ATRX silences Cartpt expression in osteoblastic cells during skeletal development
Yi-Ting Chen, … , Yangjin Bae, Brendan Lee
Yi-Ting Chen, … , Yangjin Bae, Brendan Lee
Published January 2, 2025
Citation Information: J Clin Invest. 2025;135(1):e163587. https://doi.org/10.1172/JCI163587.
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Research Article Bone biology

ATRX silences Cartpt expression in osteoblastic cells during skeletal development

Abstract

ATP-dependent chromatin remodeling protein ATRX is an essential regulator involved in maintenance of DNA structure and chromatin state and regulation of gene expression during development. ATRX was originally identified as the monogenic cause of X-linked α-thalassemia mental retardation (ATR-X) syndrome. Affected individuals display a variety of developmental abnormalities and skeletal deformities. Studies from others investigated the role of ATRX in skeletal development by tissue-specific Atrx knockout. However, the impact of ATRX during early skeletal development has not been examined. Using preosteoblast-specific Atrx conditional knockout mice, we observed increased trabecular bone mass and decreased osteoclast number in bone. In vitro coculture of Atrx conditional knockout bone marrow stromal cells (BMSCs) with WT splenocytes showed impaired osteoclast differentiation. Additionally, Atrx deletion was associated with decreased receptor activator of nuclear factor κ-B ligand (Rankl)/ osteoprotegerin (Opg) expression ratio in BMSCs. Notably, Atrx-deficient osteolineage cells expressed high levels of the neuropeptide cocaine- and amphetamine-regulated transcript prepropeptide (Cartpt). Mechanistically, ATRX suppresses Cartpt transcription by binding to the promoter, which is otherwise poised for Cartpt expression by RUNX2 binding to the distal enhancer. Finally, Cartpt silencing in Atrx conditional knockout BMSCs rescued the molecular phenotype by increasing the Rankl/Opg expression ratio. Together, our data show a potent repressor function of ATRX in restricting Cartpt expression during skeletal development.

Authors

Yi-Ting Chen, Ming-Ming Jiang, Carolina Leynes, Mary Adeyeye, Camilla F. Majano, Barakat Ibrahim, Urszula Polak, George Hung, Zixue Jin, Denise G. Lanza, Lan Liao, Brian Dawson, Yuqing Chen-Evenson, Oscar E. Ruiz, Richard J. Gibbons, Jason D. Heaney, Yangjin Bae, Brendan Lee

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Figure 5

ATRX and RUNX2 bind to the regulatory regions of Cartpt.

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ATRX and RUNX2 bind to the regulatory regions of Cartpt. (A) Genome brow...
(A) Genome browser representations of published ATRX, RUNX2, H3K4me1, H3K27Ac, and H3K9me3 ChIP-Seqs near Cartpt. ATRX, ATRX ChIP-Seq in mouse NPCs. RUNX2, RUNX2 ChIP-Seq in differentiated MC3T3 cells. H3K4me1/H3K27Ac/H3K9me3, H3K4me1, H3K27Ac, and H3K9me3 ChIP-Seqs in IDGSW3 cells. Data represented as read density in reads normalized to 108. Blue, pink, and green boxes indicate peak regions. The peak score under each color box was based on peak calling analysis. (B and C) RT-qPCR results of Atrx (B) and Cartpt (C) expression in MC3T3 cells transfected with 100 nM siControl (shown as 0 nM) or siAtrx (shown as 50 nM and 100 nM). Black, cells at the undifferentiated state; orange, cell differentiating for 3 days in osteogenic media. n = 3 per group. Data are presented as means with ± SD. Two-way ANOVA. *P < 0.05; **P < 0.01; ****P < 0.001. (D–F) ATRX ChIP-qPCR in MC3T3 cells. Primer design at the edge (P1) or peak (P2) of the ATRX binding near the Cartpt promoter region (D). ATRX ChIP-qPCR in MC3T3 cells at undifferentiated (E) and differentiated (F) (cell differentiating for 5 days) state. Tel, positive control of ATRX bindings at telomere; Rhbdf1, negative control of ATRX bindings at the Rhbdf1 intron region. n = 3 per group. (G–I) RUNX2 ChIP-qPCR in MC3T3 cells. Primer design at the edge (E1) or peak (E2) of the RUNX2 binding 10 kbp upstream of the Cartpt promoter (G). RUNX2 ChIP-qPCR in MC3T3 cells at undifferentiated (H) and differentiated (I) (cell differentiating for 5 days) state. Ocn, positive control of RUNX2 bindings at the Ocn promoter; Smad, negative control of RUNX2 bindings at the Smad4 intron region. n = 3 per group. Data are represented as means with ± SD. Two-way ANOVA. *P < 0.05; **P < 0.01; ***P < 0.005.