Intellectual disability‐associated dBRWD 3 regulates gene expression through inhibition of HIRA / YEM ‐mediated chromatin deposition of histone H3.3

Scientific Report9 February 2015free access Intellectual disability-associated dBRWD3 regulates gene expression through inhibition of HIRA/YEM-mediated chromatin deposition of histone H3.3 Wei-Yu Chen Wei-Yu Chen Institute of Molecular Medicine, College of Medicine, National Taiwan University, Taipei, Taiwan Search for more papers by this author Hsueh-Tzu Shih Hsueh-Tzu Shih Institute of Molecular Medicine, College of Medicine, National Taiwan University, Taipei, Taiwan Search for more papers by this author Kuei-Yan Liu Kuei-Yan Liu Institute of Molecular Medicine, College of Medicine, National Taiwan University, Taipei, Taiwan Search for more papers by this author Zong-Siou Shih Zong-Siou Shih Institute of Molecular Medicine, College of Medicine, National Taiwan University, Taipei, Taiwan Search for more papers by this author Li-Kai Chen Li-Kai Chen Institute of Molecular Medicine, College of Medicine, National Taiwan University, Taipei, Taiwan Search for more papers by this author Tsung-Han Tsai Tsung-Han Tsai Institute of Molecular Medicine, College of Medicine, National Taiwan University, Taipei, Taiwan Search for more papers by this author Mei-Ju Chen Mei-Ju Chen Genome and Systems Biology Degree Program, National Taiwan University and Academia Sinica, Taipei, Taiwan Search for more papers by this author Hsuan Liu Hsuan Liu Department of Cell and Molecular Biology, College of Medicine, Chang Gung University, Tao-Yuan, Taiwan Molecular Medicine Research Center, Chang Gung University, Tao-Yuan, Taiwan Search for more papers by this author Bertrand Chin-Ming Tan Bertrand Chin-Ming Tan Molecular Medicine Research Center, Chang Gung University, Tao-Yuan, Taiwan Department of Biomedical Sciences and Graduate Institute of Biomedical Sciences, College of Medicine, Chang Gung University, Tao-Yuan, Taiwan Search for more papers by this author Chien-Yu Chen Chien-Yu Chen Bio-Industrial Mechatronics Engineering, National Taiwan University, Taipei, Taiwan Search for more papers by this author Hsiu-Hsiang Lee Hsiu-Hsiang Lee Institute of Molecular Medicine, College of Medicine, National Taiwan University, Taipei, Taiwan Search for more papers by this author Benjamin Loppin Benjamin Loppin Centre de Génétique et de Physiologie Moléculaire et Cellulaire, CNRS UMR5534, Université Claude Bernard Lyon 1, Villeurbanne, France Search for more papers by this author Ounissa Aït-Ahmed Ounissa Aït-Ahmed Institute of Regenerative medicine and Biotherapy (IRMB), Inserm U1203, Saint-Eloi Hospital, CHRU Montpellier, France Search for more papers by this author June-Tai Wu Corresponding Author June-Tai Wu Institute of Molecular Medicine, College of Medicine, National Taiwan University, Taipei, Taiwan Department of Dermatology, National Taiwan University Hospital and National Taiwan University College of Medicine, Taipei, Taiwan Research Center for Developmental Biology and Regenerative Medicine, National Taiwan University, Taipei, Taiwan Search for more papers by this author Wei-Yu Chen Wei-Yu Chen Institute of Molecular Medicine, College of Medicine, National Taiwan University, Taipei, Taiwan Search for more papers by this author Hsueh-Tzu Shih Hsueh-Tzu Shih Institute of Molecular Medicine, College of Medicine, National Taiwan University, Taipei, Taiwan Search for more papers by this author Kuei-Yan Liu Kuei-Yan Liu Institute of Molecular Medicine, College of Medicine, National Taiwan University, Taipei, Taiwan Search for more papers by this author Zong-Siou Shih Zong-Siou Shih Institute of Molecular Medicine, College of Medicine, National Taiwan University, Taipei, Taiwan Search for more papers by this author Li-Kai Chen Li-Kai Chen Institute of Molecular Medicine, College of Medicine, National Taiwan University, Taipei, Taiwan Search for more papers by this author Tsung-Han Tsai Tsung-Han Tsai Institute of Molecular Medicine, College of Medicine, National Taiwan University, Taipei, Taiwan Search for more papers by this author Mei-Ju Chen Mei-Ju Chen Genome and Systems Biology Degree Program, National Taiwan University and Academia Sinica, Taipei, Taiwan Search for more papers by this author Hsuan Liu Hsuan Liu Department of Cell and Molecular Biology, College of Medicine, Chang Gung University, Tao-Yuan, Taiwan Molecular Medicine Research Center, Chang Gung University, Tao-Yuan, Taiwan Search for more papers by this author Bertrand Chin-Ming Tan Bertrand Chin-Ming Tan Molecular Medicine Research Center, Chang Gung University, Tao-Yuan, Taiwan Department of Biomedical Sciences and Graduate Institute of Biomedical Sciences, College of Medicine, Chang Gung University, Tao-Yuan, Taiwan Search for more papers by this author Chien-Yu Chen Chien-Yu Chen Bio-Industrial Mechatronics Engineering, National Taiwan University, Taipei, Taiwan Search for more papers by this author Hsiu-Hsiang Lee Hsiu-Hsiang Lee Institute of Molecular Medicine, College of Medicine, National Taiwan University, Taipei, Taiwan Search for more papers by this author Benjamin Loppin Benjamin Loppin Centre de Génétique et de Physiologie Moléculaire et Cellulaire, CNRS UMR5534, Université Claude Bernard Lyon 1, Villeurbanne, France Search for more papers by this author Ounissa Aït-Ahmed Ounissa Aït-Ahmed Institute of Regenerative medicine and Biotherapy (IRMB), Inserm U1203, Saint-Eloi Hospital, CHRU Montpellier, France Search for more papers by this author June-Tai Wu Corresponding Author June-Tai Wu Institute of Molecular Medicine, College of Medicine, National Taiwan University, Taipei, Taiwan Department of Dermatology, National Taiwan University Hospital and National Taiwan University College of Medicine, Taipei, Taiwan Research Center for Developmental Biology and Regenerative Medicine, National Taiwan University, Taipei, Taiwan Search for more papers by this author Author Information Wei-Yu Chen1,‡, Hsueh-Tzu Shih1,‡, Kuei-Yan Liu1, Zong-Siou Shih1, Li-Kai Chen1, Tsung-Han Tsai1, Mei-Ju Chen2, Hsuan Liu3,4, Bertrand Chin-Ming Tan4,5, Chien-Yu Chen6, Hsiu-Hsiang Lee1, Benjamin Loppin7, Ounissa Aït-Ahmed8 and June-Tai Wu 1,9,10 1Institute of Molecular Medicine, College of Medicine, National Taiwan University, Taipei, Taiwan 2Genome and Systems Biology Degree Program, National Taiwan University and Academia Sinica, Taipei, Taiwan 3Department of Cell and Molecular Biology, College of Medicine, Chang Gung University, Tao-Yuan, Taiwan 4Molecular Medicine Research Center, Chang Gung University, Tao-Yuan, Taiwan 5Department of Biomedical Sciences and Graduate Institute of Biomedical Sciences, College of Medicine, Chang Gung University, Tao-Yuan, Taiwan 6Bio-Industrial Mechatronics Engineering, National Taiwan University, Taipei, Taiwan 7Centre de Génétique et de Physiologie Moléculaire et Cellulaire, CNRS UMR5534, Université Claude Bernard Lyon 1, Villeurbanne, France 8Institute of Regenerative medicine and Biotherapy (IRMB), Inserm U1203, Saint-Eloi Hospital, CHRU Montpellier, France 9Department of Dermatology, National Taiwan University Hospital and National Taiwan University College of Medicine, Taipei, Taiwan 10Research Center for Developmental Biology and Regenerative Medicine, National Taiwan University, Taipei, Taiwan ‡These authors contributed equally to this work *Corresponding author. Tel: +886 2 23123456 ext 88367; E-mail: [email protected] EMBO Reports (2015)16:528-538https://doi.org/10.15252/embr.201439092 Correction(s) for this article Intellectual disability-associated dBRWD3 regulates gene expression through inhibition of HIRA/YEM-mediated chromatin deposition of histone H3.304 July 2016 PDFDownload PDF of article text and main figures. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Abstract Many causal mutations of intellectual disability have been found in genes involved in epigenetic regulations. Replication-independent deposition of the histone H3.3 variant by the HIRA complex is a prominent nucleosome replacement mechanism affecting gene transcription, especially in postmitotic neurons. However, how HIRA-mediated H3.3 deposition is regulated in these cells remains unclear. Here, we report that dBRWD3, the Drosophila ortholog of the intellectual disability gene BRWD3, regulates gene expression through H3.3, HIRA, and its associated chaperone Yemanuclein (YEM), the fly ortholog of mammalian Ubinuclein1. In dBRWD3 mutants, increased H3.3 levels disrupt gene expression, dendritic morphogenesis, and sensory organ differentiation. Inactivation of yem or H3.3 remarkably suppresses the global transcriptome changes and various developmental defects caused by dBRWD3 mutations. Our work thus establishes a previously unknown negative regulation of H3.3 and advances our understanding of BRWD3-dependent intellectual disability. Synopsis BRWD3 is mutated in patients with X-linked intellectual disability. Here, dBRWD3 is found to negatively regulate the amount of chromatin-associated H3.3, thereby regulating gene expression in the brain and several developmental processes. dBRWD3 mutation causes various developmental abnormalities by altering gene expression. HIRA/YEM-mediated H3.3 deposition is increased in dBRWD3 mutants. The transcriptomic changes and diverse developmental defects of dBRWD3 mutants are suppressed by inactivation of the HIRA/YEM–H3.3 pathway. Introduction Genomic DNA is wrapped around histone octamers and organized into chromatin. The dynamic chromatin structures instruct how a gene should be transcribed during developmental processes. Information associated with chromatin structure can be established and edited by at least four categories of epigenetic mechanisms: DNA methylation, histone tail modifications, nucleosome remodeling, and exchanges of histone variants. Among histone variants, the histone H3 variant H3.3 differs from the conventional histone H3.1 by only 5 residues, but has distinct deposition mechanisms and biological functions. Deposition of H3.3-containing nucleosomes is mediated by Daxx/ATRX or HIRA complex throughout the cell cycle (replication independent), whereas deposition of H3.1 into chromatin is mediated by histone chaperone CAF-1 mainly during DNA replication (replication dependent) 1. HIRA and its associated chaperone Yemanuclein (YEM), the fly ortholog of mammalian Ubinuclein1, deposit H3.3 to actively transcribed regions, nucleosome-free gaps 23 and the male pronucleus at fertilization 4567. In contrast, the Daxx/ATRX complex deposits H3.3 to the telomeres 8910. During development, deposition of H3.3 but not H3.1 establishes the epigenetic memory of an active gene state 11. Because H3.3 deposition is associated with gene transcription 1213 and enriched over actively transcribed regions 814, it has been assumed that H3.3 could promote gene transcription. However, HIRA/YEM-mediated H3.3 deposition at the promoter regions was also shown to be required for PRC2 recruitment and bivalent gene silencing 15. Although the diverse roles of HIRA and H3.3 on gene activation and repression have been studied extensively, how HIRA/YEM-mediated deposition of H3.3 is regulated remains unknown. Early-onset cognitive impairment, known as mental retardation or intellectual disability (ID), is defined as a reduced ability to learn new skills 16. Genetic studies suggest that ID is a disease of heterogeneous causes. For instance, in X-linked ID (XLID), which accounts for 10-12% of all forms of ID, the biological functions of the causative genes are diverse and not restricted to neurons 17. Among the XLID causative genes, CUL4B encodes the scaffold component of cullin-RING E3 ligase, CRL4B 1819. Like other CRL ubiquitin E3 ligases, CRL4B can ubiquitinate different substrates by incorporating a distinct substrate receptor through DDB1 (DNA damage-binding protein 1) 20. Interestingly, one of the CRL4 substrate receptors, BRWD3 (Bromodomain and WD repeat-containing protein 3), was mutated in three XLID families 2122. These genetic studies suggest that a BRWD3-containing CRL4 complex is critical for maintaining neural functions and structures. How the BRWD3-containing CRL4 complex functions in the nervous system is not known. Here, we report that dBRWD3, the fly homolog of XLID protein BRWD3, negatively regulates the amount of H3.3 associated with YEM and the levels of chromatin-associated H3.3. Strikingly, the diverse transcriptome changes and developmental defects found in dBRWD3 mutants were restored to normal by inactivation of the HIRA/YEM–H3.3 pathway. These findings identify dBRWD3 as a negative regulator of the HIRA/YEM–H3.3 pathway and provide a potential molecular mechanism underlying the X-linked intellectual disability. Results and Discussion dBRWD3 regulates photoreceptor differentiation To explore the molecular function of BRWD3 that underlies its implication in intellectual disability, we characterized dBRWD3, an essential gene encoding the only BRWD family protein in Drosophila 23. By immunofluorescence analysis, we confirmed that dBRWD3 protein is absent in the molecularly null dBRWD3 mutant, as previously reported 23 (Supplementary Fig S1A, dashed lines). In dBRWD3 mutant clones, the photoreceptors projected axons to brain normally (Fig 1A, arrows, n = 52, penetrance = 100%), but did not differentiate into mature neurons expressing Chaoptin, a terminal differentiation marker (Fig 1B, dashed lines, n = 56, penetrance = 92.9%). The expression of Prospero, a homeobox protein, was also markedly reduced in the mutant clones, indicating that the differentiation of R7 and cone cells is arrested (Fig 1C, dashed lines, n = 23, penetrance = 100%). To determine whether dBRWD3 functions as a subunit of the CRL4 complex in these processes, we complemented dBRWD3 mutant clones with either wild-type dBRWD3 or delta-N-dBRWD3 mutant proteins that cannot bind to the DDB1 adaptor of the CLR4 complex 24. While expression of wild-type dBRWD3 restored the expression of Prospero (Fig 1D, dashed lines, n = 69, penetrance of restoration = 100%) and rescued the lethality of dBRWD3 null mutants (Supplementary Table S1-1), expression of delta-N-dBRWD3 failed to do so (Fig 1E, n = 27, penetrance = 92.6%, and Supplementary Table S1-1), indicating that the binding to DDB1 in the CRL4 complex is essential for dBRWD3 to regulate gene expression and animal viability. It has been shown that CRL4B mediates the ubiquitination and subsequent degradation of WDR5, a core subunit of histone H3 lysine 4 (H3K4) methyltransferase complexes, thereby regulating the expression of neuronal genes 25. However, H3K4me3 was not increased in dBRWD3 mutant clones (Fig 1F, dashed lines, and G), suggesting that dBRWD3 regulates the development of photoreceptors and cone cells independently of WDR5. Figure 1. dBRWD3 is required for the differentiation of photoreceptorsdBRWD3s5349 mutant clones were generated in 3rd instar eye imaginal disks by hs-flp (A and F) or ey-flp (B to E) and marked by the presence (A, arrows) or the absence (B to F, dashed lines) of GFP. All scale bars indicate 50 μm. dBRWD3s5349 mutant clones (B, C and E) were DAPI (blue) positive. A. Projection of axons from wild-type (Chaoptin positive) and dBRWD3s5349 mutant photoreceptors (GFP positive, arrows) into the medulla layer of brain optic lobe. B, C. Expression of neuronal markers, Chaoptin (B), and Prospero (C) in wild-type (GFP positive) and dBRWD3s5349 mutant photoreceptors (GFP negative, dashed lines). D, E. Prospero expression (red) in dBRWD3s5349 mutant clones complemented with Flag-dBRWD3 (D) and Flag-delta-N-dBRWD3, encoding a mutant dBRWD3 with a deletion of a conserved HLH-box ranging from amino acid 146 to 164 (E). F. Expression of H3K4me3 in wild-type (GFP positive) and dBRWD3s5349 mutant photoreceptors (GFP negative, dashed lines). G. Quantification of H3K4me3 levels in wild-type and dBRWD3s5349 mutant photoreceptors. n = 39, P = 0.54 by Student's t-test. Download figure Download PowerPoint Identification of H3.3 as a suppressor of dBRWD3 To understand how dBRWD3 regulates photoreceptor differentiation, we conducted an RNAi screen to identify modifiers of the rough eye phenotype caused by knockdown of dBRWD3 in the eye imaginal disk (OK107-GAL4-driven UAS-dBRWD3-dsRNA, Fig 2A). From the screen, we identified H3.3B, one of the two genes expressing H3.3, as a genetic suppressor. Double knockdown of H3.3B and dBRWD3 restored normal eye development (Fig 2B). In contrast, H3.3B depletion alone did not cause any discernible eye abnormality (Fig 2C). This result prompted us to examine whether H3.3 levels are increased in dBRWD3 mutant cells. Lacking a reliable antibody for immunofluorescence studies of endogenous H3.3, we analyzed the transgenically expressed dendra2-tagged H3.3 and found that H3.3-dendra2 levels were higher in dBRWD3 mutant photoreceptors (Fig 2D, dashed lines) compared with those in wild-type twin spots. Western blot analysis confirmed an increase of both total and chromatin-associated endogenous H3.3 in dBRWD3 mutants (Fig 2E; Supplementary Fig S1B). Taken together, we concluded that dBRWD3 negatively regulates the level of chromatin-associated H3.3. To examine whether the accumulated H3.3 caused abnormal gene expression, we knocked down H3.3 in dBRWD3 mutant clones. The expression of Chaoptin (Fig 2F, arrows, n = 44, penetrance of derepression = 65.9%) and Prospero (Fig 2G, arrows, n = 33, penetrance of derepression = 84.8%) in posterior dBRWD3 mutant clones increased when H3.3 was knocked down. Thus, the increased H3.3 also underlies the deregulation of gene expression in dBRWD3 mutant clones. Because both H3.3B and H3.3A null mutants are viable 2627, we were able to investigate the impact of reducing the number of H3.3 copies on the viability of dBRWD3 mutants. Strikingly, while all homozygous dBRWD3PX2/PX2 mutant larvae died at the pupal stage, more than 40% of H3.3B−/−, dBRWD3PX2/PX2 mutants emerged as adults (Supplementary Table S1-2). Therefore, these data suggested that the increased H3.3 (likely the chromatin-associated H3.3, see below) contributes to the lethality of dBRWD3 mutants. In other words, dBRWD3 is important for viability through controlling H3.3 levels. Consistently, over-expression of H3.3-GFP reduced viability in the otherwise viable transallelic, hypomorphic dBRWD306656/GS3279, but not in wild-type flies (Supplementary Table S1-3). Together, these findings indicate that dBRWD3, through its negative regulation of H3.3, is essential for gene expression and animal viability. Figure 2. dBRWD3 negatively regulates H3.3 A–C. Suppression of dBRWD3-RNAi induced rough eye phenotype by simultaneous H3.3B depletion. Images of adult eyes: OK107-GAL4-driven UAS-dBRWD3-dsRNA (A), UAS-dBRWD3-dsRNA, UAS-H3.3B-dsRNA (B), and UAS-H3.3B-dsRNA (C). Scale bars indicate 100 μm. D. The expression of H3.3-dendra2 under the control of ubi promoter in 3rd instar dBRWD3s5349 mutant clones generated by ey-flp (dashed lines, negatively marked by GMR promoter-driven RFP). The scale bar indicates 50 μm. E. Western blot analysis of endogenous H3.3 and total H3. Lysates prepared from Canton S and dBRWD3PX2/PX2 3rd instar larvae. The levels of endogenous H3.3 and total H3 were detected by Western blot analysis. Histone H2B was used as a loading control. F, G. Chaoptin (F) and Prospero (G) expression (red) in dBRWD3s5349 mutant clones (arrows) expressing GMR-GAL4-driven UAS-H3.3B-dsRNA. Scale bars indicate 50 μm. Download figure Download PowerPoint dBRWD3 depends on HIRA/YEM to regulate H3.3 Incorporation of H3.3 into chromatin is mediated by either the HIRA/YEM or Daxx/ATRX (Dlp/XNP in fly) complex 1910. We therefore examined the functional relationship between dBRWD3 and the chaperones. Knockdown of Hira (Fig 3A) or yem (Fig 3B), but not XNP (Fig 3C), suppressed the rough eye phenotype caused by dBRWD3 knockdown (Fig 3D). As a control, knockdown of Hira (Fig 3E), yem (Fig 3F) or XNP (Fig 3G) in a wild-type background did not result in any eye phenotype. These genetic analyses suggest that HIRA/YEM rather than Dlp/XNP complex may contribute to the increase of H3.3 levels in dBRWD3 mutant cells. Consistently, in a co-immunoprecipitation experiment, Flag-tagged dBRWD3 pulled down Myc-tagged YEM as well as Myc-tagged HIRA, but not Myc-tagged XNP (Fig 3H). Domain mapping experiments revealed that HIRA directly or indirectly interacts extensively with dBRWD3 1-574a.a., 527-1214a.a., and 1049-1754a.a. fragments (Supplementary Fig S2A). In contrast, only the dBRWD3 1049-1754a.a. fragment directly or indirectly interacts with YEM (Supplementary Fig S2A). Within the dBRWD3 1049-1754a.a. fragment, bromodomain I is important since its deletion significantly reduced the interaction between dBRWD3 1049-1754a.a. and YEM (Supplementary Fig S2B). Figure 3. dBRWD3 regulates H3.3 level in a HIRA/YEM-dependent mechanism A–G. Suppression of dBRWD3-RNAi induced rough eye phenotype by simultaneous Hira or yem depletion. Images of adult eyes: OK107-GAL4-driven UAS-dBRWD3-dsRNA, UAS-Hira-dsRNA (A), UAS-dBRWD3-dsRNA, UAS-yem-dsRNA (B), UAS-dBRWD3-dsRNA, UAS-XNP-dsRNA (C), UAS-dBRWD3-dsRNA (D), UAS-Hira-dsRNA (E), UAS-yem-dsRNA (F), and UAS-XNP-dsRNA (G). Scale bars indicate 100 μm. H. Association of dBRWD3 with YEM, HIRA, and XNP. S2 cells were transfected with plasmids encoding Myc-tagged YEM (upper panel), Myc-tagged HIRA (middle panel), Myc-tagged XNP (lower panel), and Flag-tagged dBRWD3 as indicated. The expression of YEM, HIRA, XNP, and dBRWD3 were detected by Western blot analysis. dBRWD3 complex was immunopurified and analyzed by Western blot using anti-Myc antibody for the associated YEM (upper panel), HIRA (middle panel), and XNP (lower panel). I. Suppression of dBRWD3s5349 by yemGS21861 in the expression of ubi-H3.3-dendra2. Experimental settings similar to those in Figure 2D were applied to dBRWD3s5349, yemGS21861 mutants. Mutant photoreceptors are marked by the absence of RFP. The scale bar indicates 50 μm. J. Western blot analysis of endogenous H3.3 and total H3 in Canton S, dBRWD3PX2/PX2, and dBRWD3PX2/PX2 yemGS21861/GS21861 3rd instar larvae. H2B protein levels were included as a loading control. K. Quantification of endogenous H3.3 and total H3 in Canton S, dBRWD3PX2/PX2, and dBRWD3PX2/PX2 yemGS21861/GS21861 3rd instar larvae. Data shown were means ± SD from three independent experiments. *Indicates P < 0.01 versus Canton S and **indicates P < 0.005 versus dBRWD3PX2/PX2 by Student's t-test. L. A representative Western analysis of YEM-associated H3.3. YEM-Flag and HA-H3.3-RFP were transiently expressed in control knockdown, dBRWD3 knockdown, and MLN4924-treated S2 cells as indicated. The YEM-associated H3.3 was immunoprecipitated by anti-Flag antibody and analyzed by Western blot using anti-HA antibody. M. Quantification of YEM-associated H3.3 in dBRWD3 knockdown (left panel) and MLN4924-treated (right panel) S2 cells. Data shown were means ± SD from four independent experiments. *Indicates P < 0.01 by Student's t-test. Download figure Download PowerPoint The stabilization of H3.3 may be mediated either by its incorporation into chromatin or by blocking the rapid degradation of free H3.3. To identify which of the two processes is regulated by dBRWD3, we examined H3.3-dendra2 levels in dBRWD3, yem double mutant. The H3.3-dendra2 levels were similar to wild-type (Fig 3I, n = 38, penetrance = 100%), distinct from the marked increase of H3.3-dendra2 in dBRWD3 mutant photoreceptors (Fig 2D). Consistently, both the total and chromatin-associated endogenous H3.3 increased only in the dBRWD3 mutant, but not in the dBRWD3, yem double mutant (Fig 3J and K, n = 3, P < 0.01, and Supplementary Fig S1B), whereas non-chromatin-associated H3.3 appeared unchanged in dBRWD3 mutant versus dBRWD3, yem double mutant (Supplementary Fig S1C). As a control, anti-H3.3 signal intensities increased linearly when the loaded chromatin fraction proteins increased (Supplementary Fig S1D). Independently, we also evaluated the ratio of chromatin-associated H3.3 to free H3.3 in salivary glands, where the exogenous, heat shock-inducible H3.3-GFP level in the control was about 70% of endogenous H3.3 (Supplementary Fig S1E) at 8 h after a 30-min heat shock. In a randomized, blind manner, we directly measured the GFP intensities that are colocalized (arrowheads in Supplementary Fig S1F and G) and non-overlapping (arrows in Supplementary Fig S1F and G) with DAPI as indexes for chromatin-associated and free H3.3, respectively (Supplementary Fig S1H). The analysis revealed an increase of chromatin-associated H3.3 relative to free H3.3 upon knockdown of dBRWD3 (Supplementary Fig S1I), suggesting an increase of H3.3 chromatin association resulted from per unit of free protein. This increase was not because of a higher H3.3-GFP mRNA expression in dBRWD3 knockdown salivary glands (Supplementary Fig S1J). Together, these data indicate that HIRA/YEM chaperone activity is required for the increased H3.3 observed in dBRWD3 mutant cells. We therefore concluded that dBRWD3 prevents an abnormal activation of HIRA/YEM, thus negatively regulating the amount of stable, chromatin-associated H3.3. To next characterize the role of dBRWD3 on the expression of histone chaperones, we performed knockdown of dBRWD3 in the S2 cells. As a control, the nuclear dBRWD3 protein level was significantly reduced after knockdown of dBRWD3 (Supplementary Fig S3A). We next examined whether dBRWD3 depletion caused increases of HIRA, YEM, and XNP by using Western blot analyses, which revealed that loss of dBRWD3 did not change the levels of endogenous HIRA and XNP (Supplementary Fig S3B and C). In the case of YEM, although anti-YEM antibody detected overexpressed YEM well (Supplementary Fig S3D and E), it is not sensitive for endogenous YEM by either immunofluorescence or Western blot analyses (Supplementary Fig S3F and E, left panel). We therefore used an ectopic expression system—when dBRWD3 was depleted in S2 cells stably expressing YEM-Myc, YEM-Myc protein levels also did not change (Supplementary Fig S3E, right panel). To further examine HIRA, YEM, and XNP protein levels in a dBRWD3 null condition, we performed immunofluorescence studies in dBRWD3 null mutant clones. Similarly, we found that endogenous HIRA and XNP levels were not altered in dBRWD3 mutant clones (Supplementary Fig S3G and H), neither was endogenous YEM (Supplementary Fig S3I) nor the Flag-YEM (Supplementary Fig S3J) that faithfully recapitulates the expression pattern of endogenous YEM 5. Together, we concluded that dBRWD3 regulates H3.3 levels in a manner independent of degradation of H3.3 chaperones, HIRA, YEM, and XNP. We next investigated whether dBRWD3 regulates the interaction between YEM chaperone and H3.3. There was a 2.3-fold increase of the YEM-associated HA-H3.3-RFP in dBRWD3-depleted S2 cells, compared with control (Fig 3L and M, left panel, n = 4). S2 cells were then treated with MLN4924 to test whether CRL4 ligase activity leads to the same outcome. As a control, MLN4924 treatment led to an increase in cullin substrate Armadillo (Supplementary Fig S3K). A similar 2.6-fold increase of HA-H3.3-RFP associated with Flag-YEM was observed (Fig 3L and M, right panel, n = 4), suggesting that dBRWD3-containing CRL4 E3 ligase negatively regulates the binding of Flag-YEM to HA-H3.3-RFP. The increased H3.3 binding of YEM upon the loss of dBRWD3 is not due to an artifact of H3.3 overexpression as a similar increase was observed (Supplementary Fig S3M) in S2 cells stably expressing HA-H3.3-RFP at a much lower level than endogenous H3.3 (Supplementary Fig S3L, arrow and arrowhead). Moreover, we also observed a 2-fold increase of endogenous H4 associating with YEM in dBRWD3-depleted S2 cells (Supplementary Fig S3N). Together, our data suggest that dBRWD3 interacts with HIRA/YEM either directly or indirectly and ubiquitinates an unidentified substrate to reduce the amount of H3.3 associated with YEM. This represents a new CRL4-mediated regulation of histone distinct from two previously reported mechanisms: ubiquitinations of H3 and H4 upon UV-induced DNA damage 28 and an ubiquitination of newly synthesized histone H3 at lysine 122 that moves H3.1 and H3.3 from ASF1 to CAF-1 and HIRA, respectively 29. Inactivation of YEM suppresses lethality and aberrant gene expression caused by dBRWD3 mutation To investigate how much the aberrant HIRA/YEM activity contributes to dBRWD3 mutant phenotypes, we first examined how well dBRWD3, yem double-mutant photoreceptors differentiated. The expression of neuronal differentiation markers Chaoptin (Fig 4A, dashed lines) and Prospero (Fig 4B, dashed lines) was restored to the wild-type level in the dBRWD3, yem double-mutant photoreceptors (n = 31 and n = 22 respectively, penetrance of restoration = 100%). In the adult stage, the number of surviving dBRWD3 mutant ommatidia, as marked in red (white+), is much smaller than wild-type twin spots marked in white (white−) (Fig 4C, arrow). The number of dBRWD3, yem double-mutant ommatidia (red, Fig 4D, arrow) is similar to wild-type twin clones (white, Fig 4D), indicating that dBRWD3, yem double-mutant ommatidia are viable. Unlike dBRWD3, yem double mutant, the XNP, dBRWD3 double-mutant clones (carrying a deletion allele of XNP 30, Fig 4E in red, arrow) are small, again suggesting dBRWD3 specifically regulates HIRA/YEM, but not Dlp/XNP. Like H3.3B, dBRWD3 double mutant, the dBRWD3, yem double mutant could survive into the adult stage, whereas dBRWD3 homozygous, yem heterozygous mutant could not (Supplementary Table S1-2), suggest