Search

The histone methyltransferase SET domain bifurcated 1 (SETDB1) catalyzes the trimethylation of lysine 9 of histone H3, thereby regulating gene expression. In this study, we used conditional knockout mice, where Setdb1 was deleted only in neural crest cells (Setdb1 fl/fl , Wnt1 -Cre + mice), to clarify the role of SETDB1 in palatal development. Setdb1 fl/fl , Wnt1 -Cre + mice died shortly after birth due to a cleft palate with full penetration. Reduced palatal mesenchyme proliferation was seen in Setdb1 fl/fl , Wnt1 -Cre + mice, which might be a possible mechanism of cleft palate development. Quantitative RT-PCR and in situ hybridization showed that expression of the Pax9 , Bmp4, Bmpr1a, Wnt5a, and Fgf10 genes, known to be important for palatal development, were markedly decreased in the palatal mesenchyme of Setdb1 fl/fl , Wnt1 -Cre + mice. Along with these phenomena, SMAD1/5/9 phosphorylation was decreased by the loss of Setdb1. Our results demonstrated that SETDB1 is indispensable for palatal development partially through its proliferative effect. Taken together with previous reports that PAX9 regulates BMP signaling during palatal development which implies that loss of Setdb1 may be involved in the cleft palate development by decreasing SMAD-dependent BMP signaling through Pax9. • Loss of Setdb1 causes cleft palate. • Loss of Setdb1 inhibits cell proliferation in the palatal mesenchyme. • Loss of Setdb1 downregulates genes important for palatal development. • Loss of Setdb1 inhibits the SMAD-dependent BMP signaling pathway. [ABSTRACT FROM AUTHOR]

Our previous research suggested the presence of a novel SETDB1-mediated FosB pathway that could be responsible for the regulation of cell proliferation and invasiveness during anticancer treatments. In this study, we prepared FosB knock-out (FosB-KO) A549 human lung cancer cells using the CRISPR/Cas9 system and examined the physiological and molecular changes that caused. Annexin V and TUNEL assays showed that FosB-KO clones were less sensitive to doxorubicin treatment compared to the control A549 cells. Bcl2 expression and mitochondrial membrane potential were also both markedly increased in FosB-KO clones, which suggests the involvement of Bcl2 in the doxorubicin mediated increase in cell viability demonstrated the FosB-KO clones. Moreover, we identified changes in the migration and transforming activities of the FosB-KO clones that coincided with changes in the expression levels of E -cadherin, β-catenin, and Vimentin. RT-PCR and qPCR analysis showed that the expressions of Bcl2, E -cadherin, β-catenin, and Vimentin were regulated at the transcriptional level. Importantly, FosB overexpression in FosB-KO clones restored the expression of Bcl2, Akt, E-cad, β-catenin, and Vimentin, suggesting that those proteins were tightly regulated by FosB. These data suggest that the FosB gene critically regulates both drug sensitivity and invasion related genes, and does so in a manner coordinated with the function of SETDB1. Therefore, we propose that the FosB gene regulates both drug sensitivity and invasion activity related genes, and also shows coordinated function with SETDB1 for the regulation of target proteins. • FosB-KO decreases sensitivity to doxorubicin treatment through increased levels of Bcl2. • Cell migration and transforming activities were affected by alterations to E -cadherin, β-catenin, and Vimentin in FosB-KO clones. • FosB overexpression restores Bcl2, E -cadherin, β-catenin, and Vimentin expression. • FosB shows coordinated function with SETDB1 for the regulation of target proteins. [ABSTRACT FROM AUTHOR]

SETDB1 HMTase participates in various cellular processes via epigenetic transcriptional regulation. SETDB1 expression is downregulated by anticancer drug treatment in cancer cells, but we still need to verify the functional significance on SETDB1 downregulation. CRISPR/cas9 is a useful technology for doing a knockout (KO) of a target gene. It is widely used to examine the function of genes. In this study, we prepared SETDB1-KO from A549 human lung cancer cells using the CRISPR/Cas9 system, and we compared molecular changes between the A549 cells and the SETDB1-KO cells. The SETDB1-KO cell proliferation rate was slightly decreased as compared to the A549 cells, but there was no large difference in sensitivity with doxorubicin treatment. Instead, the migration activity and transforming activity were dramatically increased in SETDB-KO cells. Using a western blot analysis and an immunostaining experiment, we confirmed that SETDB1-KO downregulates the expression of E-cadherin and β-catenin. A qPCR and an RT-PCR analysis suggested that SETDB1 transcriptionally regulates E-cadherin and β-catenin. Moreover, E-cadherin expression was also detected in the cytoplasmic region of SETDB1-KO cells, indicating that functional localization of E-cadherin might be changed in SETDB1-KO cells. On the other hand, total levels of STAT3 and Akt were increased in the SETDB1-KO cells, but activation of STAT3 (pSTAT3) was not induced in doxorubicin-treated SETDB1-KO cells. SETDB1 overexpression into SETDB1-KO cells restores the expression of E-cadherin, β-catenin, STAT3, and Akt, suggesting that those proteins are tightly regulated by SETDB1. Collectively, we suggest that complex regulations on E-cadherin, β-catenin, STAT3, and Akt are correlated with the increased migration and transforming activity of SETDB1-KO cells. • SETDB1-KO has no influence on doxorubicin sensitivity but increases migration and transforming activity. • SETDB1-KO downregulates expression of E-cadherin and ꞵ-catenin, whereas upregulates expression of STAT3 and Akt. • SETDB1 overexpression restores the expression of E-cadherin, β-catenin, STAT3, and Akt. • Complex regulations on those proteins are correlated with the increased migration and transforming activity of SETDB1-KO cells. [ABSTRACT FROM AUTHOR]

We have determined a functional link to the inverse expression of SETDB1 and FosB following anticancer drug treatment. Doxorubicin treatment caused decreased SETDB1 expression and FosB overexpression both at the mRNA and protein levels. The decreased HMTase activity of SETDB1 coincided with altered occupancy across the promoter region of the FosB gene. SETDB1 overexpression decreased the luciferase reporter activity containing the FosB promoter region, but siSETDB1 increased the luciferase reporter activity, suggesting that SETDB1 directly and negatively regulated FosB expression. In addition, MEK inhibitor (PD98059) blocked the SETDB1 regulation of the FosB promoter activity via ERK2 activation during doxorubicin treatment. A microscopic analysis reveals that FosB expression was observed in living cells in spite of doxorubicin treatment. Ectopic FosB/ΔFosB expression increased the number of colonies and the migration of A549 cells compared to that in control. These results suggest that the ERK2-SETDB1-FosB signaling pathway might have an anti-therapeutic regulatory mechanism that increases the transformation and migration activity of cancer cells during anticancer drug treatment. [ABSTRACT FROM AUTHOR]

The histone methyltransferase Setdb1 represses gene expression by catalyzing lysine 9 of histone H3 trimethylation. Given that the conventional knockout of Setdb1 is embryo-lethal at the implantation stage, its role in craniofacial development is poorly understood. Here, we investigated the role of Setdb1 , using conditional knockout mice—in which Setdb1 was deleted in the Meckel's cartilage ( Setdb1 CKO)—and the mouse chondrogenic cell line ATDC5—in which Setdb1 was inhibited by siRNA. Deletion of Setdb1 in Meckel's cartilage, the supportive tissue in the embryonic mandible, led to its enlargement, instead of the degeneration that normally occurs. Chondrocytes from the Meckel's cartilage of Setdb1 CKO mice showed increased size. Furthermore, at embryonic days 16.5 and 18.5, part of the perichondrium was disrupted and mineralization was observed in the Meckel's cartilage. Proliferation analysis showed that inhibition of Setdb1 caused increased proliferation in chondrocytes in the Meckel's cartilage as well as in ATDC5 cells. Quantitative RT-PCR showed decreased expression of chondrogenic genes, such as S ox9, Mmp13, Collagen II , and Aggrecan , as a result of Setdb1 inhibition in ATDC5 cells. Along with these phenomenons, SMAD-dependent BMP signaling was significantly increased by the loss of Setdb1 in both the Meckel's cartilage of Setdb1 CKO mice and ATDC5 cells. Therefore, the abnormal development of Meckel's cartilage in Setdb1 CKO mice is partly due to the enhanced SMAD-dependent BMP signaling. Overall, to our knowledge, the present study is the first to show that epigenetic regulation by Setdb1 is indispensable for the embryonic development of Meckel's cartilage. [ABSTRACT FROM AUTHOR]

SET domain, bifurcated 1 (SETDB1) is a histone methyltransferase that methylates lysine 9 on histone H3. Although it is important to know the localization of proteins to elucidate their physiological function, little is known of the subcellular localization of human SETDB1. In the present study, to investigate the subcellular localization of hSETDB1, we established a human cell line constitutively expressing enhanced green fluorescent protein fused to hSETDB1. We then generated a monoclonal antibody against the hSETDB1 protein. Expression of both exogenous and endogenous hSETDB1 was observed mainly in the cytoplasm of various human cell lines. Combined treatment with the nuclear export inhibitor leptomycin B and the proteasome inhibitor MG132 led to the accumulation of hSETDB1 in the nucleus. These findings suggest that hSETDB1, localized in the nucleus, might undergo degradation by the proteasome and be exported to the cytosol, resulting in its detection mainly in the cytosol. [ABSTRACT FROM AUTHOR]

Highlights: [•] PTX down-regulates SETDB1 gene expression during cell death via G2/M arrest in human lung cancer cells. [•] SETDB1 gene expression could be directly regulated by p53 protein. [•] p53 Can bind to promoter regions of SETDB1, and also change H3K9me3 status in this region. [•] p53 Interacts with SUV39H1, and the increased H3K9me3 by SUV39H1 influences transcription repression of SETDB1. [ABSTRACT FROM AUTHOR]

Highlights: [•] DZNep treatment affected histone methylations, including H3K27me3 and H3K9me3 in human lung cancer cells. [•] Reduced levels of H3K27me3 and H3K9me3 were related with the decreased EZH2 and SETDB1 proteins. [•] Real time PCR analysis and luciferase assay showed that SETDB1 gene expression was decreased by DZNep treatment. [•] The present study emphasizes that mechanism of action of DZNep targets multiple HMTases during the death of lung cancer cells. [Copyright &y& Elsevier]