Kassis, J. A. | Search bioRxiv

bioRxiv2023

Polycomb protein binding and looping mediated by Polycomb Response Elements in the ON transcriptional state

Polycomb group proteins (PcG) mediate epigenetic silencing of important developmental genes and other targets. In Drosophila, canonical PcG-target genes contain Polycomb Response Elements (PREs) that recruit PcG protein complexes including PRC2 that tri- methylates H3K27 forming large H3K27me3 domains. In the OFF transcriptional state, PREs loop with each other and this looping strengthens silencing. Here we address the question of what PcG proteins bind to PREs when canonical PcG target genes are expressed, and whether PREs loop when these genes are ON. Our data show that the answer to this question is PRE-specific but general conclusions can be made. First, within a PcG-target gene, some regulatory DNA can remain covered with H3K27me3 and PcG proteins remain bound to PREs in these regions. Second, when PREs are within H3K27ac domains, PcG- binding decreases, however, this depends on the protein and PRE. The DNA binding protein GAF, and the PcG protein Ph remain at PREs even when other PcG proteins are greatly depleted. In the ON state, PREs can still loop with each other, but also form loops with presumptive enhancers. These data support the model that, in addition to their role in PcG silencing, PREs can act as "promoter-tethering elements" mediating interactions between promoter proximal PREs and distant enhancers.

genetics↗

bioRxiv2023

The activity of engrailed imaginal disc enhancers is modulated epigenetically by chromatin and autoregulation

engrailed (en) encodes a homeodomain transcription factor crucial for the proper development of Drosophila embryos and adults. Like many developmental transcription factors, en expression is regulated by many enhancers, some of overlapping function, that drive expression in spatially and temporally restricted patterns. The en embryonic enhancers are located in discrete DNA fragments that can function correctly in small reporter transgenes. In contrast, the en imaginal disc enhancers (IDEs) do not function correctly in small reporter transgenes. En is expressed in the posterior compartment of wing imaginal disks; small IDE-reporter transgenes are expressed in the anterior compartment, the opposite of what is expected. Our data show that the En protein binds to en IDEs, and we suggest that En directly represses IDE function. We identified two en IDEs, O and S. Deletion of either of these IDEs from a 79kb HA-en rescue transgene (HAen79) caused a loss-of-function en phenotype when the HAen79 transgene was the sole source of En. In contrast, flies with a deletion of the same IDEs from the endogenous en gene had no phenotype, suggesting a resiliency not seen in the HAen79 rescue transgene. Inserting a gypsy insulator in HAen79 between en regulatory DNA and flanking sequences strengthened the activity of HAen79, giving better function in both the ON and OFF transcriptional states. Altogether our data show that the en IDEs stimulate expression in the entire imaginal disc, and that the ON/OFF state is set by epigenetic regulators. Further, the endogenous locus imparts a stability to en function not seen even in a large transgene, reflecting the importance of both positive and negative epigenetic influences that act over relatively large distances in chromatin. Author SummaryGenes that control development are often used at different times and places in a developing embryo. Transcription of these important genes must be tightly regulated; therefore, these genes often have large arrays of regulatory DNA. In Drosophila, discrete fragments of DNA (enhancers) can be identified that turn genes on in patterns in the early embryo. In cells where the genes are transcriptionally ON, there are active modifications on chromatin, setting later enhancers in a transcription-permission environment. In cells where the genes are OFF, repressive chromatin marks keep later enhancers inactive. In this paper we studied two late enhancers of the Drosophila en gene. We show that the correct activity of these enhancers is dependent on being next to other, earlier acting en enhancers. Our data also show that En can repress its own expression, likely directly by acting on these late enhancers. The chromatin-regulated activity of these en late enhancers is similar to what was described for a late enhancer of another Drosophila developmental gene, Ubx. We suggest that this mode of regulation is likely to be important for many late-acting developmental enhancers in many different organisms.

developmental biology↗

bioRxiv2022

Decreasing Wapl dosage partially corrects transcriptome phenotypes in Nipbl-/+ embryonic mouse brain

Cohesin rings interact with DNA and modulate expression of thousands of genes. NIPBL loads cohesin onto chromosomes and WAPL takes it off. Heterozygous mutations in NIPBL lead to a developmental disorder called Cornelia de Lange syndrome. Nipbl heterozygous mice are a good model for this disease but mutations in WAPL were not known to cause disease or gene expression changes in mammals. Here we show dysregulation of >1000 genes in Wapl{Delta}/+ embryonic mouse brains. The patterns of dysregulation are highly similar to Nipbl heterozygotes, suggesting that Wapl mutations may also cause disease in humans. Since WAPL and NIPBL have opposite effects on cohesins association with DNA, we asked whether a heterozygous Wapl mutation could correct phenotypes seen in Nipbl heterozygous mice. In fact, both gene expression and embryonic growth are partially corrected. Our data are consistent with the view that cohesin dynamics play a key role in regulating gene expression.

genetics↗