Firre and Dxz4 loci are not required for X chromosome inactivation (XCI) in cell lines. Here, we examined the in vivo contribution of these loci by generating mice carrying a single or double deletion of Firre and Dxz4. We found that these mutants are viable, fertile and show no defect in random or imprinted XCI. However, the lack of these elements results in many dysregulated genes on autosomes in an organ-specific manner. By comparing the dysregulated genes between the single and double deletion, we identified superloop, megadomain, and Firre locus-dependent gene sets. The largest transcriptional effect was observed in all strains lacking the Firre locus, indicating that this locus is the main driver for these autosomal expression signatures. Collectively, these findings suggest that these X-linked loci are involved in autosomal gene regulation rather than XCI biology.Recent evidence has determined that the conserved X chromosome mega-structures controlled by the  In female mammals, one of the two X chromosomes is inactivated to compensate for gene dosage between males and females , a proceFirre and Dxz4  Firre that escapes XCI and plays a role in nuclear organization (Recent studies using chromosome conformation capture (3C) based methods have identified that the Xi folds into two megadomains and forms a network of long-range interactions termed superloops that are directed by the non-coding loci and Dxz4 . Deletiouperloop , while dormation . Moreovenization . Recent nization .Firre and Dxz4 and performing an extensive transcriptomic analysis in embryonic, extraembryonic and adult organs. We determined that these elements are dispensable for mouse development and XCI biology. However, the absence of these loci results in organ-specific expression changes on autosomes, suggesting that these regions are involved in autosomal gene regulation rather than XCI biology.Here, we answer this question by generating mice lacking Firre and Dxz4 loci both individually and in combination, we generated three knockout (KO) mouse strains: two carrying a single locus deletion (SKO) of either Firre (Dxz4 and one carrying a double locus deletion (DKO) of Firre in conjunction with Dxz4 (In order to test the in vivo role of er Firre  or Dxz4 ith Dxz4  and megaith Dxz4 . All fouFirre and Dxz4 loci has an impact on random or imprinted (Xp chromosome inactive) XCI in vivo, we collected embryonic day 12.5 female F1 brains (random/skewed XCI), placentas and visceral yolk sac (imprinted XCI) from reciprocal crosses between our KO strains and Mus musculus castaneus (CAST), followed by RNA sequencing (RNA-seq) and allele-specific analysis using the Allelome.PRO pipeline  to distinguish the phenotypic consequences of deletions if they are on the Xa  versus the Xi  chromosome. We did not detect Firre expression if the deletion was inherited maternally, while we detected Firre wildtype expression levels in the paternal deletion, demonstrating that Firre is exclusively expressed from the Xa , we can use these tissues to disentangle the functional roles of Firre RNA from megadomain and superloop structures that only exist on the Xi. We hypothesized that: (1) females and males carrying a maternal Firre single or Firre-Dxz4 double deletion (deletion on Xa) lack the Firre lncRNA, (2) females carrying a paternal Firre deletion (deletion on Xi) lack the superloop and (3) females carrying a paternal Dxz4 single or Firre-Dxz4 double deletion (deletion on Xi) lack both the superloop and megadomains (Firre (FDR ≤ 0.1 and |log2FC| ≥ 1), suggesting that the mega-structures and the Firre lncRNA have no impact on imprinted XCI   shared between Firre SKO and the DKO are Firre locus specific. To identify gene sets for these categories, we selected the genes dysregulated in at least one of the three KO strains where the direction of the expression change in either one of the single KO agrees with that of the DKO (|log2FC| ≥ 1). We first used this approach for the liver, an organ with a low number of differentially expressed genes in the DKO, and identified 15 megadomain, four superloop, and 4 Firre locus dependent genes  dependent genes , 26 superloop (14.8%) and 142 nt genes  sheet H.re locus .Firre SKO and DKO enriched gene sets are almost identical, sharing downregulated gene sets involved in chromosome structure and segregation , indicating that the Firre locus is the main driver for downregulation of these gene sets , we find that the top regation  sheet I.Dxz4 SKO . In addiene sets . To testene sets . Across gulation  sheet I.Firre and Dxz4 loci provide the platform for the formation of the X chromosome mega-structures and have been extensively studied in cell lines modeling random XCI  database under accession code GSE127554.Mice were housed under controlled pathogen-free conditions . C57BL/6J (Bl6), B6D2F1/J (F1 Bl6 and DBA) and CAST/Ei (CAST) mice were purchased from the Jackson Laboratory.Firre deletion mouse  was generated by inserting LoxP sites flanking the Firre gene body into JM8A ESCs (Bl6 background)  . Upon inDxz4 single deletion strain  was generated by co-injecting Cas9 mRNA (200 ng/µl) together with two guide RNA’s that span the Dxz4 locus (50 ng/µl each) into pronuclear stage 3 (PN3) zygotes isolated after mating super ovulated B6D2F1 female mice (Jackson labs) with Bl6 males as previously described , was generated by piezo-assisted Intracytoplasmic sperm injection of Firre SKO sperm into B6D2F1 oocytes , and brought to term. Protospacer sequences shown in  were ideIn order to control for strain background, all wildtype control mice were obtained from backcrossing of founder mice with C57BL/6J to match the KO strain background.Firre, Dxz4 or Firre-Dxz4 impact random or imprinted X chromosome inactivation, we collected embryonic day 12.5 brains, placentas and visceral yolk sac from reciprocal F1 crosses between the deletion strains and CAST/EiJ. For the brain and the placenta, we collected samples for all three strains from the forward cross  and reverse cross . For the placenta DKO we added an additional replicate of the maternal and paternal deletion. In addition, we collected visceral yolk sac samples from the DKO forward cross  and reverse cross . To test whether DNA methylation levels are altered in the absence of Firre and Dxz4 on either Xa or Xi, we collected placentas from the DKO forward cross  and reverse cross  to perform reduced representation bisulfite sequencing (RRBS) sequencing. To reduce the amount of maternal contamination we removed the decidua of the placentas. For the Firre-Dxz4 adult bodymap, we collected the spleen, brain, kidney, heart, lung and liver from 6 weeks old female mice carrying a homozygous double deletion (4 WT and 4 DKO replicates). To validate and classify the dysregulated genes form the DKO, we collected liver and spleen from independent generated female SKO strains (Firre 3 SKO and Dxz4 2 SKO replicates). To test whether the molecular phenotype observed in the female spleen can be uncoupled form X inactivation, we collected 6 weeks old spleens from DKO males (2 WT and 2 DKO replicates).To determine whether the deletion of + libraries from total RNA. We generated strand-specific libraries for the F1 placentas and brains (TruSeq stranded Illumina) and non-strand-specific TruSeq libraries for the adult organs and the visceral yolk sac. Libraries were quantified using a Qubit 2.0 Fluorometer, run on an Agilent 2100 Bioanalyzer to assess purity and fragment size, and sequenced on a HiSeq 2500 at Harvard University’s Bauer Sequencing Core (75 bp paired end).The collected tissues were snap frozen and stored at −80°C until further process. RNA was extracted from TRIzol lysates using RNeasy mini columns (Qiagen). The Illumina TruSeq kit was used to create polyAGenomic DNA for reduced representation bisulfite sequencing (RRBS) was quantified using a Qubit 2.0 Fluorometer, and quality-assessed on an Agilent 2200 TapeStation D1000 ScreenTape. RRBS was performed on 10 ng of each sample using the NuGen Ovation RRBS Methyl-Seq System following the manufacturer's recommendations except that barcoded adapter-ligated samples were pooled in groups of 8 immediately prior to Bisulfite Conversion with the Qiagen EpiTect Fast Bisulfite Conversion kit. Library pools were purified with a 1X Agencourt RNA XP bead clean-up and sequenced on a HiSeq 4000. Sequenced reads were aligned to the mm10 reference genome using BSmap  and methThe RNA data were aligned with STAR by using specific parameters to exclude reads mapping to multiple locations (STAR version 2.5.0 c: –outFilterMultimapNmax 1) . The reaDifferential expression analysis was performed with the assumption of negative binomial distribution of the read counts and empirical estimation of variance by using the R packages DESeq2 (version 1.22.1)  and fdrtAllele-specific expression was detected from RNA-seq by using the Allelome.PRO, as described in detail in Firre locus dependent gene sets, we selected genes dysregulated in at least one of the three KO strains (FDR ≤ 0.1) where the direction of the expression change in either one of the single KO agrees with that of the DKO (|log2FC| ≥ 1). Dysregulated gene sets that are: (1) shared across the three strains were categorized as superloop specific (2) shared between Dxz4 SKO and DKO were categorized as megadomain-dependent and (3) shared between Firre SKO and the DKO were categorized as Firre locus specific  available from MSigDB  after maO spleen , were exO spleen  sheet I  In the interests of transparency, eLife publishes the most substantive revision requests and the accompanying author responses.eLife. Your article has been reviewed by three peer reviewers, including Jeannie T Lee as the Reviewing Editor and Reviewer #1, and the evaluation has been overseen by Michael Eisen as the Senior Editor.Thank you for submitting your article "in vivo Firre and Dxz4 deletion elucidates roles for autosomal gene regulation" for consideration by The reviewers have discussed the reviews with one another and the Reviewing Editor has drafted this decision to help you prepare a revised submission.Summary:eLife. However, for publication in eLife, we would require several major revisions.The study generates single or double deletions of Firre and Dxz4 in mice, and show that despite the repeats being conserved in mammals, these mutants are viable, fertile and show no defect in random or imprinted XCI. Instead, the lack of Firre and Dxz4 results in dysregulated genes on autosomes in an organ-specific manner. Although the authors present mostly negative data, the manuscript would be of interest to the field and is significant because (1) it analyzes the phenotype in vivo in a mouse model, contrasting with previous papers that performed experiments only in cell culture. (2) The current manuscript also sheds light on a previous claim by Giorgetti et al., (2016) that Dxz4 is required for genes to escape from XCI. (3) It is revealed that the loci also do not play a role in imprinted XCI. All three reviewers believe that the scope of the work is in principle in line with 1) Better controls to rule out strain background or off-target artifacts, when looking at changes in autosomal gene expression. (See reviewer 3 notes).2) More backcrosses and RNA-seq of placenta, as it is the only tissue with non-random XCI, and therefore the only tissue where the authors can distinguish between effects stemming from Firre/Dxz4's roles on the inactive vs. the active X. (See reviewer 2).3) Direct versus indirect effects of Dxz4/Firre on autosomal expression. If the authors would like to claim a direct effect, CHART analyses would be required. Otherwise, the statements should be toned down. (See reviewer 1 notes).4) The 2 main figures should be broken up. (See reviewer 1 notes).5) Revision of statistical analyses. (See reviewer 2 notes).6) Finally, to the extent possible, please try to address the additional concerns of reviewers 2 and 3.Reviewer #1:The paper from Andergassen et al., is a succinct report made up of mostly negative findings and confirms the negative results of several past publications. Although the knockouts rule out a role in XCI, the report is significant in that (1) it analyzes the phenotype in vivo in a mouse model, contrasting with previous papers that performed experiments only in cell culture; In the majority of past reports, it was shown that the conserved X chromosome "mega-structures" controlled by the Firre and Dxz4 alleles are not required for XCI in cell lines. (2) The current manuscript also sheds light on a previous claim by Giorgetti et al., (2016) that Dxz4 is required for genes to escape from XCI. No effect on gene escape is seen by Andergassen et al., in vivo. (3) The loci also do not play a role in imprinted XCI.eLife and would be of interest to the field. Overall, the analysese are done well and the conclusions are mostly supported by the data. However, I recommend several revisions:The authors generate mice carrying a single or double deletion of Firre and Dxz4, and show that despite the repeats being conserved in mammals, these mutants are viable, fertile and show no defect in random or imprinted XCI. One positive finding is that the lack of Firre and Dxz4 results in dysregulated genes on autosomes in an organ-specific manner. By comparing the dysregulated genes between the single and double deletion, they categorize superloop, megadomain, and Firre locus-dependent gene sets and see that Firre deletion has the greatest effect on autosomal expression signatures. The manuscript is within the scope of papers published in 1) While I accept that there are changes in autosomal gene expression, I am less convinced that the effects are direct. I am also not convinced that X-chromosomal superstructures directly affect autosomal gene expression. If the authors would like to claim a direct role, they must perform additional analyses, including – for example – CHART analysis to correlate changes at specific loci with binding of the RNA, or 3C or Hi-C to determine if the loop domains interact with autosomal gene targets. Do these autosomal targets include the loci that the authors previously showed to interact with Firre?2) If the authors do not wish to include additional work, they should tone down the conclusions regarding autosomal changes and acknowledge the possibility of indirect effects, per point 1.eLife allows more than 2 display panels. At least one figure should be devoted to a more careful and deeper analysis of XCI gene escape, since the lack of effect on escape is a major point of the paper. And another figure should likewise be devoted to a deeper analysis of imprinted XCI, since this is another major point of the paper.3) The figures are very dense, difficult to read , and should be broken up into additional figures. Reviewer #2:The manuscript by Andergassen et al., describes the generation of mouse single and double knockouts of two X-linked macrosatellites, Firre and Dxz4. The authors analyze sex ratio, litter size and expression phenotypes of these three knockouts relative to wild-type, i.e. F1 hybrids of musculus x castaneous crosses. Whether and how these macrosatellites may participate in X chromosome inactivation or escape from XCI is an important question, due to previously described roles of Firre (not referenced PMID: 26048247 and PMID:25887447) and Dxz4 (not referenced PMID:26248554) in gene expression and inactive X chromosome conformation, respectively. However, as presented, in vivo defects were non-comprehensively analyzed, and gene expression experiments improperly controlled and somewhat over-interpreted:1) The authors main conclusion, that there is no sex ratio distortion in the double ko, is partially undermined by a miscalculated p-value  and only six reported litters. This table should also include the outcomes of the wildtype control and single homozygous ko matings.2) Differences in the genetic background of wild-type (not specified), single knockouts , and the double ko (variable ~1.6% BALB/C and ~6.25% DBA), are not taken into account in the gene expression analysis. This would be a non-issue with more backcrosses to C57BL/6J, at least five .3) Low and inconsistent numbers of replicates in the RNA-seq that reduce statistical power. This is especially problematic for attributing differential expression to loss of megadomains and superloop, because this analysis depended on comparing across multiple differential gene lists, which shrink with low statistical power (see only two Dxz4 ko replicates). This could be addressed by additional replicates from the single ko's, to get at least four across the board for liver, spleen and placenta.4) Homozygous animals also lack Firre RNA (and possibly Dxz4-associated transcripts) expressed from the active X. The analysis here assumes that there are no confounding interactions between differential genes attributed to one group or another. A deeper analysis of the placental data in heterozygous F1 hybrid animals, where such interactions can be excluded, would be preferable and likely yield more insight towards identifying megadomain- or superloop-specific gene expression, if any.In conclusion, while the authors present a worthwhile question and set of experiments, more careful analysis and interpretation is necessary to address functions of Firre and Dxz4 in XCI and development. At minimum, the placental RNA-seq should be repeated and fully analyzed after more than 5 backcrosses, with at least 4 replicates for each of the 4 genotypes presented. Additional novelty could come from three interesting observations reported here: (1) enrichment for differential genes implicated in faithful chromosome segregation in mice lacking Firre expression, (2) Xist upregulation by 30% in Dxz4 single and double ko's , and (3) shift in allelic ratio in the spleen of Dxz4 ko's . Are there specific genes driving this or is the deletion skewing XCI?Reviewer #3:In their study, Andergassen et al., describe mouse mutants for the noncoding RNA-gene Firre and the Dxz4 Megadomain boundary element, which are important for X-chromosome 3D-structure. It has been an important question in the X-inactivation field, what the functional significance of these elements is and what their impact is on X-chromosome inactivation. Recent studies using cell lines have already demonstrated that deletion of these elements does not affect X-chromosome inactivation (XCI) in vitro. However, the current study is the first one to assess their importance in vivo and during imprinted XCI in the placenta. The data regarding a lack of an XCI phenotype in Firre and Dxz4-deletion mice is therefore important for the X-inactivation field.The second main message of the paper is that nevertheless changes in autosomal gene expression are observed. Only few changes appear to be dependent on Dxz4-deletion, however more are observed in the Firre KO, which is expected from its role as a non-coding RNA, which could also have function away from the X-chromosome. However, as mentioned in my specific comments below, off-target or strain background effects can at the moment not be completely ruled out. Therefore, the authors should address these points, to ensure that the autosomal changes in gene expression are indeed caused by the Firre and Dxz4 mutation and are not artifacts.Essential revisions:1) The authors should perform Southern blotting and/or other assays to confirm that no autosomal off-target mutations were introduced when targeting the Firre and Dxz4 loci. PCR-screening alone as done at the moment  is not sufficient to rule that out especially considering the low number of back-crossings and the variability between replicates when looking at changes in autosomal gene expression . This would be important especially as a main message of the paper is that there are Firre/Dxz4-dependent changes in autosomal gene expression.2) Figure 2: The different knockout alleles have been generated in mixed strain backgrounds  and only relatively few backcrosses seem to have been performed to pure B6 background (>2). Where did the wildtype mice come from, to which autosomal gene expression levels were compared in the adult expression bodymap? Were they of equivalent mixed strain background, or were wildtype controls of pure strain background used? It is important that controls of matched strain background were used throughout the study, in order to avoid differences in autosomal gene expression due to strain-background effects.3) Figure 3—figure supplement 2B and Figure 4—figure supplement 4C: Xist RNA seems to be upregulated dependent on Megadomain deletion  and the allelic-ratio seems to skew random X-inactivation in the brain more towards the M Musculus (Dxz4-deleted) allele in Dxz4 and Firre/Dxz4 KO mice. The authors should check, if Xist skewing and allelic ratios of X-linked genes are significantly changed during random XCI in the Dxz4 and Firre/Dxz4 KOs.4) Figure 2D: The megadomain-specific upregulation of urinary cluster genes in liver seems to be very variable between replicates. It would be good to add a third replicate for delta Dxz4 as at the moment one replicate shows strong upregulation, while the second one doesn't. Also, in the Firre/Dxz4 double-mutants half of the replicates show an effect while others don't. How do the authors explain this discrepancy between replicates?"in vivo Firre and Dxz4 deletion elucidates roles for autosomal gene regulation" for further consideration at eLife. Your revised article has been favorably evaluated by Michael Eisen (Senior Editor), a Reviewing Editor, and three reviewers.Thank you for resubmitting your work entitled Reviewer 1 is satisfied with the revisions (no comments attached). Reviewer 3 also feels that the paper is much improved but remains concerned about strain background. We know that it may be difficult to completely rule out strain background issues, and thus do not consider this to be an impediment to publication. However, reviewer 2 has a number of remaining concerns. In particular:1) Please include a supplementary table listing all RNA-seq samples as reviewer 2 suggested.2) Please present a Venn diagram listing the CAST/BL6 -common and specific escaping genes in placental RNA-seq.3) Please add a p-value for that difference in median X-linked expression and Xist differences.4) Please report Dxz4 SKO and DKO sex ratios in Figure 1B.5) Reviewer 2 also requested that you please re-analyze genome-wide differential expression without log2 FC cutoff in the placental RNA-seq data to distinguish the Firre locus from superloop and megadomain differentially expressed genes (if any) for Figure 4.6) Resolve the incongruence between the genes listed as commonly differentially expressed in Figure 4 and the gene lists in the supplementary file. Please see comments below for more details.Reviewer #2:The revised manuscript by Andergassen et al., adds new placental RNA-seq data that was requested by reviewers so that the authors could exploit imprinted XCI to untangle the effects of Firre and Dxz4 deletions residing on the active vs. the inactive X. This is impossible to do in tissues with random XCI as analyzed in the "bodymap". The authors should state clearly that the conclusions of their bodymap data are possibly impacted by random XCI and unaccounted genetic variation (raised in the prior round of review). There appear to be very few consistently diff. expressed genes for any of the deletions in the placenta or other tissues (except the spleen). Moreover, the authors conclude that XCI and escape in the placenta are not affected, but apply a minimal log2FC cutoff of 1, the maximal differential that could be expected if XCI or escape are impacted (going from 1 to 2 active copies or vice versa). Allelic analyses which address this question directly is only presented in aggregated figures, supplementary tables, or overly compressed log-scales, obscuring gene-specific changes. These issues need to be addressed, not by new experiments, but a more in-depth and transparent gene expression analysis:1) Please include a supplementary table listing all RNA-seq samples (indicating replicate groups) with read depths and% reads aligned. Please include PCA plots of all samples in this analysis, next to or in place of supplemental heatmaps. Supplementary tables on placental RNA-seq showed some genes with high log2FC that failed the significance cutoff – is this a replicate/variance issue? Was batch-correction applied to minimize the impact of different strain backgrounds? There is insufficient information in the methods to replicate this analysis.2) Seeing more escaping genes on the CAST inactive X is an interesting observation, but not presented in meaningful depth. Allele-specific analysis of genes subject to and escaping XCI in the placental RNA-seq should be plotted on log2FC scale from -3 to +3 with Firre as an outlier . And in Figure 3B and C, there should be a Venn diagram listing the CAST/BL6 -common and specific escaping genes in placental RNA-seq.3) Both DKO and Dxz4 show increased Xist expression and DKO animals have a very significant change towards more BL6-biased random XCI . Figure 4—figure supplement 2B should be moved to main Figure 3, as Xist upregulation in the absence of Dxz4 appears to be consistent across the animal. Non-coding genes regulating Xist in cis and skewing XCI were not listed in the supplement – were any of them changed? Please include these genes the supplementary tables (use Gencode reference rather than RefSeq if necessary). In view of Xist and XCI skewing, it is essential that Dxz4 SKO and DKO sex ratios are reported in Figure 1B.4) Please re-analyze genome-wide differential expression without log2FC cutoff in the placental RNA-seq data to distinguish Firre locus from superloop and megadomain differentially expressed genes (if any). Include this info in Figure 4, and discuss in the context of the other random XCI bodymap & GSEA analysis. If Firre is only relevant in the spleen, please state so clearly.5) There is incongruence between the genes listed as commonly differentially expressed in Figure 4 and the gene lists in the supplementary file (only 8 genes match the table & the Venn diagram in 4E). And only 45 genes are diff. expressed in the spleens of both Firre and DKO mice. Please indicate clearly which GSEA-enriched gene sets originated from which comparisons , and include info listing these enriched diff. genes in the updated supplementary tables. There is insufficient information here to replicate this analysis.Reviewer #3:In their revised version the authors have added some new data  and addressed many of my concerns in changing the wording and splitting the figures, making the manuscript more readable. They have not completely addressed the concerns related to the mixed strain background, which is a main weakness of the paper. Nevertheless, the authors have explained the situation in more detail, providing indirect  arguments that the phenotypes observed are indeed due to the gene modifications and not due to strain background effects. Some additional doubts remain about the genotyping strategies used (PCR only – cannot rule out off-target mutations elsewhere in the genome).Nevertheless, the paper has generally improved from the initial version and adds an interesting piece of information to the X-inactivation field regarding functional non-requirement of Firre and X-megadomains for X-inactivation in vivo, but having rather potential functions in direct or indirect autosomal gene regulation. Summary:The study generates single or double deletions of Firre and Dxz4 in mice, and show that despite the repeats being conserved in mammals, these mutants are viable, fertile and show no defect in random or imprinted XCI. Instead, the lack of Firre and Dxz4 results in dysregulated genes on autosomes in an organ-specific manner. Although the authors present mostly negative data, the manuscript would be of interest to the field and is significant because (1) it analyzes the phenotype in vivo in a mouse model, contrasting with previous papers that performed experiments only in cell culture. (2) The current manuscript also sheds light on a previous claim by Giorgetti et al. (2016) that Dxz4 is required for genes to escape from XCI. (3) It is revealed that the loci also do not play a role in imprinted XCI. All three reviewers believe that the scope of the work is in principle in line with eLife. However, for publication in eLife, we would require several major revisions.1) Better controls to rule out strain background or off-target artifacts, when looking at changes in autosomal gene expression. (See reviewer 3 notes).We agree with the reviewer comments that the relatively low number of backcrossing (fewer than 5) of the founder mice with the C57BL/6J strain could result in strain background differences between wild type and KO strains. To control for potential strain background or off-target effects artifacts, we performed all necessary control experiments in our initial submission which we will highlight and discuss in more detail below.Firre, Dxz4 and double deletion founder mice with the C57BL/6J strain in order to ensure a matching genetic background to the KO strains and, thus, to allow for proper comparison between wildtype and KO strains. To clarify this point, we added the following sentence to the “Materials and methods” section (Mouse strains): “In order to control for strain background, all wildtype control mice were obtained from backcrossing of founder mice with C57BL/6J to match the KO strain background.”a) All the wildtype controls samples that we used for RNA-seq originate from backcrossing of the Firre-Dxz4 double KO bodymap we used four biological replicates per group, collected from different litters.b) To minimize potential strain background or off-target effects, for the independently generated SKO mice, strongly arguing for a specific effect of the deletions, rather than strain background or off-target effects. For example, the largest spleen-specific transcriptional effect on autosomes detected in the DKO bodymap was also detected in independently-generated Firre SKO but not in the Dxz4 SKO. Thus, the observed autosomal genes dysregulation can be confidently attributed to the specific deletion of the Firre locus rather than to potential background strain differences or off-target effects.c) The dysregulation of autosomal genes that was observed in the DKO mice, was confirmed in 2) More backcrosses and RNA-seq of placenta, as it is the only tissue with non-random XCI, and therefore the only tissue where the authors can distinguish between effects stemming from Firre/Dxz4's roles on the inactive vs. the active X. (See Reviewer 2)In addition to the above examples that strongly suggest background is not influencing our conclusions, it is important to note that the suggested backcrosses would take approximately a year. At the very least this would require redoing the entire sample collection and sequencing again.Firre and Dxz4 on Xa or Xi. We found similar methylation levels as in wildtype, providing further evidence that deletion of these loci does not affect X chromosome biology .In the placenta we had not observe dysregulation of X-linked genes or gene escape in the presence of the deletions on either Xa or Xi. We have now included one additional placenta biological replicate of the DKO (deletion on Xa and Xi) and confirmed that the deletion of these loci does not have an effect on the X chromosome . To further investigate whether the deletions have any impact on DNA methylation levels of the inactive X chromosome, we have now also performed reduced representation bisulfite sequencing (RRBS) on placentas lacking both Firre and Dxz4 mutants, we collected the visceral yolk sac, an extra-embryonic tissue that, similarly to the placenta, undergoes imprinted XCI. We performed RNA-seq analysis of the visceral yolk sac and as observed in the placenta, we did not detect dysregulation of X-linked genes or gene escape in the presence of the deletions either on Xa or Xi  a further evidence that the lack of the Firre lncRNA or the mega-structures does not affect imprinted X chromosome inactivation.The reviewer also commented that the placenta was the only tissue in our dataset undergoing non-random XCI and we agree that it is worth examining another tissue with non-random XCI. Thus, to further explore the role of non-random XCI in 3) Direct versus indirect effects of Dxz4/Firre on autosomal expression. If the authors would like to claim a direct effect, CHART analyses would be required. Otherwise, the statements should be toned down. (See reviewer 1 notes).Firre RNA that we reported previously , we did find that Ypel4 is downregulated in the spleen of both DKO and Firre SKO mice. We have now highlighted this finding in the revised manuscript. While this finding would suggest a direct role of Firre RNA in at least some of the expression changes, we agree that our experimental approach does not permit to discriminate between direct and indirect effects. To clarify this point, we have revised the text in the Discussion section as follow: “The major function of these dysregulated genes appears to be in ontologies associated with chromosome structure and segregation, which is in line with the known role for Firre lncRNA in nuclear organization. This relationship points to an RNA dependent role, which may be either direct or indirect, on autosomal gene regulation.”We agree with the reviewer that the effect could be either direct or indirect. Indeed, we had acknowledged that further studies are needed to establish whether the effect of the megastructures on autosomal genes regulation is direct or indirect. With regard to the autosomal targets of 4) The 2 main figures should be broken up. (See reviewer 1 notes).To reduce complexity, we broke up the two main figures into 4 figures. As suggested by the reviewers, new Figure 3 is now dedicated to the analysis of imprinted XCI and gene escape, and includes the new results from the visceral yolk sac.5) Revision of statistical analyses. (See reviewer 2 notes).Thank you very much for the observation, we now reported the correct p-value in new Figure 1B. We also added the sex ratio, and provided additional details regarding how calculations were performed in the figure legend.6) Finally, to the extent possible, please try to address the additional concerns of reviewers 2 and 3.See point by point reviewer response.Reviewer #1:The paper from Andergassen et al., is a succinct report made up of mostly negative findings and confirms the negative results of several past publications. Although the knockouts rule out a role in XCI, the report is significant in that (1) it analyzes the phenotype in vivo in a mouse model, contrasting with previous papers that performed experiments only in cell culture; In the majority of past reports, it was shown that the conserved X chromosome "mega-structures" controlled by the Firre and Dxz4 alleles are not required for XCI in cell lines. (2) The current manuscript also sheds light on a previous claim by Giorgetti et al., (2016) that Dxz4 is required for genes to escape from XCI. No effect on gene escape is seen by Andergassen et al., in vivo. (3) The loci also do not play a role in imprinted XCI.The authors generate mice carrying a single or double deletion of Firre and Dxz4, and show that despite the repeats being conserved in mammals, these mutants are viable, fertile and show no defect in random or imprinted XCI. One positive finding is that the lack of Firre and Dxz4 results in dysregulated genes on autosomes in an organ-specific manner. By comparing the dysregulated genes between the single and double deletion, they categorize superloop, megadomain, and Firre locus-dependent gene sets and see that Firre deletion has the greatest effect on autosomal expression signatures. The manuscript is within the scope of papers published in eLife and would be of interest to the field. Overall, the analysese are done well and the conclusions are mostly supported by the data. However, I recommend several revisions:1) While I accept that there are changes in autosomal gene expression, I am less convinced that the effects are direct. I am also not convinced that X-chromosomal superstructures directly affect autosomal gene expression. If the authors would like to claim a direct role, they must perform additional analyses, including – for example – CHART analysis to correlate changes at specific loci with binding of the RNA, or 3C or Hi-C to determine if the loop domains interact with autosomal gene targets. Do these autosomal targets include the loci that the authors previously showed to interact with Firre?2) If the authors do not wish to include additional work, they should tone down the conclusions regarding autosomal changes and acknowledge the possibility of indirect effects, per point 1.Firre RNA that we reported previously , we found that Ypel4 is downregulated in the spleen of both DKO and Firre SKO mice. We have now highlighted this finding in the Results section of the revised manuscript, as follow: . While this finding would suggest a direct role of Firre RNA in at least some of the expression changes, we agree that our experimental approach does not permit to discriminate between direct and indirect effects. We have now clarified this point as indicated in “Essential revision” point 3.We thank the reviewer for the comments. Indeed, we had acknowledged that the effect of the megastructures on autosomal genes regulation could be either direct or indirect (Discussion section of the first submission: “Whether these changes are directly regulated by the Barr body structure remains to be investigated”). With regard to the autosomal targets of 3) The figures are very dense, difficult to read , and should be broken up into additional figures. eLife allows more than 2 display panels. At least one figure should be devoted to a more careful and deeper analysis of XCI gene escape, since the lack of effect on escape is a major point of the paper. And another figure should likewise be devoted to a deeper analysis of imprinted XCI, since this is another major point of the paper.Please refer to “Essential revision” point 4.Reviewer #2:The manuscript by Andergassen, et al., describes the generation of mouse single and double knockouts of two X-linked macrosatellites, Firre and Dxz4. The authors analyze sex ratio, litter size and expression phenotypes of these three knockouts relative to wild-type, i.e. F1 hybrids of musculus x castaneous crosses. Whether and how these macrosatellites may participate in X chromosome inactivation or escape from XCI is an important question, due to previously described roles of Firre (not referenced PMID: 26048247 and PMID:25887447) and Dxz4 (not referenced PMID:26248554) in gene expression and inactive X chromosome conformation, respectively. However, as presented, in vivo defects were non-comprehensively analyzed, and gene expression experiments improperly controlled and somewhat over-interpreted:1) The authors main conclusion, that there is no sex ratio distortion in the double ko, is partially undermined by a miscalculated p-value  and only six reported litters. This table should also include the outcomes of the wildtype control and single homozygous ko matings.Thank you very much for catching this error. We have now corrected it as indicated in point 5 of “Essential revision”.Firre SKO, and corresponding WT, was evaluated as part of another recent study from our group , and it was found to be normal. Based on the finding in the DKO and Firre SKO strains, we reasoned that a sex ratio skewing in the Dxz4 SKO strain would also be unlikely.Given that the male-female ratio of the DKO progeny was not significantly altered, we reasoned that testing for sex ratio skewing within the WT control group was not required. With regard to the SKO strains, the sex ratio of the 2) Differences in the genetic background of wild-type (not specified), single knockouts , and the double ko (variable ~1.6% BALB/C and ~6.25% DBA), are not taken into account in the gene expression analysis. This would be a non-issue with more backcrosses to C57BL/6J, at least five .Firre SKO mouse was generated by inserting LoxP sites flanking the Firre gene body into JM8A ESCs (PMID: 19525957) which are of Bl6 background and not 129xBl6 as we indicated in the original submission. The ESCs were then injected into blastocysts to obtain chimeras that were then backcrossed to Bl6 to obtain the founder mouse. The founder mouse was then crossed with CMV-CRE mice B6.C-Tg(CMV-cre)1Cgn/J, JAX Stock no: 006054. While the CRE JAX strain was generated from BALB/c-I ESCs, stock 006054 was backcrossed to the Bl6 background for 10 generations. Thus, the background strain is Bl6 and not BALB/c as we indicated in the original submission. We apologize for the confusion. The resulting Firre SKO mouse was subsequently backcrossed three times with Bl6, more details in Lewandowski et al. bioXriv 2019. Based on this consideration the background of the Firre SKO is almost 100% Bl6 . The Dxz4 single deletion strainwas generated by co-injecting Cas9 mRNA together with two guide RNA’s that span the Dxz4 locus into pronuclear stage 3 (PN3) zygotes isolated after mating super ovulated B6D2F1 (50% Bl6 50% DBA) female mice with Bl6 males resulting in a founder mouse with 75% Bl6 background. The Founder mouse was then backcrossed two times with Bl6, and can thus be considered as 93.75% Bl6 background . The Firre-Dxz4 double deletion strain, was generated by piezo-assisted Intracytoplasmic sperm injection of Firre SKO sperm (Bl6 background) into B6D2F1 (50% Bl6 50%DBA) oocytes  The resulting DKO founder mouse was then backcrossed two times with Bl6, and can thus be considered as 93.75% Bl6 . All the wildtype controls samples that we used for RNA-seq originate from backcrossing of the Firre, Dxz4 and DKO founder mice with the Bl6 strain in order to ensure a matching genetic background to the KO strains and, thus, to allow for proper comparison between wildtype and KO strains.We agree with the reviewer of the importance to control for potential strain background differences. We addressed this point in “Essential revision” point 1. In addition, we would like to clarify that the We have now provided a more detailed description of the generation of all strains, as well as how the corresponding WT mice were obtained, in the Materials and methods section of the revised manuscript.3) Low and inconsistent numbers of replicates in the RNA-seq that reduce statistical power. This is especially problematic for attributing differential expression to loss of megadomains and superloop, because this analysis depended on comparing across multiple differential gene lists, which shrink with low statistical power (see only two Dxz4 ko replicates). This could be addressed by additional replicates from the single ko's, to get at least four across the board for liver, spleen and placenta.Firre and megadomains and superloop. However, we only used the SKO to verify the direction of dysregulation observed in the DKO bodymap, where we had enough statistical power given that we used four biological replicates per group for this analysis.We agree with the reviewer that having additional replicates from the SKO would increase the statistical power of the analysis in assigning differential expression to loss of 4) Homozygous animals also lack Firre RNA (and possibly Dxz4-associated transcripts) expressed from the active X. The analysis here assumes that there are no confounding interactions between differential genes attributed to one group or another. A deeper analysis of the placental data in heterozygous F1 hybrid animals, where such interactions can be excluded, would be preferable and likely yield more insight towards identifying megadomain- or superloop-specific gene expression, if any.Firre RNA on Xa from that of megadomain and superloop structures that only exist on the Xi. We detected only a few autosomal genes dysregulated in the DKO deletion on either Xa or Xi that were not changed in the SKO, suggesting that the mega-structures and the lncRNA Firre have no impact on autosomal gene regulation in the placenta (Supplementary file 1 sheet A-B). We have now highlighted this finding in the Results section of the revised manuscript, as follow: .”)Thank you very much for this suggestion. To address this point, we have performed an additional analysis to test whether the DKO deletion on either Xa or Xi might result in autosomal gene regulation in the placenta, a tissue where we can disentangle the functional role of In conclusion, while the authors present a worthwhile question and set of experiments, more careful analysis and interpretation is necessary to address functions of Firre and Dxz4 in XCI and development. At minimum, the placental RNA-seq should be repeated and fully analyzed after more than 5 backcrosses, with at least 4 replicates for each of the 4 genotypes presented.We have now included a fourth biological replicate of the placenta DKO as indicated in point 2 of “Essential revision”. For the strain background/backcrossing issue please refer to “Essential revision” point 1.Additional novelty could come from three interesting observations reported here: (1) enrichment for differential genes implicated in faithful chromosome segregation in mice lacking Firre expression, (2) Xist upregulation by 30% in Dxz4 single and double ko's , and (3) shift in allelic ratio in the spleen of Dxz4 ko's . Are there specific genes driving this or is the deletion skewing XCI?Firre dependent enrichment of differential genes involved in chromosome structure and segregation, however we believe that this would be outside the scope of this study and will instead be followed up by a separate study.a) It would be very interesting to follow up the Xist upregulation by 30% in Dxz4 single and DKO is an interesting observation because it suggests that the Dxz4 locus or the resulting megastructure is regulating Xist expression. Given that we don’t detect dysregulated genes on the X chromosome in the absence of Dxz4, our interpretation of this finding is that Xist upregulation occurs only from the inactive X chromosome, suggesting a role for maintaining X chromosome silencing. It would be very interesting to follow Xist expression in Dxz4 deletions to test whether Xist upregulation correlates with aging, however, that is outside the scope of this current study.b) Dxz4 and DKO brains. To test whether the additional skewing is significant we performed a t-test on the Xist allelic ratios between WT (n=17) and each of the KO strains (n=6) and observed significant skewing  in the presence of the DKO deletion , suggesting that the Bl6 chromosome is further biased towards silencing in mice lacking both Firre and Dxz4. We have now highlighted this finding in the Results section of the revised manuscript, as follow:  , suggesting that the Bl6 chromosome is further biased towards silencing in mice lacking both Firre and Dxz4.”)c) In Figure 3—figure supplement 2B we show the allelic ratio of the E12.5 F1 brains (not the spleen) and found the expected XCI skewing ratios, a well-documented effect in female cells from crosses between Bl6 and CAST that results in the predominant inactivation of the Bl6 X chromosome. As pointed out by reviewer 2 and 3, the XCI skewing was even stronger in the Reviewer #3:1) The authors should perform Southern blotting and/or other assays to confirm that no autosomal off-target mutations were introduced when targeting the Firre and Dxz4 loci. PCR-screening alone as done at the moment  is not sufficient to rule that out especially considering the low number of back-crossings and the variability between replicates when looking at changes in autosomal gene expression . This would be important especially as a main message of the paper is that there are Firre/Dxz4-dependent changes in autosomal gene expression.Please refer to point 1 of “Essential revision”.2) Figure 2: The different knockout alleles have been generated in mixed strain backgrounds  and only relatively few backcrosses seem to have been performed to pure B6 background (>2). Where did the wildtype mice come from, to which autosomal gene expression levels were compared in the adult expression bodymap? Were they of equivalent mixed strain background, or were wildtype controls of pure strain background used? It is important that controls of matched strain background were used throughout the study, in order to avoid differences in autosomal gene expression due to strain-background effects.We agreed with the reviewer comment and have addressed this in “Essential revision” point 1-2 and in the response to reviewer 2 point 2.3) Figure 3—figure supplement 2B and Figure 4—figure supplement 4C: Xist RNA seems to be upregulated dependent on Megadomain deletion  and the allelic-ratio seems to skew random X-inactivation in the brain more towards the M Musculus (Dxz4-deleted) allele in Dxz4 and Firre/Dxz4 KO mice. The authors should check, if Xist skewing and allelic ratios of X-linked genes are significantly changed during random XCI in the Dxz4 and Firre/Dxz4 KOs.Thank you very much for this observation; we have addressed this in reviewer 2 point 4c.4) Figure 2D: The megadomain-specific upregulation of urinary cluster genes in liver seems to be very variable between replicates. It would be good to add a third replicate for delta Dxz4 as at the moment one replicate shows strong upregulation, while the second one doesn't. Also, in the Firre/Dxz4 double-mutants half of the replicates show an effect while others don't. How do the authors explain this discrepancy between replicates?As noted by reviewer 2, the expression of major urinary protein genes was shown to be highly variable across individuals (PMID: 26973837) which explains the discrepancy across replicates.Reviewer 1 is satisfied with the revisions (no comments attached). Reviewer 3 also feels that the paper is much improved but remains concerned about strain background. We know that it may be difficult to completely rule out strain background issues, and thus do not consider this to be an impediment to publication. However, reviewer 2 has a number of remaining concerns. In particular:1) Please include a supplementary table listing all RNA-seq samples as Rev2 suggested.We listed all the RNA-seq samples in the supplementary table (sheet: J sample information) and updated the table legend.2) Please present a Venn diagram listing the CAST/BL6 -common and specific escaping genes in placental RNA-seq.We included a Venn diagram listing common and strain specific escaper genes in the placenta in Figure 3—figure supplement 2A.3) Please add a p-value for that difference in median X-linked expression and Xist differences.We have added the FDR-adjusted p-values for the X-linked genes.4) Please report Dxz4 SKO & DKO sex ratios in Figure 1B.Dxz4 SKO strain would be unlikely. For this reason, we kept Dxz4 SKO line to a minimum and mainly used the strain to generate crosses with CAST for the allele-specific placenta analysis. The sex-ratio of the Firre SKO progeny is also normal  and, since the Firre locus is the main driver of the observed expression changes, we feel that assessing sex-ratio in the Dxz4 SKO is outside the scope of this study.As pointed out in the previous round of revision, the male-female ratio of the DKO progeny was not significantly altered , suggesting that sex ratio skewing in the Firre and Dxz4, that provide the platform of the largest conserved chromatin structures in female mammals. We are confident that we addressed this question in the current manuscript and believe that the addition of the male-female ratios of the Dxz4 SKO progeny will not change the conclusion of this study, bearing in mind that the ratios were not altered in the DKO.The main biological question of this study was to investigate the in vivo role of the two Xlinked loci 5) Reviewer 2 also requested that you please re-analyze genome-wide differential expression without log2 FC cutoff in the placental RNA-seq data to distinguish the Firre locus from superloop and megadomain differentially expressed genes (if any) for Figure 4.Firre SKO on Xa (Firre RNA-specific), 45 genes between the DKO and Firre SKO on Xi (superloop-specific) and none between the DKO and Dxz4 SKO on Xi (megadomainspecific). Of note, in all cases (with the exception of the deleted Firre gene), the mean log2FC between the DKO and SKO’s is close to zero .As suggested, we have re-analyzed genome-wide placental RNA-seq differential expression without log2FC cutoff. By comparing differentially expressed genes between the DKO and each of the SKO we find 3 overlapping genes between the DKO and zero see . This reGiven that these results are consistent with those from our original analysis, we did not include the new analysis in the revised manuscript.6) Resolve the incongruence between the genes listed as commonly differentially expressed in Figure 4 and the gene lists in the supplementary file. Please see comments below for more details.Firre SKO spleen .The genes listed in the supplementary file (Supplementary file 1 sheet G-H) and reported in the heatmap in Figure 4D  have been identified by differential expression analysis and represent genes dysregulated in at least one of the three KO strains (FDR ≤ 0.1) where the direction of the expression change in either one of the single KO agrees with that of the DKO (|log2FC| ≥ 1). The genes in Figure 4E (MA-plot and Venn diagram), instead, are the genes within the top 5 enriched dysregulated gene sets identified by the GSEA analysis in the DKO and To make clear that these are two different analyses we have added the following header to Figure 4E “GSEA identified chromosome structure and segregation gene sets”. We have also provided additional information in the method section and included the enriched GSEA gene sets and the corresponding DESeq2 output in Supplementary file 1 sheet I (genes ranked by lfcMLE) to allow for replication of the analysis. Given that all the information of figure 4E is now provided in the supplementary table, we removed the Venn diagram and instead added the spearman correlation of the fold changes.


Pollution prevention (P2) has become an integral part of the U.S. environmental policy that emphasizes the benefits of preventing pollution generation at the source over treatment or recycling after the generation of wastes. This study extends the existing literature on the effect of voluntary adoption of P2 in reducing toxic wastes by examining the extent to which it reduces pollution substitution. We use facility panel data from the Toxics Release Inventory from 1991 to 2011 to examine the effect of the adoption of P2 techniques on the ratios of water releases to air releases, amounts of treatment to total releases, and amounts of recycling to total releases while controlling for endogeneity of the adoption of P2 techniques and facility fixed effects. We find that the adoption of P2 techniques reduces toxic air and water releases equally, but it is associated with increases in treated and recycled wastes over total releases to the environment. Pollution controls in the United States rely on command and control regulation that emphasizes end-of-pipe abatement. The problem with this method is that end-of-pipe abatement often focuses on limiting direct releases to a single environmental medium. For example, installing wet scrubbers in smokestacks often turns air pollutants to wastewater, thus solving one environmental problem by creating another –3. AdditTo provide the public a more comprehensive view of a facility’s waste management hierarchy, the PPA expanded the Toxic Release Inventory (TRI) to include additional reporting on the adoption of P2 techniques and the amount of toxic wastes recycled or reused for energy recovery (incineration) per each TRI chemical . BetweenExisting studies show that, even though many TRI chemicals are not directly regulated by environmental regulations, public access to the TRI publication has motivated polluters to voluntarily reduce toxic releases due to pressures from local communities, investors, regulators, and consumers –12. ReceThis examination is particularly important to quantify the overall environmental impact of the voluntary adoption of P2 techniques in the presence of media-specific regulation (such as the Clean Air Act). Media-specific regulation often causes pollution substitution by reducing one type of pollution  at the expense of another type of pollution  –3,14. UnHowever, it is difficult to discern substitution by observing declining releases and increasing wastes separately without econometric analysis that controls the change in output. Even if P2 techniques can reduce total production-related wastes, the relative proportions of treated and recycled wastes may still increase while holding total releases constant. In other words, the relative importance of treatment and recycling increases than what it would have otherwise been. This paper empirically examines the effect of voluntary adoption of P2 techniques on pollution substitution by examining the effect of P2 adoption on the ratios of toxic releases versus treatment or recycling of toxic wastes, and of toxic releases to water versus air. In contrast, existing studies on pollution substitution focus on command-and-control regulation, rather than voluntary adoption of P2 techniques. Studies on the adoption of P2 techniques focus on its effect on total toxic releases to the environment  or indivWe use facility panel data from the Toxics Release Inventory (TRI) for the period 1991 to 2011. The comprehensive nature of TRI allows us to examine two decades of data on P2 technique adoption, toxic releases, and treated and recycled wastes, accounting for about 50% of the U.S. manufacturing base from both public and private facilities . We modeThe adoption and diffusion of P2 techniques rely on information provisions and voluntary approaches rather than prescriptive technology standards. TRI is an information disclosure tool to help local communities prepare for potential health and environmental hazards caused by the storage, use, and release of toxic chemicals commonly used in manufacturing. Specifically, manufacturing facilities that have 10 or more full-time employees and that manufacture, process, or use any TRI-listed chemicals in amounts exceeding 25,000 pounds per year for manufacturing or processing, or 10,000 pounds per year for otherwise use, must submit annual reports to the Environmental Protection Agency (EPA) on the amounts of toxic wastes released and treated. As part of the voluntary (non-mandatory) policies to encourage P2, TRI was expanded under the PPA to include additional reporting on the adoption of new P2 techniques and the amount of toxic wastes recycled or used for energy recovery (incineration) per each TRI chemical )We model multiple pollutants  as inputs used by a TRI facility for producing output, following the previous literature , 26, 27.R and W, and another input, L (such as labor). The cost function can be expressed as C = f(Q)*C, in which f(Q) represents the output quantity, and PR, Pw, and PL represent the cost of pollution inputs R and W, and the cost of L, respectively. The facility operates in competitive input and output markets, and the cost function is homogenous degrees of one in all prices. In other words, if all prices double, the total costs will double, and the optimal allocation of inputs derived from cost minimization does not change.Following Gibson , we assuAs a result of cost-minimization, the conditional input demand for one type of pollutant can be obtained by differentiating the above equation with respect to its price, such that PL [R is expressed as the percentage change in pollutant W with respect to price change in pollutant R minus the percentage change in pollutant R in response to price change in pollutant R, holding output level fixed [Estimating the ratio of optimal inputs with respect to the price proxies reveals the net substitution between the inputs and the estimation is independent of output quantity and does not need to include the prices of all inputs, such as PL . Net subel fixed . It is mFollowing this theoretical insight, our empirical model proceeds by modeling the ratio of pollutants as described in Eq  below:lratio)it, is defined as facility i’s log ratio of two pollutants at year t. ai represents unobserved facility specific fixed effects. ppit−1 is the variable of our interests, and measures the intensity effect of P2 techniques on the implicit prices of the two pollutants by affecting costs to comply with regulations or to appeal to the public.The dependent variable, ln. ν is the percentage increase in relative prices of two inputs (β2 are expected to be zero.Gibson  shows thX′it−1 represents a vector of facility i’s covariates affecting pollution substitution in the preceding year. D′ represents a vector of fixed effects that include state and industry (defined by the 2-digit SIC codes) dummies. These dummy variables control for unobserved factors common for a given state or a given industry that affect the pollution ratios. D′×t represents the interactions between state and industry dummies and the linear year trend to control for state and industry specific year trends, such as industry’s technology change and state’s environmentally friendly efforts, which could affect pollution substitution.Additionally, cumulative P2 may still be endogenous. For example, a facility or its parent firm may have adopted environmental management systems that have been shown to increase the adoption of P2 techniques [Although facility specific fixed effects can be controlled in estimating 2) by using fixed-effects panel models, chniques ,18. Such by usingZ′it−2 represents the vector of two time-varying instrumental variables that are lagged by two years.Thus we use 2SLS panel regression with instrumental variables and multiple levels of fixed effects that takes into account the correlation of the error terms across time to estimate Eq  below, ippit−1 but are uncorrelated with time-varying unobservables, such as adoption of the environmental management system in Eq  into  for each one of the ratios: toxic releases to water versus releases to air, the amount of toxic recycling wastes versus total toxic releases, and the amount of treated toxic wastes versus total toxic releases, respectively. This analysis focuses on a group of TRI toxic chemicals (296) listed from 1988 onwards without any change in reporting requirements . We refe(x+x2+1) . To geneppit−1 to approximate the intensity of P2 techniques implemented since a facility first became an adopter of P2 techniques because TRI reporting requirement applies only for incremental (new) P2 techniques per TRI chemical in a given year. Specifically, we first aggregate the cumulative number of P2 techniques reported by a facility for the core chemicals up to one period prior, and then divide the cumulative number by the total number of reported core chemicals by each facility up to one period prior. We refer to this new variable as “cumulative P2” hereafter. Considering TRI provides annual data, we lag the cumulative P2 variable to avoid contemporaneous problems with the dependent variable. Nevertheless, a TRI facility may discontinue any previously reported P2 techniques without having to report disadoption. We conduct robustness checks on the definition of the cumulative P2 variable in the next section.We construct the variable Following the previous literature, we use environmental inspections by state and federal regulators on the facilities and each county’s attainment status as proxy variables for the regulatory pressure for a TRI facility. Hanna and Oliva  show thaWe use a county’s nonattainment status as another variable for regulatory pressure. Under the CAA, the National Ambient Air Quality Standards (NAAQS) for carbon monoxide, sulfur dioxide, total suspended particle pollution (PM), ground-level ozone, nitrogen oxide, and lead are set by the EPA, which is responsible for ensuring that ambient air quality in each county meets the NAAQS . Once a PM Nonattainment and Ozone Nonattainment as dummy variables that are equal to 1 if a county falls into nonattainment with PM or ozone, respectively, in a given year, and 0 otherwise. Following previous studies, the change in a county’s attainment status is considered exogenous to a facility’s toxic releases and the two variables are lagged by one period in the estimations to allow time for counties and TRI facilities to respond to the regulation [We focus on attainment status for two types of pollutants, particulate matter and ground-level ozone (most counties obtained attainment status for the rest of the criteria for air pollutants by 1998). We define gulation . Based ogulation .the number of employees of the facility is included in the covariate vector to control for the size of the facility. TRI does not include information regarding the level of production and the number of employees, so we use the number of employees obtained from the National Establishment Time-Series [Natural log of e-Series  to contre-Series .We apply two time-varying instrumental variables (IVs) based on findings from the previous literature. They are the lagged proportion of facilities that have adopted P2 techniques in the same industry (defined by the 2-digit SIC codes), excluding the facility itself, and the lagged average number of federal inspections on other facilities, excluding the facility itself, in the same state. Both IVs are lagged by two periods.Both IVs are expected to be strongly correlated with the number of P2 techniques. Specifically, a facility is more likely to adopt P2 if others in its industry or its state peers have adopted P2 techniques, either through information spillovers or through shared supply chains. For example, Harrington  finds thThe valid IVs have to satisfy the exclusion restriction. That is, conditional on the covariates, the IVs should have no effect on pollution substitution in the current period except through a facility’s own adoption of P2 up to the previous period. We believe these IVs satisfy the exclusion restriction because they are based on the production circumstances of other facilities two periods prior. Additionally, the number of inspections on other facilities for compliance with the Clean Air regulation two periods prior depends on their violation history, state budget of enforcements, and local complaints. While more inspections may motivate facilities from the same state to adopt P2 techniques through deterrence, they are unlikely to influence a facility’s pollution substitution after controlling for environmental inspections and other regulatory pressures on the facility itself. Summary statistics of the explanatory and instrumental variables are reported in We report the estimated effects of cumulative P2 adoption on different types of pollution substitutions , includicumulative P2 techniques was 0.083 and was statistically significant at the 5% significance level. Based on this information, one additional P2 technique adopted per chemical increased the amount of treatment in the current period by 8.3%, holding total releases and other covariates fixed. To put this into context, the average total releases were 58,235 pounds, the average quantities of treated wastes were 59,582 pounds, and the input ratio of treated wastes to total releases was 1.02 in 1991. Holding the covariates and total releases at their averages in 1991, adopting one additional P2 technique per chemical increased the ratio of the two pollutants to 1.10. Such an increase was equivalent to an increase of 4,945 pounds in the amounts of treated wastes. The positive correlation between end-of-pipe treatment and adoption of P2 techniques is also noted in Dutt and King [We find that the ratio of treated wastes versus total releases increased as facilities adopted more P2 techniques  good operation practices , (ii) inventory control, (iii) spill and leak prevention, (iv) raw material modification, (v) process modification, (vi) cleansing and decreasing modification, (vii) surface and finishing modification, and (viii) product modification. Among the eight types of P2 techniques, Ranson et al.  further cumulative P2 variable into three types of P2, following Sam [P2 PROC represents techniques related to changes in operating procedure (P2 types i and ii); variable P2 EQUIP represents techniques on the installation of environmentally friendlier equipment and processes ; and variable P2 MAT represents techniques involved in raw material and product modification (P2 types iv and viii).To investigate the heterogeneous effects by types of P2 techniques on pollution substitutions between treatment and releases and between recycling and releases, we separate the wing Sam , to examPROC, P2 EUIP, and P2MAT in the same industry two years prior, and the average number of federal inspections on other facilities in the same state two years prior.Because all three types of P2 techniques are potentially endogenous, we have recreated three additional instruments based on the percentage of other facilities in the same industry  that adopted the three specific types of P2 techniques  two years prior. For each model reported in P2 EQIP) significantly increased the ratio of treated versus total releases by 10%  and recycled versus total releases by 21.6% ((P2 PROC) did not significantly affect any type of pollution ratios. P2 techniques related to material modification (P2 MAT) only increased the ratio of recycling versus total releases .We find that P2 techniques related to equipment and process modification  P2 techniques on an annual basis. A previous study suggests that the effectiveness of P2 techniques in reducing toxic releases diminishes after 5 years . Therefocumulative P2 on the levels of releases, amounts of wastes recycled, and amounts of wastes treated, separately  has recommended that member countries establish reporting systems to track progress in pollution control and to facilitate exchanges of information on the best available techniques for P2 –45. The There are several limitations associated with this study. First, our results apply to manufacturing facilities subject to the TRI reporting requirements over the period 1991 to 2011. Since TRI reporting facilities tend to be larger polluters, this may limit the generality of our findings on P2 programs implemented by other sectors, smaller businesses below the reporting thresholds, and more recently implemented P2 techniques. Second, recycling and treatment are still the preferred pollution control approaches over direct releases to the environment. More importantly, treatment and recycling methods reduce the toxicity of wastes released to the environment through stabilization, destruction, and reuse. Due to data limitation, it is difficult to incorporate chemical toxicity by calculating toxic-weighted emissions in this analysis since chemical toxicity differs by environment medium and by specific treatment and recycling method. Third, we focus on the average effect of P2 techniques without examining the effects of P2 techniques on pollution substitution by different industries due to data limitation. Future studies can conduct industry specific analysis through surveys to develop recommendations. For example, Gaona  shows thS1 File(ZIP)Click here for additional data file.


Pin1 is unique and only catalyzes the phosphorylated Serine/Threonine-Proline (S/T-P) motifs of a subset of proteins. Since the discovery of Pin1 as a key protein in cell cycle regulation, it has been implicated in numerous diseases, ranging from cancer to neurodegenerative diseases. The main features of Pin1 lies in its two main domains: the WW (two conserved tryptophan) domain and the PPIase domain. Despite extensive studies trying to understand the mechanisms of Pin1 functions, how these two domains contribute to the biological roles of Pin1 in cellular signaling requires more investigations. The WW domain of Pin1 is known to have a higher affinity to its substrate than that of the PPIase domain. Yet, the WW domain seems to prefer the trans configuration of phosphorylated S/T-P motif, while the PPIase catalyzes the cis to trans isomerasion. Such contradicting information has generated much confusion as to the actual mechanism of Pin1 function. In addition, dynamic allostery has been suggested to be important for Pin1 function. Henceforth, in this review, we will be looking at the progress made in understanding the function of Pin1, and how these understandings can aid us in overcoming the diseases implicated by Pin1 such as cancer during drug development.Pin1 belongs to the family of the peptidyl-prolyl  This allows the cells to exert more biological functions with lesser number of proteins. There exist many types of PTMs, of which, the reversible phosphorylation is widely studied for its role in regulating many signaling cascade . Initial) domain . The WW ) domain –5. This outcomes , 6. Thisoutcomes , 8.So far, Pin1 has been extensively studied for its role in various cellular functions, particularly in cell cycle regulation , 6, 9–11cis/trans isomeric activity. This would then trigger its intended downstream cellular signals and effects. Many models have been introduced to explain the molecular mechanism of how Pin1 exert its catalytic activity. However, there has not been a model in agreement to truly explain how Pin1 acts on its biological substrates. Therefore, in this review, we will highlight the progress of Pin1 research in elucidating the actual mechanism of Pin1, with an emphasis on the structural importance of Pin1 on its function, and how the perturbation to this fundamental structure could explain for its roles in diseases such as cancer. We will also discuss how these structural features could be used in the drug development of Pin1 inhibitors.In addition, there have been many structural and protein dynamic studies to understand how Pin1 could target its biological substrates to exert its intrinsic cis/trans isomerase activity in Pin1 , and part of the PPIase domain (S138 to R142). This domain interface has been suggested to play an important role in interdomain communication that regulates the function of Pin1 upon substrate binding –62. Thesin vitro as compared to the PPIase domain  protein and splicing factor, proline-, and glutamine-rich (SFPQ) protein for its activity. As such, there seems to be two types of Pin1 interactors that requires either the binding of both WW and PPIase domains, or just the WW/PPIase domain alone.Indeed, previous studies have shown that the PPIase domain of Pin1 is able to bind to pS/T-P motif instead of just its WW domain , 58, 65.l Figure . In theitrans over cis configuration. As such, the PPIase isomerization would not have occurred with this sequential binding.These proposed four models of Pin1 interaction with its biological substrates for functional regulation might suggest two aspects. Firstly, there is still lack of concrete evidences to highlight which model(s) Pin1 may deploy for its interaction with its biological substrates. Secondly, these models demonstrate the potentially diverse interaction that Pin1 has on its already diverse biological interactors to play a role in the regulation of various biological functions. All these models do have their merits as well as potential doubts as to its suitability. For instant, the sequential binding model does fulfill the characteristics of the WW domain having a higher affinity of peptide binding to the pS/T-P motif than the PPIase domain. In proteins with multiple sites of pS/T-P motifs, this model could explain the use of the WW domain to localize the concentration of the PPIase domain for isomerization. However, for substrate with a single pS/T-P motif, the potential release of the WW domain from this motif, followed by the binding of the PPIase domain for catalytic activity does not seem to be energy favored. This is also highlighted by studies that we have mention previously , 68–70, via the WW domain of Pin1, the increased local concentration of Pin1 would allow the binding to its biological substrate in the vicinity to elicit its downstream biological processes. Furthermore, the pS/T-P motif of the kinase could be in a trans configuration state to allow for WW domain binding. In addition, the catalysis-first binding model could also be associated with the multimeric binding model where Pin1, bound to the active kinase via the WW domain, is brought to its target substrate by the active kinase. Subsequently, the active kinase would phosphorylate the Pin1 substrate, allowing for the PPIase domain of the Pin1 to bind and catalyze the cis-trans conformational change. Furthermore, the suggested simultaneous binding model could also fit the multimeric binding model, although this would occur on a single protein, instead of the proposed protein complex in the multimeric binding. Of interest, the binding of the WW domain in the trans configured pS/T-P motif could lead to conformational changes to the PPIase domain, increasing its binding capacity to the cis configured p-S/T-P motif and subsequently its catalytic efficiency. The potential role of interdomain communications between the WW domain and the PPIase domain of Pin1 has indeed been studied in recent years as discussed in the next section.For multimeric binding to an active kinase in vivo, suggesting the presence of interdomain communication. In addition, studies conducted on Pin1 and tau interactions highlighted the need of WW domain interaction with the pS/T-P motif for the effective function of the PPIase activity  studies . As highg et al. , 87, Namg et al.  found thvia a hinge-like movement, an observation echoed by a more recent study done by Campitelli et al.  in Saccharomyces cerevisiae from death  of 72.3 nM, which is 100× less than the IC50 of the first Pin1 inhibitor juglone. API-1 was found to bind to the PPIase domain of Pin1. The authors went on to show that API-1 was able to suppress the proliferation of hepatocellular carcinoma cells as well as inhibiting the tumor growth of xenografted mice. These evidences demonstrated the therapeutic value of API-1 as a cancer drug candidate. Another study by Cui et al. (via the synthesis of various pyrimidine derivatives. The authors were able to identify four potential Pin1 inhibitors with IC50 of <3 μM. Furthermore, this type of drug is only the second covalent binding Pin1 inhibitors to the phosphate-binding loop in the PPIase domain besides juglone. These studies clearly highlight the potential therapeutic value of Pin1 as a cancer drug target. Unfortunately, no drugs thus far has reached clinical trials (The development of Pin1 inhibitors has remained a challenge in the field of cancer studies as many known inhibitors of Pin1 remains unspecific enough to only block the effects of Pin1 despite a number of Pin1 inhibitors already found , 98, 99.i et al.  demonstrl trials , 98.in vivo binding affinity to Pin1. The authors went on to improve the permeability of the benzothiophene class of drug to eventually yield dihydrothiazoles as a potent Pin1 inhibitor with low micromolar inhibitory activity toward cancer cell proliferation (Of interest, most drugs identified thus far are mainly targeting the PPIase domain, especially the catalytic domain, with few drug candidates found to target the WW domain  as summaferation . Howevercis configured pS/T-P motif (Of importance, these studies only made use of the PPIase domain as the main consideration in designing of their structure-based Pin1 inhibitor. The authors did highlight some important points supporting intra-structure dynamics of the whole Pin1. Firstly, they noticed that despite the rich hydrogen bonding potential within the phosphate-binding loop in the PPIase domain of Pin1, hydrophobic interactions were central to benzothiophene binding to Pin1. Secondly, they also highlighted the presence of H-bond formation beyond the phosphate-binding loop that could be important for the Pin1 inhibitor binding infinity. Lastly, they highlighted the high degree of flexibility in Pin1 active site interaction with the inhibitors. All these observations do align with our previous discussion where interdomain communications between WW and PPIase domains could affect the side chain dynamics on the conserved hydrophobic conduit region and alters PPIase domain binding affinity , 91, 92.-P motif . Hencefoin vivo circumstances are yet to be determined and could be explored in future drug development for Pin1 inhibitor.As suggested from all the studies on the two domains of Pin1 covered in this review, the WW domain binding displays a potentially important role in driving the signaling cascade triggered by Pin1 catalytic function. To advance our development of promising Pin1 inhibitors for treatment of diseases such as cancer, there are important considerations to be taken note of. Much interest has been put forth on identifying Pin1 inhibitors that target the PPIase domain as well as its active site. As suggested in this review, the binding affinity of Pin1 inhibitors could be affected by the substrate that is bound to the WW domain. Therefore, binding affinity studies for Pin1 inhibitors screening should consider the substrate effect upon its binding to the WW domain, and how this effect could alter the Pin1 inhibitors binding to the PPIase domain or its active site. Of interest, a very recent study by Momin et al.  using MDThis potential substrate-dependent Pin1 activity could be important in future studies. This is highlighted in the lack of depth in the field of Pin1 research thus far. Most studies done on Pin1 mechanism focuses on just a few biological substrates of Pin1. As suggested in this review, the vast repertoire of Pin1 functional substrates and the different biological processes it could affect do point to the fact that Pin1 might behave differently in the presence of different substrate. This difference in behavior could affect its structural dynamics that makes identifying a functional Pin1 inhibitor difficult for different types of diseases. Therefore, to advance the research of the role of Pin1 in the pathogenesis of various diseases, more emphasis on the diversity of Pin1 substrates being tested must be done. As mentioned previously, Pin1 inhibitor screening could be done in the presence of its effector substrate to evaluate the efficacy of the Pin1 inhibitor in affecting the phenotype of the disease being studied. In addition, studies looking into how Pin1 cooperation with kinase in exerting its isomeric activity could be conducted to investigate if the proposed multimeric model holds true, and how the subsequent findings could affect the considerations when developing Pin1 inhibitors. Indeed, studies thus far do suggest that Pin1 is not a lone ranger, and it always needs a helping hand to exert its function. To advance the development of drugs to highlight the impact of Pin1 as a cancer therapy target, both domains should be considered during the drug development process for the field to realize the potential of Pin1 as a cancer therapeutic target.All authors listed have made a substantial, direct and intellectual contribution to the work, and approved it for publication.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.