Rbbp4 loss disrupts neural progenitor cell cycle regulation independent of Rb and leads to Tp53 acetylation and apoptosis

Abstract Background Retinoblastoma binding protein 4 (Rbbp4) is a component of transcription regulatory complexes that control cell cycle gene expression. Previous work indicated that Rbbp4 cooperates with the Rb tumor suppressor to block cell cycle entry. Here, we use genetic analysis to examine the interactions of Rbbp4, Rb, and Tp53 in zebrafish neural progenitor cell cycle regulation and survival. Results Rbbp4 is upregulated across the spectrum of human embryonal and glial brain cancers. Transgenic rescue of rbbp4 mutant embryos shows Rbbp4 is essential for zebrafish neurogenesis. Rbbp4 loss leads to apoptosis and γ‐H2AX in the developing brain that is suppressed by tp53 knockdown or maternal zygotic deletion. Mutant retinal neural precursors accumulate in M phase and fail to initiate G0 gene expression. rbbp4; rb1 mutants show an additive effect on the number of M phase cells. In rbbp4 mutants, Tp53 acetylation is detected; however, Rbbp4 overexpression did not rescue DNA damage‐induced apoptosis. Conclusion Rbbp4 is necessary for neural progenitor cell cycle progression and initiation of G0 independent of Rb. Tp53‐dependent apoptosis in the absence of Rbpb4 correlates with Tp53 acetylation. Together these results suggest that Rbbp4 is required for cell cycle exit and contributes to neural progenitor survival through the regulation of Tp53 acetylation.


Funding information
Mayo Foundation for Medical Education and Research; National Institute of General Medical Sciences, Grant/Award Number: GM063904; NIH Office of the Director, Grant/Award Number: R24 OD020166-05S3; Roy J. Carver Charitable Trust, Grant/Award Number:  additive effect on the number of M phase cells. In rbbp4 mutants, Tp53 acetylation is detected; however, Rbbp4 overexpression did not rescue DNA damageinduced apoptosis. Conclusion: Rbbp4 is necessary for neural progenitor cell cycle progression and initiation of G0 independent of Rb. Tp53-dependent apoptosis in the absence of Rbpb4 correlates with Tp53 acetylation. Together these results suggest that Rbbp4 is required for cell cycle exit and contributes to neural progenitor survival through the regulation of Tp53 acetylation.

K E Y W O R D S
cell cycle regulation, embryo, neural precursor, neurogenesis, Tp53

| INTRODUCTION
The WD-repeat protein retinoblastoma binding protein 4 (Rbbp4) functions as a chromatin assembly factor and adaptor for multiple chromatin remodelers and transcriptional regulators. [1][2][3] These include the H3K27 methyltransferase polycomb repressive complex PRC2 2 and the histone acetyltransferase activator p300/CREB. 4 Rbbp4 is also a member of protein complexes that play critical roles in the regulation of cell cycle gene expression. Rbbp4 was first discovered as a binding partner of the tumor suppressor Rb in yeast and shown to be required for the suppression of Ras-mediated growth. 5 The nucleosome remodeling and histone deacetylase (HDAC) complex NuRD contains Rbbp4 and its closely related homolog Rbbp7. [6][7][8] NuRD is recruited to E2F promoters by the Rb tumor suppressor and represses early S gene transcription. 9 Early studies in Caenorhabditis elegans identified the Rbbp4 homolog lin-53 as a component of the MuvB complex, which negatively regulates vulval precursor cell fate by lin-35 (Rb-like) repression of genes required for vulval induction. 10 In Drosophila and humans, MuvB functions as a core that associates with Rb-like and B-Myb transcription factors to form the multisubunit complexes DREAM/Rb-like and MMB/B-Myb. 11 DREAM and MBB complexes regulate stage-specific gene expression during cell cycle progression 12 and quiescence. 13 Together these studies indicate that Rbbp4 functions in regulation of cell cycle progression and cell fate specification through transcriptional regulation by Rb family members and NuRD, DREAM, and MMB.
Rb has been shown to be essential for embryonic and adult neurogenesis in mice. 14,15 Our previous genetic analyses of Rb and Rbbp4 mutants in zebrafish demonstrate both are required for neurogenesis during brain development. 16,17 Live brain imaging revealed that zebrafish rb1 mutant neural progenitors enter the cell cycle and fail to progress through mitosis, whereas the loss of rbbp4 leads to neural progenitor apoptosis. 17 While in yeast and cultured cells, Rbbp4 appears to function as a tumor suppressor with Rb to block cell cycle entry and inhibit cell growth, whether Rb and Rbbp4 interact to regulate the cell cycle in neural progenitors has not previously been examined. In both zebrafish Rb germline mutants and Rb-defective brain tumors, Rbbp4 is overexpressed, 17 suggesting Rbbp4 adopts oncogenic properties in the absence of Rb. In humans, Rbbp4-dependent recruitment of chromatin regulators has been demonstrated in glioblastoma multiforme cell resistance to temozolomide through p300 activation of DNA repair pathway genes. 18 Rbbp4 was also shown to be required for tumor progression in neuroblastoma xenografts by PRC2 silencing of tumor suppressors. 19 Given the important contributions of developmental and cell cycle control mechanisms in cancer, 12,20 a more detailed examination of the role of Rbbp4 in neural progenitor cell cycle regulation would provide insight into its possible oncogenic roles in brain tumors.
A previous study using mouse primary cortical neurons demonstrated that chemical inhibition of HDAC led to increased TP53 acetylation, which protected cells from DNA damage induced apoptosis by preventing TP53 association with the promoter of the pro-apoptotic gene PUMA. 21 In contrast, both NuRD 22 and Sirt1 23,24 histone deacetylase 1 complexes have been shown to deacetylate TP53 and repress TP53 transcriptional activation of apoptosis in human and mouse cell lines. Although these in vitro studies suggest opposite effects of Tp53 acetylation on apoptosis, together they indicate TP53 acetylation has a critical role in the cellular transcriptional response to stress. In vivo studies examining whether Tp53 acetylation is associated with transcriptional activation and induction of apoptosis may resolve these differences.
In this study, we use rbbp4, rb1, and tp53 zebrafish mutants to further investigate the in vivo role of Rbbp4 in neurogenesis and examine the interaction of Rbbp4, Rb, and Tp53 in neural progenitor cell cycle regulation and survival. rbbp4 mutant neural progenitors accumulate in M-phase and Tp53-dependent apoptosis occurs throughout the developing midbrain and retina. In situ analysis demonstrates that rbbp4 mutant progenitors are lost before initiating G0 cdkn1c cyclin-dependent kinase inhibitor gene expression and exiting the cell cycle. In double mutant rbbp4; rb1 midbrain and retina apoptosis still occurs, and the level of M-phase cells is additive, indicating independent functions for Rbbp4 and Rb in neural progenitor cell cycle regulation. In the absence of Tp53, apoptosis and γ-H2AX labeling is suppressed in rbbp4 mutant neural precursors, leading to increased accumulation of cells in M phase of the cell cycle. In contrast to wild-type, Tp53 acetylation can be detected in homozygous mutant rbbp4 embryos. Together these results suggest one mechanism by which Rbbp4 controls neural progenitor survival and cell cycle progression is through inhibition of Tp53 acetylation, providing new insight into an oncogenic role for Rbbp4 in brain cancer.

| Human RBBP4 is upregulated in pediatric embryonal and adult malignant brain cancers
We previously showed by RNA-Seq and qRT-PCR that rbbp4 is upregulated~8-fold in a zebrafish model of rb1defective brain tumors 17 that are similar to human primitive neuroectodermal tumors, a poorly differentiated and highly malignant pediatric brain cancer. To investigate whether human RBBP4 is upregulated in human embryonal tumors and other aggressive pediatric and adult brain cancers we examined RBBP4 expression data from 2284 human brain tumor samples from the German Cancer Research Center. Elevated RBBP4 expression was detected in tissues from many embryonal central nervous system primitive neuroectodermal tumors (CNS-PNETs) and malignant brain cancers, including ependymal, glial, oligodendroglial, and astrocytic tumors ( Figure 1). We previously used CRISPR/Cas9 gene editing to isolate a 4 bp deletion allele in zebrafish rbbp4 Δ4is60 and showed rbbp4 Δ4 is homozygous mutant larval lethal. 17 Loss of Rbbp4 leads to apoptosis throughout the 2 day postfertilization (dpf) brain and retina, resulting in microcephaly and microphthalmia. 17 Together these results show that elevated RBBP4 is a common signature in aggressive central nervous system tumors and suggest that Rbbp4 may contribute to oncogenesis by promoting brain tumor cell survival.

| Rbbp4 is essential for zebrafish neurogenesis
To demonstrate Rbbp4 is essential for cell survival during neurogenesis, we further characterized the zebrafish rbbp4 Δ4 mutant and its impact on brain development. We tested for rescue of the homozygous rbbp4 Δ4 mutant using a transgenic wild-type rbbp4 cDNA. Homozygous mutant rbbp4 Δ4/Δ4 larvae display severe defects in F I G U R E 1 RBBP4 is highly expressed in many malignant pediatric and adult brain cancers. RBBP4 Affymetrix data from 2284 human tumor samples (German Cancer Research Center DKFZ). Y axis represents MAS5.0 normalized expression values. atrt, atypical teratoid/ rhabdoid tumor; cns, central nervous system; cpc, choroid plexus carcinoma; cph, choroid plexus hyperplasia; cpp, choroid plexus papilloma; dipg, diffuse intrinsic pontine glioma; etmr, embryonal tumor with multilayered rosettes; ews, Ewing's sarcoma; mb, medulloblastoma; ptpr, papillary tumor of the pineal neurogenesis, presenting with microcephaly and microphthalmia 17 (Figure 2A-D). Alcian blue staining revealed abnormalities in formation of head cartilage structures ( Figure 2E-H), including the ceratohyal cartilage (ch), ceratobranchial cartilage (cbs), and Meckel's cartilage (m). These results suggest that Rbbp4 is necessary for the development of the central nervous system. Ubiquitous overexpression of rbbp4 cDNA by a Tol2 < ubi: rbbp4-2AGFP > transgenic line ( Figure 2I) was tested for the ability to rescue the rbbp4 Δ4/Δ4 phenotype. The transgene did not affect development in wild-type embryos or viability and fertility in adults ( Figure 2J, Table 1) and was able to rescue the gross morphological defects in rbbp4 Δ4/Δ4 mutants ( Figure 2K). Midbrain height and eye width measurements in Tol2<ubi:rbbp4-2AGFP> and rbbp4 Δ4/Δ4 ; Tol2<ubi:rbbp4-2AGFP> transgenic embryos confirmed rescued animals showed no significant difference in morphology from wild type ( Figure 2L,M).

| Rbbp4 is required for cell cycle regulation and its loss leads to neural precursor apoptosis
We previously showed that apoptosis is present throughout the midbrain and retina in 2 dpf homozygous mutant rbbp4 Δ4/Δ4 larvae, 17 which suggested that Rbbp4 is required for survival of neural precursors. To determine whether the loss of Rbbp4 affects neural precursor proliferation or survival earlier in embryogenesis we examined phosphohistone H3 and activated Caspase-3 levels at 24 and 36 hours post fertilization (hpf). At 24 hpf ( Figure 3A-E), whole mount labeled +/+ wild type ( Figure 3A,C) and rbbp4 Δ4/Δ4 mutant ( Figure 3B,D) embryos did not show a significant difference in the number of pH 3 (P = .8966) or activated Caspase-3 (P = .0960) positive cells ( Figure 3E). Like wild-type embryos, at 36 hpf, rbbp4 Δ4/Δ4 mutant embryos appear morphologically normal and have well defined brain structures, including the optic tectum, cerebellum, and hindbrain ( Figure 3F,G). Confocal microscopy was used to image pH 3 and activated Caspase-3 in the dorsal optic tectum of whole mount-labeled embryos ( Figure 3F-J). The levels of pH 3 positive cells were not significantly different between 36 hpf +/+ wild type and rbbp4 Δ4/Δ4 mutant embryos ( Figure 3J; P = .4310). However, compared to +/+ wild type ( Figure 3F,H), in the rbbp4 Δ4/Δ4 mutant embryos showed significantly elevated levels of activated Caspase-3 in the optic tectum ( Figure 3G,H arrows; J; P = .0001). These results indicate that during embryonic neurogenesis, the loss of Rbbp4 did not impact proliferation of neuroepithelial cells but was required to prevent the apoptosis in the optic tectum.
The effect of loss of Rbbp4 on brain morphology was first evident in 2 dpf embryos ( Figure 4A-D). Compared to wild type ( Figure 4A,C, in rbbp4 Δ4/Δ4 ( Figure 4B,D) the size of the optic tectum and midbrain-hindbrain structures appeared reduced ( Figure 4A,B arrows; C,D brackets). Labeling of 2 and 3 dpf sectioned head tissue with pH 3 or activated Caspase-3 ( Figure 4E-P) revealed differences in both proliferation and apoptosis. pH 3 positive cells can be detected in the midbrain and retina in   Figure 4E,I,M) and rbbp4 Δ4/Δ4 mutants ( Figure 4F,J,N). The number of pH 3 positive cells at 2 dpf was significantly elevated in both rbbp4 Δ4/Δ4 mutant midbrain (Q P = .0058) and retina (R 0.0258). At 3 dpf, there was no difference in the midbrain (Q P = .4904), but the level of pH 3 cells in the retina remained significantly higher in the rbbp4 Δ4/Δ4 mutants (R P = .0324). At 2 and 3 dpf in wild type ( Figure 4G,K, O), there are very few activated Caspase-3 positive cells in the midbrain or retina. In contrast, as we reported previously, at 2 dpf activated Caspase-3 labeling was significantly increased in rbbp4 Δ4/Δ4 mutants ( Figure 4H,L,P) in both the midbrain ( Figure 4S; P = .0022) and retina ( Figure 4T; P = .0010). Activated Caspase-3 labeling and fragmented nuclei were detected throughout the inner nuclear layer of the retina ( Figure 4L). HuC/D labeling in the midbrain and retinal ganglion cell layer indicated that earlier born neurons were able to differentiate and survive in the absence of Rbbp4, possibly due to maternal contribution of Rbbp4. By 3 dpf, the number of activated Caspase-3 positive cells in rbbp4 Δ4/Δ4 mutant midbrain tissue varied but was not significantly different than wild type ( Figure 4S; P = .2569) and became restricted to the proliferative zones in the dorsal tectum. In the 3 dpf rbbp4 Δ4/Δ4 mutant retina, the level of activated Caspase-3 positive cells remained significantly high ( Figure 4T; P = .0358). Together, these results suggest that the loss of Rbbp4 leads to a failure in neural precursor cell cycle progression and induction of apoptosis in the midbrain and retina.
To examine which retinal cell populations fail to survive after loss of Rbbp4, BrdU pulse-chase labeling was used to follow the fate of rbbp4 Δ4/Δ4 neural precursors in the post-embryonic retina. Retinal neurogenesis proceeds in a conveyor belt pattern, with stem cells at the ciliary marginal zone generating progenitors that become progressively more committed as they move inward in the growing retina. 25,26 The 2 dpf embryos were exposed to a 3-hour BrdU pulse and collected immediately or chased until 3 and 5 dpf. At the end of 2 dpf in wild-type retina, BrdU is detected in stem cells at the retina ciliary marginal zone (CMZ) and progenitors in the inner nuclear and newly developing photoreceptor layers ( Figure 5A). A similar pattern of BrdU incorporation was detected in the rbbp4 Δ4/Δ4 mutant retina ( Figure 5B). At 3 dpf, BrdUlabeled cells were found in the region adjacent to the CMZ in both wild type and rbbp4 Δ4/Δ4 mutants ( Figure 5C,D outlines); however, many fewer BrdU positive cells were present in the mutant and the retina tissue was severely reduced. By 5 dpf, in wild-type retina, the CMZ was devoid of BrdU positive cells, and BrdU positive cells were incorporated into the ganglion and inner nuclear layers of the laminated region of the wild-type retina ( Figure 5E). In the rbbp4 Δ4/Δ4 mutant cells at the CMZ were present but were not labeled with BrdU ( Figure 5F arrow). Together, these results show that rbbp4 Δ4/Δ4 mutant retinal stem cells at the ciliary marginal zone survive and continue to divide, but the neural progenitor and/or newborn neuron cell populations undergoes apoptosis.

| rbbp4 mutant retinal neural precursors are lost prior to initiation of quiescence and differentiation
Neurogenesis in the zebrafish larval retina can be visualized at 3 dpf by expression of genes in discreet sectors that mark progressive steps in neural progenitor commitment 27 ( Figure 6A). To identify the stage at which rbbp4 Δ4/Δ4 neural precursors are lost we performed in situ hybridization on 3 dpf wild type and rbbp4 Δ4/Δ4 retina with probes to collagen type XV alpha 1b (mz98), 28 cyclin D1 (ccnD1), 29 atonal 7 (atoh7) 30 and G1 cyclindependent kinase inhibitor 1Ca (cdkn1c) 31 ( Figure 6B-I).
In wild type, cells at the periphery of the ciliary marginal zone express stem cell marker mz98 (Figure B arrow), followed by proliferating progenitors that express ccnD1 ( Figure 6C bracket), and then atoh7-expressing committed neural precursors ( Figure 6D bracket). Lastly, cdkn1c expression marks precursors arrested in G1 that will initiate quiescence and differentiation ( Figure 6E arrow). In the rbbp4 Δ4/Δ4 mutant retina, mz98-expressing cells were present in the ciliary marginal zone ( Figure 6F arrow). The population of ccnd1-labeled progenitors and atoh7expressing committed precursors was significantly expanded in the mutant compared to wild type ( Figure 6G,H brackets). However, cdkn1c-expression was not detected in the next adjacent sector in the rbbp4 Δ4/Δ4 mutants ( Figure 6I arrow). These results indicate that in the absence of Rbbp4, loss of committed retinal precursors adjacent to the ciliary marginal zone occurs before the cells transition to cell cycle exit and initiation of quiescence and differentiation. The expansion of the ccnD1 and atoh7 domains also suggests a failure in terminal differentiation.
In the midbrain only rbbp4 Δ4/Δ4 mutants at 2 dpf showed a significant elevation in activated Caspase-3 labeling in comparison to wild type (P = .0053) ( Figure 8Q,R). Apoptosis was not detected in the ciliary marginal zone of the retina at 2 or 3 dpf in any genotype ( Figure 8E-H CMZ, M-P CMZ brackets) but could be detected in the inner nuclear layer and the progenitor region of the CMZ ( Figure 8N,P dashed lines). The level of activated Caspase-3 cells in rbbp4 Δ4/Δ4 single and rbbp4 Δ4/Δ4 ; rb1 Δ7/ Δ7 double mutant retinas was not significantly different at  dpf (P > .9999) or 3 dpf (P = .0859). "These results show that loss of Rbbp4 can induce apoptosis in rb1 mutant neural precursors, further supporting a role for Rbbp4 in neural progenitor survival that is independent of Rb." 2.7 | tp53 morpholino knockdown suppresses rbbp4 mutant neural precursor apoptosis and γ-H2AX To test whether apoptosis in rbbp4 Δ4/Δ4 mutants was dependent on Tp53, tp53 expression was knocked down by injection of an antisense translation blocking tp53 morpholino into 1-cell stage embryos ( Figure 9A). Control un-injected and injected wild type and rbbp4 Δ4/Δ4 mutant embryos were sectioned at 2 and 3 dpf and double labeled with antibodies to activated Caspase-3 and neuronal differentiation marker HuC/D ( Figure 9B-I). Compared to wildtype un-injected control embryos, Tp53 knock down did not affect development or cell viability in the wild-type midbrain or retina at 2 dpf ( Figure 9B,C) and 3 dpf ( Figure 9F,G). In comparison to un-injected rbbp4 Δ4/Δ4 embryos, Tp53 knock down suppressed activated Caspase-3 labeling and apoptosis in the rbbp4 Δ4/Δ4 midbrain and retina at 2 dpf ( Figure 9D,E) and 3 dpf ( Figure 9D,E). Suppression of apoptosis in the midbrain was significant at 2 dpf, but not at 3 dpf, possibly since the overall number of apoptotic cells in the rbbp4 Δ4/Δ4 mutant midbrain was reduced at this later stage ( Figure 9J). In the retina, tp53 knockdown significantly reduced the number of activated Caspase-3 cells at both 2 and 3 dpf ( Figure 9K). Together these results demonstrate that the activation of programmed cell death in rbbp4 Δ4/Δ4 mutant neural progenitors is dependent on tp53 signaling. γ-H2AX is routinely used as a marker for DNA damage associated with induction of TP53-dependent apoptosis but also shows extensive labeling throughout cells and degrading nuclei at late stages of apoptosis. We examined γ-H2AX labeling in rbbp4 mutant larvae with and without tp53 morpholino knockdown in 2 dpf ( Figure 9L-O) and 3 dpf larvae ( Figure 9P-S). At 2 dpf intense γ-H2AX labeling can be detected in the rbbp4 Δ4/Δ4 midbrain and retina ( Figure 9N) where activated Caspase-3 is present and becomes similarly restricted at 3 dpf to proliferative zones ( Figure 9R). Morpholino knockdown of tp53 nearly completely suppressed γ-H2AX labeling in rbbp4 Δ4/Δ4 2 and 3 dpf larvae ( Figure 9O,S,T,U). The lack of γ-H2AX labeling in rbbp4 Δ4/Δ4 mutants after suppression of apoptosis by tp53 knockdown suggests that the γ-H2AX signal in the mutants may not represent DNA damage, but the end stage of cells undergoing apoptosis. This indicates loss of Rbbp4 may activate TP53 through a mechanism or pathway distinct from the canonical DNA damage response.

| Maternal zygotic tp53Δ is55 deletion suppresses rbbp4 mutant neural precursor apoptosis
To demonstrate a genetic requirement for Tp53 in apoptosis after loss of Rbbp4, we used two TALEN pairs targeting sites near the 5 0 and 3 0 ends of the tp53 gene to generate the deletion allele tp53Δ is55 . The left TALEN pair target site was located in intron 1, and the right TALEN pair site was located in the 3 0 UTR downstream of exon 11 ( Figure 10A). The sequences of the TALENs (Table 2) were previously reported by Ignatius et al., in which a tp53 del deletion allele was isolated; however, that allele was generated with a different 5 0 TALEN pair that sits upstream of tp53 exon 1. 33 The tp53Δ is55 allele deletes~13 kb and is homozygous viable ( Figure 10B). Analysis of the deletion junction shows sequences upstream of the 5 0 TALEN Left cut site and downstream of the 3 0 TALEN Right cut site fused together, with a single C inserted in between ( Figure 10C).

| Rbbp4 loss leads to Tp53 acetylation, while overexpression fails to rescue apoptosis after chemically induced DNA damage
To investigate the role of Rbbp4 in activation of Tp53-dependent apoptosis, we examined how the loss of Rbbp4 impacts Tp53 acetylation. Tp53 transcriptional activity is known to be activated by p300-mediated acetylation of C-terminal lysine residues and repressed by histone deacetylase 1. 34 Since Rbbp4 is a component of the histone deacetylase 1 NuRD complex, we examined whether the loss of Rbb4 leads to increased Tp53 acetylation at a lysine amino acid p300 target ( Figure 12A). Western blotting with anti-Tp53-acetyl-K370 antibody of human HEK293 cells, human HCT116 cells, and wildtype +/+ 24 hpf zebrafish embryos treated with the histone deacetylase inhibitor Trichostatin A (TSA) show high levels of Tp53-acetyl-K370 in comparison to untreated cells and embryos. In contrast to wild type, Tp53-acetyl-K370 is present in homozygous mutant rbbp4 Δ4/Δ4 embryos. These results show that the loss of Rbbp4 leads to increased Tp53 acetylation.
The suppression of γ-H2AX labeling in rbbp4 Δ4/Δ4 mutants after tp53 knockdown suggested induction of apoptosis may occur through a pathway independent of the DNA damage response. To examine whether Rbbp4 functions in the DNA damage response pathway, we tested whether overexpression of Rbbp4 by mRNA injection would rescue DNA-damage induced apoptosis. Wild-type WIK embryos were injected with 100 pg of in vitro transcribed rbbp4 mRNA. At 24 hpf, the embryos were treated with 100 μm camptothecin for 5 hours and labeled with acridine orange to visualize cells undergoing apoptosis ( Figure 12B-E). Compared to control uninjected WIK embryos treated with camptothecin ( Figure 12B-B',D-D'), injection of 100 pg rbbp4 mRNA did not appear to reduce the amount of acridine orange labeling in the neural tube ( Figure 12C-C', E-E'). Quantification of the level of acridine orange in the neural tube showed no significant difference between control camptothecin-treated embryos and those injected with rbbp4 mRNA ( Figure 12F). A western blot of control, camptothecin treated, injected, and injected plus camptothecin-treated embryos probed with an anti-Rbbp4/7 antibody showed a slight elevation in the level of Rbbp4 protein after rbbp4 mRNA injection. These results suggest that overexpression of Rbbp4 alone is not sufficient to rescue induction of apoptosis caused by DNA damage.
Together, the increase in Tp53 acetylation in rbbp4 Δ4/Δ4 mutants, and lack of rescue of apoptosis after Rbbp4 overexpression, suggest one mechanism driving apoptosis after loss of Rbbp4 is disruption of Rbbp4-containing histone deacetylase complexes that regulate Tp53 acetylation and transcriptional activation.
Our analysis of zebrafish rbbp4 mutants revealed Rbbp4 is necessary for cell survival as well as cell cycle control during neurogenesis in the developing midbrain and retina. Apoptosis of midbrain neural progenitors could be detected as early as 36 hpf in the rbbp4 mutant optic tectum, but the number of mitotic M phase cells was not significantly different than wild type. By 2 dpf, the number of apoptotic cells and M-phase cells had increased significantly in the midbrain and retina. One interpretation of this observation is that it reflects the requirement for Rbbp4 in regulation of cell cycle entry as a component of the NuRD complex, which cooperates with the Rb tumor suppressor to block expression of E2Fregulated early S phase genes. 9 However, in rbbp4; rb1 double mutants, the number of cells in M phase was enhanced but additive, indicating independent roles for Rb and Rbbp4 in cell cycle regulation. These differences may reflect vertebrate or neural specific requirements for Rbbp4 in progenitor cell cycle progression. Examining the requirement for Rbbp4 in cell cycle gene expression will lend new insight into the mechanism by which Rbbp4 regulates the neural progenitor cell cycle.
The observation that the loss of Rbbp4 leads to accumulation of cells in M-phase is more consistent with a role for Rbbp4 in regulating cell cycle exit and differentiation. Rbbp4 is a component of the MuvB complex which regulates multiple stages of cell cycle gene expression. 12 MuvB is part of the larger DREAM complex which represses cell cycle gene expression in G0/G1. 35 MuvB associates with B-Myb to form the MBB complex and activate gene expression necessary for progression through S/G2. 36 At the G2/M transition, MuvB loses association with B-Myb and forms a complex with the transcription factor FoxM1 to drive transcriptional activation of late cell cycle genes. 36 Our analysis of pH 3 labeling does not determine whether Rbbp4 is required for MBB regulation of S/G2 progression, but it is consistent with a role for Rbbp4 in the MuvB/FoxM1 complex that drives progression through M phase. This interpretation is supported by the absence of expression of the G1 to G0 transition cell cycle inhibitor cdkn1c.
Our study demonstrates that the loss of Rbbp4 leads to Tp53-dependent apoptosis in zebrafish neural progenitors and Tp53 acetylation. Tp53-mediated transcriptional activation in response to DNA damage is stimulated by p300/CBP and PCAF acetylation. 37 40 The observation that loss of Rbbp4 stimulates Tp53-dependent apoptosis and Tp53 acetylation suggests a defect in NuRD-mediated Tp53 deacetylation, which would increase acetylated-Tp53 and stimulate Tp53-dependent apoptosis. Preventing Tp53 activation through NuRD may provide a mechanism by which Rbbp4 promotes neural progenitor proliferation and contributes to brain tumor oncogenesis.
In the rbbp4 mutant retina, the expansion of the atoh7-positive progenitor population and the absence of cdkn1c-expressing cells suggest that progenitors fail to transition to a differentiated state and initiate G0. The elevated level of M-phase cells in rbbp4 and rbbp4; maternal zygotic tp53 double mutants suggests the G1/G0 transition defect is due to a failure of progenitors to exit the cell cycle. Loss of Rbbp4 may lead to altered transcriptional regulation of late cell cycle genes by the FoxM1/ MuvB complex, leading to arrest in M phase. Alternatively, loss of Rbbp4 could affect PRC2-mediated cell cycle gene silencing and heterochromatin formation required for cell cycle exit and differentiation. 41 In Arabidopsis, the Rbbp4 homolog AtMSI1 is necessary for persistent growth of reproductive tissues through expression of homeotic genes and heterochromatin assembly. 42 Knockdown of Rbbp4 in cultured chicken DT40 cells leads to defects in DNA synthesis, nucleosome assembly, heterochromatin formation, and accumulation of cells in G2/M leading to cell death. 43 Our results are consistent with these observations that Rbbp4 is required for cell cycle exit and terminal differentiation. The ability to suppress apoptosis by Tp53 knockdown or genetic deletion in zebrafish rbbp4 mutants provides an opportunity to examine in vivo how Rbbp4 loss impacts cell cycle gene expression and heterochromatin formation required for progression of neural precursors to quiescence.
BrdU lineage tracing showed loss of Rbbp4 lead to cell death in the inner part of the retina but did not affect the proliferation or survival of retinal stem cells located at the periphery of the ciliary marginal zone. This observation contrasts with the requirement for Rbbp4 to maintain embryonic stem cell pluripotency, 44 which suggests that after early embryogenesis Rbbp4 may no longer be required in tissue specific stem cell populations later in development. Conditional inactivation of Rbbp4 in retinal neural stem and progenitor populations in vivo would address whether Rbbp4 has a role in neural stem cell maintenance that is distinct from its function in neural progenitor cell cycle regulation and survival.
In summary, our study indicates that Rbbp4 may promote proliferation and survival of neural precursors by driving cell cycle progression independent of Rb and preventing activation of Tp53-dependent apoptosis. While these results do not rule out the possibility that the loss of Rbbp4 leads to Tp53 activation through the DNA damage response pathway, they suggest one of the mechanisms driving apoptosis is disruption of chromatin remodeling complexes that directly regulate Tp53 acetylation and transcriptional activity. Together, our analysis provides the foundation for future studies examining the mechanisms by which Rbbp4 and its associated chromatin remodelers promote cell cycle progression and survival during neurogenesis.

| Zebrafish care and husbandry
Zebrafish were reared in an Aquatic Habitat system (Aquatic Ecosystems, Inc., Apopka, FL). Fish were maintained on a 14-hours light/dark cycle at 27 C. Transgenic lines were established in a WIK wild-type strain obtained from the Zebrafish International Research Center (http://zebrafish.org/zirc/home/guide.php). The zebrafish rbbp4 and rb1 alleles used in this study were isolated by CRISPR-Cas9 or TALEN targeting and described previously: rbbp4 4 base pair (bp) deletion allele rbbp4 Δ4is6017 ; rb1 7 bp deletion allele rb1 Δ7is54 . 16 For in situ hybridization and immunohistochemistry experiments, embryos were collected and maintained at 28.5 C in embryo media 45 until harvesting. Embryos were staged according to published guidelines. 46 All experimental protocols were approved by the Iowa State University Institutional Animal Care and Use Committee (IACUC-18-379, IBC-18-177) in compliance with American Veterinary Medical Association and the National Institutes of Health guidelines for the humane use of laboratory animals in research.

| Larval genotyping and tp53 morpholino injections
Primers to amplify rbbp4 exon 2 containing the rbbp4 Δ4is60 mutation: Forward 5 0 GCGTGATGACAGATCTCATATTG TTTTCCC 3 0 ; Reverse 5 0 CTGGTGACATCTGGCAA CCACT 3 0 . Primers to amplify rb1 exon 2 containing the rb1 Δ7is54 mutation: forward 5'-TTTCCAGACACAAGGAC AAGGATCC-3 0 ; reverse 5 0 -GCAGATATCAGAAGAAA GAGTACATTTGTCTT-3 0 . To inhibit Tp53-dependent apoptosis, embryos were injected at the one cell stage with 2 ng of tp53 translation blocking morpholino GCGCCATT GCTTTGCAAGAATTG 47 (Gene Tools; ZDB-MRPHLNO-070126-7). 4.3 | Isolation of deletion allele tp53 Δis55 and transgenic line Tg(Tol2<ubi: rbbp4-2AGFP>) is61 The previously described TALEN pairs for targeted deletion of tp53 (Table 2) were designed to a site in intron 1 and a site 2 kb downstream of exon 11. 33 pT3TS-TALEN vectors were linearized using SacI and mRNA was in vitro transcribed using the mMessage mMachine T3 kit (ThermoFisher AM1348). The mRNA was purified using Qiagen RNeasy MinElute Cleanup Kit (Qiagen 74 204). A 40 pg of each TALEN mRNA was co-injected into 1-cell WIK embryos. Ten pairs of adults were screened for transmission of the 13 kb deletion allele by PCR of embryo genomic DNA with primers that span the deletion junction (Table 2). A single founder was identified and the deletion allele junction fragment confirmed by sequencing. A single heterozygous F1 adult was outcrossed to WIK to generate F2s, and a single heterozygous F2 was outcrossed to WIK to establish an F3 family of the tp53 Δis55 /+ line.
The rbbp4 1275 bp cDNA minus the translation termination codon was amplified by reverse transcription PCR with forward 5 0 -catgTCTAGATGTGGAGTCGT TATGGCTG-3 0 and reverse 5 0 -catgGGATCCTCCCTGAA CCTCAGTGTCTG-3 0 primers that included 5 0 XbaI and 3 0 BamHI sites, respectively. The Tol2<ubi:rbbp4-2AGFP> transgene was assembled using the NEBuilder HiFi DNA Assembly protocol and mix (New England Biolabs E2621S) in the mini-pTol2 vector. 48 The zebrafish ubiquitin promoter 49 was cloned into the vector followed by the rbbp4 cDNA, an in-frame 2A viral peptide GFP cassette 50 and the zebrafish β-actin 3'UTR (Dr. Darius Balciunas, Temple University 51 ). A 1 μg of linearized pT3TS-Tol2 transposase plasmid 48 was used as template for in vitro synthesis of Tol2 transposase capped, polyadenylated mRNA with T3 mMessage mMachine High Yield Capped RNA transcription kit (ThermoFisher AM1348). Synthesized mRNA was precipitated with LiCl and resuspended in RNase, DNase-free molecular grade water. The Tg(Tol2<ubi: rbbp4-2AGFP>) is61 transgenic line was isolated by coinjection into 1-cell WIK zebrafish embryos of 25 pg Tol2 vector plus 100 pg Tol2 mRNA. Fourteen founder fish were screened for germline transmission of ubiquitously expressed GFP. A single F1 adult that showed Mendelian transmission of the Tol2<ubi:rbbp4-2AGFP> transgene to the F2 generation was used to establish the line.

| Embryo rbbp4 mRNA injections and camptothecin treatment
The rbbp4 cDNA was cloned into the expression vector pT3TS. A 1 μg linearized vector was purified with the PureYield Plasmid Miniprep System (Promega, A1223) and used as template for in vitro synthesis of capped mRNA with the Ambion mMessage Machine T3 Transcription Kit (Thermo Fisher, AM1348). In vitro synthesized mRNA was purified with the RNA Clean and Concentrator Kit RCC (Zymo, R1013). Single-cell zebrafish embryos were injected with 100 pg or 300 pg rbbp4 mRNA. At 24 hpf, the embryos were incubated for 5 hours with 100 μm Camptothecin (Sigma-Aldrich, C9911) or DMSO as a control. Embryos were incubated in 10ug/ml acridine orange (Thermo Fisher Scientific, AC423340010) in embryo media for 30 minutes. Acridine orange-labeled living embryos were rinsed with embryo media and immediately imaged on a Zeiss Discovery.V12 stereomicroscope using a Cannon Rebel digital camera and EOS Utility software.

| QUANTIFICATION AND STATISTICAL ANALYSIS
Statistical analyses using unpaired, two tailed Student's t-test; unpaired, two tailed Welch's t-test; one-way ANOVA Sidak's multiple comparisons test; and two-ANOVA multiple comparison test, were performed using GraphPad Prism software. Statistical parameters are included in the figure legends. Data plots represent mean ± s.e.m.