control. Larvae were first frozen at −80°C. To prepare the chromatin, they were first ground to a powder on dry ice using a mortar and pestle, then homogenized in nuclear isolation buffer ). Subsequent steps were as described in Mulvey et al. [Chromatin was prepared as in . For they et al. .. Louis, MO, USA) with 30 μl of Protein A beads  in 0.9 ml of RIPA buffer. For the anti-HP1 ChIPs, washes were performed as described in [ChIPs were performed using 0.24 ml clarified chromatin ), 2.5 μg anti-H3K27me3 , 3.5 μg anti-H3K9me3  anti-Ser5-P (H14) , or non-immune serum  and had at least two biological replicates.Negative controls used NI serum . The whole cell extracts were pre-cleared in 5 μl Protein A/G beads for 1 h, diluted to approximately 0.6 ml and incubated with 2–9 μl of antibody for 2 h at RT and 2 h at 4°C. Antibody and extract were incubated with 5 μl of protein A or G beads for 2 h at RT and at 4°C overnight with rotation. IPs were rinsed and washed once for 5 min, then two more times for 10 min with rotation, each with 500-μl Buffer C. IPs were denatured and solubilized in 1x sample loading buffer, boiled, fractionated, blotted, and probed as described below.One-3.5 h Western blot experiments were performed by standard methods. Antibodies were used at: HP1 (1:7000 rabbit), (1:1000 mouse C1A9), E(Z) (1:600 rabbit), SU(Z)12 (1:500 rabbit), PC (1:500 rabbit), and Suppressor of Fused Su(Fu) (1:5 mouse). Blots were incubated with secondary antibodies  and imaged by Odyssey Infrared Imaging System .


Pcdh isoforms can be disctinguished. The individual duplicated Pcdh-α isoforms showed diverse expression level with stochastic expression manner, even though those have an identical promoter sequence. Interestingly, the 5′-located duplicated Pcdh-αc2, which is constitutively expressed in the wild-type brain, shifted to stochastic expression accompanied by increased DNA methylation. These results demonstrate that tandem duplication in the Pcdh cluster expands the stochastic expression repertoire irrespective of sequence divergence.Tandem duplications are concentrated within the Pcdh cluster throughout vertebrate evolution and as copy number variations (CNVs) in human populations, but the effects of tandem duplication in the Pcdh cluster remain elusive. To investigate the effects of tandem duplication in the Pcdh cluster, here we generated and analyzed a new line of the Pcdh cluster mutant mice. In the mutant allele, a 218-kb region containing the Pcdh-α2 to Pcdh-αc2 variable exons with their promoters was duplicated and the individual duplicated  Genetic variations play critical roles in animal evolution and human diseases128Although detailed analyses of these gene clusters have revealed their sophisticated gene regulation mechanisms, for example, somatic DNA rearrangements in the immunoglobulin and T-cell receptor clustersRecently, the protocadherin (Pcdh) cluster was shown to be rich in tandem duplications111314173The diversity and sophisticated gene regulation exhibited by the Pcdh cluster genes are important for normal development of the nervous system1112212324252611122829111221To better understand the evolutionary origin of the stochastic expression and CNVs' effect in the Pcdh cluster, here we focused on the effect of tandem duplication in the mouse Pcdh-α cluster. In the present study, we engineered a targeted tandem duplication within the mouse Pcdh-α cluster, a situation somewhat comparable to that occurring during vertebrate evolution and in current human populations. The individual Pcdh-α isoforms transcribed from each duplicate exon can be distinguished in the mutant mice, enabling us to determine the manner by which their expression was regulated. The individual duplicated Pcdh-α isoforms showed diverse expression level with stochastic expression manner, even though those have an identical promoter sequence. Surprisingly, the duplicated Pcdh-αc2 isoform, which shows constitutive expression in the wild-type allele, shifted to stochastic expression accompanied by increased DNA methylation. Our results demonstrate that tandem duplication in the Pcdh cluster expands the stochastic expression repertoire irrespective of sequence divergence.To study the consequences of tandem duplication in the Pcdh gene cluster, we generated a targeted tandem duplication in the mouse Pcdh-α cluster using the inter-strain targeted meiotic recombination (iTAMERE) systemBefore using the iTAMERE system, we individually inserted two loxP sites into the variable region of the Pcdh-α cluster with the same orientation. First, the “G16Neo” alleledup(2-c2)/dup(2-c2)) were obtained by crossing heterozygous (Pcdhαwt/dup(2-c2)) parents.To duplicate the sequence between the loxP site of the G16Neo allele and that of the SR allele using the Cre-loxP system, we obtained male mice that possessed the G16Neo and SR alleles (G16Neo/SR) and the Sycp1-Cre transgene, which elicits Cre recombinase expression specifically in the testisdup(2-c2)/dup(2-c2) pups were born with the expected Mendelian distribution (dup(2-c2)/dup(2-c2) mouse brain (dup(2-c2)/dup(2-c2) mice  mice . Furtherc2) mice . Finallyc2) mice . These fdup(2-c2)/dup(2-c2) mice. The analyzed non-Pcdh genes included Wdr55, Dnd1, Hars, Zmat2, and Vault, located about 130-kb ~ 170-kb upstream from the duplication's 5′ boundary and Slc25a2, Taf7, Diap1, and Hdac3, located about 480-kb ~ 780-kb downstream from the duplication's 3′ boundary /dup(2-c2) mice by in situ hybridization (ISH). We analyzed the expression of all the Pcdh-α genes (using Pcdh-αCR probe), Pcdh-α1, Pcdh-α3, Pcdh-αc2, Pcdh-β22, and all the Pcdh-γ genes (using Pcdh-γCR probe). Similar positive signals were observed for all the genes examined between the wild-type and the Pcdhαdup(2-c2)/dup(2-c2) cerebellum  analysis, we focused on these exons and on Pcdh-α1, which was not duplicated. We amplified the cDNA fragments of these Pcdh-α genes using specific primer combinations, and the resultant amplicons were sequenced directly . The anadup(2-c2)/dup(2-c2) mice. There were no significant differences in the expression levels of most of the 3′-located duplicated Pcdh-αs  compared to wild-type. In contrast, the expression levels of all the 5′-located duplicated Pcdh-αs and the 3′-located duplicated Pcdh-α5 and Pcdh-α9 genes were significantly reduced compared to wild-type. These observations revealed that the expression level of the total Pcdh-α genes was maintained, while that of individual isoforms was altered, indicating that expressional re-allocation occurred in the dup(2-c2) allele.Next, the expression levels of these duplicated Pcdh-αs were quantified by qRT-PCR and cloning-mediated SNP analysis, which is highly sensitive and yields quantitative data . The expInterestingly, the expression levels of the 5′-located duplicated Pcdh-αs were significantly lower than those of their duplicated 3′-located counterparts. This observation suggested that, despite their identical promoter sequences, each 5′-located and 3′-located duplicated Pcdh-α receives distinct gene-regulation influences. Taken together, these findings indicate that tandem duplication alters the manner of gene regulation in the Pcdh-α cluster.JF1/dup(2-c2) mice. The PcdhαJF1/dup(2-c2) mice were F1 mice from a JF1 × Pcdhαdup(2-c2)/dup(2-c2) cross. To distinguish the 5′-located exons (B6) and 3′-located exons (CBA) in the dup(2-c2) allele, and exons in the wild-type allele (JF1) by SNP analysis, we focused on Pcdh-α3, Pcdh-α5, Pcdh-α7  mice were picked up by glass capillary. Complementary DNA of Pcdh-α3, Pcdh-α5, Pcdh-α7, and Pcp-2 (a marker for Purkinje cells) was synthesized from the single-cell samples in the same tube, and the resulting cDNA was then divided into three tubes and subjected to separate, first-round multiplex PCR analysis. The second round of PCR amplification was carried out individually for each tube and used nested primers for the Pcdh-α3, Pcdh-α5, Pcdh-α7 genes, and for Pcp-2. Finally, each PCR product was subjected to direct sequencing to determine from which exon the transcript was derived: i.e., the wild-type (JF1) allele or the 5′-located (dup-5′) or 3′-located (dup-3′) exons in the dup(2-c2) allele.To analyze the expression of Pcdh-α3, Pcdh-α5, and Pcdh-α7 in the dup(2-c2) allele, single Purkinje cells from 4-week-old PcdhαOf the 163 single Purkinje cells analyzed, 45 yielded PCR amplicons of Pcdh-α3, Pcdh-α5, or Pcdh-α7 from the same exons in all three tubes, and all 45 cells were positive for Pcp-2, confirming that they were differentiated Purkinje cells. In addition to three of three specific transcripts from the same exon, some cells showed one or two of three transcripts ; these findings suggested that the amounts of corresponding transcripts in these cells were low, and therefore we excluded these cells from the following analysis.For Pcdh-α3, the transcripts were derived from the wild-type exon, 5′-located exon, and 3′-located exon in 1 cell, 2 cells, and 5 cells, respectively: the wild-type (#1-138), 5′-located , and 3′-located exon . For Pcdh-α5, the transcripts were derived from the wild-type exon, 5′-located exon, and 3′-located exon in 14 cells, 5 cells, and 5 cells, respectively: the wild-type , 5′-located , and 3′-located exon . For Pcdh-α7, the transcripts were derived from the wild-type exon, 5′-located exon, and 3′-located exon in 13 cells, 2 cells, and 4 cells, respectively: the wild-type , 5′-located , and 3′-located exon . Only one cell expressed both the wild-type and the 3′-located exon for the same Pcdh-α isoform (Pcdh-α5 in cell #1-53) simultaneously, indicating that Pcdh-α genes are not always expressed monoallelically. Importantly, although it cannot rule out the possibility of small number of analyzed cells and possible lower expression level of the 5′-located exons may reduce the chance of detection, no cell expressed both the 5′-located and the 3′-located exon for the same Pcdh-α isoform simultaneously. These results strongly suggested that tandem duplication maintained the stochastic expression of the Pcdh-α isoforms, as seen in the wild-type brain.To investigate whether the 5′-located and 3′-located duplicated Pcdh-αc2 retained its constitutive expression at the single-neuron level, single-cell RT-PCR and SNP analysis was carried out . Of the To our surprise, the Pcdh-αc2 transcript from the 5′-located duplicated exon was detected in three tubes for only two of the 16 cells (#2-4 and #2-13). Notably, 8 of the 16 cells  showed no Pcdh-αc2 transcript from the 5′-located duplicated exon . For thodup(2-c2)/dup(2-c2) mouse cerebellum (343536dup(2-c2)/dup(2-c2) mouse cerebellum using bisulfite sequencing (dup(2-c2)/dup(2-c2) mouse (11.0%) compared with the wild-type mouse (0.3%).The 5′-located Pcdh-αc2 was down-regulated in the Pcdhαrebellum  and acqurebellum , suggestquencing . The resBecause bisulfite sequencing was unable to discriminate between the two Pcdh-αc2s in the dup(2-c2) allele, we further analyzed the DNA methylation using HpaII digestion-mediated DNA methylation analysis . The Hpadup(2-c2)/dup(2-c2) mouse cerebellum. Similar to Pcdh-αc2, bisulfite sequencing showed higher DNA methylation of Pcdh-αc1 in the Pcdhαdup(2-c2)/dup(2-c2) mouse (17.2%) than in the wild-type mouse (0.5%) (We further examined the DNA methylation of Pcdh-αc1 in the Pcdhαe (0.5%) . Taken tWe next extended the DNA methylation analysis to the stochastically expressed Pcdh-α isoforms. To distinguish the 5′-located from the 3′-located exons in the dup(2-c2) allele by SNP analysis after the bisulfite reaction, we focused on the Pcdh-α1 (promoter), Pcdh-α6 (exon 5′-region), and Pcdh-α12 (promoter) . A mosaiTo identify the region with increased DNA methylation more precisely, DNA methylation around the 5′-located Pcdh-α12 promoter was examined . No signTo gain further insight into the cluster-dependent regulation of the DNA methylation, we next examined the methylation during development . InteresTaken together, these results suggest that 3′-located genes in the wild-type Pcdh-α cluster, Pcdh-α12, Pcdh-αc1, and Pcdh-αc2, are hypomethylated in a cluster-structure dependent manner, and that these DNA hypomethylations were masked by position shift to 5′-location, which is caused by tandem duplication in the dup(2-c2) allele. This mechanism probably underlies the lower expression level of the the 5′-located Pcdh-α12 duplicate and the stochastic expression of the 5′-located Pcdh-αc2 duplicate in the dup(2-c2) allele.Tandem duplications are concentrated within the Pcdh cluster throughout vertebrate evolution and as CNVs in human populations319What are the mechanisms by which individual duplicated Pcdh-αs, each of which has an identical promoter sequence, are expressed stochastically? The present data are consistent with our previous findings, which is that the stochastic expression of Pcdh-α cluster genes is governed by enhancer and promoter DNA methylation253940Stochastic promoter competition has also been suggested for other gene clusters, such as the olfactory receptor MOR28 cluster108Here we found that the 5′-located duplicated Pcdh-αc2, which is constitutively expressed in the wild-type brain, acquired stochastic expression upon tandem duplication, accompanied by increased DNA methylation. These results provide supportive evidence for previous findings that mosaic DNA methylation states are correlated with the stochastic expression of Pcdh-α isoforms in wild-type mouse brain343640The experiments described here reveal some role of tandem duplication on gene regulatory differentiation, that include expression level divergences of the duplicated Pcdh-α genes and changes from constitutive to stochastic manner of the 5′-located duplicated Pcdh-αc2 gene. The data suggest that the current state of the Pcdh-α cluster in mammals, in terms of how it is expressed, was shaped by tandem duplication and distance from promoter to enhancer. Furthermore, although it is not clear that Pcdh-α gene number or stochastic expression have been strongly selected during vertebrate evolution, the present results may imply the evolutionary history of Pcdh cluster gene regulation. Previous reports have suggested that Pcdh cluster evolution included successive tandem duplications and sequence divergences1617http://dgv.tcag.ca/gb2/gbrowse/dgv2_hg18/?name=chr5:140050001..141050000). The present study showed that the tandem duplication resulted in healthy mice with a macroscopically normal brain. This result can be explained in part by the maintenance of the expression levels and distribution patterns of the total Pcdh-α transcript, by the re-allocation of Pcdh-α isoform expression, and by the maintenance of the expression levels of neighboring genes, upon duplication. Thus, it is likely that following various CNV events in the human PCDH cluster, the total expression level, dual gene-regulatory mechanisms, and stochastic expression of the human PCDH genes are maintained. This robustness may provide the predominant reason for the frequent CNVs in the human PCDH cluster. One study reported that there is no phenotypic link between a CNV in the human PCDH cluster, a 16.7-kb deletion affecting PCDHA8-A10, and psychiatric disordersde novo gene disruption in PCDHA13 was reported in autismet al. reported strong genetic evidence of PCDHA as a potential candidate gene for autismThe human PCDH cluster is particularly rich in CNVs, including duplications and deletions320Recent human genome analyses revealed that tandem duplications contribute to human phenotypes, including many psychiatric disorders, color vision, Parkinson's disease, and Rheumatoid arthritis13dup(2-c2)/dup(2-c2) mice will improve our understanding how tandem duplication in the Pcdhα cluster contribute to brain function.The vertebrate Pcdh cluster shows remarkable similarity to the Drosophila Dscam1 gene, within which tandem duplication is frequent throughout its evolutionary history11B6 mice were purchased from Charles River Japan. The wild-type mouse strain JF1 was obtained from the National Institute for Genetics . All animals were maintained in a specific pathogen-free space under a 12-h light/dark regimen. Experimental procedures were performed in accordance with the Guide for the Care and Use of Laboratory Animals of the Science Council of Japan and approved by the Animal Experiment Committee of Gunma University and Osaka University.SR/G16Neo, Sycp1-Cre). We then crossed PcdhαSR/G16Neo, Sycp1-Cre male and B6 female, and genotyped the pups using genomic DNA extracted from the tail. Some of these pups carried the duplicated [dup(2-c2)] or deleted [del(2-c2)] allele as a result of TAMERE in the testiswt/dup(2-c2) mice, we performed Southern blotting and PCR analyses (dup(2-c2)/dup(2-c2) mice were obtained by crossing Pcdhαwt/dup(2-c2) parents, which were backcrossed with B6 for more than three generations.By crossing SR mice see  with Sycanalyses . The BamIn situ hybridization (ISH) was performed essentially as described previouslyThe cerebellum was dissected from 4-week-old mice and immediately frozen in liquid nitrogen. The tissue was homogenized with a Polytron homogenizer. Total RNA was isolated using RNeasy (Qiagen), according to the supplier's recommendations. To obtain cDNA, 2.5 μg of the total RNA was treated with DNase I (Takara) and reverse transcribed with SuperscriptIII reverse transcriptase (Invitrogen) using random primers in a 40-μl reaction volume.For the SNP analyses, PCR for each Pcdh-α isoform was performed using 0.4 μl of cDNA from the cerebellum of a 4-week-old mouse as a template. The primer sequences used for the SNP analyses are shown in The qRT-PCR was performed with SYBR Premix ExTaq II (Takara) using the 7500Fast Real-Time PCR system (Applied Biosystems) or LightCycler480 (Roche). The primer sequences used for qRT-PCR are shown in Single-cell RT-PCR was performed essentially as described previously, with small modifications24wt/dup(2-c2) parents. The morning of the vaginal plug was designated E0.5. Bisulfite conversion was performed with the EpiTect Plus DNA Bisulfite Kit (Qiagen), according to the supplier's recommendations. We used “MethPrimer” (http://www.urogene.org/methprimer/) to design primers for use on bisulfite-treated DNAhttp://quma.cdb.riken.jp/)The genomic DNA was prepared with the QIAamp DNA Micro Kit (Qiagen) from the cerebellum of a 4-week-old or 3-month-old mouse, or from sperm, or with the EpiTect Plus LyseAll Kit (Qiagen) from the brain of an E12.5 embryo, head of an E9.5 embryo, E7.5 whole embryo, or blastocyst (E3.5), according to the supplier's recommendations. Sperm was obtained from the cauda epididymides of adult male mice. Embryos were obtained by the natural breeding of PcdhαIn the HpaII digestion-mediated DNA methylation analysis, HpaII was used to distinguish between methylated (undigested) and unmethylated (digested) HpaII/MspI sites, whereas MspI digested both methylated and unmethylated HpaII/MspI sites. The HpaII/MspI-digested DNA was subjected to PCR analysis to amplify the SNP-containing regions. We mixed 500 ng of DNA with 10 U HpaII or 10 U MspI, and restriction buffer in a 10-μl reaction volume. Samples were incubated at 37°C overnight, and we used 40 ng of the digested DNA for the PCR reaction. The primers are listed in Statistical analysis was performed using GraphPad Prism Version 6.0  and was performed using one-way ANOVA followed by Bonferroni's post hoc test, or using an unpaired two-tailed Student's t test if applicable. All data are expressed as the mean ± S.E.M.R.K. and T.Y. conceived and designed the experiments, analyzed the data and wrote the manuscript. R.K. and M.A. performed research; R.K., M.A., T.H., A.U., K.S. and Y.Y. contributed unpublished reagents/analytic tools.Supplementary information


SA-1 are regulatory proteins involved in a number of critical cellular processes including transcription, maintenance of chromatin domain architecture, and insulator function. To assess changes in the CTCF and cohesinSA-1 interactomes during erythropoiesis, chromatin immunoprecipitation coupled with high throughput sequencing and mRNA transcriptome analyses via RNA-seq were performed in primary human hematopoietic stem and progenitor cells (HSPC) and primary human erythroid cells from single donors.CTCF and cohesinSA-1 co-occupancy were enriched in gene promoters in HSPC and erythroid cells compared to single CTCF or cohesin sites. Cell type-specific CTCF sites in erythroid cells were linked to highly expressed genes, with the opposite pattern observed in HSPCs. Chromatin domains were identified by ChIP-seq with antibodies against trimethylated lysine 27 histone H3, a modification associated with repressive chromatin. Repressive chromatin domains increased in both number and size during hematopoiesis, with many more repressive domains in erythroid cells than HSPCs. CTCF and cohesinSA-1 marked the boundaries of these repressive chromatin domains in a cell-type specific manner.Sites of CTCF and cohesinSA-1 have multiple roles in the regulation of gene expression during erythropoiesis including transcriptional regulation at gene promoters and maintenance of chromatin architecture. These data from primary human erythroid cells provide a resource for studies of normal and perturbed erythropoiesis.These genome wide data, changes in sites of protein occupancy, chromatin architecture, and related gene expression, support the hypothesis that CTCF and cohesin The dynamic interplay between DNA methylation, histone modification, and chromatin structure are critical for establishing and maintaining appropriate patterns of mammalian gene expression. In vertebrates, the highly conserved, multifunctional CCTC-binding factor CTCF binds throughout the genome in a sequence- and DNA SA-1 and cohesin.SA-2 [The cohesin complex plays numerous roles in mammalian gene regulation including promoting transcription factor binding at enhancers , 6 and psin.SA-2 , 19 The SA-1, and their association with gene expression and chromatin domain organization in erythroid development. Chromatin immunoprecipitation coupled with high throughput sequencing and mRNA transcriptome analyses via RNA-seq were performed in primary human hematopoietic stem and progenitor cells (HSPC) and primary human erythroid cells from single donors. Changes in sites of CTCF and cohesinSA-1 occupancy and their association with gene expression were observed. Cell type-specific CTCF sites in erythroid cells were linked to highly expressed genes. Repressive chromatin domains increased in both number and size during hematopoiesis, with many more repressive domains in erythroid cells than HSPCs. CTCF and cohesinSA-1 marked the boundaries of these repressive chromatin domains in a cell-type specific manner. These genomic data support the hypothesis that CTCF and cohesinSA-1 have multiple roles in the regulation of gene expression during erythropoiesis including transcriptional regulation at gene promoters and maintenance of chromatin architecture.The goal of these studies was to gain insight into the roles of CTCF, cohesinet al.[Human CD34+-selected hematopoietic stem and progenitor cells  isolated at >95% purity were obtained from the Yale Cooperative Center for Excellence in Molecular Hematology from unused clinical specimens. Erythroid progenitor cells were cultured and isolated as described. Immunomaet al. at >95% SA1, were performed using CD34+ and erythroid cells derived from the same donor.To avoid donor-to-donor variability observed in hematopoietic cells, including differences in age, gender, genetic background, etc., –24 studiRNA was isolated and prepared for RNA-seq analyses as described. Samples SA1 data sets from individual donors were analyzed together.ChIP assays were performed as previously described., 25, 26 http://broadinstitute.github.io/picard), Phantompeakqualtools and the DiffBind package.[SA1 binding sites relative to known genes was done using the BEDTools software package.[http://genome.ucsc.edu/). Motif finding was done using the Homer software package.[cis-regulatory regions identified by ChIP-seq.[p-values were obtained by randomization of genomic intervals within the human genome excluding gap regions for 1000 iterations.The MACS program version 1.4.0rc2 was used to identify peaks with a p-value<10e-5 and a fold enrichment >6 for erythroid SA1 and >8 for the other samples. Quality  package., 30 The  package. Comparis package. Motifs d package. The GenoChIP-seq. Broad reChIP-seq. Regions SA-1 in the target genes identified by the MACS program (Table A in Primers were designed for representative binding regions for both CTCF and cohesinhttp://www.ncbi.nlm.nih.gov/geo/ Reference series number GSE67893).The raw data files generated by RNA-seq and ChIP-seq analyses have been submitted to Gene Expression Omnibus  to generate genome-wide maps of CTCF and cohesinSA-1 binding in primary human HSPC and erythroid cell chromatin. Quality control analyses of Chip-seq data for read duplication, strand cross correlation, and principal components clustering demonstrated the data were of high quality , while in erythroid cells 12,790 transcripts were detected. Five thousand two hundred thirty two transcripts were differentially expressed by more than 2 fold between HSPC and erythroid cells, with 2289 genes up regulated in erythroid cells and 2943 down regulated in erythroid cells.SA-1 occupancy were analyzed in HSPC and erythroid cells . In contrast, in HSPCs, the majority of CTCF sites lacked cohesinSA-1 co-occupancy .Overlap of sites of CTCF and cohesinSA-1 binding sites were enriched in 5’ flanking regions and promoter regions, and, intergenic regions were underrepresented relative to genome composition  of the CTCF sites in HSPC cells were invariant, i.e. present in all 7 data sets compared to 39%  in erythroid cells. Typical cell-type specific and invariant CTCF binding sites are shown at several gene loci in erythroid cells , lymphoblastoid (G17828), embryonic stem cell (H1ES), human cardiac myocytes (HCM), human mammary fibroblasts (HMF), human umbilical vein endothelial (HUVEC), and normal human epidermal keratinocytes (NHEK) . Cell tyid cells .p-value < 2.2e-16). In contrast, in HSPCs, genes linked to cell type-specific CTCF sites were expressed at significantly lower levels than genes linked to invariant CTCF sites (Levels of mRNA expression were assessed in genes within 1kb of cell type-specific or invariant CTCF sites. Genes linked to erythroid-specific CTCF sites were expressed at significantly higher levels than those with invariant CTCF sites ( 2.2e-16). A serieThese studies were performed in primary human hematopoietic cells rather than in cells from transformed lines. K562 erythroleukemia cells have been utilized as a model of erythroid cell genetics and epigenetics by ENCODE. When comparing CTCF occupancy in human primary erythroid cells to K562 cells, only 69% of sites were shared .vs. HSPC cell chromatin . Thus a large number of tissue-specific repressive chromatin domains are found in differentiated erythroid cells. There was a strong anti-correlation of H3K27me3 domains with gene expression. This difference was much greater in erythroid cells than HSPCs  were specific to erythroid cells  and 56% of CTCF sites at boundaries  binding both proteins in HSPC and erythroid cell chromatin, respectively (SA-1 at an erythroid-specific boundary is shown at the ankyrin-1 (ANK1) locus in ANK1 gene which all join in frame to exon 2, creating cDNA transcripts with unique 5’ ends. In erythroid cells, the sequence surrounding and including a neural-specific ANK1 exon 1, located 5’ of the erythroid exon 1, is in a region of repressive chromatin, heavily modified by H3K27me3  Motif analysis using the Homer algorithm in HSPC. . Motif analysis using the Homer algorithm in erythroid cells.  Correlation of repressive domains and gene expression in HSPCs and erythroid cells. Analytic flow activated cell sorting analyses of cultured human primary erythroid cells.  Correlation heat map using affinity (read count) data.  Principal components analysis of affinity (read count) data.  Heat map of affinity (read count) data for individual sites in individual ChIP-seq samples.  Sites of CTCF and cohesin(PDF)Click here for additional data file.S2 FileSA-1 validation. (Table B) Read Count, Duplication and Strand Cross Correlation Analyses. (Table C) Quantitative ChIP Validation of CTCF binding Sites. (Table D) Quantitative ChIP Validation of cohesinSA-1 binding sites. (Table E) Summary of ChIP seq results.(Table A) PCR primers for CTCF and cohesin(DOCX)Click here for additional data file.


Escherichia coli to study how antisense transcription can change the expression of a gene and tune the response characteristics of a regulatory circuit. We developed a new genetic part that consists of a unidirectional terminator followed by a constitutive antisense promoter and demonstrate that this part represses gene expression proportionally to the antisense promoter strength. Chip‐based oligo synthesis was applied to build a large library of 5,668 terminator–promoter combinations that was used to control the expression of three repressors  in a simple genetic circuit (NOT gate). Using the library, we demonstrate that antisense promoters can be used to tune the threshold of a regulatory circuit without impacting other properties of its response function. Finally, we determined the relative contributions of antisense RNA and transcriptional interference to repressing gene expression and introduce a biophysical model to capture the impact of RNA polymerase collisions on gene repression. This work quantifies the role of antisense transcription in regulatory networks and introduces a new mode to control gene expression that has been previously overlooked in genetic engineering.A surprise that has emerged from transcriptomics is the prevalence of genomic antisense transcription, which occurs counter to gene orientation. While frequent, the roles of antisense transcription in regulation are poorly understood. We built a synthetic system in  Larger designs consisting of multiple genes are often organized similarly, where transcription is designed to proceed in one direction. This organization avoids potential interference between promoters that can arise due to RNA polymerase (RNAP) collisions  that is generated, which can regulate gene expression by binding to the mRNA to change its stability or translation, or act as a transcriptional regulator . Downstream of rfp, there is a constitutive antisense promoter PR whose RNAPs will be fired toward Ptac. The full cassette was placed on a plasmid containing the p15A origin . Notably, the strongest promoter tested (apFAB96) is unable to completely repress expression. This promoter is among the strongest from a large synthetic library . Interestingly, the shape of the induction curve remains similar for the different antisense promoters  is also unaffected by different antisense promoters. Thresholds and Hill coefficients were calculated by fitting the response functions in Fig x is the concentration of IPTG, y is the activity of POUT, n is the Hill coefficient, and K is the threshold level of input where the output is half‐maximal. Escherichia coli growth rates were also unaffected by the addition of antisense promoters  is used to sort the cells into bins, the contents of which are determined using next‐generation sequencing  . The high fluorescence bins (bins 3 and 4) contain constructs with greater median antisense promoter strength than lower fluorescence bins (bins 1 and 2)  Fig  and S4.Ts antisense > 10), the composite parts are incapable of shifting circuit thresholds  most likely facilitate greater shifts in gate thresholds than bidirectional terminators by allowing more RNAPs fired from the antisense promoter to proceed. Terminators that are predicted to destabilize mRNA or contain cryptic antisense promoters also facilitate large shifts in gate thresholds   from the set Fig F. Then,  0.9275) .E. coli along with the original RFP reporter plasmid  and expressed at a higher level (due to the absence of RNAP collisions and the higher copy number plasmid). Fold repression generated by transcriptional interference θTI was then determined by dividing the total fold repression θ by θasRNA. Thus, θasRNA and θTI reflect the relative contributions of asRNA and transcriptional interference to total repression generated by antisense transcription. Plotting θ, θasRNA, and θTI versus inducer concentration shows that asRNA and transcriptional interference generate approximately equivalent contributions to repression  and gene length N. The model and parameters are shown in Fig F transcribe a gene at a constant velocity v unless they collide with polymerases from the interfering promoter PR on the opposing strand of DNA. In the event of a collision, polymerases may dissociate from the DNA.A differential equation model was developed to explore collision interference and parameterize the repression that arises for different forward and antisense promoter firing rates (ϕCF and reverse CR strands as a function of the distance from the start site x (in bp): Collision interference can be captured by two differential equations that track the steady‐state concentration of polymerases on the forward εF and εR are parameters that reflect the possibility that RNAPs fired from the forward and antisense promoters encounter collisions and dissociate from the DNA. Several factors may influence the frequency with which RNAPs encounter collisions and dissociate from the DNA. For example, transcriptional bursting could prevent RNAPs from encountering head‐on collisions by increasing the time between transcription events initiated at the opposing promoter. In vitro experiments show that head‐on RNAP collisions result in stalling and backtracking of the enzymes  = ϕF/v and reverse CR(x = N) = ϕR/v promoters. The polymerase velocity v = 40 bp/s is held constant  transcripts produced from PF with and without an antisense promoterθ is calculated and compared with that derived from experiments.Boundary conditions are defined by the rates that polymerases are fired, that is, begin elongating, at the forward bination . We chosequation . For eacθ, which assumes that there is no contribution from asRNA to the observed repression. Next results were fit to θTI, which represents the minimum amount of repression attributable to transcriptional interference. These bounds were then used to fit the underlying biophysical parameters and provide a range of values that reflect the possible contribution of transcriptional interference to gene repression.Repression due to asRNA is not included in the model. Rather, our approach was to fit the model predictions to the two bounds that we measured. First, results were fit to total repression εF = εR = 1, since previous models of transcriptional interference assume that actively transcribing polymerases never survive head‐on collisions  fit to either θ or θTI. The optimal values of εF and εR produced behavior that closely matches the experimental data . Values of ε < 1 are interpreted as cases where polymerases either avoid or survive collision and continue transcribing. Importantly, the model does not assume that one RNAP must dissociate in order for the RNAP on the opposing strand of DNA to survive collision. This ability to bypass is supported by in vitro experiments done with viral  as compared to those from the forward promoter (<εF> = 7–15%)  Fig A and B. oter Fig D. This iet al, et al, et al, et al, et al, et al, et al, et al, et al, et al, This work demonstrates that antisense promoters can be reliably used to tune gene expression. The degree of repression is proportional to the strength of the antisense promoter over a > 30‐fold range. This builds on the modern revisiting of the classical “expression cassette” to incorporate additional non‐canonical parts to tune expression, insulate against context, and provide for rapid debugging via omics techniques . This imbalance may be due to the differences in the kinetic properties of the two promoters, for example, burstiness and differential dissociation of the RNAPs. Single‐molecule experiments that measure polymerase survival rates directly could be done to differentiate between these two mechanisms. Additional experiments can also refine our understanding of antisense transcription by parameterizing additional mechanisms. The model presented here is limited to collision interference as the mechanism of repression in 70—have relatively information poor binding sites, promoters are expected to frequently arise spontaneously during evolution 7697 araD139 fhuA ΔlacX74 galK16 galE15 e14‐ ϕ80dlacZΔM15 recA1 relA1 endA1 nupG rpsL (StrR) rph spoT1 Δ(mrr‐hsdRMS‐mcrBC)) and DH10B (F‐ Δ(ara‐leu)7697 araD139 ΔlacX74 galE15 ϕ80dlacZΔM15 recA1 endA1 nupG rpsL mcrA Δ(mrr‐hsdRMS‐mcrBC) λ‐) were used for all experiments. Cells were grown in either LB Miller broth (Becton Dickinson 244630) or M9 minimal medium supplemented with glucose , 2 mM MgSO4 (Affymetrix 18651), 100 μM CaCl2 (Sigma C1016), 0.4% glucose (Fisher scientific M10046), 0.2% casamino acids (Becton Dickinson 223050), 340 mg/ml thiamine (vitamin B1) (Alfa Aesar A19560). Carbenicillin (100 μg/ml) (Gold Bio C‐103), kanamycin (50 μg/ml) (Gold Bio K‐120), and/or chloramphenicol (35 μg/ml) (USB Corporation 23660) were added to growth media to maintain plasmids when appropriate. Isopropyl β‐D‐1‐thiogalactopyranoside (IPTG) (Gold Bio I2481C) was used as the inducer for all constructs.Escherichia coli strains were grown for 16 h in LB media containing antibiotics in 96‐deep well blocks (USA Scientific 1896–2000) at 37°C and 250 rpm in an INFORS‐HT Multitron Pro. After 16 h, the cultures were diluted 1:200 into M9 medium with antibiotics and grown for 3 h with the same shaking and temperature settings as the overnight growth. Next, the cultures were diluted 1:700 into fresh M9 medium with antibiotics and different concentrations of isopropyl β‐D‐1‐thiogalactopyranoside (IPTG). These cultures were grown for 6 h and then diluted 1:5 into phosphate‐buffered saline (PBS) containing 2 mg/ml kanamycin or 35 μg/ml chloramphenicol to arrest protein production, and fluorescence was measured using a flow cytometer.Cells were analyzed by flow cytometry using a BD Biosciences Fortessa flow cytometer with blue (488‐nm) and red (640‐nm) lasers. An injection volume of 10 μl and flow rate of 0.5 μl/s were used. Cytometry data were analyzed using FlowJo , and populations were gated on forward and side scatter heights. The gated populations consisted of at least 30,000 cells. The median fluorescence of the gated populations was used calculated using FlowJo and used for all reporting. Autofluorescence of white cells (NEB10β without plasmids) was subtracted from all fluorescence measurements.bla  were estimated using NEB10β cells harboring one of the following plasmids: pJBTI26, pJBTI264, pJBTI265, pJBTI266, pJBTI267, pJBTI136 . Fluorestac) activity was measured using plasmid pJBTI26 (Relative expression units (REUs) were calculated using DH10B cells harboring one of the following plasmids: 0RFP2, pAN1717 . Strains pJBTI26  and convet al . Strong terminators resulted in a large drop in RFP fluorescence relative to a control plasmid with no terminator (pGR). Chen et al measured several terminators in both the forward and reverse orientation, which allowed us to identify unidirectional terminators for this study. We classified terminators as unidirectional if they have termination strength < 10 in the reverse orientation and > 10 in the forward direction.Terminators that encode cryptic antisense promoters or destabilize mRNA when placed at the 3′‐end were identified by analyzing data from Chen et al's library of terminators were used to identify terminators that encode cryptic antisense promoters or destabilize mRNA. Terminators that decreased GFP expression relative to the average were assumed to destabilize mRNA. Similarly, terminators that increased RFP expression when measured in the reverse direction were assumed to encode cryptic antisense promoters. We classified terminators that decreased GFP expression more than one standard deviation below the mean as destabilizing mRNA and terminators that increased RFP expression more than one standard deviation as encoding cryptic antisense promoters.Average levels of GFP and RFP fluorescence produced by plasmids carrying Chen et al, et al, et al, et al, et al, E. coli K12 genome that were previously characterized by Chen et al , resulting in three libraries  of ~160,000 clones each and > 20‐fold coverage of the designed sequence space. Each library was scraped from solid media plates and frozen at −80°C in 200 μl aliquots with 15% glycerol for subsequent analysis.The oligonucleotide library was constructed by CustomArray, Inc., using their CMOS semiconductor technology. The library was delivered as a 1 fM oligonucleotide pool and amplified using specific PCR primers: oj1299 and oj1300 . The PCR600 in 25 ml of M9 medium with carbenicillin and either 0 or 100 μM IPTG. These cultures were grown for 6 h to obtain exponential phase growth. At the end of 6 h, cultures were diluted to OD600 ~0.05 into PBS containing 35 μg/ml chloramphenicol to arrest cell growth and protein production until sorting. Aliquots of each library were also frozen at −80°C with 15% glycerol (VWR BDH1172) to serve as “unsorted” controls.To grow libraries for flow cytometry analysis or cell sorting, one aliquot of each library was thawed and 10 μl of the sample was added to 3 ml of LB media supplemented with carbenicillin in 15‐ml culture tubes (Fischer Scientific 352059). Once thawed, the remaining library aliquot was discarded to avoid cell death from repeated freeze–thaw cycles. The inoculated libraries were grown for 12 h at 30°C and 250 rpm in a New Brunswick Scientific Innova 44. NEB10β control strains containing unmodified NOT gate plasmids  were inoCell sorting was done on a BD Biosciences FACSAria II with a blue (488‐nm) laser. Each NOT gate library was sorted into four non‐adjacent log‐spaced bins based on YFP fluorescence. Control strains grown with 0 and 100 μM IPTG defined the upper and lower boundaries for bin placement, respectively. One million cells were sorted into the lowest fluorescence bin  by thawing one aliquot of each frozen sorted bin and using the entire sample as input to the kit. For deep sequencing, 30 ng of each miniprepped sample was amplified for thirty cycles of PCR with Phusion High‐Fidelity Polymerase Master Mix (New England Biolabs M0531). This PCR step added barcodes to each sample using primers oj1302, 1334 – oj1348 . AmplifiCustom software .To calculate enrichment for each composite part, we normalized the counts of each composite part in a bin to the total number of perfect sequences in that bin. We defined the frequency of a composite part fiux = 0), ciux was set to one, indicating one count of the part in the unsorted library. This correction was used to ensure that none of the enrichments were infinite. Enrichment Eijx for each composite part was then averaged by bin across all three libraries. We defined the average enrichment Eij¯ for a composite part asN = 3, the total number of libraries. To ensure that composite part behavior is consistent across all three libraries, any composite parts that did not appear in all three libraries for a given bin were assigned an enrichment of zero for that bin, that is, if Eijx = 0 for any x, Eij¯ = 0.If a composite part did not appear in the unsorted library at least once  at 37°C and 250 rpm in an INFORS‐HT Multitron Pro. After 16 h, the cultures were diluted 1:200 into M9 medium with antibiotics and grown for 3 h with the same shaking and temperature settings as the overnight growth. Next, the cultures were diluted to OD600 = 0.001 into fresh M9 medium with antibiotics and 100 μM IPTG; 150 μl of these cultures was grown in black 96‐well optical bottom plates (Thermo scientific 165305) at 37°C and 1 mm orbital shaking in a BioTek Synergy H1 plate reader. Optical density measurements at 600‐nm wavelength (OD600) were made every 20 min for 12 h.Custom MATLAB software was written to solve the model ODEs (equations CF(x = 0) = ϕF/v and CR(x = N) = ϕR/v. εF and εR were input directly into the model for εF = εR = 1 or parameter sweep experiments. Model results were reported as polymerase concentrations CF(x) and CR(x). Full‐length transcript production is assumed to be proportional to CF|εF = 0, which should be a measure of polymerases fired from PF that successfully transcribe the entire stretch of DNA between promoters. Fold repression θ is calculated using equation CF in the absence of interference (CF|εF = 0) to CF with an interfering promoter (CF|εF > 0). Model results for each forward/interfering promoter pair were scored by simple comparison to experimental data: s is the score for a specific promoter pairing, θTI_m and θTI_p are measured and predicted repression, respectively, for that pair. The best εF and εR values were calculated for each forward/interfering promoter pair using a weighted average, where each ε was weighted by its score.Integration constants were approximated using boundary conditions Brophy, JAN, Voigt, CA (2015). Antisense library. NCBI Sequence Read Archive SRP065456.CAV and JANB conceived of the study, designed the experiments, and wrote the manuscript. JANB performed the experiments and analyzed the data.The authors declare that they have no conflict of interest.AppendixClick here for additional data file.Expanded View Figures PDFClick here for additional data file.Code EV1Click here for additional data file.Source Data for AppendixClick here for additional data file.Review Process FileClick here for additional data file.Source Data for Figure 1Click here for additional data file.Source Data for Figure 2Click here for additional data file.Source Data for Figure 4Click here for additional data file.