The C-terminal binding protein (CtBP) is a NADH-dependent transcriptional repressor that links carbohydrate metabolism to epigenetic regulation by recruiting diverse histone modifying complexes to chromatin. Here, global profiling of CtBP in breast cancer cells reveals that it drives epithelial to mesenchymal transition, stem cell pathways, and genome instability. CtBP expression induces mesenchymal and stem cell-like features while CtBP depletion or caloric restriction reverses gene repression and increases DNA repair. Multiple members of the CtBP-targeted gene network are selectively down-regulated in aggressive breast cancer subtypes. Differential expression of CtBP-targeted genes predicts poor clinical outcome in breast cancer patients, and elevated levels of CtBP in patient tumors predict shorter median survival. Finally, both CtBP promoter targeting and gene repression can be reversed by small molecule inhibition. These findings define broad roles for CtBP in breast cancer biology and suggest novel chromatin-based strategies for pharmacologic and metabolic intervention in cancer.

Studies in Saccharomyces cerevisiae indicate the histone variant H2A.Z is deposited at promoters by the chromatin remodeling protein Swr1 and plays a critical role in the regulation of transcription. In higher eukaryotes, however, little is known about the distribution, method of deposition, and function of H2A.Z at promoters. Using biochemical studies, we demonstrated previously that SRCAP (SNF-2-related CREB-binding protein activator protein), the human ortholog of Swr1, could catalyze deposition of H2A.Z into nucleosomes. To address whether SRCAP directs H2A.Z deposition in vivo, promoters targeted by SRCAP were identified by a chromatin immunoprecipitation (ChIP)-on-chip assay. ChIP assays on a subset of these promoters confirmed the presence of SRCAP on inactive and active promoters. The highest levels of SRCAP were observed on the active SP-1, G3BP, and FAD synthetase promoters. Detailed analyses of these promoters indicate sites of SRCAP binding overlap or occur adjacent to the sites of H2A.Z deposition. Knockdown of SRCAP levels using siRNA resulted in loss of SRCAP at these promoters, decreased deposition of H2A.Z and acetylated H2A.Z, and a decrease in levels of SP-1, G3BP, and FAD synthetase mRNA. Thus, these studies provide the first evidence that SRCAP is recruited to promoters and is critical for the deposition of H2A.Z.Chromatin remodeling has emerged as a key mechanism for gene regulation in development and cancer. The histone variant H2A.Z is a universally conserved intrinsic component of eukaryotic chromatin (1). Studies in Saccharomyces cerevisiae indicate that H2A.Z is required for normal gene expression, is distributed throughout the genome, and appears to be required for proper recruitment of RNA polymerase II (RNAP II) 2 and TATA-binding protein (TBP) (2). The highest levels of H2A.Z in S. cerevisiae occur within nucleosomes located at inactive promoters where it has been postulated to provide the correct promoter architecture to facilitate activation of transcription (3-5). Activation of transcription results in decreased levels of H2A.Z and an increase in acetylated H2A.Z, which has been proposed to facilitate disassembly/reassembly of nucleosomes (6 -8).In higher eukaryotes, the genomic distribution and the biological function(s) of H2A.Z are poorly defined. In mammals, H2A.Z is essential for embryonic development and chromosome segregation, and increased H2A.Z expression is implicated in cardiac hypertrophy (9 -11). Studies done in chicken cells suggest that deposition of both H2A.Z and acetylated H2A.Z in higher eukaryotes differs from that observed in S. cerevisiae and occurs at active promoters but not at inactive promoters (12, 13). The specific role that H2A.Z plays at active promoters in higher eukaryotes has not been established.The exchange of H2A.Z into nucleosomes in S. cerevisiae has been demonstrated by genetic and biochemical approaches to be carried out by the catalytic subunit of the SWR-C complex, termed Swr1 (14, 15). A SRCAP complex, which is the human ortholog of the...

The two best-characterized types of CD4+ regulatory T cells (Tregs) are Foxp3+ Tregs and Foxp3− type 1 regulatory (Tr1) cells. The ability of Foxp3+ Tregs and Tr1 cells to suppress adaptive immune responses is well known, but how these cells regulate innate immunity is less defined. We discovered that CD44hiFoxp3− T cells from unmanipulated mice are enriched in Tr1 cell precursors, enabling differentiation of cells that express IL-10, as well as Tr1-associated cell surface markers, CD49b and LAG-3, and transcription factors, cMaf, Blimp-1, and AhR. We compared the ability of Tr1 cells versus Foxp3+ Tregs to suppress IL-1β production from macrophages following LPS and ATP stimulation. Surprisingly, Tr1 cells, but not Foxp3+ Tregs, inhibited the transcription of pro–IL-1β mRNA, inflammasome-mediated activation of caspase-1, and secretion of mature IL-1β. Consistent with the role for IL-10 in Tr1 cell–mediated suppression, inhibition of inflammasome activation and IL-1β secretion was abrogated in IL-10R–deficient macrophages. Moreover, IL-1β production from macrophages derived from Nlrp3A350V knockin mice, which carry a mutation found in cryopyrin-associated periodic syndrome patients, was suppressed by Tr1 cells but not Foxp3+ Tregs. Using an adoptive transfer model, we found a direct correlation between Tr1 cell engraftment and protection from weight loss in mice expressing a gain-of-function NLRP3. Collectively, these data provide the first evidence for a differential role of Tr1 cells and Foxp3+ Tregs in regulating innate immune responses. Through their capacity to produce high amounts of IL-10, Tr1 cells may have unique therapeutic effects in disease-associated inflammasome activation.

Cell therapy with autologous donor-specific regulatory T cells (Tregs) is a promising strategy to minimize immunosuppression in transplant recipients. Chimeric antigen receptor (CAR) technology has recently been used successfully to generate donor-specific Tregs and overcome the limitations of enrichment protocols based on repetitive stimulations with alloantigens. However, the ability of CAR-Treg therapy to control alloreactivity in immunocompetent recipients is unknown. We first analyzed the effect of donor-specific CAR Tregs on alloreactivity in naive, immunocompetent mice receiving skin allografts. Tregs expressing an irrelevant or anti-HLA-A2-specific CAR were administered to Bl/6 mice at the time of transplanting an HLA-A2 + Bl/6 skin graft.Donor-specific CAR-Tregs, but not irrelevant-CAR Tregs, significantly delayed skin rejection and diminished donor-specific antibodies (DSAs) and frequencies of DSAsecreting B cells. Donor-specific CAR-Treg-treated mice also had a weaker recall DSA response, but normal responses to an irrelevant antigen, demonstrating antigen-specific suppression. When donor-specific CAR Tregs were tested in HLA-A2-sensitized mice, they were unable to delay allograft rejection or diminish DSAs. The finding that donor-specific CAR-Tregs restrain de novo but not memory alloreactivity has important implications for their use as an adoptive cell therapy in transplantation. K E Y W O R D S alloantigen, B cell biology, basic (laboratory) research/science, cellular biology, cellular transplantation (non-islet), immunosuppression/immune modulation, T cell biology, tolerance, translational research/science | 1563 SICARD et Al.

Insulin-like growth factor 2 mRNA-binding protein 1 (IGF2BP1) is an oncofetal protein expressed in various cancers including leukemia. In this study, we assessed the role of IGF2BP1 in orchestrating leukemia stem cell properties. Tumor-initiating potential, sensitivity to chemotherapeutic agents and expression of cancer stem cell markers were assessed in a panel of myeloid, B-, and T-cell leukemia cell lines using gain-and loss-of-function systems, cross-linking immunoprecipitation (CLIP), and photoactivatable ribonucleoside-enhanced crosslinking and immunoprecipitation (PAR-CLIP) techniques. Here we report that genetic or chemical inhibition of IGF2BP1 decreases leukemia cells' tumorigenicity, promotes myeloid differentiation, increases leukemia cell death, and sensitizes leukemia cells to chemotherapeutic drugs. IGF2BP1 affects proliferation and tumorigenic potential of leukemia cells through critical regulators of self-renewal HOXB4 and MYB and through regulation of expression of the aldehyde dehydrogenase, Users may view, print, copy, and download text and data-mine the content in such documents, for the purposes of academic research, subject always to the full Conditions of use:

BackgroundEpithelial ovarian cancer is a significant cause of mortality both in the United States and worldwide, due largely to the high proportion of cases that present at a late stage, when survival is extremely poor. Early detection of epithelial ovarian cancer, and of the serous subtype in particular, is a promising strategy for saving lives. The low prevalence of ovarian cancer makes the development of an adequately sensitive and specific test based on blood markers very challenging. We evaluated the performance of a set of candidate blood markers and combinations of these markers in detecting serous ovarian cancer.Methods and FindingsWe selected 14 candidate blood markers of serous ovarian cancer for which assays were available to measure their levels in serum or plasma, based on our analysis of global gene expression data and on literature searches. We evaluated the performance of these candidate markers individually and in combination by measuring them in overlapping sets of serum (or plasma) samples from women with clinically detectable ovarian cancer and women without ovarian cancer. Based on sensitivity at high specificity, we determined that 4 of the 14 candidate markers-MUC16, WFDC2, MSLN and MMP7-warrant further evaluation in precious serum specimens collected months to years prior to clinical diagnosis to assess their utility in early detection. We also reported differences in the performance of these candidate blood markers across histological types of epithelial ovarian cancer.ConclusionsBy systematically analyzing the performance of candidate blood markers of ovarian cancer in distinguishing women with clinically apparent ovarian cancer from women without ovarian cancer, we identified a set of serum markers with adequate performance to warrant testing for their ability to identify ovarian cancer months to years prior to clinical diagnosis. We argued for the importance of sensitivity at high specificity and of magnitude of difference in marker levels between cases and controls as performance metrics and demonstrated the importance of stratifying analyses by histological type of ovarian cancer. Also, we discussed the limitations of studies (like this one) that use samples obtained from symptomatic women to assess potential utility in detection of disease months to years prior to clinical detection.

The putative ATPase chromatin-remodeling machine SRCAP was identified in a yeast two-hybrid protein screen by interaction with the histone acetylase CBP. SRCAP is implicated in the transcriptional coactivation of cyclic AMP-and steroid-dependent promoters, but no natural chromosomal targets for SRCAP regulation have been identified. DOM is the unique SRCAP homolog in Drosophila melanogaster. The goal of this study was to test whether SRCAP is a functional homolog of DOM and to identify potential activities and targets of SRCAP in vivo. We show that human SRCAP complements recessive domino mutant phenotypes. This rescue depends on an intact ATPase homology domain. SRCAP colocalizes extensively with DOM on Drosophila polytene chromosomes and is recruited to sites of active transcription, such as steroid-regulated loci, but not to activated heat shock loci. We show that SRCAP recruits Drosophila CBP to ectopic chromosomal sites, providing the first evidence to suggest that SRCAP and CBP interact directly or indirectly on chromosomes. We show that DOM is a Notch pathway activator in Drosophila and that wild-type SRCAP-but not an ATPase domain mutant-can substitute for DOM in Notch-dependent wing development. We show that SRCAP potentiates Notch-dependent gene activation in HeLa cells. Taken together, these data implicate SRCAP and DOM in developmental gene activation.Most regulated gene expression in eukaryotes is the result of transcriptional regulation. Current models of transcriptional regulation invoke the targeted recruitment of enzymes that make covalent changes in histone and/or DNA structure, in ATPases that remodel histone-DNA interactions, and, in cases of gene activation, in RNA polymerase itself. A major challenge is to identify the diverse roles of chromatin modifiers in transcriptional regulation.SRCAP (SNF2-related CBP-activating protein) shares homology with members of the SNF2/SWI2 class of chromatinremodeling enzymes (20). SRCAP was originally identified in a yeast two-hybrid protein screen for proteins that interact with the histone acetyltransferase CBP (CREB-binding protein). Subsequent studies using transiently transfected reporters have implicated SRCAP as a coactivator for CREB-and nuclear hormone receptor-mediated transcriptional activation (30, 31). The mechanism by which SRCAP activates transcription has not been completely elucidated. Results from a number of studies, including mammalian two-hybrid and coimmunoprecipitation experiments, indicate SRCAP may function in part by recruitment of CBP or other coactivators, such as the arginine methyltransferase CARM-1 and the nuclear hormone receptor coactivator GRIP-1 (20, 31). A role for the conserved ATPase/helicase homology domain in regulation of transcription has not been established, since this activity is dispensable for SRCAP activity in transient transfection assays, nor have the natural chromosomal promoters that utilize SRCAP to regulate transcription been identified.In mammals, the protein most homologous to SRCAP is p400. p400 was ...

Profiling the dynamic interaction of p300 with proximal promoters of human T cells identified a class of genes that rapidly coassemble p300 and RNA polymerase II (pol II) following mitogen stimulation. Several of these p300 targets are immediate early genes, including FOS, implicating a prominent role for p300 in the control of primary genetic responses. The recruitment of p300 and pol II rapidly transitions to the assembly of several elongation factors, including the positive transcriptional elongation factor (P-TEFb), the bromodomain-containing protein (BRD4), and the elongin-like eleven nineteen lysine-rich leukemia protein (ELL). However, transcription at many of these rapidly induced genes is transient, wherein swift departure of P-TEFb, BRD4, and ELL coincides with termination of transcriptional elongation. Unexpectedly, both p300 and pol II remain accumulated or ''bookmarked'' at the proximal promoter long after transcription has terminated, demarking a clear mechanistic separation between the recruitment and elongation phases of transcription in vivo. The bookmarked pol II is depleted of both serine-2 and serine-5 phosphorylation of its C-terminal domain and remains proximally positioned at the promoter for hours. Surprisingly, these p300/pol II bookmarked genes can be readily reactivated, and elongation factors can be reassembled by subsequent addition of nonmitogenic agents that, alone, have minimal effects on transcription in the absence of prior preconditioning by mitogen stimulation. These findings suggest that p300 is likely to play an important role in biological processes in which transcriptional bookmarking or preconditioning influences cellular growth and development through the dynamic priming of genes for response to rechallenge by secondary stimuli.gene regulation ͉ histone acetylation ͉ transcription ͉ ELL ͉ epigenetics