RNA polymerase (pol) III transcribes various untranslated RNAs including 5S rRNA and tRNAs. RNA pol III-derived transcripts play essential roles in several processes, including protein synthesis and secretion (Dieci et al., 2013; Dieci et al., 2007). In addition to RNA pol III, transcription of tRNAs requires the recruitment of TFIIIC and TFIIIB to the promoter. TFIIIB consists of Brf1, TATA-binding protein (TBP) and B-double prime (Orioli et al., 2012). RNA pol III-dependent transcription is also tightly regulated through either direct or indirect mechanisms that control TFIIIB recruitment to the promotor (Gomez-Roman et al., 2003; Kenneth et al., 2007; Felton-Edkins et al., 2003; Sriskanthadevan-Pirahas et al., 2018; Crighton et al., 2003; Sutcliffe et al., 2000; White et al., 1996; Woiwode et al., 2008). MAF1 is a key repressor of RNA pol III-dependent transcription ( Graczyk et al., 2015; Johnson et al., 2007). It binds to RNA pol III and prevents the interaction between RNA pol III and TFIIIB (Vorländer et al., 2019; Vannini et al., 2010). MAF1 has also been shown to regulate a variety of RNA pol II-transcribed targets (Johnson et al., 2007; Palian et al., 2014; Khanna et al., 2014; Li et al., 2016; Lee et al., 2015), and acts as a tumor suppressor (Palian et al., 2014; Li et al., 2016), regulates metabolism (Willis et al., 2018; Bonhoure et al., 2015), and longevity (Shetty et al., 2020; Cai and Wei, 2016).

RNA pol III-mediated transcription has been shown to play an important role during development and cellular differentiation. Two RNA pol III isoforms are differentially expressed between pluripotent and differentiated embryonic stem cells (ESCs) (Haurie et al., 2010; Wong et al., 2011; Wang et al., 2020). RNA pol III-dependent transcription also modulates the formation of hematopoietic lineages in zebrafish (Wei et al., 2016) and transcription is downregulated during skeletal muscle differentiation of Xenopus tropicalis (McQueen et al., 2019). MAF1 enhances the formation of mesoderm in embryonic stem cells, and the downregulation of RNA pol III-dependent transcription enhances the differentiation of ESCs and 3T3-L1 cells into adipocytes (Chen et al., 2018).

Diseases associated with ribosomal disfunctions, ribosomopathies, are commonly associated with bone marrow, skeletal and craniofacial disorders (Trainor and Merrill, 2014). This surprising tissue specificity suggests that these tissues may be particularly sensitive to alterations in protein synthesis. For example, Treacher Collins syndrome is caused by mutations in POLR1C, POLR1D, or TCOF1, which affect rDNA transcription by RNA pol I and ribosome biogenesis (Noack Watt et al., 2016; Dauwerse et al., 2011). RNA pol III-derived transcripts may also play a role as POLR1C and POLR1D are common subunits of both RNA pol I and RNA pol III. In yeast, Treacher Collins syndrome-related mutations in POLR1D result in altered functions of both RNA pol I and III (Walker-Kopp et al., 2017). Furthermore, RNA pol III-dependent transcription may play a role in cerebellofaciodental syndrome which is associated with mutations in the RNA pol III-specific transcription factor, Brf1. This syndrome is characterized by a neurodevelopmental phenotype as well as changes in the facial and dental structure and delayed bone age (Borck et al., 2015; Jee et al., 2017; Honjo et al., 2021). Additionally, bone phenotypes have been described in a subgroup of patients with mutations in RNA pol III subunits (Borck et al., 2015; Jee et al., 2017; Honjo et al., 2021; Terhal et al., 2020; Ghoumid et al., 2017).

It is known that MAF1 regulates mesoderm formation and adipocyte differentiation (Chen et al., 2018). Osteoblasts and adipocytes are both derived from the mesenchymal lineage (Chen et al., 2016) and mutations in BRF1 and RNA pol III subunits are associated with bone-related phenotypes. These facts led us to hypothesize that regulation of RNA pol III-dependent transcription by MAF1 may play a fundamental role in osteoblast differentiation, bone formation and hence, bone mass. Here, we show that both whole body deletion of MAF1 in mice and tissue-specific overexpression of MAF1 in stromal cells of the long bones enhances bone mass in vivo, while MAF1 induces osteoblast differentiation in vitro. To further examine the role of MAF1 and RNA pol III-mediated transcription in osteoblast differentiation, we attempted to study the effect on osteoblast differentiation by repressing transcription through different approaches. Surprisingly, while MAF1 induced osteoblast differentiation, repression of RNA pol III-dependent transcription, by either chemical inhibition of RNA pol III or by Brf1 knockdown, decreased osteoblast differentiation. Thus, changes in MAF1 expression produce an opposing effect on osteoblast differentiation compared with other approaches that repress RNA pol III-mediated transcription. We further show that these three different approaches to decrease RNA pol III-dependent transcription result in divergent gene expression changes. Altered MAF1 expression affects the expression of the osteoblast differentiation gene program. Together, these findings reveal that MAF1 plays a key role in osteoblast differentiation and bone mass regulation and that its ability to regulate RNA pol III-dependent transcription contributes to the observed phenotypic outcomes.

MAF1 is a key repressor of transcription by RNA pol III (Johnson et al., 2007; Vorländer et al., 2019). Changes in MAF1 expression have been shown to enhance adipocyte differentiation (Chen et al., 2018; Figure 2—figure supplement 1 and Figure 3—figure supplement 1). Maf1^-/- mice are shorter than WT mice and exhibit a lean phenotype with resistance to diet-induced obesity, decreased fertility and fecundity, and increased longevity/healthspan (Bonhoure et al., 2015). Here, we demonstrate that these mice display an increase in bone volume and bone formation. However, Maf1^-/- derived primary stromal cells showed a decrease in osteoblast formation in ex vivo cultures. While it is possible this is due to different culture conditions, this latter finding was consistent with the effects of MAF1 overexpression specifically in the mesenchyme of long bones, which resulted in enhanced osteoblast differentiation and an increase in bone mass. In these latter mice, compared with Maf1^-/- mice, any confounding actions due to the absence of MAF1 in other tissues, such as possible endocrine or paracrine effects, are limited. Thus, our results indicate that the ability of MAF1 to regulate bone mass involves both cell autonomous and non-cell-autonomous actions. This idea is further corroborated by our in vitro results showing that osteoblast differentiation is regulated by increasing or decreasing MAF1 expression in ST2 cells, suggesting that MAF1 promotes osteoblast differentiation and mineralization. Our findings are summarized in Table 1. As MAF1 also enhances adipogenesis (Chen et al., 2018; Figure 2—figure supplement 1, Figure 3—figure supplement 1), we conclude that MAF1 is an important regulator in the development and differentiation of mesenchymal cells into multiple lineages.

MAF1 is a well-established repressor of RNA pol III-mediated transcription through its direct interaction with RNA pol III (Vorländer et al., 2019; Vannini et al., 2010). To further determine whether MAF1 functions to promote osteoblast differentiation through its ability to regulate RNA pol III-dependent transcription, we used complementary approaches to repress this transcription process. During differentiation, we observed an overall increase in tRNA gene transcripts. This increase was repressed by MAF1 overexpression, Brf1 downregulation, or chemical inhibition of RNA pol III. Surprisingly, however, in contrast to the positive regulation of osteoblast differentiation by MAF1, chemical inhibition of RNA pol III or Brf1 knockdown resulted in a decrease in osteoblast differentiation (Table 1). Thus, while different perturbations in RNA pol III-dependent transcription all affect the differentiation process, MAF1-mediated changes produce an opposing action compared with chemical inhibition of RNA pol III or decreased Brf1 expression. This is in contrast to what we observed for adipogenesis, where all three approaches to repress RNA pol III transcription similarly increased adipocyte formation (Figure 2—figure supplement 1, Figure 3—figure supplement 1, Figure 4—figure supplement 2, Figure 5—figure supplement 1 and Table 1; Chen et al., 2018).

To understand the basis of the different osteoblast differentiation outcomes, we examined changes in gene expression that resulted from the different perturbations of RNA pol III-dependent transcription. RNA seq revealed that alterations in RNA pol III-mediated transcription prior to, and during differentiation, result in a limited number of overlapping changes, with the four conditions largely producing distinct changes in gene expression profiles. Changes in several established regulators of osteoblast formation and function were identified that correlated with the differentiation outcomes. Overall, the different gene expression profiles likely contribute to the differences in osteoblast differentiation that we observe. However, the precise mechanism underlying the difference between MAF1-regulated changes compared with the alternate approaches to repress RNA pol III-mediated transcription remains unclear. Unlike RNA pol III and Brf1, MAF1 is not an essential RNA pol III transcription factor, and it functions to repress this transcription process by directly interacting with RNA pol III (Vorländer et al., 2019). Given that MAF1 is recruited to certain RNA pol II promoters (Johnson et al., 2007; Palian et al., 2014; Khanna et al., 2014; Li et al., 2016), it is conceivable that the ability of MAF1 to regulate both RNA pol III- and RNA pol II-transcribed genes contributes to its ability to drive osteoblast differentiation. This may also explain its differential effect on osteoblast differentiation when compared with the alternative approaches used to selectively repress RNA pol III-dependent transcription. However, RNA seq did not detect changes in genes previously reported to be regulated by MAF1, such as Tbp and Fasn. The same finding was obtained in an RNA-seq analysis in Maf1^-/- liver (Bonhoure et al., 2020). Thus, the regulation of RNA pol II-dependent gene expression by MAF1 is likely context dependent. Additional studies suggest that the regulation of RNA pol II targets by MAF1 is limited (Orioli et al., 2016). Therefore, we cannot exclude a possible role for MAF1 regulation of RNA pol II transcription in the regulation of osteoblast differentiation. However, since other approaches used to modulate RNA pol III-mediated transcription also alter osteoblast differentiation, MAF1 functions, at least in part, to regulate osteoblastogenesis through its effect on RNA pol III.

In addition to different effects on gene expression by manipulation of MAF1 expression, RNA-seq analysis revealed distinct changes in gene expression exhibited by chemical inhibition of RNA pol III and Brf1 knockdown, despite similar outcomes on osteoblast differentiation. These data indicate that manipulating RNA pol III transcription using different approaches results in disparate outcomes on gene expression. This could be due to differential changes in the tRNA population. The prevalence of specific tRNAs has been correlated with codon-biased translation in multiple tissues (Dittmar et al., 2006; Gobet et al., 2020; Kutter et al., 2011; Rak et al., 2018). Our analysis of mRNA changes during osteoblast differentiation showed that a significant codon bias emerges during this process. Thus, it is conceivable that during osteoblast differentiation specific changes in tRNAs are required to efficiently drive codon-biased translation of mRNAs needed for differentiation to proceed. Thus, this may be a mechanism in which RNA pol III transcription affects osteoblast differentiation. There are several other mechanisms that could contribute to the observed differences in gene regulation by RNA pol III-mediated transcription. RNA pol III transcribes a variety of untranslated RNAs and changes in any of these RNA pol III-derived transcripts may potentially play a role. Additionally, the generation of tRNA fragments (Schimmel, 2018; Su et al., 2020), or the regulation of RNA pol II genes through the recruitment of RNA pol III to nearby SINE sites such as described for CDKN1a (Lee et al., 2015), could all potentially contribute to the differential expression of osteoblast genes when RNA pol III-mediated transcription is altered. Future work will be needed to identify the specific mechanisms by which MAF1 and RNA pol III-mediated transcription alter gene expression to regulate osteoblast differentiation.

In all, our results describe a novel role for MAF1 and RNA pol III in bone biology. Given that different approaches used to modulate RNA pol III-dependent transcription also affect osteoblast differentiation, albeit in an opposing direction from MAF1-mediated effects, the findings support the idea that MAF1 functions, at least in part, to regulate osteoblast development and bone mass through its ability to control RNA pol III-mediated transcription. Distinct qualitative or quantitative changes resulting from different perturbations in RNA pol III-dependent transcription may also play a role in developmental disorders. Interestingly, several different syndromes relating to mutations in RNA pol III subunits show very heterogeneous phenotypes. This includes POLR3-related hypomyelinating leukodystrophies (Lata et al., 2021; Yeganeh and Hernandez, 2020; Thomas and Thomas, 2019), Wiedemann-Rautenstrauch syndrome, a neonatal progeroid syndrome (Wu et al., 2021b; Wambach et al., 2018; Paolacci et al., 2017; Beauregard-Lacroix et al., 2020), and cerebellar hypoplasia with endosteal sclerosis (Terhal et al., 2020; Ghoumid et al., 2017). Additionally, Brf1 mutations have been shown to be causative for cerebellofaciodental syndrome (Borck et al., 2015; Jee et al., 2017; Honjo et al., 2021; Valenzuela et al., 2020). Some patients with RNA pol III-related mutations, but not all, show bone-related phenotypes (Borck et al., 2015; Jee et al., 2017; Honjo et al., 2021; Terhal et al., 2020; Ghoumid et al., 2017). The considerable heterogeneity of these syndromes has been suggested to be related to differential changes in RNA pol III-dependent transcription (Yeganeh and Hernandez, 2020). Thus, some cells and tissues, including bone and its cells, may be more sensitive to disruption of RNA pol III-mediated transcription. Together, these collective findings and our current study indicate that the exquisite regulation of RNA pol III plays an essential role in a variety of biological and developmental processes.

Sequencing data will be deposited in GEO Source data files will be provided All data will be made available to the public. Raw and processed data from the RNA sequencing experiment determining gene expression before and during osteoblast differentiation has been uploaded to the GEO data base with accession nr. GSE203308.

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Author details

1. Ellen Phillips

   Department of Molecular and Cellular Biology, Baylor College of Medicine, Houston, United States

   Contribution

   Conceptualization, Data curation, Formal analysis, Investigation, Methodology, Validation, Visualization, Writing – original draft, Writing – review and editing

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0003-3654-2066

2. Naseer Ahmad

   Departments of Medicine and Pharmacological Sciences and Center for translational Medicine and Pharmacology, Icahn School of Medicine at Mount Sinai, New York, United States

   Contribution

   Data curation, Investigation, Methodology

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0001-9514-2703

3. Li Sun

   Departments of Medicine and Pharmacological Sciences and Center for translational Medicine and Pharmacology, Icahn School of Medicine at Mount Sinai, New York, United States

   Contribution

   Data curation, Investigation, Methodology

   Competing interests

   No competing interests declared

4. James Iben

   Molecular Genomics Core, Eunice Kennedy Shriver National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, United States

   Contribution

   Formal analysis, Software, Validation, Writing – review and editing

   Competing interests

   No competing interests declared

5. Christopher J Walkey

   Department of Molecular and Cellular Biology, Baylor College of Medicine, Houston, United States

   Contribution

   Conceptualization, Investigation, Resources

   Competing interests

   No competing interests declared

6. Aleksandra Rusin

   Department of Molecular and Cellular Biology, Baylor College of Medicine, Houston, United States

   Contribution

   Methodology, Supervision

   Competing interests

   No competing interests declared

7. Tony Yuen

   Departments of Medicine and Pharmacological Sciences and Center for translational Medicine and Pharmacology, Icahn School of Medicine at Mount Sinai, New York, United States

   Contribution

   Validation, Writing – review and editing

   Competing interests

   No competing interests declared

8. Clifford J Rosen

   Center for Clinical and Translational Research, Maine Medical Center Research Institute, Scarborough, United States

   Contribution

   Data curation, Formal analysis, Investigation

   Competing interests

   No competing interests declared

9. Ian M Willis

   Departments of Biochemistry and Systems and Computational Biology, Albert Einstein College of Medicine, Bronx, United States

   Contribution

   Project administration, Resources, Supervision, Writing – review and editing

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0001-6599-2395

10. Mone Zaidi

    Departments of Medicine and Pharmacological Sciences and Center for translational Medicine and Pharmacology, Icahn School of Medicine at Mount Sinai, New York, United States

    Contribution

    Investigation, Methodology, Supervision, Writing – review and editing

    Competing interests

    Senior editor, eLife

    "This ORCID iD identifies the author of this article:" 0000-0001-5911-9522

11. Deborah L Johnson

    Department of Molecular and Cellular Biology, Baylor College of Medicine, Houston, United States

    Contribution

    Conceptualization, Formal analysis, Funding acquisition, Methodology, Project administration, Resources, Supervision, Writing – original draft, Writing – review and editing

    For correspondence

    deborah.johnson@bcm.edu

    Competing interests

    No competing interests declared

    "This ORCID iD identifies the author of this article:" 0000-0003-1745-1580

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