Epigenetic silencing of RASSF (Ras association domain family) genes RASSF1 and RASSF5 (also called NORE1) by CpG hypermethylation is found frequently in many cancers. Although the physiological roles of RASSF1 have been studied in some detail, the exact functions of RASSF5 are not well understood. Here, we show that RASSF5 plays an important role in mediating apoptosis in response to death receptor ligands, TNF-␣ and TNF-related apoptosis-inducing ligand. Depletion of RASSF5 by siRNA significantly reduced TNF-␣-mediated apoptosis, likely through its interaction with proapoptotic kinase MST1, a mammalian homolog of Hippo. Consistent with this, siRNA knockdown of MST1 also resulted in resistance to TNF-␣-induced apoptosis. To further study the role of Rassf5 in vivo, we generated Rassf5-deficient mouse. Inactivation of Rassf5 in mouse embryonic fibroblasts (MEFs) resulted in resistance to TNF-␣-and TNF-related apoptosis-inducing ligandmediated apoptosis. Importantly, Rassf5-null mice were significantly more resistant to TNF-␣-induced apoptosis and failed to activate Mst1. Loss of Rassf5 also resulted in spontaneous immortalization of MEFs at earlier passages than the control MEFs, and Rassf5-null immortalized MEFs, but not the immortalized wild type MEFs, were fully transformed by K-RasG12V. Together, our results demonstrate a direct role for RASSF5 in death receptor ligand-mediated apoptosis and provide further evidence for RASSF5 as a tumor suppressor.A small family of genes termed RASSF (Ras association domain family) has been recently described, the members of which are characterized by the presence of a Ras association (RA) 4 domain and a novel motif named the SARAH (Salvador, Rassf, Hippo) domain at the C terminus (reviewed in Refs. 1-3). Among the members of the RASSF family, RASSF1 and RASSF5 (also known as NORE1, for novel Ras effector 1) share the closest homology, displaying 49% identity (66% similarity) at the protein level, and are frequently inactivated by CpG hypermethylation in human cancer cell lines and primary tumors (4 -6). RASSF1 has been studied extensively (reviewed in Ref. 7) and shown to play important roles in mitosis (8), microtubule and genomic stability (9 -11), apoptosis (12, 13), and cell cycle (14), which are consistent with its function as a tumor suppressor. However, not much is known about the physiological roles of RASSF5.RASSF5 encodes multiple isoforms because of dual promoter usage and alternative splicing (15). The longest isoform, designated RASSF5A (NORE1A), is transcribed from the 5Ј-most promoter region containing CpG island and encodes a 418-amino acid protein containing the cysteine-rich diacylglycerol/ phorbol ester-binding domain (also called protein kinase C conserved region 1, C1), the RA domain, and the C-terminal SARAH domain. Through alternative splicing, another isoform RASSF5B (NORE1A␤), lacking the SARAH domain is produced. The shortest isoform, RASSF5C (NORE1B, also called RAPL), lacking the N-terminal C1 domain, is produced from a downstream CpG-containing ...

Summary A recent surge in obesity has given impetus to better understand the mechanisms of adipogenesis, particularly brown adipose tissue (BAT) due to its potential utilization for anti-obesity therapy. Postnatal brown adipocytes arise from early muscle-progenitors but how brown fat lineage is determined is not completely understood. Here, we show that a multifunctional protein EWS (Ewing Sarcoma) is essential for determining brown fat lineage during development. BATs from Ews-null embryos and newborns are developmentally arrested. Ews mutant brown preadipocytes fail to differentiate due to loss of Bmp7 expression, a critical early brown adipogenic factor. We demonstrate that EWS, along with its binding partner YBX1 (Y-box binding protein 1), activates Bmp7 transcription. Depletion of either Ews or Ybx1 leads to loss of Bmp7 expression and brown adipogenesis. Remarkably, Ews-null BATs and brown preadipocytes ectopically express myogenic genes. These results demonstrate that EWS is essential for early brown fat lineage determination.

Promyelocytic leukemia zinc finger (PLZF) protein is a sequence-specific DNA-binding protein that represses the transcriptional activity of target genes such as those for cyclin A and the interleukin-3 receptor ␣ chain. The PLZF gene becomes fused to the retinoic acid receptor ␣ gene as a result of the t(11, 17)(q23;q21) chromosomal translocation that is associated with acute promyelocytic leukemia. We now show that endogenous PLZF in human promyelocytic leukemia HL-60 cells is modified by conjugation with SUMO-1 (small ubiquitin-related modifier-1) and that PLZF colocalizes with SUMO-1 in the nucleus of transfected human embryonic kidney 293T cells. Site-directed mutagenesis identified lysine 242 in the RD2 domain of human PLZF as the sumoylation site. A luciferase reporter gene assay suggested that SUMO-1 modification of this residue is required for transcriptional repression by PLZF, and an electrophoretic mobility shift assay showed that this modification increases the DNA binding activity of PLZF. PLZFmediated regulation of the cell cycle and transcriptional repression of the cyclin A2 gene were also dependent on sumoylation of PLZF on lysine 242. These results demonstrate that PLZF is modified by SUMO-1 conjugation and that this modification regulates the biological functions of PLZF.Promyelocytic leukemia zinc finger protein (PLZF) 1 is a sequence-specific DNA-binding transcriptional regulator. It comprises 673 amino acids and contains nine Kruppel-like C 2 H 2 zinc finger domains and an NH 2 -terminal POZ (Pox virus and zinc finger) or BTB (broad complex, tramtrack, bric-a-brac) domain (1, 2). PLZF is expressed in human CD34 ϩ myeloid progenitor cells and primitive multipotent cell lines, and its gene is fused to that of retinoic acid receptor ␣ (RAR␣) in a variant of acute promyelocytic leukemia (APL) associated with the t(11, 17)(q23;q21) chromosomal translocation (2). Whereas RAR␣ activates key genes required for normal myelopoiesis, the PLZF-RAR␣ fusion protein represses the expression of some of these genes in a dominant negative manner. PLZF colocalizes with the PML (promyelocytic leukemia) protein, suggesting a possible link with the pathogenesis of the more common t(15, 17)-associated form of APL (3-5).In its role as a transcriptional repressor, PLZF binds to the promoter of target genes, including those for cyclin A2 and the interleukin-3 receptor ␣ chain (IL-3R␣), and interacts with the corepressors N-CoR, SMRT, and Sin3A as well as histone deacetylases. The POZ/BTB domain overlaps with a transcriptional repression domain (RD1) and mediates PLZF self-association (2, 4) and interaction with histone deacetylases. A second transcriptional repression domain (RD2) is located downstream of the POZ domain and interacts with the ETO-AML1 protein in t(8, 21)-associated acute myelocytic leukemia; this protein may function as a PLZF corepressor (6, 7).During development, PLZF contributes to patterning of the limb and axial skeleton by acting as an upstream regulator of Hox gene expression (8 -10). PLZF is...

The oncogenic fusion gene EWS-WT1 is the defining chromosomal translocation in desmoplastic small roundcell tumors (DSRCT), a rare but aggressive soft tissue sarcoma with a high rate of mortality. EWS-WT1 functions as an aberrant transcription factor that drives tumorigenesis, but the mechanistic basis for its pathogenic activity is not well understood. To address this question, we created a transgenic mouse strain that permits physiologic expression of EWS-WT1 under the native murine Ews promoter. EWS-WT1 expression led to a dramatic induction of many neuronal genes in embryonic fibroblasts and primary DSRCT, most notably the neural reprogramming factor ASCL1. Mechanistic analyses demonstrated that EWS-WT1 directly bound the proximal promoter of ASCL1, activating its transcription through multiple WT1-responsive elements. Conversely, EWS-WT1 silencing in DSRCT cells reduced ASCL1 expression and cell viability. Notably, exposure of DSRCT cells to neuronal induction media increased neural gene expression and induced neurite-like projections, both of which were abrogated by silencing EWS-WT1. Taken together, our findings reveal that EWS-WT1 can activate neural gene expression and direct partial neural differentiation via ASCL1, suggesting agents that promote neural differentiation might offer a novel therapeutic approach to treat DSRCT. Cancer Res; 74(16); 4526-35. Ó2014 AACR.