Kit/SCF signaling and Mitf-dependent transcription are both essential for melanocyte development and pigmentation. To identify Mitf-dependent Kit transcriptional targets in primary melanocytes, microarray studies were undertaken. Among identified targets was BCL2, whose germline deletion produces melanocyte loss and which exhibited phenotypic synergy with Mitf in mice. BCL2's regulation by Mitf was verified in melanocytes and melanoma cells and by chromatin immunoprecipitation of the BCL2 promoter. Mitf also regulates BCL2 in osteoclasts, and both Mitf(mi/mi) and Bcl2(-/-) mice exhibit severe osteopetrosis. Disruption of Mitf in melanocytes or melanoma triggered profound apoptosis susceptible to rescue by BCL2 overexpression. Clinically, primary human melanoma expression microarrays revealed tight nearest neighbor linkage for MITF and BCL2. This linkage helps explain the vital roles of both Mitf and Bcl2 in the melanocyte lineage and the well-known treatment resistance of melanoma.
The amino acid antiporter system Xc − is important for the synthesis of glutathione (GSH) that functions to prevent lipid peroxidation and protect cells from nonapoptotic, iron-dependent death (i.e., ferroptosis). While the activity of system Xc − often positively correlates with the expression level of its light chain encoded by SLC7A11, inhibition of system Xc − activity by small molecules (e.g., erastin) causes a decrease in the intracellular GSH level, leading to ferroptotic cell death. How system Xc − is regulated during ferroptosis remains largely unknown. Here we report that activating transcription factor 3 (ATF3), a common stress sensor, can promote ferroptosis induced by erastin. ATF3 suppressed system Xc − , depleted intracellular GSH, and thereby promoted lipid peroxidation induced by erastin. ATF3 achieved this activity through binding to the SLC7A11 promoter and repressing SLC7A11 expression in a p53-independent manner. These findings thus add ATF3 to a short list of proteins that can regulate system Xc − and promote ferroptosis repressed by this antiporter.
SUMMARY Increased activation of the serine-glycine biosynthetic pathway is an integral part of cancer metabolism that drives macromolecule synthesis needed for cell proliferation. Whether this pathway is under epigenetic control is unknown. Here we show that the histone H3 lysine 9 (H3K9) methyltransferase G9A is required for maintaining the pathway enzyme genes in an active state marked by H3K9 monomethylation and for the transcriptional activation of this pathway in response to serine deprivation. G9A inactivation depletes serine and its downstream metabolites, triggering cell death with autophagy in cancer cell lines of different tissue origins. Higher G9A expression, which is observed in various cancers and is associated with greater mortality in cancer patients, increases serine production and enhances the proliferation and tumorigenicity of cancer cells. These findings identify a G9A-dependent epigenetic program in the control of cancer metabolism, providing a rationale for G9A inhibition as a therapeutic strategy for cancer.
Renal fibrosis is the final, common pathway of end-stage renal disease. Whether and how autophagy contributes to renal fibrosis remains unclear. Here we first detected persistent autophagy in kidney proximal tubules in the renal fibrosis model of unilateral ureteral obstruction (UUO) in mice. UUOassociated fibrosis was suppressed by pharmacological inhibitors of autophagy and also by kidney proximal tubule-specific knockout of autophagy-related 7 (PT-Atg7 KO). Consistently, proliferation and activation of fibroblasts, as indicated by the expression of ACTA2/a-smooth muscle actin and VIM (vimentin), was inhibited in PT-Atg7 KO mice, so was the accumulation of extracellular matrix components including FN1 (fibronectin 1) and collagen fibrils. Tubular atrophy, apoptosis, nephron loss, and interstitial macrophage infiltration were all inhibited in these mice. Moreover, these mice showed a specific suppression of the expression of a profibrotic factor FGF2 (fibroblast growth factor 2). In vitro, TGFB1 (transforming growth factor b 1) induced autophagy, apoptosis, and FN1 accumulation in primary proximal tubular cells. Inhibition of autophagy suppressed FN1 accumulation and apoptosis, while enhancement of autophagy increased TGFB1-induced-cell death. These results suggest that persistent activation of autophagy in kidney proximal tubules promotes renal interstitial fibrosis during UUO. The profibrotic function of autophagy is related to the regulation on tubular cell death, interstitial inflammation, and the production of profibrotic factors.
Activation of oncogenes underlies the pathogenesis of most human cancers. In neuroblastoma, amplification of the oncogene MYCN occurs in ϳ22% of cases and is associated with advanced stages of the disease and poor prognosis. Identification of other oncogenes that are consistently mutated or overexpressed in neuroblastoma is crucial for a molecular understanding of the pathogenic process. Here, we report that the oncogene Bmi-1 is highly expressed in human neuroblastoma cell lines and primary tumors. Neuroblastoma development in MYCN transgenic mice, an animal model for the human disease, was associated with a marked increase in the levels of Bmi-1 expression. Bmi-1 cooperated with MYCN in transformation of benign S-type neuroblastoma cells and avian neural crest cells by inhibiting the apoptotic activity of MYCN. Importantly, down-regulation of Bmi-1 impaired the ability of neuroblastoma cells to grow in soft agar and induce tumors in immunodeficient mice. Moreover, Bmi-1-knockdown neuroblastoma xenografts were characterized by a significant increase in the amount of Schwannian stroma, a histological feature associated with clinically favorable neuroblastomas. These findings suggest a crucial role for Bmi-1 in neuroblastoma pathogenesis and provide insights into the molecular basis of neuroblastoma heterogeneity.
Histone H1 promotes the generation of a condensed, transcriptionally inactive, higher-order chromatin structure. Consequently, histone H1 activity must be antagonized in order to convert chromatin to a transcriptionally competent, more extended structure. Using simian virus 40 minichromosomes as a model system, we now demonstrate that the nonhistone chromosomal protein HMG-14, which is known to preferentially associate with active chromatin, completely alleviates histone H1-mediated inhibition of transcription by RNA polymerase II. HMG-14 also partially disrupts histone H1-dependent compaction of chromatin. Both the transcriptional enhancement and chromatin-unfolding activities of HMG-14 are mediated through its acidic, C-terminal region. Strikingly, transcriptional and structural activities of HMG-14 are maintained upon replacement of the C-terminal fragment by acidic regions from either GAL4 or HMG-2. These data support the model that the acidic C terminus of HMG-14 is involved in unfolding higher-order chromatin structure to facilitate transcriptional activation of mammalian genes.In mammalian cells, genomic DNA is highly condensed, being organized in the nucleoprotein complex constituting chromatin (65,70). The packaging of genomic DNA into chromatin can inhibit gene expression in multiple ways: restricting the access of transcription factors to their promoter elements, blocking assembly and initiation by the general transcriptional machinery, and inhibiting elongation by the RNA polymerase. As a corollary, activation of transcription requires remodeling of the chromatin structure of the gene in order to relieve the repressive effects of chromatin on transcription (34,38,45,71,72).The building block of chromatin is the nucleosome, consisting of DNA wrapped twice around a histone octamer comprising the core histones H2A, H2B, H3, and H4. A linker histone, generally histone H1, interacts with DNA at several sites simultaneously (3, 51, 55, 58): (i) with DNA at or close to the entry and exit points of the nucleosome, (ii) with nucleosomal DNA over the dyad axis, after one wrap around the octamer, and (iii) with linker DNA between adjacent nucleosomal core particles. Within the interphase nucleus of higher eukaryotic cells, the linear array of nucleosomes is folded into a higherorder structure, called the 30-nm chromatin fiber (65, 70). Histone H1 plays a key role in the formation of such a higherorder chromatin structure (4, 37, 52, 59, 60).Biochemical and genetic studies have identified two distinct systems capable of remodeling the nucleosome structure for transcriptional activation. The multisubunit SWI/SNF complex, which is conserved from yeasts to humans (49, 69), can perturb the structure of a nucleosome in an ATP-dependent manner (15,29,35). This promotes the binding in vitro of either specific or general transcription factors, such as GAL4 and TBP, to their sites on nucleosomal DNA. A distinct nucleosome-remodeling factor (NURF), originally purified from Drosophila embryo extracts (63), is also capable ...
SUMMARY The histone lysine demethylase KDM4C is often overexpressed in cancers primarily through gene amplification. The molecular mechanisms of KDM4C action in tumorigenesis are not well defined. Here we report that KDM4C transcriptionally activates amino acid biosynthesis and transport, leading to a significant increase in intracellular amino acid levels. Examination of the serine-glycine synthesis pathway reveals that KDM4C epigenetically activates the pathway genes under steady-state and serine deprivation conditions by removing the repressive histone modification H3 lysine 9 (H3K9) trimethylation. This action of KDM4C requires ATF4, a transcriptional master regulator of amino acid metabolism and stress responses. KDM4C activates ATF4 transcription and interacts with ATF4 to target serine pathway genes for transcriptional activation. We further present evidence for KDM4C in transcriptional coordination of amino acid metabolism and cell proliferation. These findings suggest a molecular mechanism linking KDM4C-mediated H3K9 demethylation and ATF4-mediated transactivation in reprogramming amino acid metabolism for cancer cell proliferation.
Cells contain a large pool of nonpumping Na/K-ATPase that participates in signal transduction. Here, we show that the expression of ␣1 Na/K-ATPase is significantly reduced in human prostate carcinoma as well as in several human cancer cell lines. This down-regulation impairs the ability of Na/KATPase to regulate Src-related signaling processes. A supplement of pNaKtide, a peptide derived from ␣1 Na/K-ATPase, reduces the activities of Src and Src effectors. Consequently, these treatments stimulate apoptosis and inhibit growth in cultures of human cancer cells. Moreover, administration of pNaKtide inhibits angiogenesis and growth of tumor xenograft. Thus, the new findings demonstrate the in vivo effectiveness of pNaKtide and suggest that the defect in Na/K-ATPase-mediated signal transduction may be targeted for developing new anticancer therapeutics. Na/K-ATPase was originally discovered as an ion pump that is essential for cell vitality and provides a means for epithelium to secrete and/or absorb solutes and nutrients (1, 2). Recent studies have revealed that in addition to pumping ions across the cell membrane, Na/K-ATPase, specifically the ␣1 isoform, conducts many nonpumping functions, including scaffolding and signal transduction. As a signaling protein, it is involved in the formation of membrane structures such as tight junction and caveolae (3, 4). Moreover, a large fraction of cellular Na/KATPase is involved in tethering and regulating multiple protein and lipid kinases as well as membrane receptors (e.g. Src, human epidermal growth factor receptor, and PI3K) in a cellspecific manner (5, 6). Recently, the ␣1 Na/K-ATPase-Src receptor complex has been identified as one of the central components of ␣1 Na/K-ATPase-mediated signaling transduction (7). In this receptor complex, the Src SH2 domain binds to the second cytosolic domain, whereas the Src kinase domain interacts with the nucleotide binding (N) domain 4 of the ␣1 subunit. The latter interaction keeps Src in an inactive state (7). It is important to note that normal epithelial cells express approximately one million ␣1 Na/K-ATPase molecules (roughly five times the amount of Src). Thus, ␣1 Na/K-ATPase could provide at least two ways of regulating cellular Src activity. First, it could bind and keep Src in an inactive state. Consistently, when knock-out of one copy of the ␣1 gene caused a 20 -30% reduction in cellular ␣1 Na/K-ATPase, it produced a Ͼ2-fold increase in Src and ERK activities in tissues of ␣1 ϩ/Ϫ mice (8). Second, formation of the Na/K-ATPase-Src complex provides a functional receptor for endogenous cardiotonic steroids such as ouabain to regulate cellular signaling via Src and Src effectors (7, 9). Thus, changes in cellular ␣1 Na/K-ATPase would have a significant effect on cellular signaling events induced by either cardiotonic steroids or other growth factors through Src-related pathways.Based on the fact that the N domain of the ␣1 subunit binds and inhibits Src, we have recently made pNaKtide from the N domain of the human ␣1 subunit of...
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