Loss Of Transcription Factor Binding Research Articles

Identification and Targeting of the Developmental Blockade in Extranodal Natural Killer/T-cell Lymphoma.

Through epigenetic and transcriptomic analyses of ENKTL, a rare, aggressive malignancy, along with normal NK-cell developmental intermediates, we identified that extreme DNA hypermethylation targets genes required for NK-cell development. Disrupting this epigenetic blockade in novel PDX models led to ENKTL differentiation and improved survival. This article is highlighted in the In This Issue feature, p. 85.

Thrombospondin-1 Gene Polymorphism Is Associated with Estimated Pulmonary Artery Pressure in Patients with Sickle Cell Anemia

Thrombospondin-1 (TSP1) is a multifunctional glycoprotein present in platelet α-granules that contains domains for adhesive proteins, enzymes, and cell receptors. It is released by activated platelets and is increased in the plasma of patients with sickle cell anemia (SCA). TSP1 is known to mediate adherence of sickle reticulocytes to endothelial cells. Furthermore, TSP1 also inhibits the NO signaling pathway through its binding to the CD47 cell receptor on endothelial cells and platelets. Thus, we hypothesized that genetic variants in TSP1 may contribute to the marked phenotypic variation seen in SCA.We tested whether genetic variants of the TSP1 gene are associated with biomarkers of SCA severity and specific complications in a cohort of 452 patients with homozygous HbSS disease from the multicenter walk-PHaSST (Treatment of Pulmonary Hypertension and Sickle Cell Disease with Sildenafil Therapy) trial. Genetic data from 406 patients were included in the analysis. Patients ranged in age from 12 to 70 years, and 51% were females. 307 patients were enrolled at various sites in the United States, while 99 patients were enrolled at Hammersmith Hospital in the UK. A total of 10 SNPs were selected, including haplotype tagged SNPs, coding variants, and SNPs potentially regulating gene expression and alternative splicing. DNA isolated from peripheral blood was used for genotyping.We found that the TSP1 SNPs rs1478604 and rs1478605 were significantly positively correlated with tricuspid regurgitation velocity (TRV) >2.5 m/sec, an echocardiographic marker of pulmonary hypertension associated with increased mortality, under a recessive model (r=0.10, p=0.0288 and r=0.13, p=0.0047 respectively). TRV was also analyzed as a categorical variable using regression analysis, and subjects were grouped based on the following clinically relevant cut-off points, 2.5 m/sec, 2.7 m/sec, and 3 m/sec. Univariate regression analyses showed TRV>2.5 m/sec was independently associated with the recessive genotypes of rs1478604 and rs1478605 (p=0.069 and 0.017 respectively). As previously published, we confirmed the association of TRV with age, gender, NT-proBNP and creatinine, and we adjusted for these variables in multivariate regression analyses for the association of TRV and each SNP. The results showed that the two SNPs were significantly associated with TRV>2.5 m/sec (rs1478604, OR 2.54, 95% CI (1, 6.44), p<0.05 and rs1478605, OR 4.20, 95% CI (1.41, 12.49), p<0.01) even after controlling for these variables.The results suggest that genetic variations in the TSP1 gene are associated with variations in estimated pulmonary artery pressure in patients with SCA. Both rs1478604 and rs1478605 are localized to the 5' untranslated region. Bioinformatic analysis of these SNPs for putative transcription factor binding sites demonstrated that the two SNPs are associated with both gain and loss of transcription factor binding compared to the wild type. Therefore, both SNPs are potentially functional in TSP1 regulation. These data also suggest possible translational implications in light of earlier pre-clinical reports that showed that mice lacking TSP1 were protected from hypoxia-mediate pulmonary and right ventricular remodeling and showed less alterations in PA pressure.In populations without SCA, these alleles have been independently linked to risk of malaria, corneal immune function, and gastric cancer. TSP1 activity has also been linked to fibrotic renal disease in a rat model and to IRI-mediated AKI in a murine model. The mechanism implicated in some of these cases involves disordered TSP1 regulation of transforming growth factor beta (TGF-beta), whose superfamily of receptors is dysregulated in the pathogenesis of pulmonary hypertension and kidney disease.Our findings support a hypothetical model of TSP1 involvement in the pathophysiology of SCA. Efforts to seek validation of the genetic findings from this study in an independent SCA cohort are currently underway. DisclosuresIsenberg:RCTI, Inc.: Equity Ownership, Patents & Royalties: US Patent No. 8236313; Vasculox, Inc.: Equity Ownership, Patents & Royalties: US Patent No. 8236313.

The limited contribution of DNA methylation to PML-RARα induced leukemia

The limited contribution of DNA methylation to PML-RARα induced leukemia

DNA methylation changes are a late event in acute promyelocytic leukemia and coincide with loss of transcription factor binding

AbstractThe origin of aberrant DNA methylation in cancer remains largely unknown. In the present study, we elucidated the DNA methylome in primary acute promyelocytic leukemia (APL) and the role of promyelocytic leukemia–retinoic acid receptor α (PML-RARα) in establishing these patterns. Cells from APL patients showed increased genome-wide DNA methylation with higher variability than healthy CD34+ cells, promyelocytes, and remission BM cells. A core set of differentially methylated regions in APL was identified. Age at diagnosis, Sanz score, and Flt3-mutation status characterized methylation subtypes. Transcription factor–binding sites (eg, the c-myc–binding sites) were associated with low methylation. However, SUZ12- and REST-binding sites identified in embryonic stem cells were preferentially DNA hypermethylated in APL cells. Unexpectedly, PML-RARα–binding sites were also protected from aberrant DNA methylation in APL cells. Consistent with this, myeloid cells from preleukemic PML-RARα knock-in mice did not show altered DNA methylation and the expression of PML-RARα in hematopoietic progenitor cells prevented differentiation without affecting DNA methylation. Treatment of APL blasts with all-trans retinoic acid also did not result in immediate DNA methylation changes. The results of the present study suggest that aberrant DNA methylation is associated with leukemia phenotype but is not required for PML-RARα–mediated initiation of leukemogenesis.

The mechanism of repression of the myeloid-specific c-fms gene by Pax5 during B lineage restriction

The transcription factor Pax5 (BSAP) is required for the expression of a B-cell-specific genetic program and for B-cell differentiation, and also to suppress genes of alternative lineages. The molecular mechanism by which repression of myeloid genes occurs during early B-lineage restriction is unknown and in this study we addressed this question. One of the genes repressed by Pax5 in B cells is the colony-stimulating factor receptor 1 gene (csf1r or c-fms). We examined the changes in chromatin caused by Pax5 activity, and we show that Pax5 is directly recruited to c-fms resulting in the rapid loss of RNA polymerase II binding, followed by loss of transcription factor binding and DNaseI hypersensitivity at all cis-regulatory elements. We also show that Pax5 targets the basal transcription machinery of c-fms by interacting with a binding site within the major transcription start sites. Our results support a model by which Pax5 does not lead to major alterations in chromatin modification, but inhibits transcription by interfering with the action of myeloid transcription factors.

Regulation of astrocyte nitric oxide synthase type II expression by ATP and glutamate involves loss of transcription factor binding to DNA.

Pretreatment of astrocytes with either glutamate or ATP, acting via specific receptors, suppresses subsequent cytokine-induced expression of type II nitric oxide synthase (NOS). This effect is downstream from the activation and translocation of nuclear factor (NF)-kappaB, as western blotting revealed no difference in accumulation of the p50 subunit in nuclear extracts from agonist-treated vs. nontreated cells. However, evidence from gel-shift assay suggests that the binding of nuclear protein from agonist-treated cells to NF-kappaB and activator protein-1 consensus oligonucleotides is markedly reduced. Selective inhibitors of protein kinases A and C could not restore either binding of transcription factors to DNA or type II NOS mRNA expression in agonist-pretreated cells. The modulation of proinflammatory cytokine-evoked type II NOS expression by ATP and glutamate may play an important role in CNS pathologies associated with stroke and trauma.

Dissection of progesterone receptor-mediated chromatin remodeling and transcriptional activation in vivo.

We have investigated whether constitutive binding by the progesterone receptor (PR) to a promoter is required for the maintenance of an open chromatin structure in vivo. For these experiments, we used human T47D breast cancer cells in which the mouse mammary tumor virus (MMTV) promoter, stably assembled as chromatin, is constitutively hypersensitive to endonucleolytic cleavage. In vivo footprinting revealed that transcription factors nuclear factor 1 and the PR were constitutively bound to the MMTV promoter in these cells. Treatment of these cells for 1 hr with the steroid antagonist ZK98299 prevented PR binding to chromatin in vivo and reversed hypersensitivity, leading to the loss of transcription factor binding. The reduction in hypersensitivity induced by ZK98299 was readily reversed by treatment with the progestin R5020. The chromatin organization of the promoter could be cycled between the open and closed states by consecutive treatments with agonist or antagonist. The antagonist RU486 also blocked activation of transcription and the assembly of a transcription preinitiation complex, but in contrast to ZK98299, maintained the hypersensitive chromatin state. Taken together, these results suggest that PR binding to chromatin is sufficient to induce hypersensitivity to endonucleolytic cleavage. Furthermore, they indicate that the PR binding to DNA and the resulting chromatin hypersensitivity is functionally separate from transcriptional activation in vivo.