Modular UBE2H-CTLH E2-E3 complexes regulate erythroid maturation

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    This work, which will be of interest to scientists in the field of hematology and ubiquitin biology, identifies previously unrecognized functions and regulatory mechanisms of an E3 ubiquitin ligase during erythrocyte progenitor maintenance and differentiation. This work has the potential to reveal that the exchange of scaffold proteins of a modular E3 ligase can have an effect on cell fate and reveal a novel mechanism of E2 enzyme regulation during differentiation. However, additional work is needed to support the major claims.

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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.

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  1. eLife assessment

    This work, which will be of interest to scientists in the field of hematology and ubiquitin biology, identifies previously unrecognized functions and regulatory mechanisms of an E3 ubiquitin ligase during erythrocyte progenitor maintenance and differentiation. This work has the potential to reveal that the exchange of scaffold proteins of a modular E3 ligase can have an effect on cell fate and reveal a novel mechanism of E2 enzyme regulation during differentiation. However, additional work is needed to support the major claims.

  2. Reviewer #1 (Public Review):

    Modular E3 ligase complexes play important roles in controlling cell proliferation and differentiation. As had been illustrated by Cullin-RING-ligase complexes that are associated at specific stages of neuronal differentiation, regulated formation of E3 ligase complexes can strongly impact cell fate specification, but only very few examples of such regulation have been reported. Whether E3 ligase composition impacts the global proteome is not known. Providing additional examples of E3 ligase complexes, whose composition is regulated during differentiation processes, would be an important contribution to our understanding of how the ubiquitin system controls cell fate.

    In this manuscript, Sherpa and coworkers used quantitative proteomics in in vitro models of erythrocyte differentiation to discover changes in the composition of the CTLH E3 ligase. Rather than finding altered association of substrate adaptors, including GID4, the authors noted the exchange of the scaffold subunits RanBP9 and RanBP10. Structural analyses suggested that this exchange does not have major impacts on CTHL conformation, but may lead to a reduction in E3 ligase activity towards a model substrate. The authors also deleted the enzymatic CTLH subunit MAEA and the E2 UBE2H from cell lines that served as in vitro models of erythrocyte differentiation. They found that loss of MAEA caused a strong decrease in UBE2H, which interestingly required the catalytic activity of CTLH. This observation suggested that CTLH complex composition is actively regulated. The loss of CTLH activity led to an increase protein abundance of hemoglobin subunits and accelerated erythrocyte differentiation, suggesting that CTLH might restrict cell fate specification until proper signals have been received by precursor cells.

    While the observation of altered CTLH composition during differentiation is interesting, this study does not establish whether it is functionally important. The authors should assess whether deletion of RanBP9 or RanBP10 has functional consequences onto erythrocyte differentiation, which would indicate that the observations made here are significant in the context of this differentiation program. Furthermore, how MAEA loss causes a depletion in UBE2H has not been addressed beyond a simple rescue experiment using a single MAEA mutant, and the specificity and importance of this regulatory circuit therefore remains somewhat unclear. I do believe that especially the first issue needs to be addressed by the authors in order to establish the importance of the findings reported in this manuscript.

  3. Reviewer #2 (Public Review):

    In this study, the authors investigate the ubiquitin-mediated mechanisms underlying erythroid maturation. They first investigated proteome changes of CD34+ cells and HUDEP2 cells (an immortalized CD34+-derived line) which can be induced to undergo differentiation into different erythroblast stages. They identified that protein members of the E3 ubiquitin ligase complex called CTLH complex were globally increased during differentiation. They also found that the expression of several E2 enzymes including UBE2H, which partners with the CTLH complex, increase in later stages of erythroid maturation. Interestingly, they found that the 2 subunits of the CTLH complex, RanBP9 and RanBP10 which are structurally very similar, display opposite changes of expression, with RanBP9 decreasing and RanBP10 increasing during differentiation. They then show that both RanBP9 and RanBP10 can support complex formation in vitro and result in ubiquitin transfer competent complexes using ubiquitination with a model substrate peptide in vitro.

    In the second part of the study, they created CRISPR-Cas9 knock out of UBE2H and the CTLH complex subunit MAEA in HUDEP2 cells to investigate the effect on proteome changes and erythroid cell differentiation. They found that both UBE2H and MAEA knockout cells display pronounced proteome-wide changes in erythroid-specific factors. They also show that the knockout of UBE2H and MAEA cause aberrant differentiation, with accelerated maturation, altogether suggesting that these 2 factors are required to maintain cells in progenitor state. Finally, they identify that MAEA expression is required to maintain UBE2H expression and that this regulation occurs at the post-translational level.

    The authors clearly demonstrate that the CTLH complex and its associated E2 enzyme play important roles in erythroid differentiation. They also generated a wealth of data that document erythroid differentiation and point out very interesting co-regulatory mechanisms regarding ubiquitin machineries underlying this process. Notably, the authors identify an intriguing regulation of two CTLH complex members, RanBP9 and RanBP10 during erythroid maturation that correlates with, and suggests that the replacement of RanBP9 and RanBP10 during the process may be involved in regulating pathways that lead to erythroid maturation.

    Unfortunately, while the above-mentioned regulation of the two CTLH complex members, RanBP9 and RanBP10 is suggested to play a role in erythroid maturation, it is not investigated further. It is genuinely surprising that the authors did not investigate the proteome of the RanBP9 and RanBP10 knockout HUDEP2 cells they generated, to figure out the effect the differential expression of these factors on erythrocyte development.

    Instead, the study changes direction to focus on another CTLH complex subunit, MAEA, and how that subunit may function to regulate the expression of UBE2H, the E2 enzyme associated with the CTLH complex, in a manner seemingly independent of the other complex members. Overall, the work is interesting and advance our knowledge of the erythroid differentiation process, but there are some main issues including over-interpretation of data and experimental issues limiting data interpretation that would need to be addressed or the authors would need to revise their conclusions since as it stands now, some of the conclusions are not supported by the data.

  4. Reviewer #3 (Public Review):

    Sherpa, Müller et al. utilize temporal global proteome analysis of human erythropoiesis models to identify dynamic differential expression of RANBP9 and RANBP10, two homologous subunits of the multi-subunit ubiquitin E3 ligase CTLH. Through elegant biochemical and structural approaches, the authors provide compelling evidence that RANBP9 and RANBP10 form distinct, but structurally similar, catalytically competent CTLH E3 ligase complexes, that are differentially enriched in different stages of erythrocyte differentiation. Using CRISPR/Cas mediated knock outs, the authors inactivate the catalytic subunit of the CTLH E3 ligase, MAEA, or its cognate E2 enzyme UBE2H and show that this leads to spontaneous differentiation in erythrocyte progenitors under maintenance conditions and provide evidence that loss of these two proteins also accelerates differentiation. Interestingly, in these experiments the authors find that loss of MAEA leads to proteasomal degradation of UBE2H, which can be rescued by wildtype, but not catalytically inactive MAEA, demonstrating that UBE2H stability is coupled to cognate E3 ligase activity.

    This study confirms previously known transcriptional regulation and functions of UBE2H and CTHL E3 ligase components during erythrocyte differentiation and identifies a previously unrecognized role for CTHL E3s during erythrocyte progenitor maintenance. In addition, the authors identify two new regulatory mechanisms impinging on the UBE2H-CTLH E3 that might be important for erythrocyte differentiation: differentiation stage-specific assembly of RANBP9-CTHL and RANBP10-CTHL complexes and coupling of UBE2H stability to catalytic activity of the CTLH E3 ligase.

    While the newly identified regulatory mechanisms are interesting, the major weakness of the study is that there is no evidence that these regulatory processes are functionally relevant for erythrocyte differentiation. In addition, the described phenotypes of UBE2H and MAEA deletion on erythrocyte differentiation could be analyzed in more detail, in particular addressing whether the accelerated differentiation reported is yielding functional progeny. Also the study could be strengthened by more quantitative assessment of the differentiation stage-dependent RANBP9-CTLH and RANBP9-CTLH E3 ligase complexes.