scholarly journals Maf1, a repressor of RNA polymerase III-dependent transcription, regulates bone mass

2021 ◽  
Author(s):  
Ellen Busschers ◽  
Naseer Ahmad ◽  
Li Sun ◽  
James R Iben ◽  
Christopher J. Walkey ◽  
...  
Keyword(s):  
Gene Expression ◽  
Bone Mass ◽  
Rna Polymerase ◽  
Osteoblast Differentiation ◽  
Rna Polymerase Iii ◽  
High Bone Mass ◽  
Polymerase Iii ◽  
High Bone ◽  
High Bone Mass Phenotype ◽  
Pol Iii

Maf1, a key repressor of RNA polymerase III-mediated transcription, has been shown to promote mesoderm formation in vitro. Here, we show for the first time 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 global deletion of Maf1 (Maf1-/- mice), as well as paradoxically, in mice that overexpressed MAF1 in cells of the osteoblast lineage (Prx-Cre;LSL-Maf1 mice). Osteoblasts isolated from Maf1-/- mice unexpectedly showed reduced osteoblastogenesis ex vivo. Prx-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. Expectedly, MAF1 overexpression promoted osteoblast differentiation and shRNA-mediated Maf1 downregulation inhibited differentiation of ST2 cells, indicating an overall positive action of Maf1 on osteoblast formation. We also found that, in contrast to MAF1 overexpression, other perturbations that repress RNA pol III transcription, including Brf1 knockdown and the chemical inhibition of RNA pol III by ML-60218, paradoxically inhibited osteoblast differentiation. RNA-seq was used to determine the basis for these opposing actions. The three modalities used to perturb RNA pol III transcription resulted in distinct changes gene expression, indicating that this transcription process is highly sensitive and triggers diverse gene expression programs and phenotypic outcomes. Specifically, MAF1 overexpression in ST2 cells induced genes known to promote osteoblast differentiation. A subset of these genes was altered in an opposite manner with Brf1 downregulation or treatment with ML-60218, both of which also inhibit RNA pol III-mediated transcription. All these perturbations, however, enhanced adipogenesis in ST2 cell cultures. Furthermore, codon bias was observed in a subset of genes expressed during osteoblast differentiation. 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.

scholarly journals Functions of vasopressin and oxytocin in bone mass regulation

2015 ◽  
Vol 113 (1) ◽  
pp. 164-169 ◽  
Author(s):  
Li Sun ◽  
Roberto Tamma ◽  
Tony Yuen ◽  
Graziana Colaianni ◽  
Yaoting Ji ◽  
...  
Keyword(s):  
Gene Expression ◽  
Bone Formation ◽  
Bone Mass ◽  
High Bone Mass ◽  
Nuclear Extracts ◽  
High Bone ◽  
High Bone Mass Phenotype ◽  
Opposing Effects ◽  
G Protein Coupled

Prior studies show that oxytocin (Oxt) and vasopressin (Avp) have opposing actions on the skeleton exerted through high-affinity G protein-coupled receptors. We explored whether Avp and Oxtr can share their receptors in the regulation of bone formation by osteoblasts. We show that the Avp receptor 1α (Avpr1α) and the Oxt receptor (Oxtr) have opposing effects on bone mass: Oxtr−/− mice have osteopenia, and Avpr1α−/− mice display a high bone mass phenotype. More notably, this high bone mass phenotype is reversed by the deletion of Oxtr in Oxtr−/−:Avpr1α−/− double-mutant mice. However, although Oxtr is not indispensable for Avp action in inhibiting osteoblastogenesis and gene expression, Avp-stimulated gene expression is inhibited when the Oxtr is deleted in Avpr1α−/− cells. In contrast, Oxt does not interact with Avprs in vivo in a model of lactation-induced bone loss in which Oxt levels are high. Immunofluorescence microscopy of isolated nucleoplasts and Western blotting and MALDI-TOF of nuclear extracts show that Avp triggers Avpr1α localization to the nucleus. Finally, a specific Avpr2 inhibitor, tolvaptan, does not affect bone formation or bone mass, suggesting that Avpr2, which primarily functions in the kidney, does not have a significant role in bone remodeling.

scholarly journals Lytic infection with murine gammaherpesvirus 68 activates host and viral RNA polymerase III promoters and enhances non-coding RNA expression

2021 ◽  
Author(s):  
Ashley N. Knox ◽  
Alice Mueller ◽  
Eva M. Medina ◽  
Eric T. Clambey ◽  
Linda F. van Dyk
Keyword(s):  
Gene Expression ◽  
Flow Cytometry ◽  
Rna Polymerase ◽  
Rna Polymerase Iii ◽  
Viral Gene Expression ◽  
Viral Gene ◽  
Murine Gammaherpesvirus ◽  
Polymerase Iii ◽  
Gammaherpesvirus 68 ◽  
Pol Iii

RNA polymerase III (pol III) transcribes multiple non-coding (nc) RNAs that are essential for cellular function. Pol III-dependent transcription is also engaged during certain viral infections, including the gammaherpesviruses (γHVs), where pol III-dependent viral ncRNAs promote pathogenesis. Additionally, several host ncRNAs are upregulated during γHV infection and play integral roles in pathogenesis by facilitating viral establishment and gene expression. Here, we sought to investigate how pol III promoters and transcripts are regulated during gammaherpesvirus infection using the murine gammaherpesvirus 68 (γHV68) system. To compare the transcription of host and viral pol III-dependent ncRNAs, we analyzed a series of pol III promoters for host and viral ncRNAs using a luciferase reporter optimized to measure pol III activity. We measured promoter activity from the reporter gene at the translation level via luciferase activity and at the transcription level via RT-qPCR. We further measured endogenous ncRNA expression at single cell-resolution by flow cytometry. These studies demonstrated that lytic infection with γHV68 increased the transcription from multiple host and viral pol III promoters, and further identified the ability of accessory sequences to influence both baseline and inducible promoter activity after infection. RNA flow cytometry revealed the induction of endogenous pol III-derived ncRNAs that tightly correlated with viral gene expression. These studies highlight how lytic gammaherpesvirus infection alters the transcriptional landscape of host cells to increase pol III-derived RNAs, a process that may further modify cellular function and enhance viral gene expression and pathogenesis. IMPORTANCE Gammaherpesviruses are a prime example of how viruses can alter the host transcriptional landscape to establish infection. Despite major insights into how these viruses modify RNA polymerase II-dependent generation of messenger RNAs, how these viruses influence the activity of host RNA polymerase III remains much less clear. Small non-coding RNAs produced by RNA polymerase III are increasingly recognized to play critical regulatory roles in cell biology and virus infection. Studies of RNA polymerase III dependent transcription are complicated by multiple promoter types and diverse RNAs with variable stability and processing requirements. Here, we characterized a reporter system to directly study RNA polymerase III-dependent responses during gammaherpesvirus infection and utilized single-cell flow cytometry-based methods to reveal that gammaherpesvirus lytic replication broadly induces pol III activity to enhance host and viral non-coding RNA expression within the infected cell.

scholarly journals Involvement of RNA Polymerase III in Immune Responses

2015 ◽  
Vol 35 (10) ◽  
pp. 1848-1859 ◽  
Author(s):  
Damian Graczyk ◽  
Robert J. White ◽  
Kevin M. Ryan
Keyword(s):  
Transcription Factor ◽  
Immune Responses ◽  
Rna Polymerase ◽  
Rna Polymerase Iii ◽  
Trna Genes ◽  
Malignant Cells ◽  
Polymerase Iii ◽  
Mouse Macrophages ◽  
Tightly Coupled ◽  
Pol Iii

Inflammation in the tumor microenvironment has many tumor-promoting effects. In particular, tumor-associated macrophages (TAMs) produce many cytokines which can support tumor growth by promoting survival of malignant cells, angiogenesis, and metastasis. Enhanced cytokine production by TAMs is tightly coupled with protein synthesis. In turn, translation of proteins depends on tRNAs, short abundant transcripts that are made by RNA polymerase III (Pol III). Here, we connect these facts by showing that stimulation of mouse macrophages with lipopolysaccharides (LPS) from the bacterial cell wall causes transcriptional upregulation of tRNA genes. The transcription factor NF-κB is a key transcription factor mediating inflammatory signals, and we report that LPS treatment causes an increased association of the NF-κB subunit p65 with tRNA genes. In addition, we show that p65 can directly associate with the Pol III transcription factor TFIIIB and that overexpression of p65 induces Pol III-dependent transcription. As a consequence of these effects, we show that inhibition of Pol III activity in macrophages restrains cytokine secretion and suppresses phagocytosis, two key functional characteristics of these cells. These findings therefore identify a radical new function for Pol III in the regulation of macrophage function which may be important for the immune responses associated with both normal and malignant cells.

Eukaryotic transcription termination factor La mediates transcript release and facilitates reinitiation by RNA polymerase III

1994 ◽  
Vol 14 (3) ◽  
pp. 2147-2158
Author(s):  
R J Maraia ◽  
D J Kenan ◽  
J D Keene
Keyword(s):  
Rna Polymerase ◽  
Rna Binding ◽  
Transcription Termination ◽  
Rna Polymerase Iii ◽  
Class Iii ◽  
Ample Evidence ◽  
Polymerase Iii ◽  
Transcription Complexes ◽  
Pol Iii

Ample evidence indicates that Alu family interspersed elements retrotranspose via primary transcripts synthesized by RNA polymerase III (pol III) and that this transposition sometimes results in genetic disorders in humans. However, Alu primary transcripts can be processed posttranscriptionally, diverting them away from the transposition pathway. The pol III termination signal of a well-characterized murine B1 (Alu-equivalent) element inhibits RNA 3' processing, thereby stabilizing the putative transposition intermediary. We used an immobilized template-based assay to examine transcription termination by VA1, 7SL, and Alu class III templates and the role of transcript release in the pol III terminator-dependent inhibition of processing of B1-Alu transcripts. We found that the RNA-binding protein La confers this terminator-dependent 3' processing inhibition on transcripts released from the B1-Alu template. Using pure recombinant La protein and affinity-purified transcription complexes, we also demonstrate that La facilitates multiple rounds of transcription reinitiation by pol III. These results illustrate an important role for La in RNA production by demonstrating its ability to clear the termination sites of class III templates, thereby promoting efficient use of transcription complexes by pol III. The role of La as a potential regulatory factor in transcript maturation and how this might apply to Alu interspersed elements is discussed.

scholarly journals Sex dimorphic regulation of osteoprogenitor progesterone in bone stromal cells

2017 ◽  
Vol 59 (4) ◽  
pp. 351-363 ◽  
Author(s):  
Alexander Kot ◽  
Zhendong A Zhong ◽  
Hongliang Zhang ◽  
Yu-An Evan Lay ◽  
Nancy E Lane ◽  
...  
Keyword(s):  
Mouse Model ◽  
Bone Mass ◽  
Gene Transcription ◽  
Conditional Knockout ◽  
Sequencing Analysis ◽  
Wild Type ◽  
High Bone Mass ◽  
Matrix Interaction ◽  
High Bone ◽  
High Bone Mass Phenotype

Increasing peak bone mass is a promising strategy to prevent osteoporosis. A mouse model of global progesterone receptor (PR) ablation showed increased bone mass through a sex-dependent mechanism. Cre-Lox recombination was used to generate a mouse model of osteoprogenitor-specific PR inactivation, which recapitulated the high bone mass phenotype seen in the PR global knockout mouse mode. In this work, we employed RNA sequencing analysis to evaluate sex-independent and sex-dependent differences in gene transcription of osteoprogenitors of wild-type and PR conditional knockout mice. PR deletion caused marked sex hormone-dependent changes in gene transcription in male mice as compared to wild-type controls. These transcriptional differences revealed dysregulation in pathways involving immunomodulation, osteoclasts, bone anabolism, extracellular matrix interaction and matrix interaction. These results identified many potential mechanisms that may explain our observed high bone mass phenotype with sex differences when PR was selectively deleted in the MSCs.

Sign in / Sign up

Export Citation Format

Share Document