Premature translation of the zygotic genome activator Zelda is not sufficient to precociously activate gene expression

Following fertilization, the unified germ cells rapidly transition to a totipotent embryo. Maternally deposited mRNAs encode the proteins necessary for this reprogramming as the zygotic genome remains transcriptionally quiescent during the initial stages of development. The transcription factors required to activate the zygotic genome are among these maternally deposited mRNAs and are robustly translated following fertilization. In Drosophila, the mRNA encoding Zelda, the major activator of the zygotic genome, is not translated until one hour after fertilization. Here we demonstrate that zelda translation is repressed following fertilization by the TRIM-NHL protein Brain tumor (BRAT). BRAT also regulates Zelda levels in the larval neuroblast lineage. In the embryo, BRAT-mediated translational repression is regulated by the Pan Gu (PNG) kinase, which is triggered by egg activation. The PNG kinase phosphorylates translational regulators, suggesting that PNG kinase activity alleviates translational repression of zelda by BRAT and coupling translation of zelda with that of other regulators of early embryonic development. Using the premature translation of zelda in embryos lacking BRAT activity, we showed that early translation of a zygotic genome activator is not sufficient to drive precocious gene expression. Instead, Zelda-target genes showed increased expression at the time they are normally activated. We propose that transition through early development requires the integration of multiple processes, including the slowing of the nuclear division cycle and activation of the zygotic genome. These processes are coordinately controlled by PNG kinase-mediated regulation of translation.


Introduction 25
Regulated gene expression allows a single-celled zygote to divide and differentiate into 26 all the cell types of the adult organism. Transcription factors bind in a sequence-specific 27 manner to the DNA genome to drive these changes in gene expression. Nonetheless, the 28 packaging of the genome into chromatin can limit access to the DNA, with nucleosomes 29 acting as a barrier to transcription-factor binding (Zaret 2020). A specialized subclass of 30 transcription factors, called pioneer factors, can bind to DNA in nucleosomes. This 31 functionality allows pioneer factors to access regions of the genome that are inaccessible 32 to other transcription factors. These pioneer factors facilitate chromatin opening, enabling 33 other factors to bind and activate gene expression (Zaret 2020). Because of these unique 34 characteristics, pioneer factors are instrumental in cellular reprogramming, and 35 misexpression can be detrimental to the animal (Larson et al. 2021b). Thus, expression 36 of pioneer factors must be precisely controlled during development. 37

38
In all animals studied to date, pioneer factors are essential for reprogramming 39 differentiated germ cells to the totipotent embryo following fertilization (Schulz and 40 Harrison 2019). This early developmental transition is initially controlled by maternally 41 provided mRNAs and proteins deposited into the embryo. During this time, the zygotic 42 genome is transcriptionally silent. Gradual activation of the zygotic genome is coordinated 43 with the degradation of maternally deposited products during the maternal-to-zygotic 44 transition (MZT) (Vastenhouw et al. 2019). Activation of zygotic transcription requires 45 maternally encoded pioneer factors to reprogram the genome. Since the initial 46 identification of Zelda (ZLD) as the major activator of the zygotic genome in Drosophila 47 melanogaster (Liang et al. 2008), pioneer factors have been similarly found to be required 48 to activate zygotic transcription in frogs, zebrafish, mice and humans (Schulz and 49 Harrison 2019). Expression of these factors in tissues apart from the early embryo can 50 lead to defects in development and cancer (Dobersch et al. 2019;Larson et al. 2021b). 51 52 Early Drosophila development is characterized by a series of 13 rapid, synchronous 53 nuclear divisions with durations on the order of minutes. The rapidity of these divisions is 54 ensured by the absence of any gap phases. Each division is comprised of a DNA 55 We initially planned to use embryos that were heterozygous for the brat-mutant alleles 149 (brat hets) as controls (brat fs1/+ and brat Df/+ ). However, upon further analysis we 150 discovered that these heterozygous embryos display differential gene expression as 151 compared to His2AvRFP embryos (Supplemental Figure 1). At NC10 over half of the 152 genes that were mis-regulated in the brat-mutant embryos were also mis-regulated in the 153 embryos laid by brat hets (Supplemental Figure 1A). Not one particular allele contributes 154 to these changes as when we overlapped the genes that were mis-regulated in the 155 brat fs1/+ and brat Df/+ NC10 embryos, a large percentage of them are shared (Supplemental 156 Figure 1C,D). Additionally, we found that many genes were already mis-regulated in a 157 brat-mutant oocyte. And many of these mis-regulated genes were similarly mis-regulated 158 in the brat het oocytes (Supplemental Figure 1B). Based on the observed gene 159 expression changes in the embryos laid by heterozygotes, we decided to use His2AvRFP 160 flies (HisRFP) as controls. Technologies, Coralville, IA). BREs were mutated from UGUU to CGCU. These 167 transgenes were integrated at ZH-86Fb site on chromosome 3 using φC31-mediated 168 integration (Bischof and Basler 2008)  used to obtain data in each experiment. Antibodies used include rabbit anti-ZLD (1:500) 195 (Harrison et al. 2010), rat anti-DPN Antibody (1:2) (clone 11D1BC7.14) (Lee et al. 2006), 196 To assess zld transcript levels in wild-type and brat-mutant embryos, total RNA was 201 isolated (see RNA-seq) from 10 embryos collected from w 1118 or brat-mutant females (as 202 described above). The total RNA was then used to prepare single-stranded cDNA by 203 reverse transcription using random primers and Superscript IV reverse transcriptase 204 (Invitrogen, Waltham, MA). The single-stranded cDNA was used to perform quantitative 205 real-time PCR with primers specific to the zld transcript, as well as Act5C (see reagents 206

RNA-seq analysis 229
Raw reads were aligned to the BDGP D. melanogaster genome release 6 (dm6) using 230 hisat2 v2.1.0 with the following parameters: -k 2 -very-sensitive (Kim et al. 2015). Reads 231 aligning to multiple locations were discarded. SAMtools was used to filter (-q 30) and 232 convert file formats (Li et al. 2009). Reads were assigned to annotated genes using 233 featureCounts v1.5.3 (Liao et al. 2014) using default parameters and the UCSC 234 annotation (r6.20). The resultant table of read counts was imported into R v4.1.0, and 235 differential expression was determined using DESeq2 v1.33.5. (Love et al. 2014). Genes 236 with < 50 reads across all samples were filtered out. Differential expression (adjusted p 237 value < 0.05 and log2(fold change) > 1) was determined using the standard DESeq2 238 analysis. Read counts were z-score normalized for visualization of the time course line 239 graphs. To identify ZLD-target genes, previously determined ZLD-ChIP peaks were 240 assigned to the nearest gene (ChIP from Harrison et al. 2011, reanalyzed by Larson et 241 al. 2021a. Previously identified BRAT-regulated transcripts and BRAT-bound transcripts 242 were from Laver et al. 2015. Zygotically and maternally expressed genes, as well as the 243 timing of zygotic gene expression, were previously defined (Lott et al. 2011;Li et al. 2014;244 Strong et al. 2020 (Harrison et al. 2010;Nien et al. 2011). To understand how ZLD protein levels 256 are precisely controlled, we determined whether the timing of zld translation depended 257 on fertilization of the oocyte. Immunoblots on both fertilized and unfertilized embryos 258 harvested in one-hour increments after egg laying (AEL) showed that ZLD protein levels 259 increased regardless of whether the oocytes were fertilized ( Figure 1A). Thus, a 260 maternally regulated process controls the timing of zld translation. 261 262 Translation in the early embryo is broadly regulated by the Pan Gu (PNG) kinase, which 263 phosphorylates and inactivates translational repressors (Kronja et al. 2014a(Kronja et al. , 2014b264 Eichhorn et al. 2016;Hara et al. 2018). PNG is comprised of three subunits (Pangu 265 (PNG), Giant nuclei (GNU) and Plutonium (PLU)) and is subject to precise control such 266 that it is active in a short window following egg activation (Hara et al. 2017). Embryos with 267 mutations in PNG subunits arrest in early development with failures in nuclear division 268 resulting from the failure to translate cyclin B (Shamanski and Orr-Weaver 1991;Fenger 269 et al. 2000;Lee et al. 2003;Vardy and Orr-Weaver 2007). We performed immunoblots 270 for ZLD in embryos lacking functional PNG subunits, PNG or PLU. ZLD levels were 271 reduced in embryos laid by png [50] mutant mothers during the three hours AEL as 272 compared to w 1118 control embryos ( Figure 1B). Similarly, in 2-3 hr AEL embryos laid by 273 plu [1]/[3] mutant mothers, ZLD levels were lower than controls ( Figure 1C). Because ZLD 274 translation occurs in the absence of fertilization, we can conclude that the decrease in 275 Figure 1: Translational upregulation of ZLD is reduced in PNG kinase complex mutants. A. Immunoblots with anti-ZLD antibody on unfertilized or fertilized embryos collected at one-hour time points after egg laying (AEL). Non-specific background (*) band serves as loading control. B. Immunoblots on control (w 1118 ) or png-mutant embryos at one-hour intervals after egg laying (AEL). Tubulin is shown as a loading control. C. Immunoblots on control (w 1118 ) or plu-mutant embryos at 2-3 hours after egg laying (AEL). Tubulin is shown as a loading control.
ZLD levels in these mutants is due to defects in PNG kinase activity rather than the failure 276 of the embryos to develop. 277 278 BRAT regulates ZLD levels in larval neuroblasts through consensus binding sites 279 As in the early embryo, ZLD levels in the type II neuroblast lineage of the larval brain are 280 tightly controlled (Reichardt et al. 2018;Larson et al. 2021a). In the neuroblast lineage, 281 ZLD levels are controlled by Brain Tumor (BRAT), which binds to zld mRNA and has been 282 suggested to degrade it (Reichardt et al. 2018). In wild-type animals, ZLD is rapidly 283 eliminated from the partially differentiated progeny (immature intermediate neuroblast 284 progenitor (immINP)) following asymmetric neuroblast division ( brat results in extra neuroblasts, and this can be partially suppressed by knockdown of 292 zld (Reichardt et al. 2018). We recently demonstrated that in wild-type brains 293 overexpression of zld with the SV40 3'UTR, which is commonly used for transgene 294 expression and stabilizes mRNA, results in extra stem cells (61.5±15.9 type II 295 neuroblasts) ( Figure 2D) (Larson et al. 2021a). We therefore used this quantitative system 296 to determine whether BRAT mediates regulation of ZLD levels through binding to the 297 3'UTR. Expression of zld with the endogenous 3'UTR resulted in fewer extra neuroblasts 298 (34.7±8.5 type II neuroblasts) as compared to zld with the SV40 3'UTR ( Figure 2D), 299 supporting a role for the 3'UTR in regulating ZLD activity in the neuroblasts. We then 300 mutated all 16 identified BREs in the zld 3'UTR. Overexpression of zld regulated by the 301 3'UTR containing mutations in all 16 putative BREs resulted in significantly more 302 neuroblasts (55.1±10.9 type II neuroblasts) than the wild-type zld 3'UTR (p-value = 303 0.0006, ANOVA with post hoc Tukey's multiple comparisons test) ( Figure 2D). 304 RNAcompete predicted that two of the BREs in the zld 3'UTR were high-affinity (strong) 305 BREs ( Figure 2A) (Laver et al. 2015). To test the relative contribution of these two BREs, 306 we created transgenes in which only these two BREs were mutated in the zld 3'UTR. 307 When compared to the number of neuroblasts induced when all 16 BREs were mutated, 308 mutation of only the strong BREs did not result in a significant difference (44.3±7.8 type 309 II neuroblasts). Together these data support a role for BRAT in regulating ZLD levels in 310 the neuroblast lineage by binding preferentially to a subset of BREs in the 3'UTR. 311 312 BRAT regulates ZLD levels in the early embryo 313 In the early embryo, BRAT regulates both mRNA translation and stability and is bound to 314 zld mRNA (Laver et al. 2015). This, together with our demonstration of the role of BRAT 315 in regulating zld in the neuroblasts, suggested that BRAT may regulate zld translation in 316 the early embryo. To test this, we immunoblotted for ZLD in staged control (w 1118 ) and 317 brat-mutant embryos (embryos laid by brat Df/fs1 mothers) ( Figure 3A). brat-mutant 318 embryos are devoid of functional BRAT and do not survive past embryogenesis 319 Immunostaining of wild-type or brat 150 clones in the third instar larval brains for Deadpan (DPN) (a marker of neuroblasts and INPs), ZLD and GFP to mark the clone. Dashed yellow lines mark the clone boundary. White arrows indicate type II neuroblasts. White arrowheads represent Asense negative (ASE-) immature INPs, and yellow arrowheads indicate mature INPs. Scale bar = 10mm. C. Schematic of the zld 3'UTR with previously identified BRAT (UGUU) motifs highlighted. Asterisks indicate two strong binding sites as determined by RNAcompete. D. Quantification of type II neuroblasts when ZLD is overexpressed from reporters with the 3'UTRs indicated below. Error bars indicate standard deviation. ** = p < 0.005, *** = p < 0.0001 and n.s. = non-significant as determined by a one-way ANOVA test with post hoc Tukey's multiple comparisons test. (Schüpbach and Wieschaus 1991;Stathakis et al. 1995;Arama et al. 2000;Sonoda and 320 Wharton 2001;Loedige et al. 2014). In w 1118 embryos ZLD levels increased normally from 321 0-3 hr AEL. By contrast, at 0-1 hr AEL in brat-mutant embryos ZLD protein levels were 322 already at levels similar to controls at 1-2 hr AEL, after zld translation increased ( Figure  323 3A). This premature increase in ZLD levels in brat-mutant embryos is not due to changes 324 in the total RNA present. RT-qPCR for zld in control (w 1118 ) and brat-mutant embryos at 325 two time points, stage 2-3 and stage 5, showed that zld mRNA levels remained relatively 326 constant as compared to an internal control, Act5C ( Figure 3B). Together with previously 327 published data, this suggests that BRAT binds to zld mRNA and represses translation 328 immediately following fertilization.  half-way into NC12 (NC12 +5min ), half-way into NC13 (NC13 +8min ), at the beginning of NC14 361 (NC14 +5min ) and half-way into NC14 (NC14 +30min ) ( Figure 4A). To validate the data 362 collected, we performed principal component analysis (PCA). We included previously 363 published single-embryo RNA-seq data from wild-type embryos for comparison (Lott et 364 al. 2011). The PCA demonstrated that the oocyte and embryo replicates collected at each 365 time point cluster together and cluster with independently collected, previously published 366 data from wild-type embryos ( Figure 4B). Further supporting the robustness of the data 367 collected, the replicates from each time point of each genotype were highly correlated 368 (Supplemental Figure 2). 369

370
We identified gene expression changes in brat-mutant embryos as compared to control 371 embryos at NC14 +30min , which is most similar to the prior time point analyzed by Laver et 372 al. 2015. We identified 582 genes with significantly higher mRNA signal in the brat-mutant 373 embryos as compared to the controls ( Figure 4C). These genes are comprised of those 374 that were previously found to be increased in the published microarray data ( Figure 4C) 375 (Laver et al. 2015). Many of the genes that have increased mRNA levels are bound by 376 BRAT in wild-type embryos and are stabilized in the brat mutant (Laver et al. 2015). These 377 are exemplified by rolling stone (rost), stambha A (stmA) and CG7488, which are directly 378 regulated by BRAT binding ( Figure 4D). These analyses provided confidence in the data 379 generated and allowed us to interrogate these data to determine if ZLD-target genes were 380 precociously expressed. 381  (Lott et al. 2011). C. Volcano plot highlighting genes mis-regulated at NC14 in brat Df/fs1 embryos as compared to HisRFP controls. Blue dots indicate genes that are were also increased in brat mutants at 1.5 -3 hr AEL (Laver et al. 2015). Black dots indicate genes not identified in Laver et al. 2015 that change in expression in brat Df/fs1 embryos as compared to controls (log2(fold change) >1, padj <0.05). D. Expression pattern of three genes bound by BRAT (Laver et al. 2015) with increased expression in brat Df/fs1 embryos as compared to controls at all developmental timepoints analyzed. Points indicate the average z score, and the surrounding region indicates the standard deviation of the replicates for each timepoint.
We initially focused our analysis on genes that were increased in the brat-mutant embryos 383 as compared to controls at any of the embryonic time points collected (NC10 through 384 NC14 +30min ). We performed k-means clustering on the expression of this set of genes in 385 the control embryos and identified six clusters with distinct expression patterns ( Figure  386 5A). Clusters 1-4 decreased in expression over the time course. These transcripts were 387 enriched for maternally deposited genes and most were bound by BRAT (Lott et al. 2011;388 Laver et al. 2015;Strong et al. 2020). Thus, these clusters are largely comprised of 389 maternal genes that are direct BRAT targets and are normally degraded during the MZT. 390 By contrast, clusters 5-6 increased in expression over the time course. The transcripts in 391 these clusters were enriched for zygotically expressed genes and the majority were not 392 bound by BRAT ( Figure 5A) (Lott et al. 2011;Laver et al. 2015;Strong et al. 2020). As 393 might be expected for zygotically expressed genes, the genes in clusters 5 and 6 are 394 located proximally to regions occupied by ZLD in the early embryo (Harrison et al. 2011). 395 Thus, these clusters contain ZLD-target genes that are indirectly increased in the brat 396 mutants and therefore are likely to include any genes that might be precociously activated 397 by the premature translation of ZLD in the brat-mutant embryos. 398

399
Having identified those genes most likely to reflect increased activation due to precocious 400 ZLD expression, we leveraged our tightly staged RNA-seq data to determine the 401 dynamics of gene expression that generated the observed increase in transcript levels. If 402 these genes are precociously activated, they would be expected to have increased 403 transcript levels in the brat mutant as compared to controls at time points preceding 404 widespread gene activation at NC14 (NC12 +5min , NC13 +8min , NC14 +5min ). In contrast to this 405 prediction, genes in clusters 5 and 6 do not exhibit increased transcript abundance at 406 these early time points ( Figure 5A). Instead, the largest increase in expression of genes 407 in a brat-mutant embryo is evident at NC14 +30min . ZLD-target genes teashirt (tsh), zinc 408 finger homeodomain 2 (zfh2) and FER exemplify this robust hyperactivation at NC14 409 ( Figure 5B). 410

411
To further investigate whether a small subset of zygotically transcribed genes might be 412 precociously activated, we classified genes based on their timing of transcriptional 413 activation, early (NC10-11), mid (NC12-13), late (early NC14) or later (late NC14) (Li et 414 al. 2014) and plotted their expression in brat-mutant and control embryos ( Figure 5C). 415 Genes that initiated transcription prior to the major wave at NC14 (early, mid, and late) 416 showed relatively little difference in expression levels between the control and brat mutant 417 ( Figure 5C). By contrast for the genes activated later in NC14, transcript levels were 418 increased in the brat-mutant embryos as compared to controls ( Figure 5C). Similar to 419 what we observed with our k-means clustering analysis, zygotic ZLD-target genes were 420 A. kmeans clustering of all genes significantly increased in brat Df/fs1 embryos as compared to HisRFP controls (log2(fold change) >1, padj <0.05). Points indicate the average z score, and the surrounding region indicates the standard deviation of the replicates for each timepoint. n = number of genes in each cluster. B. Expression for three representative genes that are bound by ZLD, not bound by BRAT, and significantly increased in brat Df/fs1 as compared to HisRFP control embryos (cluster 5). C. Expression profiles of classes of zygotically expressed genes (Li et al. 2014). D. Length in minutes for S phase of nuclear cycles 11-13 in brat Df/fs1 embryos and wild-type embryos as determined by Shermoen et al. 2010. Error bars are standard deviation of 3 biological replicates. not precociously activated in the brat-mutant embryos. Instead, an increased level of 421 zygotic transcripts was evident at NC14. We conclude that the premature increase in ZLD 422 protein in brat-mutant embryos results in increased, rather than precocious, expression 423 of zygotic ZLD-target genes. 424

425
Our data demonstrate that precocious expression of a major activator of the zygotic 426 genome is not sufficient to drive gene expression early. Thus, additional factors are 427 required for timing the transcriptional activation of the zygotic genome. During early 428 development, the nuclei are dividing rapidly (~every 10 minutes). It is not until NC14 that 429 this division cycle slows. Because the division cycle is known to disrupt transcription, we 430 hypothesized that these rapid divisions may keep precocious ZLD from activating 431 transcription globally until NC14. Indeed, we timed the length of S phase in brat-mutant 432 embryos at NC11, NC12 and NC13 and confirmed that the nuclear division cycles 433 occurred in these mutants with the same timing as wild-type ( Figure 5D) (Shermoen et al. 434 2010). Together our data demonstrate that precocious expression of an essential 435 activator of the zygotic genome is not capable of driving early gene expression, and we 436 suggest that the slowing of the cell cycle is essential for the activator to promote robust 437 gene expression. Here we demonstrate that BRAT regulates levels of the pioneer transcription factor ZLD 442 in both the larval neuroblasts and in the early embryo. In both tissues, this regulation is 443 required for normal development. In the neuroblasts, BRAT functions to ensure that ZLD 444 levels are rapidly reduced following asymmetric division, enabling cells to exit the stem-445 cell fate and begin to differentiate. We previously demonstrated that following asymmetric 446 neuroblast division misexpression of ZLD in the differentiating progeny results in a 447 reversion to the neuroblast fate and a tumor-like phenotype (Larson et al. 2021a). 448 Furthermore, knockdown of zld can partially rescue a brat-mutant phenotype in the larval 449 brain (Reichardt et al. 2018). Our data from transgenes in which zld is expressed with 450 wild-type or mutated 3'UTRs show that BRAT-binding sites in the 3'UTR function to 451 suppress ZLD activity following asymmetric division. BRAT is localized to the 452 differentiating progeny and promotes deadenylation and subsequent degradation of the 453 target gene deadpan (Komori et al. 2014;Reichardt et al. 2018  However, BRAT also more directly regulates translation initiation through interactions with 473 the cap binding protein, 4EHP (Connacher and Goldstrohm 2021). In addition to the role 474 of BRAT in repressing zld translation, BRAT is also required during the MZT for 475 degradation of maternally provided mRNAs (Laver et al. 2015). Thus, the function of 476 BRAT may depend both on the target mRNAs bound and developmental stage. Indeed, 477 this duality is not limited to BRAT as ME31B represses translation early during the MZT, 478 but transitions to promoting mRNA destruction as development proceeds in a manner 479 regulated by PNG (Wang et al. 2017). 480

481
Having demonstrated precocious expression of ZLD in brat-mutant embryos, we used 482 this system to test whether early expression of a zygotic genome activator could 483 prematurely drive transcriptional activation. Using RNA-seq on precisely staged embryos, 484 we showed that while transcript levels of ZLD-target genes were increased in the brat 485 mutant this occurred only at the time at which these genes would normally be activated. 486

487
Early models suggested that the ratio of nuclear content to cytoplasmic content was 488 essential for genome activation and that titration of a maternally provided repressor was 489 required for genome activation (Newport and Kirschner 1982;Schulz and Harrison 2019). 490 Work from zebrafish and Xenopus identified histones as maternally provided repressors 491 and showed that increased concentrations of pioneer factors allow these genome 492 activators to outcompete histones to activate gene expression (Amodeo et al. 2015; 493 Figure 6: PNG kinase activity alleviates translational repression of zld by BRAT. In wild-type embryos BRAT represses zld translation during the earliest stages of embryogenesis, and this repression is alleviated by the PNG kinase complex. In a brat-mutant embryo, zld is precociously translated. This results in increased transcription of ZLDtarget genes at stage 5, the time in which they are normally expressed. When PNG activity is lost, zld translation is inhibited throughout the MZT, and ZLD expression fails to reach wild-type levels. We propose PNG activation coordinates multiple essential processes during the MZT including increased levels of the zygotic genome activator, ZLD. Joseph et al. 2017). In contrast to these models, our data indicate that increased 494 concentration of pioneer factors alone is not sufficient to precociously activate zygotic 495 transcription. Instead, developmental features aside from the increased levels of the 496 transcriptional activators are required for widespread activation of the zygotic genome. 497 We propose that one of these features is the slowing of the division cycle. As in many 498 other organisms, the initial division cycles in the Drosophila embryo are extremely rapid, 499 only lasting about 10 minutes. During these rapid cycles, transcription is limited to S-500 phase as mitosis interrupts transcription (McCleland et al. 2009). It is not until NC14 that 501 the division cycle slows and provides ample time for robust gene expression. Further 502 supporting a process independent from nuclear to cytoplasmic ratio in timing activation of 503 the zygotic genome, recent studies in Drosophila and zebrafish showed that zygotic 504 genome activation of a large subset of genes can occur in the absence of increased 505 nuclear content (Chan et al. 2019;Strong et al. 2020). Instead, this activation requires 506 both translation and time after fertilization (Chan et al. 2019;Strong et al. 2020). In 507 zebrafish, as in flies, the levels of the major activators of the zygotic genome Pou5f3, 508 Sox19b, and Nanog are controlled by robust translation following fertilization (Lee et al. 509 2013). Together with our data, this suggests that translation of genome activators coupled 510 with a slowing of the division cycle is essential for zygotic genome activation. 511

512
Our data support a model in which activation of the zygotic genome requires a tight 513 coordination of multiple processes, including increased levels of zygotic genome 514 activators and slowing of the division cycle. We propose that in Drosophila activation of 515 the PNG kinase coordinately regulates these processes and couples them to egg 516 activation, allowing for robust control of this process. PNG kinase is essential for 517 translation of cyclin B, which encodes a key regulator of the division cycle in the early 518 embryo, and smaug, which regulates maternal mRNA stability (Tadros et al. 2007;Vardy 519 and Orr-Weaver 2007). Here we show that zld translation is similarly dependent on PNG 520 activity ( Figure 6). PNG phosphorylates key regulators of translation, ME31B, BIC-C and 521 Trailer hitch, and, in the case of TRAL, blocks it's repressive effects on translation (Hara 522 et al. 2018). Indeed, translation of hundreds of transcripts are mis-regulated in the 523 absence of PNG activity (Kronja et al. 2014b). Coupled with the fact that PNG kinase 524 activity is limited to a precise developmental time window (Hara et al. 2017   A. Immunoblots with anti-ZLD antibody on unfertilized or fertilized embryos collected at one-hour time points after egg laying (AEL). Non-specific background (*) band serves as loading control. B. Immunoblots on control (w 1118 ) or png-mutant embryos at one-hour intervals after egg laying (AEL). Tubulin is shown as a loading control. C. Immunoblots on control (w 1118 ) or plu-mutant embryos at 2-3 hours after egg laying (AEL). Tubulin is shown as a loading control.   Developmental timing of oocytes and embryos collected for RNA-seq. B. PCA plot of RNA-seq from embryos/oocytes. Previously published single-embryo, RNA-seq data from wild-type embryos are included as verification of developmental staging (Lott et al. 2011). C. Volcano plot highlighting genes mis-regulated at NC14 in brat Df/fs1 embryos as compared to HisRFP controls. Blue dots indicate genes that are were also increased in brat mutants at 1.5 -3 hr AEL (Laver et al. 2015). Black dots indicate genes not identified in Laver et al. 2015 that change in expression in brat Df/fs1 embryos as compared to controls (log2(fold change) >1, padj <0.05). D. Expression pattern of three genes bound by BRAT (Laver et al. 2015) with increased expression in brat Df/fs1 embryos as compared to controls at all developmental timepoints analyzed. Points indicate the average z score, and the surrounding region indicates the standard deviation of the replicates for each timepoint. Points indicate the average z score, and the surrounding region indicates the standard deviation of the replicates for each timepoint. n = number of genes in each cluster. B. Expression for three representative genes that are bound by ZLD, not bound by BRAT, and significantly increased in brat Df/fs1 as compared to HisRFP control embryos (cluster 5). C. Expression profiles of classes of zygotically expressed genes (Li et al. 2014). D. Length in minutes for S phase of nuclear cycles 11-13 in brat Df/fs1 embryos and wild-type embryos as determined by Shermoen et al. 2010. Error bars are standard deviation of 3 biological replicates. In wild-type embryos BRAT represses zld translation during the earliest stages of embryogenesis, and this repression is alleviated by the PNG kinase complex. In a bratmutant embryo, zld is precociously translated. This results in increased transcription of ZLD-target genes at stage 5, the time in which they are normally expressed. When PNG activity is lost, zld translation is inhibited throughout the MZT, and ZLD expression fails to reach wild-type levels. We propose PNG activation coordinates multiple essential processes during the MZT including increased levels of the zygotic genome activator, ZLD.
Supplemental Table 1: Lists of differentially expressed genes. Lists of differentially expressed genes at each time point collected (late stage oocyte, NC10, NC12, NC13, early NC14 and later NC14) between the brat mutant and HisRFP embryos including the log2(foldchange,) p value, adjusted p value, MZT class, whether or not the gene is bound by ZLD, whether or not the gene is bound by BRAT, whether or not the gene was upregulated in Laver et al. 2015, the zygotic class, whether or not the gene was considered zygotically expressed by Strong et al. 2020 and whether or not the gene determined to be differentially expressed using the criteria log2(fold change) >1, padj <0.05. Comparisons between the brat hets and HisRFP, and clusters from Figure 5A and classes from Figure 5C are also included. Figure 1: Embryos laid by mothers heterozygous for brat-mutant alleles (brat het) have differences in gene expression as compared to controls. A. Volcano plot of differentially expressed genes (log 2 fold change >1, padj <0.05) in NC10 embryos laid by brat Df/fs1 mothers as compared HisRFP NC10 embryos. Genes that are also misexpressed in NC10 embryos laid by mothers heterozygous for a brat-mutant allele (brat het) as compared to HisRFP cotrols are in blue. All other significantly differentially expressed genes are in black and non-significant genes are in gray. B. Volcano plot of differentially expressed genes (log 2 fold change >1, padj <0.05) in brat Df/fs1 oocytes compared HisRFP oocytes. Genes that are also misexpressed in brat het oocytes compared to HisRFP cotrols are in blue. All other significantly differentially expressed genes are in black and non-significant genes are in gray. C. Overlap of individual genes that show increased expression in NC10 embryos laid from either brat fs1/+ or brat Df/+ heterozygous mothers. D. Overlap of individual genes that show decreased expression in NC10 embryos laid from either brat fs1/+ or brat Df/+ heterozygous mothers.