Author response: Modular UBE2H-CTLH E2-E3 complexes regulate erythroid maturation (2022)

Article Figures and data Abstract Editor's evaluation Introduction Results Discussion Materials and methods Data availability References Decision letter Author response Article and author information Metrics Abstract The development of haematopoietic stem cells into mature erythrocytes – erythropoiesis – is a controlled process characterized by cellular reorganization and drastic reshaping of the proteome landscape. Failure of ordered erythropoiesis is associated with anaemias and haematological malignancies. Although the ubiquitin system is a known crucial post-translational regulator in erythropoiesis, how the erythrocyte is reshaped by the ubiquitin system is poorly understood. By measuring the proteomic landscape of in vitro human erythropoiesis models, we found dynamic differential expression of subunits of the CTLH E3 ubiquitin ligase complex that formed maturation stage-dependent assemblies of topologically homologous RANBP9- and RANBP10-CTLH complexes. Moreover, protein abundance of CTLH’s cognate E2 ubiquitin conjugating enzyme UBE2H increased during terminal differentiation, and UBE2H expression depended on catalytically active CTLH E3 complexes. CRISPR-Cas9-mediated inactivation of CTLH E3 assemblies or UBE2H in erythroid progenitors revealed defects, including spontaneous and accelerated erythroid maturation as well as inefficient enucleation. Thus, we propose that dynamic maturation stage-specific changes of UBE2H-CTLH E2-E3 modules control the orderly progression of human erythropoiesis. Editor's evaluation This paper will be of interest to scientists in the field of hematology and ubiquitin biology. The work identifies previously unrecognized functions of and regulatory mechanisms impinging on CTLH E3 ubiquitin ligases during erythrocyte progenitor maintenance and differentiation. It provides new insights into the dynamic formation of E3 ubiquitin ligases during development, suggesting that rather than simply exchanging substrate adaptors, scaffolding proteins and collaborating E2 enzymes are also tightly regulated. The experiments are of high quality and a wealth of data supports the conclusions. https://doi.org/10.7554/eLife.77937.sa0 Decision letter Reviews on Sciety eLife's review process Introduction Cellular differentiation in multicellular organisms is often accompanied by programmed proteome reshaping and cellular reorganization to accomplish cell-type-specific functions. For instance, during myogenesis proliferative myoblasts undergo a differentiation programme with induction of specialized cytoskeletal proteins to form myofibrils in terminally differentiated myofibers (Chal and Pourquie, 2017; Le Bihan et al., 2015), whereas adipose stem cells induce differentiation cues controlling expression of proteins involved in lipid storage and lipid synthesis (Tsuji et al., 2014). Recently, global temporal proteomic analysis during neurogenesis of human embryonic stem cells revealed large-scale proteome and organelle remodelling via selective autophagy (Ordureau et al., 2021). A striking example of proteome remodelling is mammalian erythropoiesis, which is required for the generation of disc-shaped enucleated erythrocytes, whose unique topology dictates function of efficient red blood cell movement through the vasculature (Figure 1A). After several specialized cell divisions, erythroid progenitors progress through morphologically distinct differentiation stages known as pro-erythroblasts (ProE), early and late basophilic erythroblasts (EBaso and LBaso, respectively), polychromatic erythroblasts (Poly), and orthochromatic erythroblasts (Ortho), a process associated with erythroid-specific gene expression (Cantor and Orkin, 2002; Cross and Enver, 1997; Perkins et al., 1995; Pevny et al., 1991; Shivdasani et al., 1995), reduction of cell volume (Dolznig et al., 1995), chromatin condensation (Zhao et al., 2016), and haemoglobinization. Ejection of the nucleus at the reticulocyte stage (Keerthivasan et al., 2011) is followed by the elimination of all remaining organelles such as Golgi, mitochondria, endoplasmic reticulum, peroxisomes, and ribosomes (Moras et al., 2017; Nguyen et al., 2017). The progression of erythroid maturation must be tightly controlled, although the molecular regulation of this process is not fully understood. Figure 1 with 1 supplement see all Download asset Open asset Stage-dependent expression of UBE2H and CTLH complex subunits during erythropoiesis. (A) Cartoon indicating key features of mammalian erythropoiesis. (B) Heat map of z-scored protein abundance (log2 data-independent acquisition (DIA) intensity) of differentially expressed E2 enzymes in differentiated HUDEP2 cells. (C) Heat map of z-scored protein abundance (log2 DIA intensity) of differentially expressed E2 enzymes in differentiated CD34+ cells. (D) HUDEP2 cells were differentiated in vitro and analysed by immunoblotting with indicated antibodies. (E) CD34+ cells were differentiated in vitro, cell populations enriched by FACS, and analysed by immunoblotting with indicated antibodies. (F) Heat map of z-scored protein abundance (log2 DIA intensity) of differentially expressed CTLH complex subunits in differentiated HUDEP2 cells. (G) Heat map of z-scored protein abundance (log2 DIA intensity) of differentially expressed CTLH complex subunits in differentiated CD34+ cells. Figure 1—source data 1 Original, uncropped scans of immunoblots. https://cdn.elifesciences.org/articles/77937/elife-77937-fig1-data1-v1.pdf Download elife-77937-fig1-data1-v1.pdf Our current knowledge of protein dynamics during erythropoiesis has been deduced largely from epigenetic and transcriptomic studies (reviewed in An et al., 2015), which used in vitro differentiation systems where erythroid progenitors, such as primary multipotent CD34+ haematopoietic stem and progenitor cells (HSPC) or immortalized CD34+-derived lines (known as HUDEP2 and BEL-A) (Kurita et al., 2013; Trakarnsanga et al., 2017), possess an autonomous differentiation programme with a capacity to complete terminal differentiation when cultured with cytokines and other factors (Seo et al., 2019). However, the dynamics of mRNA expression during erythropoiesis does not accurately predict protein expression (Gautier et al., 2016). The erythroid proteome landscape of defined precursors generated by in vitro differentiation of normal donor CD34+ cells was recently mapped (Karayel et al., 2020; Peng et al., 2022; Gautier et al., 2016), and the findings provided insight into cellular remodelling at protein resolution and indicated high level of post-transcriptional regulation. Ubiquitin (UB)-mediated protein degradation pathways are likely to play prominent roles in post-transcriptional regulation of erythropoiesis. Ubiquitin conjugating enzymes (E2), components of the E3 ubiquitin ligase machinery, and deubiquitylases have been implicated in regulating protein stability and turnover in erythroid cell proliferation and maturation (Feng et al., 2022; Liang et al., 2019; Maetens et al., 2007; Mancias et al., 2015; Minella et al., 2008; Nguyen et al., 2017; Randle et al., 2015; Thom et al., 2014; Xu et al., 2020). The E2 enzyme UBE2O is greatly upregulated in reticulocytes and required for clearing ribosomes (Nguyen et al., 2017). Recently, a functional role of the multiprotein C-terminal to LisH (CTLH) E3 ubiquitin ligase was implicated in mammalian erythropoiesis. The CTLH subunits MAEA and WDR26 are expressed in a differentiation stage-dependent manner and implicated in maintaining erythroblastic islands in the bone marrow and regulating nuclear condensation in developing erythroblasts, respectively (Wei et al., 2019; Zhen et al., 2020). The tight correlation between protein abundance and functionality in differentiation suggested that large-scale proteome profiling is a potential way to identify proteins that are important for the functional specialization of erythroid cells. Here, we profiled protein abundance of E2-E3 modules in erythroid differentiation uncovering a dynamic regulation of CTLH E3 ligase subunits and its cognate E2 conjugating enzyme UBE2H. Interestingly, UBE2H amounts are dependent on active CTLH E3, suggesting a coupled E2/E3 regulation. We further show that CTLH complex composition is remodelled and complex assemblies are formed in a maturation stage-dependent manner. Our study indicates that unique UBE2H-CTLH assemblies are organized and co-regulated in functional E2-E3 modules and are required for the orderly progression of terminal erythroid maturation. Results Stage-dependent expression of UBE2H and CTLH complex subunits during erythropoiesis Reshaping of the erythropoietic proteome is thought to be regulated in part by the transient presence of stage-specific E2-E3 ubiquitin targeting machineries. To identify potential E2-E3 components, we applied a system-wide approach and established differentiation stage-specific proteomes of human erythropoiesis from two in vitro erythropoiesis cell model systems: CD34+ and HUDEP2 cells (Figure 1—figure supplement 1A and B). We recently described the stage-specific proteomes from in vitro differentiated CD34+ cells (Karayel et al., 2020). HUDEP2 cells proliferate in immature progenitor state and can be induced to undergo terminal erythroid differentiation by modulating cell culture conditions (Figure 1—figure supplement 1B; Kurita et al., 2013). In this study, HUDEP2 cells were shifted to differentiation conditions and semi-synchronous bulk cell populations were obtained at different time points (days 0, 3, 6, 9, and 12) corresponding to maturation stages spanning from proerythroblast to orthochromatic stages. Each population was processed in three biological replicates, and their proteomes were acquired by measuring single 100 min gradient runs for each sample/replicate in data-independent acquisition (DIA) mode (Aebersold and Mann, 2016; Gillet et al., 2012; Karayel et al., 2020; Ludwig et al., 2018). DIA raw files were searched with direct DIA (dDIA), yielding 6727 unique proteins and quantitative reproducibility with Pearson correlation coefficients greater than 0.9 between the biological replicates of all populations (Figure 1—figure supplement 1C and D). When we clustered the 2771 differentially expressed proteins (ANOVA, false discovery rate [FDR] < 0.01 and S0 = 0.1), we observed dynamic changes of the proteome between early (day 0) and late (day 12) time points across erythroid differentiation (Figure 1—figure supplement 1E). The majority of proteins cluster into two co-expression profiles: continuous decrease or increase of protein levels that ultimately resulted in a reshaped erythrocyte-specific proteome. We next examined our proteome data for all (~40) annotated human E2 ubiquitin conjugating enzymes. The levels of most E2s detected in HUDEP2 cells varied across maturation, with a cluster of six enzymes progressively accumulating until day 12 (Figure 1B). Included among them was UBE2O, which mediates ribosomal clearance in reticulocytes (Nguyen et al., 2017). We expanded the analysis to stage-specific proteomes from in vitro differentiated CD34+ cells (Figure 1C; Karayel et al., 2020), which revealed a similar cluster of E2s upregulated at poly- and orthochromatic stages. Notably, UBE2B, UBE2O, CDC34 (aka UBE2R1), and UBE2H enzymes exhibited similar protein abundance profiles during HUDEP2 and CD34+ maturation, suggesting roles for these E2s during terminal erythropoiesis. CDC34, the cognate E2 for cullin-1 RING ligase (CRL1) complexes, is essential for cell cycle regulation (Kleiger et al., 2009; Skaar and Pagano, 2009). UBE2B (aka RAD6B) regulates DNA repair pathways, histone modifications, and proteasomal degradation (Kim et al., 2009; Varshavsky, 1996; Watanabe et al., 2004). We focused on UBE2H because it is transcriptionally regulated by the essential erythroid nuclear protein TAL1 and accumulates to high levels during terminal maturation (Lausen et al., 2010; Nguyen et al., 2017; Wefes et al., 1995). Immunoblotting confirmed UBE2H protein upregulation during maturation of HUDEP2 and CD34+ cells, which was paralleling induction of the erythroid membrane protein CD235a (glycophorin A [GYPA]) and haemoglobin expression (Figure 1D and E, Figure 1—source data 1). The stage-dependent regulation of UBE2H suggested that a cognate E3 partnering with UBE2H would have a similar expression profile during erythropoiesis. In vitro ubiquitylation reactions indicate that UBE2H is the preferred E2 of the CTLH E3 ubiquitin ligase (Lampert et al., 2018; Sherpa et al., 2021). The multiprotein CTLH complex, orthologue of the yeast GID complex, consists of at least RANBP9, and/or RANBP10 (yeast Gid1), TWA1 (yeast Gid8), ARMC8 (yeast Gid5), WDR26, and/or MKLN1 (yeast Gid7), the catalytic module – MAEA and RMND5A (yeast Gid9 and Gid2) that mediate ubiquitin transfer, and the substrate receptor GID4 (yeast Gid4) (Kobayashi et al., 2007; Lampert et al., 2018; Liu and Pfirrmann, 2019; Maitland et al., 2022; Mohamed et al., 2021; Salemi et al., 2017; Sherpa et al., 2021; Umeda et al., 2003). Our analyses of the stage-dependent proteomes of differentiated HUDEP2 (Figure 1F) and CD34+ cells (Figure 1G) revealed that protein levels of most annotated CTLH subunits increased during erythroid maturation, in parallel with UBE2H. Interestingly, the homologues RANBP9 and RANBP10 showed an inverse expression pattern: RANBP9 levels were high at progenitor stages and dropped at later stages, whereas RANBP10 levels exhibited the opposite pattern. Taken together, analyses of differentiation-resolved proteomes revealed stage-dependent expression of CTLH subunits and UBE2H suggesting a dynamic assembly of distinct CTLH complexes linked to erythrocyte development. Erythroid maturation stage-dependent modulation of RANBP9- and RANBP10-assembled CTLH complexes Recent cryo-EM maps of human CTLH sub- and supramolecular complexes revealed that RANBP9 is part of a core scaffold module of the CTLH complex (Figure 2A; Sherpa et al., 2021). Beyond an N-terminal extension unique to RANBP9, both homologues RANBP9 and RANBP10 have a common domain architecture (Figure 2—figure supplement 1). Hence, we reasoned that RANBP10 may replace RANBP9 and also form topologically similar complexes and, depending on abundance and availability of RANBP9 and RANBP10, distinct RANBP9-, ‘mixed’ RANBP9/RANBP10-, and/or RANBP10-CTLH complexes may assemble (Figure 2B). To test this hypothesis, we monitored CTLH complexes by fractionating whole-cell lysates from non-differentiated (day 0) or differentiated (day 6) HUDEP2 cells on 5–40% sucrose density gradients and detecting CTLH subunits by immunoblot analysis. All CTLH subunits sedimented at ≥670 kDa, corresponding to the shift observed for the supramolecular CTLH assemblies we previously described (Figure 2C, Figure 2—source data 1; Sherpa et al., 2021). However, RANBP9 amounts in the CTLH fraction were higher at day 0 compared to day 6, while RANBP10 amounts had the opposite pattern, suggesting stage-specific modulation of CTLH complex composition and/or stoichiometry during differentiation. To further test stage-specific modulation of CTLH, we established a sequential immunoprecipitation (IP) protocol allowing us to determine relative proportions of RANBP9-, RANBP9/RANBP10-, and RANBP10-CTLH complexes in cell lysates (Figure 2D). In step 1, RANBP9-assembled complexes were immunoprecipitated from lysates of differentiation days 0, 4, and 8 using RANBP9-specific antibody. Subsequently, RANBP9-depleted supernatants were subjected to step 2 for immunoprecipitation with an ARMC8-specific nanobody (Figure 2—figure supplement 2) to precipitate remaining RANBP10-CTLH complexes. Immunoblot analysis of pellet 1 samples revealed a progressive decrease of precipitated RANBP9-CTLH in differentiating HUDEP2 cells (Figure 2E, Figure 2—source data 2). Notably, RANBP10 was co-precipitated and relative RANBP10 amounts increased towards differentiation day 8, indicating a shift from RANBP9- to RANBP9/RANBP10-assembled CTLH complexes. Moreover, the amount of precipitated RANBP10-CTLH complexes in pellet 2 samples increased, indicating the predominant assembly of RANBP10-CTLH complexes at late stages of differentiation (Figure 2B and E). To further test whether RANBP9 and RANBP10 can independently form CTLH complexes, we deleted either RANBP9 or RANBP10 in HUDEP2 cells using CRISPR-Cas9 editing (Figure 2F, Figure 2—source data 2, Figure 2—figure supplement 3). Whole-cell lysates of parental and KO lines were analysed using sucrose density gradients and immunoblot analysis to assess the sedimentation of RANBP9 and RANBP10 along with other CTLH subunits WDR26 and MAEA (Figure 2G, Figure 2—source data 3). The sedimentation of the supramolecular CTLH complex containing RANBP9 was similar in parental and RANBP10-/- cells. Likewise, RANBP10-CTLH assemblies sedimented similarly in parental and RANBP9-/- cells. These data indicate a dynamic modulation of RANBP9- to RANBP10-assembled supramolecular CTLH complexes during the process of erythroid maturation. Figure 2 with 3 supplements see all Download asset Open asset Erythroid maturation stage-dependent modulation of RANBP9- and RANBP10-assembled CTLH complexes. (A) Cartoon of the supramolecular RANBP9-CTLH assembly indicating the catalytic (blue), core (grey/yellow), and supramolecular assembly (orange) modules. (B) Model of remodelling RANBP9- and RANBP10-CTLH complexes. (C) HUDEP2 cell lysates from differentiation days 0 and 6 were separated on sucrose gradients, and fractions analysed by immunoblotting with indicated antibodies. Fractions containing supramolecular CTLH assemblies are boxed in red. (D) Workflow of the sequential immunoprecipitation (IP) to determine RANBP9- and RANBP10-CTLH complexes. (E) HUDEP2 cell lysates from differentiation days 0, 4, and 8 were subjected to sequential IPs with RANBP9 antibody and ARMC8-specific nanobody as described in (D) followed by immunoblot analysis with indicated antibodies. IgG: unspecific antibody; mock: absence of nanobody. (F) Immunoblots of lysates of HUDEP2 parental, RANBP9-/-, and RANBP10-/- cells probing for RANBP9 and RANBP10. Actin serves as loading control. (G) Sucrose gradient fractionation of HUDEP2 cell lysates from RANBP9-/- or RANBP10-/- knock out lines, fractions were analysed by immunoblotting with indicated antibodies. Fractions containing supramolecular CTLH assemblies are boxed in red. Figure 2—source data 1 Original, uncropped scans of immunoblots. https://cdn.elifesciences.org/articles/77937/elife-77937-fig2-data1-v1.pdf Download elife-77937-fig2-data1-v1.pdf Figure 2—source data 2 Original, uncropped scans of immunoblots. https://cdn.elifesciences.org/articles/77937/elife-77937-fig2-data2-v1.pdf Download elife-77937-fig2-data2-v1.pdf Figure 2—source data 3 Original, uncropped scans of immunoblots. https://cdn.elifesciences.org/articles/77937/elife-77937-fig2-data3-v1.pdf Download elife-77937-fig2-data3-v1.pdf Figure 3 with 1 supplement see all Download asset Open asset RANBP9 and RANBP10 form similar CTLH complex structures that cooperate with UBE2H to promote ubiquitin transfer. (A) Chromatograms (top) and Coomassie-stained SDS-PAGE gels (bottom) from size-exclusion chromatography of recombinant RANBP10-CTLHSR4 and RANBP9-CTLHSR4 complexes. (B) Cryo-EM map of RANBP10-CTLHSR4 (EMDB: EMD-16242, left) and RANBP9-CTLHSR4 (EMDB: EMD-12537) (right) with Cat-module and SRS-module indicated. (C) Focused refined map of the RANBP10-CTLH SRS-module with coloured subunits: ARMC8, purple; TWA1, salmon;GID4(Δ1–99), red; RANBP10 SPRY-domain, green. (D) Immunoprecipitation (IP) from HUDEP2 cell lysates with IgG control and UBE2H-specific antibody and immunoblot analysis. (E) IP from K562 cell lysates with IgG control and UBE2H-specific antibody and immunoblot analysis. IgG light chain (IgG-LC), IgG heavy chain (IgG-HC). (F) Fluorescence scan of SDS-PAGE gels presenting time course of in vitro ubiquitylation assay with fluorescently labelled model substrate peptide PGLW(X)n-23K with lysine at position 23 (pep*) in the presence of UBE2H, RANBP10-CTLH or RANBP9-CTLH, and GID4. Figure 3—source data 1 Original, uncropped scans of Coomassie-stained SDS-PAGEs and immunoblots. https://cdn.elifesciences.org/articles/77937/elife-77937-fig3-data1-v1.pdf Download elife-77937-fig3-data1-v1.pdf RANBP9 and RANBP10 form similar CTLH complex structures that cooperate with UBE2H to promote ubiquitin transfer As RANBP9 and RANBP10 can independently assemble in supramolecular CTLH complexes in cells, we next asked whether RANBP10 forms a similar molecular CTLH structure as described previously for RANBP9 (Sherpa et al., 2021). To test this, we expressed and purified a recombinant version of the core CTLH subcomplex, containing a scaffold module (RANBP10, TWA1, α-ARMC8), the catalytic module (MAEA and RMND5A), and the substrate receptor GID4 (named hereafter RANBP10-CTLHSR4). In parallel, we generated the previously described homologous complex where RANBP9 replaced RANBP10 (RANBP9-CTLHSR4) (Mohamed et al., 2021; Sherpa et al., 2021). The two complexes eluted at similar range in size-exclusion chromatography (SEC), indicating they had comparable subunit stoichiometry (Figure 3A, Figure 3—source data 1). Cryo-EM analysis of the RANBP10-CTLHSR4 peak fraction yielded a reconstitution at ~12 Å resolution (EMDB: EMD-16242) (Figure 3B). Comparison to the previously determined RANBP9-CTLHSR4 map (EMDB: EMD-12537) revealed overall structural similarity, including the clamp-like assembly of substrate receptor scaffolding (SRS) and catalytic (Cat) modules conserved in related yeast GID complexes, albeit with differences in the extent of the catalytic modules visible in the maps (Qiao et al., 2020; Sherpa et al., 2021; Figure 3B, Figure 3—figure supplement 1). Importantly, atomic coordinates of α-ARMC8,GID4, and TWA1 derived from the RANBP9-CTLHSR4 structure (PDB: 7NSC), along with crystal structure of RANBP10-SPRY domain (PDB: 5JIA), fit into the 7.6 Å resolution-focused refined map of RANBP10-CTLHSR4 (Figure 3C). To position RANBP10, the crystal structure of the RANBP10’s SPRY domain (PDB: 5JIA; Hong et al., 2016) was superimposed to the structure of the RANBP9’s SPRY domain from recently published RANBP9-CTLHSR4 (PDB: 7NSC; Sherpa et al., 2021; Figure 2—figure supplement 1B). Thus, at an overall level, the core RANBP10-CTLHSR4 and RANBP9-CTLHSR4 complexes are structurally homologous. We next asked whether the structural similarity of the core RANBP10-CTLHSR4 and RANBP9-CTLHSR4 complexes extended to the mechanism of ubiquitin transfer activity. First, we assessed the physical association between UBE2H and CTLH complex subunits in HUDEP2 or erythroleukemia K562 cells using anti-UBE2H IPs (Figure 3D and E, Figure 3—source data 1; Andersson et al., 1979). Endogenous UBE2H specifically co-precipitated the Cat-module subunit MAEA in whole-cell lysates from both cell lines, indicating that UBE2H can form a reasonably stable E2-E3 enzyme module. Next, we tested ubiquitin transfer activity by in vitro ubiquitylation assays with a fluorescently labelled model peptide substrate (Sherpa et al., 2021). This model substrate consisted of an N-terminal PGLW that human GID4 et al., 2020; et al., and a with the lysine towards the at position 23 (Figure Figure 3—source data 1). In a with UBE2H, both RANBP9-CTLH and RANBP10-CTLH of the model substrate peptide in a although RANBP10-CTLH was active these conditions than the homologous RANBP9-CTLH and structural data revealed that RANBP9 and RANBP10 can assemble in distinct homologous CTLH complexes of ubiquitin transfer activity. CTLH may a of E3 ligase complexes generated by assembly of different with core scaffold CTLH E3 complexes and UBE2H erythroid maturation To potential functional roles of CTLH E3 and UBE2H in erythroid maturation, we used K562 cells as a erythroid cell model that erythroid including and haemoglobin with the histone et al., 1979). We generated K562 and cells by CRISPR-Cas9 editing (Figure supplement 1; Sherpa et al., 2021). of part of the catalytic RING subunit in all CTLH E3 would in a complete of all CTLH E3 ligase and MAEA or UBE2H cell lines were either or and erythroid maturation was assessed by maturation expression via cell conditions and lines showed increased cells comparable to parental cells (Figure supplement immunoblot analysis showed increased expression in whole-cell lysates of and lines at of suggesting that MAEA and UBE2H promote erythroid differentiation (Figure supplement 2B and Figure supplement 2—source data 1). To further the we expanded the analysis to and HUDEP2 cell lines (Figure Figure supplement 1A and B). analysis revealed an of cells in MAEA and or UBE2H and indicating spontaneous erythroid maturation in (Figure Figure supplement 2D). As terminal erythropoiesis is characterized by a stage-dependent proteome remodelling (Gautier et al., 2016; Karayel et al., 2020), we next asked whether spontaneous erythroid maturation of and cells is in with global proteomic analysis of parental, and HUDEP2 cells unique proteins in (Figure 1—figure supplement 1C and with < and S0 = We found that and of all proteins were in parental and parental respectively with < and S0 = (Figure supplement and Notably, of these proteins were differentially in a and manner. These several erythroid-specific proteins including haemoglobin subunits and and erythroid maturation in both indicating an remodelled proteome (Figure In proteins associated with related to erythropoiesis such as and were enriched in both and cells compared to parental cells (Figure Figure with supplements see all Download asset Open asset CTLH E3 complexes and UBE2H erythroid maturation. (A) Immunoblots of lysates of HUDEP2 parental, and probing for MAEA and UBE2H. Actin serves as loading control. (B) of of indicated HUDEP2 cell lines fraction of cells in of = biological (C) between proteins with abundance differences of parental and parental with abundance differences of parental proteins with abundance differences parental enriched proteins are (D) analyses of upregulated protein in parental and parental using test (E) of of indicated HUDEP2 cell lines fraction of cells at differentiation day of = biological (F) HUDEP2 cell lines, cultured in were for followed by induction of erythroid maturation. of indicated HUDEP2 cell lines expression at day induced erythroid maturation. (G) of with fraction of cells. of = 2 biological analysis of erythroid differentiation stages (day 0, day 3, purple; day 6, day 9, day of HUDEP2 parental and cell lines with their biological replicates on expression profiles of erythroid Immunoblot analysis of CRISPR-Cas9-mediated targeting of MAEA in CD34+ cells. serves as loading control. of of cell lines fraction of enucleated cells. of = 2 biological Immunoblot analysis of CRISPR-Cas9-mediated targeting of UBE2H in CD34+ cells. serves as loading