Rapid embryonic cell cycles defer the establishment of heterochromatin by Eggless/SetDB1 inDrosophila

Abstract

5 6 7Rapid embryonic cell cycles defer the establishment of heterochromatin by Eggless/SetDB1 in Drosophila.Acquisition of chromatin modifications during embryogenesis distinguishes different regions of an initially 11 naïve genome. In many organisms, repetitive DNA is packaged into constitutive heterochromatin that is 12 marked by di/tri methylation of histone H3K9 and the associated protein HP1a. These modifications enforce 13 the unique epigenetic properties of heterochromatin. However, in the early Drosophila melanogaster embryo 14 the heterochromatin lacks these modifications which only appear later when rapid embryonic cell cycles slow 15 down at the Mid-Blastula Transition or MBT. Here we focus on the initial steps restoring heterochromatic 16 modifications in the embryo. We describe the JabbaTrap, a technique for inactivating maternally provided 17 proteins in embryos. Using the JabbaTrap we reveal a major requirement for the methyltransferase 18Eggless/SetDB1 in the establishment of heterochromatin. In contrast, other methyltransferases contribute 19 minimally. Live-imaging reveals that endogenous Eggless gradually accumulates on chromatin in interphase, 20 but then dissociates in mitosis and its accumulation must restart in the next cell cycle. Cell cycle slowing as the 21 embryo approaches the MBT permits increasing accumulation and action of Eggless at its targets. 22 (Janssen et al. 2018). Constitutive heterochromatin displays conserved genetic and molecular properties that are 32 stably transmitted throughout development and across generations (Allshire and Madhani 2018). However, 33 embryogenesis interrupts this stability, and the early embryos of many animals lack true heterochromatin. Thus, 34 embryogenesis involves the restoration of heterochromatin, and this restoration can serve as a paradigm for 35 how epigenetic control of the genome arises during development. 36In general, constitutive heterochromatin is transcriptionally silent, late replicating, and low in 37 recombination. The chromosomes of many species contain large megabase sized arrays of simple repeated 38 sequences, known as satellite DNA, surrounding their centromeres, and it is this pericentric repetitive DNA that 39 composes the bulk of the constitutive heterochromatin. Although heterochromatin was originally defined 40 cytologically -it remains condensed throughout the cell cycle -modern studies often emphasize the set of 41 conserved modifications to its chromatin. Heterochromatic nucleosomes are hypoacetylated and di/tri-42 methylated at lysine 9 on the N-terminal tail of Histone H3 (hereafter H3K9me2/3). The deposition of 43 H3K9me2/3 creates a binding site for HP1 proteins (Eissenberg and Elgin 2014) and HP1 can oligomerize and 44 recruit additional heterochromatin factors which could then generate a distinct compartment in the nucleus. 45Studies in Drosophila have historically made major contributions to our understanding of 46 heterochromatin (Elgin and Reuter 2013). Many key regulators of heterochromatin were discovered ...

Cell cycle dynamics in the reprogramming of cellular identity

“…Specifically, the length of interphase limits the access of Eggless to chromatin, leading to insufficient Eggless activity on the chromatin and failure in heterochromatic foci formation. However, as cell cycle lengthens at the MBT stage, the effect of Eggless becomes sufficient to establish and maintain heterochromatic foci . This mechanism supports the hypothesis that cell cycle length could regulate the epigenome by controlling the time available for establishing/maintaining epigenetic marks.…”

“…Seller et al . found that Eggless, the Drosophila H3K9 methyltransferase, mediates this process. Specifically, the length of interphase limits the access of Eggless to chromatin, leading to insufficient Eggless activity on the chromatin and failure in heterochromatic foci formation.…”

Reflections on the use of protein binders to study protein function in developmental biology

“…Upon binding to the GFP‐tagged POI, GrabFPs have been shown to relocalize the GFP‐tagged POI from the endogenous localization domain to a novel, ectopic location (Figure f). In a similar way, a GFP nanobody fused to a lipid droplet‐associated scaffold has been successfully employed to relocalize GFP‐tagged nuclear proteins to lipid droplets to block their function (JabbaTrap, Seller, Cho, & O'Farrell, , Figure 1g).…”

“…Recently, tools that re‐localize GFP‐tagged POIs to specific intracellular compartments in developing organisms have been reported (GrabFP, Harmansa et al, ; JabbaTrap, Seller et al, ). In both cases, the GFP nanobody has been fused to a protein scaffold with biased subcellular localization (plasma membrane, lipid droplets).…”

“…It thus is indispensable to check the presence of non‐tagged POI in the original localization. The relocalization of nuclear proteins out of the nucleus has been proven a successful method (Seller et al, ). It is thus important to relocalize the POI to a subcellular location in which it cannot function.…”