Polycomb Group (PcG) proteins form memory of transient transcriptional repression that is necessary for development. In Drosophila, DNA elements termed Polycomb Response Elements (PREs) recruit PcG proteins. How PcG activities are targeted to PREs to maintain repressed states only in appropriate developmental contexts has been difficult to elucidate. PcG complexes modify chromatin, but also interact with both RNA and DNA, and RNA is implicated in PcG targeting and function. Here we show that R-loops form at many PREs in Drosophila embryos, and correlate with repressive states. In vitro, both PRC1 and PRC2 can recognize R-loops and open DNA bubbles. Unexpectedly, we find that PRC2 drives formation of RNA-DNA hybrids, the key component of R-loops, from RNA and dsDNA. Our results identify R-loop formation as a feature of Drosophila PREs that can be recognized by PcG complexes, and RNA-DNA strand exchange as a PRC2 activity that could contribute to R-loop formation.

The human chromosomal region 17q12-q21 is one of the best replicated genome-wide association study loci for childhood asthma. The associated SNPs span a large genomic interval that includes several protein-coding genes. Here, we tested the hypothesis that the zona pellucida-binding protein 2 (ZPBP2) gene residing in this region contributes to asthma pathogenesis using a mouse model. We tested the lung phenotypes of knock-out (KO) mice that carry a deletion of the Zpbp2 gene. The deletion attenuated airway hypersensitivity (AHR) in female, but not male, mice in the absence of allergic sensitization. Analysis of the lipid profiles of their lungs showed that female, but not male, KO mice had significantly lower levels of sphingosine-1-phosphate (S1P), very long-chain ceramides (VLCCs), and higher levels of long-chain ceramides compared to wild-type controls. Furthermore, in females, lung resistance following methacholine challenge correlated with lung S1P levels (Pearson correlation coefficient 0.57) suggesting a link between reduced AHR in KO females, Zpbp2 deletion, and S1P level regulation. In livers, spleens and blood plasma, however, VLCC, S1P, and sphingosine levels were reduced in both KO females and males. We also find that the Zpbp2 deletion was associated with gain of methylation in the adjacent DNA regions. Thus, we demonstrate that the mouse ortholog of ZPBP2 has a role in controlling AHR in female mice. Our data also suggest that Zpbp2 may act through regulation of ceramide metabolism. These findings highlight the importance of phospholipid metabolism for sexual dimorphism in AHR.

Genome-wide association study (GWAS) loci for several immunity-mediated diseases (early onset asthma, inflammatory bowel disease (IBD), primary biliary cholangitis, and rheumatoid arthritis) map to chromosomal region 17q12-q21. The predominant view is that association between 17q12-q21 alleles and increased risk of developing asthma or IBD is due to regulatory variants. ORM sphingolipid biosynthesis regulator (ORMDL3) residing in this region is the most promising gene candidate for explaining association with disease. However, the relationship between 17q12-q21 alleles and disease is complex suggesting contributions from other factors, such as trans-acting genetic and environmental modifiers or circadian rhythms. Circadian rhythms regulate expression levels of thousands of genes and their dysregulation is implicated in the etiology of several common chronic inflammatory diseases. However, their role in the regulation of the 17q12-q21 genes has not been investigated. Moreover, the core clock gene nuclear receptor subfamily 1, group D, member 1 (NR1D1) resides about 200 kb distal to the GWAS region. We hypothesized that circadian rhythms influenced gene expression levels in 17q12-q21 region and conversely, regulatory elements in this region influenced transcription of the core clock gene NR1D1 in cis. To test these hypotheses, we examined the diurnal expression profiles of zona pellucida binding protein 2 (ZPBP2/Zpbp2), gasdermin B (GSDMB), and ORMDL3/Ormdl3 in human and mouse tissues and analyzed the impact of genetic variation in the ZPBP2/Zpbp2 region on NR1D1/Nr1d1 expression. We found that Ormdl3 and Zpbp2 were controlled by the circadian clock in a tissue-specific fashion. We also report that deletion of the Zpbp2 region altered the expression profile of Nr1d1 in lungs and ileum in a time-dependent manner. In liver, the deletion was associated with enhanced expression of Ormdl3. We provide the first evidence that disease-associated genes Zpbp2 and Ormdl3 are regulated by circadian rhythms and the Zpbp2 region influences expression of the core clock gene Nr1d1.

Epigenetic regulation is conveyed through information encoded by specific chromatin 20 features. Non-canonical nucleic acid structures could in principle also convey biological 21 information but their role(s) in epigenetic regulation is not known. Polycomb Group (PcG) 22 proteins form memory of transient transcriptional repression events that is necessary for 23 development. In Drosophila, PcG proteins are recruited to specific DNA sequences, Polycomb 24 Response Elements (PREs). PREs are switchable memory elements that can exist in repressed, 25 active, or unengaged states 1,2 . How PcG activities are targeted to PREs to maintain repressed 26 states only in appropriate developmental contexts has been difficult to elucidate. Biochemically, 27PcG protein complexes modify chromatin to maintain gene repression 1,3,4 . However, PcG 28 proteins also interact with both RNA and DNA, and RNA is implicated in the targeting of PcG 29 function. We find that R-loops, three-stranded nucleic acid structures formed when an RNA 30 hybridizes to its complementary DNA and displaces the other DNA strand 5 , form at many PREs 31 in Drosophila embryos, and correlate with the repressive state. R-loops are recognized by the 32 PcG complex PRC1 in vitro. Unexpectedly, we find that the PcG complex PRC2 has RNA strand 33 invasion activity, which can drive formation of RNA-DNA hybrids, the key component of R-34 loops. Our results suggest a new mechanism for targeting PcG function through R-loop 35 formation by PRC2 and recognition by PRC1. More generally, our findings suggest formation 36 and recognition 6 of non-canonical nucleic acid structures as an epigenetic mechanism.37 38 39 40 41 Main Text: 42 During Drosophila embryogenesis, transiently expressed transcription factors activate 43 homeotic (Hox) genes in certain regions of the embryo and repress them in others to dictate the 44 future body plan 7 . Polycomb Group (PcG) proteins form a memory of these early cues by 45 maintaining patterns of Hox gene repression for the rest of development 1,7,8 . This paradigm for 46 transcriptional memory is believed to be used by the PcG at many genes in Drosophila, and to 47 underlie the conserved and essential functions of PcG proteins in cell differentiation and 48 development from plants to mammals 9,10 . Polycomb Response Elements (PREs) are DNA 49 elements that can recruit PcG proteins, but they also recapitulate the memory function of the PcG-50 when combined with early acting, region-specific enhancers in transgenes, they maintain transgene 51 repression in a PcG-dependent manner only in regions where the early enhancer was not active 52 1,11,12 . PREs contain a high density of binding sites for transcription factors that can recruit PcG 53 proteins through physical interactions 12 . However, the widespread expression, binding pattern, 54 and properties of factors that bind PREs cannot explain how PREs can exist in alternate, 55 transcription-history dependent states to maintain restricted patterns of gene expression, or how 56