SOX4 reversibly induces phenotypic changes by suppressing the epithelial marker genes in human keratinocytes.

BACKGROUND: SOX4 is a transcription factor belonging to the SOX (Sry-related High Mobility Group [HMG] box) family and plays a pivotal role in various biological processes at various stages of life. SOX4 is also expressed in the skin in adults and has been reported to be involved in wound healing, tumor formation, and metastasis. METHODS AND RESULTS: In this study, we investigated the role of SOX4 in keratinocyte phenotypic changes. We generated a SOX4-overexpressing keratinocyte cell line that expresses SOX4 in a doxycycline (DOX)-inducible manner. DOX treatment induced a change from a paving stone-like morphology to a spindle-like morphology under microscopic observation. Comprehensive gene analysis by RNA sequencing revealed increased expression of genes related to anatomical morphogenesis and cell differentiation as well as decreased expression of genes related to epithelial formation and keratinization, suggesting that SOX4 induced EMT-like phenotype in keratinocytes. Differentially expressed genes (DEGs) obtained by RNA-seq were confirmed using qRT-PCR. DOX-treated TY-1 SOX4 showed a decrease in the epithelial markers (KRT15, KRT13, KRT5, and CLDN1) and an increase in the mesenchymal marker FN1. Protein expression changes by Western blotting also showed a decrease in the epithelial marker proteins keratin 15, keratin 13, and claudin 1, and an increase in the mesenchymal marker fibronectin. Removal of DOX from DOX-treated cells also restored the epithelial and mesenchymal markers altered by SOX4. CONCLUSION: Our results indicate that SOX4 reversibly induces an EMT-like phenotype in human keratinocytes via suppression of epithelial marker genes.

SNAI2 is induced by transforming growth factor-ß1, but is not essential for epithelial-mesenchymal transition in human keratinocyte HaCaT cells.

Epithelial-mesenchymal transition (EMT) is a cellular process in which epithelial cells lose their epithelial traits and shift to the mesenchymal phenotype, and is associated with various biological events, such as embryogenesis, wound healing and cancer progression. The transcriptional program that promotes phenotype switching is dynamically controlled by transcription factors during EMT, including Snail (SNAI1), twist family bHLH transcription factor (TWIST) and zinc finger E-box binding homeobox 1 (ZEB1). The present study aimed to investigate the molecular mechanisms underlying EMT in squamous epithelial cells. Western blot analysis and immunocytochemical staining identified Slug (SNAI2) as a transcription factor that is induced during transforming growth factor (TGF)-ß1-mediated EMT in the human keratinocyte cell line HaCaT. The effect of SNAI2 overexpression and knockdown on the phenotypic characteristics of HaCaT cells was evaluated. Filamentous actin staining and western blot analysis revealed that the overexpression of SNAI2 did not induce the observed EMT-related phenotypic changes. In addition, SNAI2 knockdown demonstrated almost no impact on the EMT phenotypes induced by TGF-ß1. Notably, DNA microarray analysis followed by comprehensive bioinformatics analysis revealed that the differentially expressed genes upregulated by TGF-ß1 were significantly enriched in cell adhesion and extracellular matrix binding, whereas the genes downregulated in response to TGF-ß1 were significantly enriched in the cell cycle. No enriched gene ontology term and biological pathways were identified in the differentially expressed gene sets of SNAI2-overexpressing cells. In addition, the candidates for master transcription factors regulating the TGF-ß1-induced EMT were identified using transcription factor enrichment analysis. In conclusion, the results of study demonstrated that SNAI2 does not play an essential role in the EMT of HaCaT cells and identified candidate transcription factors that may be involved in EMT-related gene expression induced by TGF-ß1. These findings may enhance the understanding of molecular events in EMT and contribute to the development of a novel therapeutic approach against EMT in cancers and wound healing.

SMARCAD1-mediated recruitment of the DNA mismatch repair protein MutLα to MutSα on damaged chromatin induces apoptosis in human cells.

The mismatch repair (MMR) complex is composed of MutSα (MSH2-MSH6) and MutLα (MLH1-PMS2) and specifically recognizes mismatched bases during DNA replication. O6-Methylguanine is produced by treatment with alkylating agents, such as N-methyl-N-nitrosourea (MNU), and during DNA replication forms a DNA mismatch (i.e. an O6-methylguanine/thymine pair) and induces a G/C to A/T transition mutation. To prevent this outcome, cells carrying this DNA mismatch are eliminated by MMR-dependent apoptosis, but the underlying molecular mechanism is unclear. In this study, we provide evidence that the chromatin-regulatory and ATP-dependent nucleosome-remodeling protein SMARCAD1 is involved in the induction of MMR-dependent apoptosis in human cells. Unlike control cells, SMARCAD1-knockout cells (ΔSMARCAD1) were MNU-resistant, and the appearance of a sub-G1 population and caspase-9 activation were significantly suppressed in the ΔSMARCAD1 cells. Furthermore, the MNU-induced mutation frequencies were increased in these cells. Immunoprecipitation analyses revealed that the recruitment of MutLα to chromatin-bound MutSα, observed in SMARCAD1-proficient cells, is suppressed in ΔSMARCAD1 cells. Of note, the effect of SMARCAD1 on the recruitment of MutLα exclusively depended on the ATPase activity of the protein. On the basis of these findings, we propose that SMARCAD1 induces apoptosis via its chromatin-remodeling activity, which helps recruit MutLα to MutSα on damaged chromatin.

Intramolecular Binding of the Rad9 C Terminus in the Checkpoint Clamp Rad9-Hus1-Rad1 Is Closely Linked with Its DNA Binding.

The human checkpoint clamp Rad9-Hus1-Rad1 (9-1-1) is loaded onto chromatin by its loader complex, Rad17-RFC, following DNA damage. The 120-amino acid (aa) stretch of the Rad9 C terminus (C-tail) is unstructured and projects from the core ring structure (CRS). Recent studies showed that 9-1-1 and CRS bind DNA independently of Rad17-RFC. The DNA-binding affinity of mutant 9(ΔC)-1-1, which lacked the Rad9 C-tail, was much higher than that of wild-type 9-1-1, suggesting that 9-1-1 has intrinsic DNA binding activity that manifests in the absence of the C-tail. C-tail added in trans interacted with CRS and prevented it from binding to DNA. We narrowed down the amino acid sequence in the C-tail necessary for CRS binding to a 15-aa stretch harboring two conserved consecutive phenylalanine residues. We prepared 9-1-1 mutants containing the variant C-tail deficient for CRS binding, and we demonstrated that the mutant form restored DNA binding as efficiently as 9(ΔC)-1-1. Furthermore, we mapped the sequence necessary for TopBP1 binding within the same 15-aa stretch, demonstrating that TopBP1 and CRS share the same binding region in the C-tail. Indeed, we observed their competitive binding to the C-tail with purified proteins. The importance of interaction between 9-1-1 and TopBP1 for DNA damage signaling suggests that the competitive interactions of TopBP1 and CRS with the C-tail will be crucial for the activation mechanism.

Interaction between Rad9-Hus1-Rad1 and TopBP1 activates ATR-ATRIP and promotes TopBP1 recruitment to sites of UV-damage.

The checkpoint clamp Rad9-Hus1-Rad1 (9-1-1) interacts with TopBP1 via two casein kinase 2 (CK2)-phosphorylation sites, Ser-341 and Ser-387 in Rad9. While this interaction is known to be important for the activation of ATR-Chk1 pathway, how the interaction contributes to their accumulation at sites of DNA damage remains controversial. Here, we have studied the contribution of the 9-1-1/TopBP1 interaction to the assembly and activation of checkpoint proteins at damaged DNA. UV-irradiation enhanced association of Rad9 with chromatin and its localization to sites of DNA damage without a direct interaction with TopBP1. TopBP1, as well as RPA and Rad17 facilitated Rad9 recruitment to DNA damage sites. Similar to Rad9, TopBP1 also localized to sites of UV-induced DNA damage. The DNA damage-induced TopBP1 redistribution was delayed in cells expressing a TopBP1 binding-deficient Rad9 mutant. Pharmacological inhibition of ATR recapitulated the delayed accumulation of TopBP1 in the cells, suggesting that ATR activation will induce more efficient accumulation of TopBP1. Taken together, TopBP1 and Rad9 can be independently recruited to damaged DNA. Once recruited, a direct interaction of 9-1-1/TopBP1 occurs and induces ATR activation leading to further TopBP1 accumulation and amplification of the checkpoint signal. Thus, we propose a new positive feedback mechanism that is necessary for successful formation of the damage-sensing complex and DNA damage checkpoint signaling in human cells.

Two serine phosphorylation sites in the C-terminus of Rad9 are critical for 9-1-1 binding to TopBP1 and activation of the DNA damage checkpoint response in HeLa cells.

A heteromeric proliferating cell nuclear antigen-like ring complex 9-1-1 is comprised of Rad9, Hus1 and Rad1. When assembled, 9-1-1 binds to TopBP1 and activates the ATR-Chk1 checkpoint pathway. This binding in vitro depends on the phosphorylation of Ser-341 and Ser-387 in Rad9 and is reduced to 70% and 20% by an alanine substitution for Ser-341 (S341A) and Ser-387 (S387A), respectively, and to background level by their simultaneous substitution (2A). Here, we show the importance of phosphorylation of these two serine residues in vivo. siRNA-mediated knockdown of Rad9 in HeLa cells impaired UV-induced phosphorylation of checkpoint kinase, Chk1, and conferred hypersensitivity to UV irradiation and to methyl methane sulfonate or hydroxyurea treatments. Either siRNA-resistant wild-type Rad9 (Rad9R(r)) or Rad9R(r) harboring the S341A substitution restored the phosphorylation of Chk1 and damage sensitivity, whereas Rad9R(r) harboring S387A or 2A did not. However, high expression of S387A restored Chk1 phosphorylation and partially suppressed the hypersensitivity. Thus, the affinity of Rad9 to TopBP1 correlates with the activation of the cellular DNA damage response and survival after DNA damage in HeLa cells, and phosphorylation of Ser-341 and Ser-387 of Rad9 is critical for full activation of the checkpoint response to DNA damage.

Casein kinase 2-dependent phosphorylation of human Rad9 mediates the interaction between human Rad9-Hus1-Rad1 complex and TopBP1.

The checkpoint clamp Rad9-Hus1-Rad1 (9-1-1) is loaded by the Rad17-RFC complex onto chromatin after DNA damage and plays a key role in the ATR-dependent checkpoint activation. Here, we demonstrate that in vitro casein kinase 2 (CK2) specifically interacts with human 9-1-1 and phosphorylates serines 341 and 387 (Ser-341 and Ser-387) in the C-terminal tail of Rad9. Interestingly, phosphorylated Ser-387 has previously been reported to be required for interacting with a checkpoint mediator TopBP1. Indeed, 9-1-1 purified from Escherichia coli and phosphorylated in vitro by CK2 physically interacts with TopBP1. Further analyses showed that phosphorylation at both serine residues occurs in vivo and is required for the efficient interaction with TopBP1 in vitro. Furthermore, when over-expressed in HeLa cells, a mutant Rad9 harboring phospho-deficient substitutions at both Ser-341 and Ser-387 residues causes hypersensitivity to UV and methyl methane sulfonate (MMS). Our observations suggest that CK2 plays a crucial role in the ATR-dependent checkpoint pathway through its ability to phosphorylate Ser-341 and Ser-387 of the Rad9 subunit of the 9-1-1 complex.