In preparation for dramatic morphogenetic events of gastrulation, rapid embryonic cell cycles slow at the mid-blastula transition (MBT). In Drosophila melanogaster embryos, down-regulation of cyclin-dependent kinase 1 (Cdk1) activity initiates this slowing by delaying replication of heterochromatic satellite sequences and extending S phase. We found that Cdk1 activity inhibited the chromatin association of Rap1 interacting factor 1 (Rif1), a candidate repressor of replication. Furthermore, Rif1 bound selectively to satellite sequences following Cdk1 down-regulation at the MBT. In the next S phase, Rif1 dissociated from different satellites in an orderly schedule that anticipated their replication. Rif1 lacking potential phosphorylation sites failed to dissociate and dominantly prevented completion of replication. Loss of Rif1 in mutant embryos shortened the post-MBT S phase and rescued embryonic cell cycles disrupted by depletion of the S phase–promoting kinase, cell division cycle 7 (Cdc7). Our work shows that Rif1 and S phase kinases compose a replication timer controlling first the developmental onset of late replication and then the precise schedule of replication within S phase. In addition, we describe how onset of late replication fits into the progressive maturation of heterochromatin during development.

No abstract

Although histone acetylation and deacetylation machineries (HATs and HDACs) regulate important aspects of cell function by targeting histone tails, recent work highlights that non-histone protein acetylation is also pervasive in eukaryotes. Here, we use quantitative mass-spectrometry to define acetylations targeted by the sirtuin family, previously implicated in the regulation of non-histone protein acetylation. To identify HATs that promote acetylation of these sites, we also performed this analysis in gcn5 (SAGA) and esa1 (NuA4) mutants. We observed strong sequence specificity for the sirtuins and for each of these HATs. Although the Gcn5 and Esa1 consensus sequences are entirely distinct, the sirtuin consensus overlaps almost entirely with that of Gcn5, suggesting a strong coordination between these two regulatory enzymes. Furthermore, by examining global acetylation in an ada2 mutant, which dissociates Gcn5 from the SAGA complex, we found that a subset of Gcn5 targets did not depend on an intact SAGA complex for targeting. Our work provides a framework for understanding how HAT and HDAC enzymes collaborate to regulate critical cellular processes related to growth and division. Molecular & Cellular

At the Mid-Blastula Transition (MBT), externally developing embryos refocus from increasing cell number to elaboration of the body plan. Studies in Drosophila reveal a sequence of changes in regulators of Cyclin:Cdk1 that increasingly restricts the activity of this cell cycle kinase to slow cell cycles during early embryogenesis. By reviewing these events, we provide an outline of the mechanisms slowing the cell cycle at and around the time of MBT. The perspectives developed should provide a guiding paradigm for the study of other MBT changes as the embryo transits from maternal control to a regulatory program centered on the expression of zygotic genes.

Acquisition of chromatin modifications during embryogenesis distinguishes different regions of an initially naïve genome. In many organisms, repetitive DNA is packaged into constitutive heterochromatin that is marked by di/ trimethylation of histone H3K9 and the associated protein HP1a. These modifications enforce the unique epigenetic properties of heterochromatin. However, in the early Drosophila melanogaster embryo, the heterochromatin lacks these modifications, which appear only later, when rapid embryonic cell cycles slow down at the midblastula transition (MBT). Here we focus on the initial steps restoring heterochromatic modifications in the embryo. We describe the JabbaTrap, a technique for inactivating maternally provided proteins in embryos. Using the JabbaTrap, we reveal a major requirement for the methyltransferase Eggless/SetDB1 in the establishment of heterochromatin. In contrast, other methyltransferases contribute minimally. Live imaging reveals that endogenous Eggless gradually accumulates on chromatin in interphase but then dissociates in mitosis, and its accumulation must restart in the next cell cycle. Cell cycle slowing as the embryo approaches the MBT permits increasing accumulation and action of Eggless at its targets. Experimental manipulation of interphase duration shows that cell cycle speed regulates Eggless. We propose that developmental slowing of the cell cycle times embryonic heterochromatin formation.

SUMMARY We have developed a technique, called Ubiquitin Ligase Substrate Trapping, for the isolation of ubiquitinated substrates in complex with their ubiquitin ligase (E3). By fusing a ubiquitin associated (UBA) domain to an E3 ligase, we were able to selectively purify the polyubiquitinated forms of E3 substrates. Using Ligase Traps of eight different F-box proteins (SCF specificity factors) coupled with mass spectrometry, we identified known, as well as previously uncharacterized substrates. Polyubiquitinated forms of candidate substrates associated with their cognate F-box partner, but not other Ligase Traps. Interestingly, the four most abundant candidate substrates identified for the F-box protein Saf1 were all vacuolar/lysosomal proteins. Analysis of one of these substrates, Prb1, showed that Saf1 selectively promotes ubiquitination of the unprocessed form of the zymogen. This suggests that Saf1 is part of a pathway that targets protein precursors for proteasomal degradation.

SUMMARY Background In eukaryotes, ribosome biosynthesis involves the coordination of rRNA and ribosomal protein (RP) production. In S. cerevisiae, the regulation of ribosome biosynthesis occurs largely at the level of transcription. The transcription factor Ifh1 binds at RP genes and promotes their transcription when growth conditions are favorable. Although Ifh1 recruitment to RP genes has been characterized, little is known about the regulation of promoter-bound Ifh1. Results We used a novel whole-cell-extract screening approach to identify Spt7, a member of the SAGA transcription complex, and the RP transactivator Ifh1 as highly acetylated non-histone species. We report that Ifh1 is modified by acetylation specifically in an N-terminal domain. These acetylations require the Gcn5 histone acetyltransferase and are reversed by the sirtuin deacetylases Hst1 and Sir2. Ifh1 acetylation is regulated by rapamycin treatment and stress, and limits the ability of Ifh1 to act as a transactivator at RP genes. Conclusions Our data suggest a novel mechanism of regulation whereby Gcn5 functions to titrate the activity of Ifh1 following its recruitment to RP promoters to provide more than an all-or-nothing mode of transcriptional regulation. We provide insights into how the action of histone acetylation machineries converges with nutrient sensing pathways to regulate important aspects of cell growth.

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 ...