Project description:Osteoblast differentiation leading to bone formation requires a coordinated transcriptional program. We have recently demonstrated that microtubule actin crosslinking factor 1 (MACF1) promotes osteoblast differentiation, suggesting a key role in regulating early-phase osteoblast differentiation. Here, we showed that the early-phase osteoblast differentiation transcriptome dynamics was regulated by MACF1 and the transcription of TCF7/LEF1, key effectors of Wnt signaling that is important for osteoblast differentiation was suppressed by MACF1 knockdown. Co-IP and Protein mass spectrometry revealed that MACF1 interacted with a known and two previously unknown repressors of TCF7/LEF1, DKK1, CDK12 and MEAF6. ChIP-seq analysis of MACF1-associated promoters further revealed that MACF1 interacted with transcription factors TCF12 and E2F6, which also suppressed the transcription of TCF7/LEF1. Furthermore, all these four MACF-interacted proteins inhibited osteoblast differentiation. By studying the underlying mechanism, we found that cytoplasmic-nuclear localization of MACF1 was dependent on its level and the cytoplasmic-nuclear localization of TCF12 and E2F6 was regulated by MACF1 localization. In addition, MACF1 oppositely regulated the transcription activity of TCF12 and TCF7. Current study, for the first time to our knowledge, suggest that MACF1 acts as a sponge of osteoblast differentiation repressors to promote osteoblast differentiation, and indicate a novel mechanism for regulating the cellular location of transcription factors by a protein associated with microtubule and actin.
Project description:Osteoblast differentiation leading to bone formation requires a coordinated transcriptional program. We have recently demonstrated that microtubule actin crosslinking factor 1 (MACF1) promotes osteoblast differentiation, suggesting a key role in regulating early-phase osteoblast differentiation. Here, we showed that the early-phase osteoblast differentiation transcriptome dynamics was regulated by MACF1 and the transcription of TCF7/LEF1, key effectors of Wnt signaling that is important for osteoblast differentiation was suppressed by MACF1 knockdown. Co-IP and Protein mass spectrometry revealed that MACF1 interacted with a known and two previously unknown repressors of TCF7/LEF1, DKK1, CDK12 and MEAF6. ChIP-seq analysis of MACF1-associated promoters further revealed that MACF1 interacted with transcription factors TCF12 and E2F6, which also suppressed the transcription of TCF7/LEF1. Furthermore, all these four MACF-interacted proteins inhibited osteoblast differentiation. By studying the underlying mechanism, we found that cytoplasmic-nuclear localization of MACF1 was dependent on its level and the cytoplasmic-nuclear localization of TCF12 and E2F6 was regulated by MACF1 localization. In addition, MACF1 oppositely regulated the transcription activity of TCF12 and TCF7. Current study, for the first time to our knowledge, suggest that MACF1 acts as a sponge of osteoblast differentiation repressors to promote osteoblast differentiation, and indicate a novel mechanism for regulating the cellular location of transcription factors by a protein associated with microtubule and actin.
Project description:MC3T3-E1 cells were transfected with short hairpin RNA (shRNA) specifically targeting the murine MACF1 lentivirus vector or with scrambled shRNA, and the stably transfected cell lines were selected using puromycin. After 15 days of selection, all cells were collected for further study.
Project description:Our preliminary data shows that dietary ACNs increased osteoblast differentiation and function in human osteoblasts, as well as increased osteoblast differentiation and reduced bone resorption in transgenic medaka. In cultured human osteoblasts, ACNs and resveratrol enhanced growth, reduced apoptosis and increased the expression of HDACs and PGC-1, suggesting that ACNs alter epigenetic regulation and mitochondrial biogenesis