[PMC free article] [PubMed] [Google Scholar]Sawai CM, Freund J, Oh P, Ndiaye-Lobry D, Bretz JC, et al. analyses revealed that D-cyclins repress the expression of the death receptor, Fas and its ligand, FasL. These findings provide a mechanism linking a cyclin protein to cell survival in normal homeostasis following ablation of D-cyclins (Figure 5C). Open in a separate window Figure 4 Molecular Analyses of Hematopoietic Cells Following Cyclin D Shutdown(A) Strategy for the identification of genes with altered expression upon ablation of D-cyclins. Bone marrows from MxTKO and MxCntrl mice (n = 4 per group) were collected 48 hrs after one dose of pI-pC (to delete D-cyclins in MxTKO mice) and analyzed for the expression levels of apoptotic genes by RT-PCR-based array (See Supplemental BMY 7378 Experimental Procedures). (B) Volcano plot analysis using web-based software for Apoptosis RT2 Profiler? Apoptosis PCR SArray PAMM-3012 (Qiagen) for 4 biological replicates from MxTKO and MxCntrl mice. The volcano plot displays significance of changes (-Log10 p-values) versus fold-change (Log2 fold change TKO/Cntrl) on the y- and x-axes, respectively. Eight genes encircled in red satisfied the following criteria: genes that had >2-fold increased (red vertical line) or decreased Rabbit polyclonal to ZNF624.Zinc-finger proteins contain DNA-binding domains and have a wide variety of functions, mostof which encompass some form of transcriptional activation or repression. The majority ofzinc-finger proteins contain a Krppel-type DNA binding domain and a KRAB domain, which isthought to interact with KAP1, thereby recruiting histone modifying proteins. Zinc finger protein624 (ZNF624) is a 739 amino acid member of the Krppel C2H2-type zinc-finger protein family.Localized to the nucleus, ZNF624 contains 21 C2H2-type zinc fingers through which it is thought tobe involved in DNA-binding and transcriptional regulation (blue vertical line) expression in MxTKO, as compared to MxCntrl; The significance of changes had p-value <0.05 (thin horizontal line). (C) Results of RT-PCR analysis for eight genes which were identified by Apoptosis PCR Array as having altered expression (>2 fold change, p<0.05) in cyclin D-deleted bone marrows. Each dot shows the fold-change expression in MxTKO versus MxCntrl bone marrows, for each of the 4 independent biological replicates. Horizontal lines denote mean fold-change for the indicated genes, red dashed line = no change (ratio of 1 1). See also Table S1. Open in a separate window Figure 5 Fas/FasL Signaling Is a Critical Mediator of Apoptosis Induced by Cyclin D Ablation(A) FACS analysis of Fas and FasL surface expression in MxTKO and MxCntrl bone marrow cells 2 days after ablation of D-cyclins. Note increased staining in MxTKO as compared to MxCntrl. (B) Western blot analysis of Fas protein levels in bone marrow 2 days after ablation of D-cyclins. (C) Upregulation of Fas and FasL mRNA levels in hematopoietic stem/progenitor cells (HSPC) 24 hours after ablation of D-cyclins (MxTKO). N=4 mice for each genotype, error bars, SD. (D) Intracellular staining for cleaved caspase 8 in bone marrow cells 2 days after cyclin D-deletion. Note increased staining in MxTKO as compared to MxCntrl. (E) Western blot analysis of the levels of cleaved caspase 8 in bone marrow cells of MxCntrl and MxTKO mice, three days after pI-pC injection BMY 7378 (to delete D-cyclins in MxTKO). (F and G) MxTKO and MxCntrl mice were injected with neutralizing anti-FasL antibody (FasL), or with isotype control (IgG) concomitantly with administration of pI-pC (to delete D-cyclins in MxTKO mice). Two days later, hematopoietic stem/progenitor cells, HSPC (F), or hematopoietic stem cells, HSC (G) were flow sorted and stained with Annexin V and 7AAD to mark apoptotic (Annexin V+/7AAD+) cells. Note that inhibition of FasL abrogated apoptosis of HSC and HSPC triggered by ablation of D-cyclins (compare MxTKO-IgG vs. MxTKO-FasL). (F) Shows representative staining with Annexin V 7AAD. (G) Mean percentage of Annexin V+ HSC (n=3 mice for MxCntrl and n=5 MxTKO). Error bars, SD. See also Figure S3. Interaction of the death receptor Fas with its ligand, FasL triggers cell death via an extrinsic apoptotic process that involves activation of caspase 8 (Strasser et al., 2009). Indeed, we observed activation of caspase 8 following cyclin D deletion in hematopoietic cells (Figures 5D and 5E). At these early BMY 7378 time-points we detected no changes in mitochondrial membrane.