Myocardin-related transcription factor B (MRTFB) is a candidate tumor-suppressor gene identified in transposon mutagenesis screens of the intestine, liver, and pancreas. Using a combination of cell-based assays, in vivo tumor xenograft assays, and Mrtfb knockout mice, we demonstrate here that MRTFB is a human and mouse colorectal cancer (CRC) tumor suppressor that functions in part by inhibiting cell invasion and migration. To identify possible MRTFB transcriptional targets, we performed whole transcriptome RNA sequencing in MRTFB siRNA knockdown primary human colon cells and identified 15 differentially expressed genes. Among the top candidate tumor-suppressor targets were melanoma cell adhesion molecule (MCAM), a known tumor suppressor, and spindle apparatus coiled-coil protein 1 (SPDL1), which has no confirmed role in cancer. To determine whether these genes play a role in CRC, we knocked down the expression of MCAM and SPDL1 in human CRC cells and showed significantly increased invasion and migration of tumor cells. We also showed that Spdl1 expression is significantly down-regulated in Mrtfb knockout mouse intestine, while lower SPDL1 expression levels are significantly associated with reduced survival in CRC patients. Finally, we show that depletion of MCAM and SPDL1 in human CRC cells significantly increases tumor development in xenograft assays, further confirming their tumor-suppressive roles in CRC. Collectively, our findings demonstrate the tumor-suppressive role of MRTFB in CRC and identify several genes, including 2 tumor suppressors, that act downstream of MRTFB to regulate tumor growth and survival in CRC patients.
UNC-45B is a multidomain molecular chaperone that is essential for the proper folding and assembly of myosin into muscle thick filaments in vivo. We have previously demonstrated that its UCS domain is responsible for the chaperone-like properties of UNC-45B. In order to better understand the chaperoning function of the UCS domain we engineered mutations designed to: i) disrupt chaperone-client interactions by removing and altering the structure of the putative client-interacting loop and ii) disrupt chaperone-client interactions by changing highly conserved residues in the putative client-binding groove. We tested the effect of these mutations by using a novel combination of complementary biophysical (circular dichroism, intrinsic tryptophan fluorescence, chaperone activity, and SAXS) and in vivo tools (C. elegans sarcomere structure). Removing the client-holding loop had a pronounced effect on the secondary structure, thermal stability, solution conformation and chaperone function of the UCS domain. These results are consistent with previous in vivo findings that this mutation neither rescue the defect in C. elegans sarcomere organization nor bind to myosin. We found that mutating several conserved residues in the client-binding groove do not affect UCS domain secondary structure or structural stability but reduced its chaperoning activity. We found that these groove mutations also significantly altered the structure and organization of the worm sarcomeres. We also tested the effect of R805W, a mutation distant from the client-binding region. Our in vivo data show that, to our surprise, the R805W mutation appeared to have the most drastic effect on the structure and organization of the worm sarcomeres. In humans, the R805W mutation segregates with human congenital/infantile cataract, indicating a crucial role of R805 in UCS domain stability and/or client interaction. Hence, our experimental approach combining biophysical and biological tools facilitates the study of myosin/chaperone interactions in mechanistic detail.
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