MAF1, a repressor of RNA polymerase III-dependent transcription, regulates bone mass
- Baylor College of Medicine, United States
- Ican School of Medicine at Mount Sinai, United States
- Eunice Kennedy Shriver National Institute of Child Health and Human Development, United States
- Icahn School of Medicine at Mount Sinai, United States
- Maine Medical Center Research Institute, United States
- Albert Einstein College of Medicine, United States
Abstract
MAF1, a key repressor of RNA polymerase III-mediated transcription, has been shown to promote mesoderm formation in vitro. Here, we show that MAF1 plays a critical role in the regulation of osteoblast differentiation and bone mass. A high bone mass phenotype was noted in mice with a global deletion of Maf1 (Maf1-/- mice). However, osteoblasts isolated from Maf1-/- mice showed reduced osteoblastogenesis ex vivo. Therefore, we determined the effect of MAF1 overexpression specifically in cells from the mesenchymal lineage (Prx1-Cre;LSL-MAF1 mice). These mice showed increased bone mass. Ex vivo, cells from Prx1-Cre;LSL-MAF1 mice showed enhanced osteoblastogenesis concordant with their high bone mass phenotype. Thus, the high bone mass phenotype in Maf1-/- mice is likely due to the confounding effects of the global absence of Maf1 in Maf1-/- mice. MAF1 overexpression promoted osteoblast differentiation and shRNA-mediated Maf1 downregulation inhibited differentiation of ST2 cells, overall indicating MAF1 enhances osteoblast formation. We also found that, in contrast to MAF1 overexpression, other perturbations that repress RNA pol III transcription, including Brf1 knockdown and chemical inhibition of RNA pol III by ML-60218, inhibited osteoblast differentiation. All perturbations that decrease RNA pol III transcription, however, enhanced adipogenesis in ST2 cell cultures. RNA-seq was used to determine the basis for these opposing actions on osteoblast differentiation. The 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 in ST2 cells 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 and differentiation and bone mass regulation.
Data availability
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.
Article and author information
Author details
Funding
National Cancer Institute (CA108614)
- Tony Yuen
- Clifford J Rosen
- Ian M Willis
- Mone Zaidi
National Cancer Institute (CA74138)
- Li Sun
The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.
Reviewing Editor
- Subburaman Mohan, Jerry L. Pettis Memorial VA Medical Center, United States
Ethics
Animal experimentation: This study was performed in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health. All of the mice were handled according to approved institutional animal care and use committee (IACUC) protocol AN-6370 ofBaylor College of Medicine.
Version history
- Received: October 15, 2021
- Preprint posted: November 11, 2021 (view preprint)
- Accepted: April 26, 2022
- Accepted Manuscript published: May 25, 2022 (version 1)
- Version of Record published: June 21, 2022 (version 2)
Copyright
This is an open-access article, free of all copyright, and may be freely reproduced, distributed, transmitted, modified, built upon, or otherwise used by anyone for any lawful purpose. The work is made available under the Creative Commons CC0 public domain dedication.
Metrics
-
- 995
- views
-
- 298
- downloads
-
- 6
- citations
Views, downloads and citations are aggregated across all versions of this paper published by eLife.
Download links
A two-part list of links to download the article, or parts of the article, in various formats.
Downloads (link to download the article as PDF)
Open citations (links to open the citations from this article in various online reference manager services)
Cite this article (links to download the citations from this article in formats compatible with various reference manager tools)
MAF1, a repressor of RNA polymerase III-dependent transcription, regulates bone mass
eLife 11:e74740.
https://doi.org/10.7554/eLife.74740
Further reading
-
- Biochemistry and Chemical Biology
- Cell Biology
Numerous lipids are heterogeneously distributed among organelles. Most lipid trafficking between organelles is achieved by a group of lipid transfer proteins (LTPs) that carry lipids using their hydrophobic cavities. The human genome encodes many intracellular LTPs responsible for lipid trafficking and the function of many LTPs in defining cellular lipid levels and distributions is unclear. Here, we created a gene knockout library targeting 90 intracellular LTPs and performed whole-cell lipidomics analysis. This analysis confirmed known lipid disturbances and identified new ones caused by the loss of LTPs. Among these, we found major sphingolipid imbalances in ORP9 and ORP11 knockout cells, two proteins of previously unknown function in sphingolipid metabolism. ORP9 and ORP11 form a heterodimer to localize at the ER-trans-Golgi membrane contact sites, where the dimer exchanges phosphatidylserine (PS) for phosphatidylinositol-4-phosphate (PI(4)P) between the two organelles. Consequently, loss of either protein causes phospholipid imbalances in the Golgi apparatus that result in lowered sphingomyelin synthesis at this organelle. Overall, our LTP knockout library toolbox identifies various proteins in control of cellular lipid levels, including the ORP9-ORP11 heterodimer, which exchanges PS and PI(4)P at the ER-Golgi membrane contact site as a critical step in sphingomyelin synthesis in the Golgi apparatus.
-
- Cell Biology
- Neuroscience
Like other volume electron microscopy approaches, automated tape-collecting ultramicrotomy (ATUM) enables imaging of serial sections deposited on thick plastic tapes by scanning electron microscopy (SEM). ATUM is unique in enabling hierarchical imaging and thus efficient screening for target structures, as needed for correlative light and electron microscopy. However, SEM of sections on tape can only access the section surface, thereby limiting the axial resolution to the typical size of cellular vesicles with an order of magnitude lower than the acquired xy resolution. In contrast, serial-section electron tomography (ET), a transmission electron microscopy-based approach, yields isotropic voxels at full EM resolution, but requires deposition of sections on electron-stable thin and fragile films, thus making screening of large section libraries difficult and prone to section loss. To combine the strength of both approaches, we developed ‘ATUM-Tomo, a hybrid method, where sections are first reversibly attached to plastic tape via a dissolvable coating, and after screening detached and transferred to the ET-compatible thin films. As a proof-of-principle, we applied correlative ATUM-Tomo to study ultrastructural features of blood-brain barrier (BBB) leakiness around microthrombi in a mouse model of traumatic brain injury. Microthrombi and associated sites of BBB leakiness were identified by confocal imaging of injected fluorescent and electron-dense nanoparticles, then relocalized by ATUM-SEM, and finally interrogated by correlative ATUM-Tomo. Overall, our new ATUM-Tomo approach will substantially advance ultrastructural analysis of biological phenomena that require cell- and tissue-level contextualization of the finest subcellular textures.
