FACT maintains nucleosomes during transcription and stem cell viability in adult mice.

Preservation of nucleosomes during replication has been extensively studied, while the maintenance of nucleosomes during transcription has gotten less attention. The histone chaperone FACT has a role in transcription elongation, although whether it disassembles or assembles nucleosomes during this process is unclear. To elucidate the function of FACT in mammals, we deleted the Ssrp1 subunit of FACT in adult mice. FACT loss is lethal, possibly due to the loss of the earliest progenitors in bone marrow and intestine, while more differentiated cells are not affected. Using cells isolated from several tissues, we show that FACT loss reduces the viability of stem cells but not of cells differentiated in vitro. FACT depletion increases chromatin accessibility in a transcription-dependent manner in adipose mesenchymal stem cells, indicating that nucleosomes are lost in these cells during transcription in the absence of FACT. We also observe activation of interferon (IFN) signaling and the accumulation of immunocytes in organs sensitive to FACT loss. Our data indicate that FACT maintains chromatin integrity during transcription in mammalian adult stem cells, suggesting that chromatin transcription in stem cells and differentiated cells is different.

Ras alters epithelial-mesenchymal transition in response to TGFbeta by reducing actin fibers and cell-matrix adhesion.

TGF-beta and Ras regulate epithelial-mesenchymal transition (EMT), a process that contributes to tumor invasion and metastasis. The interaction of these pathways in EMT is still poorly understood. Here, we show that TGF-beta induces EMT but limits cell invasion whereas hyperactivated Ras (H-RasV12) does not cause EMT but enhances cell invasion, alleviating the inhibitory effect of TGF-beta. TGF-beta disrupts cell junctions and induces tropomyosin-mediated actin fibers and matrix adhesion. Smad transcription factors mediate both steps of the TGF-beta-induced EMT whereas RasV12 inhibits the second step by blocking the induction of tropomyosins (TPM1) and reducing cell-matrix adhesion and integrin signaling. RasV12 prevents binding of Smads to the TPM1 promoter by forcing CRM1-dependent nuclear export of Smad4. Soft agar and animal studies demonstrate that RasV12 confers the metastatic potential in epithelial cells, whereas tropomyosin suppresses tumor growth and metastases. Thus, TGF-beta-induced EMT is not sufficient for the acquisition of the invasive potential and activated Ras alters this TGF-beta response, conferring the tumorigenic and invasive potential.

Silencing of the Tropomyosin-1 gene by DNA methylation alters tumor suppressor function of TGF-beta.

Loss of actin stress fibers has been associated with cell transformation and metastasis. TGF-beta induction of stress fibers in epithelial cells requires high molecular weight tropomyosins encoded by TPM1 and TPM2 genes. Here, we investigated the mechanism underlying the failure of TGF-beta to induce stress fibers and inhibit cell migration in metastatic cells. RT-PCR analysis in carcinoma cell lines revealed a significant reduction in TPM1 transcripts in metastatic MDA-MB-231, MDA-MB-435 and SW620 cell lines. Treatment of these cells with demethylating agent 5-aza-2'-deoxycytidine (5-aza-dC) increased mRNA levels of TPM1 with no effect on TPM2. Importantly, 5-aza-dC treatment of MDA-MB-231 cells restored TGF-beta induction of TPM1 and formation of stress fibers. Forced expression of TPM1 by using Tet-Off system increased stress fibers in MDA-MB-231 cells and reduced cell migration. A potential CpG island spanning the TPM1 proximal promoter, exon 1, and the beginning of intron 1 was identified. Bisulfite sequencing showed significant cytosine methylation in metastatic cell lines that correlated with a reduced expression of TPM1. Together these results suggest that epigenetic suppression of TPM1 may alter TGF-beta tumor suppressor function and contribute to metastatic properties of tumor cells.