Polyamine depletion is associated with altered chromatin structure in HeLa cells.

HeLa cells depleted of polyamines by treatment with alpha-difluoromethylornithine (DFMO), methylglyoxal bis(guanylhydrazone) (MGBG) or a combination of the two, were examined for sensitivity to micrococcal nuclease, DNAase I and DNAase II. The degrees of chromatin accessibility to DNAase I and II appeared enhanced somewhat in all three treatment groups, and the released digestion products differed from those in non-depleted cells. DNA released from MGBG- and DFMO/MGBG-treated cells by DNAase II digestion was enriched 4-7-fold for Mg2+-soluble species relative to controls. DNA released by micrococcal nuclease digestion from all three treatment groups was characterized as consisting of higher-order nucleosomal structure than was DNA released from untreated cells. At least some of the altered chromatin properties were abolished by a brief treatment of cells with polyamines, notably spermine. These studies provide the first demonstration in vivo of altered chromatin structure in cells treated with inhibitors of polyamine biosynthesis.     

Repression of the IL-6 gene is associated with hypermethylation.

The expression of the IL-6 gene is usually tightly controlled and may be induced in specific tissues after treatment with appropriate stimuli. Although much is known about the inducible expression of the IL-6 gene, the molecular mechanisms responsible for its repression in specific tissues or cell types remain poorly defined. To address this question we have studied two human breast carcinoma cell lines, MDA-MB-231, in which the IL-6 gene is expressed, and, MCF-7, in which the IL-6 message is undetectable by Northern blot assay even in the presence of inducers. The expression of the IL-6 message was estimated after treatment with 5-aza-2'deoxycytidine and the methylation state of the IL-6 gene was analyzed. We show herein that treatment of MCF-7 cells with an agent which reduces DNA methylation correlates with IL-6 gene hypomethylation and increases the level of its expression.     

Bromodomain protein Brd4 associated with acetylated chromatin is important for maintenance of higher-order chromatin structure

Chromatin structure organization is crucial for regulating many fundamental cellular processes. However, the molecular mechanism that regulates the assembly of higher-order chromatin structure remains poorly understood. In this study, we demonstrate that Brd4 (bromodomain-containing protein 4) protein participates in the maintenance of the higher-order chromatin structure. Brd4, a member of the BET family of proteins, has been shown to play important roles in cellular growth control, cell cycle progression, and cancer development. We apply in situ single cell chromatin imaging and micrococcal nuclease (MNase) assay to show that Brd4 depletion leads to a large scale chromatin unfolding. A dominant-negative inhibitor encoding the double bromodomains (BDI/II) of Brd4 can competitively dissociate endogenous Brd4 from chromatin to trigger severely fragmented chromatin morphology. Mechanistic studies using Brd4 truncation mutants reveal that the Brd4 C-terminal domain is crucial for maintaining normal chromatin structure. Using bimolecular fluorescence complementation technology, we demonstrate that Brd4 molecules interact intermolecularly on chromatin and that replacing Brd4 molecules by BDI/II causes abnormal nucleosome aggregation and chromatin fragmentation. These studies establish a novel structural role of Brd4 in supporting the higher chromatin architecture.     

Interleukin-10-1082 gene polymorphism is associated with papillary thyroid cancer

The etiopathogenesis of thyroid cancer has not been clearly elucidated although the role of chronical inflammation and the imbalance between pro- and anti-inflammatory cytokines may play a role in the etiology. The aim of the present study was to investigate whether cytokine gene polymorphisms are associated with papillary thyroid cancer (PTC), and to evaluate the relationship between genotypes and clinical/laboratory manifestation of PTC. Tumor necrosis factorα (TNFα) G-308A (rs 1800629), interleukin-6 (IL-6) G-174C (rs 1800795) and IL-10 A-1082G (rs 1800896) single nucleotide polymorphisms in DNA from peripheral blood leukocytes of 190 patients with thyroid cancer and 216 healthy controls were investigated by real-time PCR combined with melting curve analysis. There was no notable risk for PTC afflicted by TNFα-308 and IL-6-174 alone. However, IL-10-1082 G allele frequency were higher among PTC patients than healthy controls (p = 0.009). The patients with IL-10-1082 GG geotype have twofold increased risk of developing thyroid cancer according to AA genotype (OR 2.07, 95 % CI 1.21–3.55). In addition, the concomitant presence of IL-10-1082 G allele (GG + AG genotypes) together with IL-6 -174 GG genotype has a nearly twofold increased risk for thyroid cancer (OR 1.75 with 95 % CI 1.00–3.05, p = 0.049). We suggest that IL-10-1082 G allele is associated with an increased risk of PTC. The polymorphism of IL-10 gene can improve our knowledge about the pathogenesis of PTC, and could provide to estimate people at the increased risk for PTC.     

MYC-induced apoptosis in mammary epithelial cells is associated with repression of lineage-specific gene signatures

Apoptosis caused by deregulated MYC expression is a prototype example of intrinsic tumor suppression. However, it is still unclear how supraphysiological MYC expression levels engage specific sets of target genes to promote apoptosis. Recently, we demonstrated that repression of SRF target genes by MYC/MIZ1 complexes limits AKT-dependent survival signaling and contributes to apoptosis induction. Here we report that supraphysiological levels of MYC repress gene sets that include markers of basal-like breast cancer cells, but not luminal cancer cells, in a MIZ1-dependent manner. Furthermore, repressed genes are part of a conserved gene signature characterizing the basal subpopulation of both murine and human mammary gland. These repressed genes play a role in epithelium and mammary gland development and overlap with genes mediating cell adhesion and extracellular matrix organization. Strikingly, acute activation of oncogenic MYC in basal mammary epithelial cells is sufficient to induce luminal cell identity markers. We propose that supraphysiological MYC expression impacts on mammary epithelial cell identity by repressing lineage-specific target genes. Such abrupt cell identity switch could interfere with adhesion-dependent survival signaling and thus promote apoptosis in pre-malignant epithelial tissue.     

Interleukin-6 genotype is associated with high-density lipoprotein cholesterol responses to exercise training

BACKGROUND: High-density lipoprotein cholesterol (HDL-C) and its subfractions are modifiable with exercise training and these responses are heritable. The interleukin-6 (IL6)-174G/C polymorphism may be associated with HDL-C levels. We hypothesized that the IL6-174G/C polymorphism would be associated with plasma HDL-C response to exercise training. METHODS AND RESULTS: Sixty-five 50- to 75-year-olds on a standardized diet were studied before and after 24 weeks of aerobic exercise training. Significant differences existed among genotype groups for change with exercise training in HDL-C, HDL3-C, integrated HDL4,5NMR-C, and HDLsize. The CC genotype group increased HDL-C more than the GG (7.0 +/- 1.3 v. 1.0 +/- 1.1 mg/dL, p = 0.001) and GC groups (3.3 +/- 0.9 mg/dL, p = 0.02); for HDL3-C, the CC group increased more than the GG (6.1 +/- 1.0 v. 0.9 +/- 0.9, mg/dL p < 0.001) and GC groups (2.5 +/- 0.7 mg/dL, p = 0.006). Integrated HDL4,5NMR-C increased more in the CC than GG group (6.5 +/- 1.6 mg/dL v. 1.0 +/- 1.3 mg/dL, p = 0.01), as did HDLsize compared to the GG (CC: 0.3 +/- 0.1 v. GG: 0.1 +/- 0.1 nm, p = 0.02) and GC (0.0 +/- 0.0 nm, p = 0.007) groups. CONCLUSIONS: IL6 genotype is associated with HDL-C response to exercise training.     

Developmental switch in chromatin structure associated with alternate promoter usage in the Drosophila melanogaster alcohol dehydrogenase gene.

During the development of Drosophila melanogaster a switch in alcohol dehydrogenase gene promoter usage occurs, such that proximally initiated mRNA is replaced by mRNA initiated from a more distal location. Investigation of the nucleo-protein organization at this gene in cells inactive for Adh expression, or derived from tissues active at either the proximal or distal promoter, reveals distinct changes in patterns of nucleosome organization and regions of nuclease sensitivity that are strongly correlated with the activity of the gene and its promoter usage. A positioned array of nucleosomes covers the coding region of the inactive gene, but is partially disassembled on gene activation. A series of proximally located hypersensitive sites, detected in early third instar larval fat body cells, are replaced by new, distally located regions of hypersensitivity in late third instar larval fat body, the change apparently coinciding with the promoter switch. Further developmental stage differences are detected in regions over 1 kb upstream of the distal start site. In addition, for both proximally and distally expressing cells, separate and different regions of apparent resistance to DNase I cleavage in chromatin are detected in locations that, in some instances, were previously demonstrated to bind specific factors in vitro.     

Interleukin-6 is a centrally acting endogenous pyrogen in the rat.

Intracerebroventricular (i.c.v.) injection of human recombinant interleukin-6 (IL-6; 20-100 ng) caused significant increases in colonic temperature and resting oxygen consumption (VO2) in conscious rats. These effects were prevented by pretreatment with a cyclooxygenase inhibitor (flurbiprofen, 1 mg/kg, i.p.) or a corticotrophin-releasing factor antagonist (alpha-helical CRF9-41, 25 micrograms, i.c.v.). Higher doses of IL-6 (i.c.v.) caused only small changes in VO2 and temperature, and very high doses given intravenously (i.v.) (4 micrograms/kg) were required to stimulate these parameters. Central injection of anti-rat IL-6 antibody inhibited the effects of interleukin-1 beta (i.c.v.) or endotoxin injection (i.p.) on colonic temperature and VO2 in conscious rats. These data indicate that IL-6 is an important endogenous pyrogen that acts within the central nervous system.     

The chromatin structure of specific genes: II. Disruption of chromatin structure during gene activity.

We have compared the chromatin structure in the active and inactive states at loci encoding the major heat shock protein in Drosophila. DNAase I and micrococcal nuclease were used as probes of higher order organization and nucleosomal integrity. Such integrity is gauged here by the characteristic pattern of discrete DNA fragments produced at specific chromosomal loci by nucleolytic cleavage. The specific fragment patterns are visualized by gel electrophoresis, Southern blotting onto nitrocellulose sheets, hybridization with 32P-labeled cloned DNA containing the heat shock genes and autoradiography. Using this criterion, a disruption in nucleosomal and possibly in higher order organization are observed as indicated by a relative loss or smearing of the characteristic discrete DNA fragment patterns from the heat shock loci in the active state. The fragment patterns are restored when cells are allowed to recover from heat shock and these loci return to the inactive state.