MAF1, a repressor of RNA polymerase III-dependent transcription, regulates bone mass
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Evaluation Summary:
Johnson et al. have used several complementary in vivo and in vitro approaches to analyze the effects of regulated MAF1 expression or inhibition of RNA pol III transcription on osteogenesis and adipocyte differentiation. The data are well controlled and of excellent quality, providing novel insights into Maf1 and RNA polymerase-mediated transcriptions in skeleton biology.
(This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. The reviewers remained anonymous to the authors.)
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Abstract
MAF1, a key repressor of RNA polymerase (pol) III-mediated transcription, has been shown to promote mesoderm formation in vitro. Here, we show that MAF1 plays a critical role in regulating osteoblast differentiation and bone mass. Global deletion of MAF1 ( Maf1 -/- mice) produced a high bone mass phenotype. However, osteoblasts isolated from Maf1 -/- mice showed reduced osteoblastogenesis ex vivo. Therefore, we determined the phenotype of mice overexpressing MAF1 in cells from the mesenchymal lineage ( Prx1 -Cre;LSL- MAF1 mice). These mice showed increased bone mass. Ex vivo, cells from these mice showed enhanced osteoblastogenesis concordant with their high bone mass phenotype. Thus, the high bone mass phenotype in Maf1 -/- mice is likely due to confounding effects from the global absence of MAF1. MAF1 overexpression promoted osteoblast differentiation of ST2 cells while MAF1 downregulation inhibited differentiation, indicating MAF1 enhances osteoblast formation. However, other perturbations used to repress RNA pol III transcription, inhibited osteoblast differentiation. However, decreasing RNA pol III transcription through these perturbations enhanced adipogenesis in ST2 cells. RNA-seq analyzed the basis for these opposing actions on osteoblast differentiation. The different modalities used to perturb RNA pol III transcription resulted in distinct gene expression changes, indicating that this transcription process is highly sensitive and triggers diverse gene expression programs and phenotypic outcomes. Specifically, MAF1 induced genes known to promote osteoblast differentiation. Furthermore, genes that are induced during osteoblast differentiation displayed codon bias. Together, these results reveal a novel role for MAF1 and RNA pol III-mediated transcription in osteoblast fate determination, differentiation, and bone mass regulation.
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Author Response:
Reviewer #2 (Public Review):
In this manuscript, Busschers et al. present data demonstrating a function of the RNA polymerase III transcriptional repressor and tumor suppressor MAF1 in regulating bone mass. By combining in vivo and in vitro experiments, they provide results that are sometimes difficult to reconcile. For example, general KO of MAF1 in mice but also tissue-specific overexpression of MAF1 in stromal cells resulted in increased bone mass, suggesting that both positive and negative regulation of RNA polymerase III transcription may contribute to enhanced osteoblast differentiation and osteogenesis. Interestingly, primary stromal cells derived from the bone marrow of MAF1-/- mice showed enhanced osteoclastogenesis and decreased osteoblastogenesis, which is in contrast to the results obtained in the mice …
Author Response:
Reviewer #2 (Public Review):
In this manuscript, Busschers et al. present data demonstrating a function of the RNA polymerase III transcriptional repressor and tumor suppressor MAF1 in regulating bone mass. By combining in vivo and in vitro experiments, they provide results that are sometimes difficult to reconcile. For example, general KO of MAF1 in mice but also tissue-specific overexpression of MAF1 in stromal cells resulted in increased bone mass, suggesting that both positive and negative regulation of RNA polymerase III transcription may contribute to enhanced osteoblast differentiation and osteogenesis. Interestingly, primary stromal cells derived from the bone marrow of MAF1-/- mice showed enhanced osteoclastogenesis and decreased osteoblastogenesis, which is in contrast to the results obtained in the mice from which these cells were derived. Suppression of RNA pol III transcription by RNA interference-induced decrease in BRF1 expression or by inhibition of RNA pol III itself using ML-60218 treatment resulted in decreased osteoblast differentiation and thus bone mass. When bone mineralization was analyzed by ALP and alizarin red staining, distinct methods of inhibiting RNA pol III transcription produced different results. Overexpression of MAF1 enhanced ALP and alizarin red staining, whereas treatment with ML-60218 or suppression of BRF1 resulted in less staining. Finally, overexpression of MAF1 in ST2 cells also resulted in decreased osteoblast differentiation. In contrast to these sometimes contradictory results, adipocyte differentiation was consistently affected by different types of regulation of RNA pol III transcription. Overexpression of MAF1 or repression of RNA pol III transcription by BRF1 knockdown or ML-60218 treatment resulted in enhanced adipocyte differentiation. RNA sequencing of samples derived from the different approaches to regulate bone differentiation in this work revealed approach-specific regulation of mRNA expression. Overall, the data presented in this manuscript paint a complex picture of the regulation of bone differentiation by different influences on the activity of the RNA polymerase III transcriptional apparatus.
Strengths: The work contains several complementary in vivo and in vitro approaches to analyze The effects of regulated MAF1 expression or inhibition of RNA pol III transcription on osteogenesis and adipocyte differentiation.
The data are well controlled and of excellent quality.
The experiments suggest that bone differentiation is regulated by the activity of the RNA polymerase III transcription system, as any condition affecting this system influences osteogenesis.
Weaknesses: No clear conclusions can be drawn regarding the mechanisms underlying the observed, sometimes contradictory, effects.
While there is more to be done to uncover the complex mechanisms that produce these phenotypes, this study represents a novel and comprehensive study that demonstrates a clear role for Maf1 and RNA pol III-dependent transcription in osteoblast differentiation and bone biology.
Consideration of the RNA pol III transcription system focuses exclusively on the expression of type 1 and type 2 promoters, thus neglecting possible effects of gene-external type 3 promoters.
It is possible that other RNA pol III-produced transcripts play a role in osteoblast differentiation. For this initial study, however, we were unable to determine the role of each of the many RNA pol III transcripts. However, since the knockdown of Brf1 does affect osteoblast differentiation, and Brf1 is not used by type III RNA pol III promoters, such as U6 snRNA, we focused on the effect of type II promoters, the majority of which are tRNA genes.
RNA sequencing results are presented only as GO analyses, but the genuine results of the sequencing were not reported.
We are unsure what the reviewer is suggesting with the presentation of the sequencing results. However, we have uploaded our differential expression data as excel spreadsheets in supporting data, and additionally will deposit our data to GEO.
The authors have clearly achieved their goal of showing that MAF1 and RNA polymerase III gene transcription affect bone differentiation. This work broadens the spectrum of processes affected by RNA polymerase III gene transcription. Because of the complexity of the results observed, a tabular summary might be helpful for the reader to quickly and comprehensively grasp the most important findings. Such a table could include the various experimental analyses, the effects on osteogenesis, the effects on adipocyte differentiation, the effects on RNA Pol III activity, and possibly the effects on gene expression determined by RNA-seq.
We added a table. Table 1 is referenced in the first paragraph of the discussion.
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Evaluation Summary:
Johnson et al. have used several complementary in vivo and in vitro approaches to analyze the effects of regulated MAF1 expression or inhibition of RNA pol III transcription on osteogenesis and adipocyte differentiation. The data are well controlled and of excellent quality, providing novel insights into Maf1 and RNA polymerase-mediated transcriptions in skeleton biology.
(This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. The reviewers remained anonymous to the authors.)
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Reviewer #1 (Public Review):
RNA polymerase III and it transcribes are essential for eukaryotic cell growth and survival. Moreover, mutations in RNA polymerase III and its specific transcription factor BRF1 lead to human diseases associated with bone phenotypes, indicating that RNA polymerase III mediated transcription plays a critical role in regulating skeletal cells during development and homeostasis. In work reported by this study, the authors aimed to elucidate the role of Maf1, a repressor of RNA polymerase III, in bone homeostasis using the loss-of-function (Maf1 germline deletion mice) and the gain-of-function (Maf1 bone specific overexpression mice) genetically modified mouse models and in vitro complemental approaches. The authors found that loss of Maf1 and inhibition of RNA polymerase III transcription resulted in decreased …
Reviewer #1 (Public Review):
RNA polymerase III and it transcribes are essential for eukaryotic cell growth and survival. Moreover, mutations in RNA polymerase III and its specific transcription factor BRF1 lead to human diseases associated with bone phenotypes, indicating that RNA polymerase III mediated transcription plays a critical role in regulating skeletal cells during development and homeostasis. In work reported by this study, the authors aimed to elucidate the role of Maf1, a repressor of RNA polymerase III, in bone homeostasis using the loss-of-function (Maf1 germline deletion mice) and the gain-of-function (Maf1 bone specific overexpression mice) genetically modified mouse models and in vitro complemental approaches. The authors found that loss of Maf1 and inhibition of RNA polymerase III transcription resulted in decreased osteoblast differentiation in vitro, whereas overexpression of Maf1 promoted this process. In contrast, both Maf1 knockout and overexpression mice exhibited increased trabecular bone mass, suggesting amplified bone formation and/or attenuated bone resorption. The strength of is manuscript is the novelty in that it is the first comprehensive investigation on the role of Maf1 and RNA polymerase III in skeleton biology. The major weakness is the lack of bone histology and histomorphometric analysis of number and function of osteoblasts and osteoclast in vivo. It is especially important when in vitro findings are contradictory with the in vivo results. Overall, the study provides the first and novel insights of Maf1 and RNA polymerase-mediated transcriptions in skeleton biology.
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Reviewer #2 (Public Review):
In this manuscript, Busschers et al. present data demonstrating a function of the RNA polymerase III transcriptional repressor and tumor suppressor MAF1 in regulating bone mass. By combining in vivo and in vitro experiments, they provide results that are sometimes difficult to reconcile. For example, general KO of MAF1 in mice but also tissue-specific overexpression of MAF1 in stromal cells resulted in increased bone mass, suggesting that both positive and negative regulation of RNA polymerase III transcription may contribute to enhanced osteoblast differentiation and osteogenesis. Interestingly, primary stromal cells derived from the bone marrow of MAF1-/- mice showed enhanced osteoclastogenesis and decreased osteoblastogenesis, which is in contrast to the results obtained in the mice from which these cells …
Reviewer #2 (Public Review):
In this manuscript, Busschers et al. present data demonstrating a function of the RNA polymerase III transcriptional repressor and tumor suppressor MAF1 in regulating bone mass. By combining in vivo and in vitro experiments, they provide results that are sometimes difficult to reconcile. For example, general KO of MAF1 in mice but also tissue-specific overexpression of MAF1 in stromal cells resulted in increased bone mass, suggesting that both positive and negative regulation of RNA polymerase III transcription may contribute to enhanced osteoblast differentiation and osteogenesis. Interestingly, primary stromal cells derived from the bone marrow of MAF1-/- mice showed enhanced osteoclastogenesis and decreased osteoblastogenesis, which is in contrast to the results obtained in the mice from which these cells were derived. Suppression of RNA pol III transcription by RNA interference-induced decrease in BRF1 expression or by inhibition of RNA pol III itself using ML-60218 treatment resulted in decreased osteoblast differentiation and thus bone mass. When bone mineralization was analyzed by ALP and alizarin red staining, distinct methods of inhibiting RNA pol III transcription produced different results. Overexpression of MAF1 enhanced ALP and alizarin red staining, whereas treatment with ML-60218 or suppression of BRF1 resulted in less staining. Finally, overexpression of MAF1 in ST2 cells also resulted in decreased osteoblast differentiation. In contrast to these sometimes contradictory results, adipocyte differentiation was consistently affected by different types of regulation of RNA pol III transcription. Overexpression of MAF1 or repression of RNA pol III transcription by BRF1 knockdown or ML-60218 treatment resulted in enhanced adipocyte differentiation. RNA sequencing of samples derived from the different approaches to regulate bone differentiation in this work revealed approach-specific regulation of mRNA expression.
Overall, the data presented in this manuscript paint a complex picture of the regulation of bone differentiation by different influences on the activity of the RNA polymerase III transcriptional apparatus.Strengths:
The work contains several complementary in vivo and in vitro approaches to analyze the effects of regulated MAF1 expression or inhibition of RNA pol III transcription on osteogenesis and adipocyte differentiation.The data are well controlled and of excellent quality.
The experiments suggest that bone differentiation is regulated by the activity of the RNA polymerase III transcription system, as any condition affecting this system influences osteogenesis.
Weaknesses:
No clear conclusions can be drawn regarding the mechanisms underlying the observed, sometimes contradictory, effects.The complexity of MAF1-dependent regulation of gene expression by all three nuclear RNA polymerases has not been adequately considered or discussed.
Consideration of the RNA pol III transcription system focuses exclusively on the expression of type 1 and type 2 promoters, thus neglecting possible effects of gene-external type 3 promoters.
RNA sequencing results are presented only as GO analyses, but the genuine results of the squencing were not reported.
The authors have clearly achieved their goal of showing that MAF1 and RNA polymerase III gene transcription affect bone differentiation. This work broadens the spectrum of processes affected by RNA polymerase III gene transcription. Because of the complexity of the results observed, a tabular summary might be helpful for the reader to quickly and comprehensively grasp the most important findings. Such a table could include the various experimental analyses, the effects on osteogenesis, the effects on adipocyte differentiation, the effects on RNA Pol III activity, and possibly the effects on gene expression determined by RNA-seq.
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Reviewer #3 (Public Review):
Johnson and colleagues present a study linking Maf1, a negative regulator of RNA polymerase III, to osteoblast differentiation and mineralization.
A major strength of this study is the use of in vivo and ex vivo models where Maf1 expression is manipulated and separately, models where RNA polymerase III activity is modulated independently of Maf1. A weakness of the study in its current form is the absence of combining these models to discern the dependency of the Maf1 influence on RNApolIII activity for the phenotypes presented.
The authors have clearly demonstrated that Maf1 is important for bone biology in both the in vivo and ex vivo models, which is an important contribution to the field. However, the opposite roles that Maf1 plays in these two models is confusing, despite being biologically interesting …
Reviewer #3 (Public Review):
Johnson and colleagues present a study linking Maf1, a negative regulator of RNA polymerase III, to osteoblast differentiation and mineralization.
A major strength of this study is the use of in vivo and ex vivo models where Maf1 expression is manipulated and separately, models where RNA polymerase III activity is modulated independently of Maf1. A weakness of the study in its current form is the absence of combining these models to discern the dependency of the Maf1 influence on RNApolIII activity for the phenotypes presented.
The authors have clearly demonstrated that Maf1 is important for bone biology in both the in vivo and ex vivo models, which is an important contribution to the field. However, the opposite roles that Maf1 plays in these two models is confusing, despite being biologically interesting and certainly worth further study.
The authors present a very interesting hypothesis that codon usage plays an important role in osteoblast differentiation. This is important as translation rates will be influenced by the available pool of tRNAs which are made by RNApolymeraseIII and regulated by Maf1.
A major impact of this work is defining the importance of RNA polymerase III on osteoblast differentiation and mineralization, which should be taken into consideration when future studies investigate the biology underlying bone development.
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