Derangement of a Factor Upstream of RARα Triggers the Repression of a Pleiotropic Epigenetic Network

Chromatin adapts and responds to extrinsic and intrinsic cues. We hypothesize that inheritable aberrant chromatin states in cancer and aging are caused by genetic/environmental factors. In previous studies we demonstrated that either genetic mutations, or loss, of retinoic acid receptor alpha (RARalpha), can impair the integration of the retinoic acid (RA) signal at the chromatin of RA-responsive genes downstream of RARalpha, and can lead to aberrant repressive chromatin states marked by epigenetic modifications. In this study we tested whether the mere interference with the availability of RA signal at RARalpha, in cells with an otherwise functional RARalpha, can also induce epigenetic repression at RA-responsive genes downstream of RARalpha.To hamper the availability of RA at RARalpha in untransformed human mammary epithelial cells, we targeted the cellular RA-binding protein 2 (CRABP2), which transports RA from the cytoplasm onto the nuclear RARs. Stable ectopic expression of a CRABP2 mutant unable to enter the nucleus, as well as stable knock down of endogenous CRABP2, led to the coordinated transcriptional repression of a few RA-responsive genes downstream of RARalpha. The chromatin at these genes acquired an exacerbated repressed state, or state "of no return". This aberrant state is unresponsive to RA, and therefore differs from the physiologically repressed, yet "poised" state, which is responsive to RA. Consistent with development of homozygosis for epigenetically repressed loci, a significant proportion of cells with a defective CRABP2-mediated RA transport developed heritable phenotypes indicative of loss of function.Derangement/lack of a critical factor necessary for RARalpha function induces epigenetic repression of a RA-regulated gene network downstream of RARalpha, with major pleiotropic biological outcomes

Differential expression of circulating microRNAs according to severity of colorectal neoplasia

There is a need to develop a colorectal cancer (CRC) screening test that is noninvasive, cost effective, and sensitive enough to detect preneoplastic lesions. This case-control study examined the feasibility of using circulating extracellular microRNAs (miRNAs) to differentiate a spectrum of colorectal neoplasia of various severity and hence for early detection of colorectal neoplasia. Archived serum samples of 10 normal controls and 31 cases, including 10 with nonadvanced adenoma, 10 with advanced adenoma, and 11 with CRC, were profiled for circulating miRNAs using next-generation sequencing. Multiple linear regression, adjusting for age, gender, and smoking status, compared controls and the 3 case groups for levels of 175 miRNAs that met stringent criteria for miRNA sequencing analysis. Of the 175 miRNAs, 106 miRNAs were downregulated according to severity of neoplasia and showed a relative decrease in the expression from controls to nonadvanced adenoma to advanced adenoma to CRC (Ptrend \u3c 0.05). Pairwise group comparisons showed that 39 and 80 miRNAs were differentially expressed in the advanced adenoma and CRC groups compared with the controls, respectively. Differences in miRNA levels between the nonadvanced adenoma group and controls were modest. Our study found that expression of many miRNAs in serum was inversely correlated with the severity of colorectal neoplasia, and differential miRNA profiles were apparent in preneoplastic cases with advanced lesions, suggesting circulating miRNAs could serve as potential biomarkers for CRC screening

Effective DNA/RNA Co-Extraction for Analysis of MicroRNAs, mRNAs, and Genomic DNA from Formalin-Fixed Paraffin-Embedded Specimens

Background: Retrospective studies of archived human specimens, with known clinical follow-up, are used to identify predictive and prognostic molecular markers of disease. Due to biochemical differences, however, formalin-fixed paraffinembedded (FFPE) DNA and RNA have generally been extracted separately from either different tissue sections or from the same section by dividing the digested tissue. The former limits accurate correlation whilst the latter is impractical when utilizing rare or limited archived specimens. Principal Findings: For effective recovery of genomic DNA and total RNA from a single FFPE specimen, without splitting the proteinase-K digested tissue solution, we optimized a co-extraction method by using TRIzol and purifying DNA from the lower aqueous and RNA from the upper organic phases. Using a series of seven different archived specimens, we evaluated the total amounts of genomic DNA and total RNA recovered by our TRIzol-based co-extraction method and compared our results with those from two commercial kits, the Qiagen AllPrep DNA/RNA FFPE kit, for co-extraction, and the Ambion RecoverAll TM Total Nucleic Acid Isolation kit, for separate extraction of FFPE-DNA and-RNA. Then, to accurately assess the quality of DNA and RNA co-extracted from a single FFPE specimen, we used qRT-PCR, gene expression profiling and methylation assays to analyze microRNAs, mRNAs, and genomic DNA recovered from matched fresh and FFPE MCF10A cells. These experiments show that the TRIzol-based co-extraction method provides larger amounts of FFPE-DNA and –RNA tha

Cyp26b1 Regulates Retinoic Acid-Dependent Signals in T Cells and Its Expression Is Inhibited by Transforming Growth Factor-β

The vitamin A metabolite, retinoic acid (RA), plays important roles in the regulation of lymphocyte properties. Dendritic cells in gut-related lymphoid organs can produce RA, thereby imprinting gut-homing specificity on T cells and enhancing transforming growth factor (TGF)-β-dependent induction of Foxp3+ regulatory T cells upon antigen presentation. In general, RA concentrations in cells and tissues are regulated by its degradation as well. However, it remained unclear if T cells could actively catabolize RA.We assessed the expression of known RA-catabolizing enzymes in T cells from mouse lymphoid tissues. Antigen-experienced CD44+ T cells in gut-related lymphoid organs selectively expressed Cyp26b1, a member of the cytochrome P450 family 26. However, T cells in the spleen or skin-draining lymph nodes did not significantly express Cyp26b1. Accordingly, physiological levels of RA (1-10 nM) could induce Cyp26b1 expression in naïve T cells upon activation in vitro, but could not do so in the presence of TGF-β. Overexpression of Cyp26b1 significantly suppressed the RA effect to induce expression of the gut-homing receptor CCR9 on T cells. On the other hand, knocking down Cyp26b1 gene expression with small interfering RNA or inhibiting CYP26 enzymatic activity led to enhancement of the RA-induced CCR9 expression.Our data demonstrate a role for CYP26B1 in regulating RA-dependent signals in activated T cells but not during TGF-β-dependent differentiation to Foxp3+ regulatory T cells. Aberrant expression of CYP26B1 may disturb T cell trafficking and differentiation in the gut and its related lymphoid organs

Physiogenomic comparison of human fat loss in response to diets restrictive of carbohydrate or fat

<p>Abstract</p> <p>Background</p> <p>Genetic factors that predict responses to diet may ultimately be used to individualize dietary recommendations. We used physiogenomics to explore associations among polymorphisms in candidate genes and changes in relative body fat (Δ%BF) to low fat and low carbohydrate diets.</p> <p>Methods</p> <p>We assessed Δ%BF using dual energy X-ray absorptiometry (DXA) in 93 healthy adults who consumed a low carbohydrate diet (carbohydrate ~12% total energy) (LC diet) and in 70, a low fat diet (fat ~25% total energy) (LF diet). Fifty-three single nucleotide polymorphisms (SNPs) selected from 28 candidate genes involved in food intake, energy homeostasis, and adipocyte regulation were ranked according to probability of association with the change in %BF using multiple linear regression.</p> <p>Results</p> <p>Dieting reduced %BF by 3.0 ± 2.6% (absolute units) for LC and 1.9 ± 1.6% for LF (p < 0.01). SNPs in nine genes were significantly associated with Δ%BF, with four significant after correction for multiple statistical testing: rs322695 near the retinoic acid receptor beta (<it>RARB</it>) (p < 0.005), rs2838549 in the hepatic phosphofructokinase (<it>PFKL</it>), and rs3100722 in the histamine N-methyl transferase (<it>HNMT</it>) genes (both p < 0.041) due to LF; and the rs5950584 SNP in the angiotensin receptor Type II (<it>AGTR2</it>) gene due to LC (p < 0.021).</p> <p>Conclusion</p> <p>Fat loss under LC and LF diet regimes appears to have distinct mechanisms, with <it>PFKL </it>and <it>HNMT </it>and <it>RARB </it>involved in fat restriction; and <it>AGTR2 </it>involved in carbohydrate restriction. These discoveries could provide clues to important physiologic mechanisms underlying the Δ%BF to low carbohydrate and low fat diets.</p

Cryptic Patterning of Avian Skin Confers a Developmental Facility for Loss of Neck Feathering

Vertebrate skin is characterized by its patterned array of appendages, whether feathers, hairs, or scales. In avian skin the distribution of feathers occurs on two distinct spatial levels. Grouping of feathers within discrete tracts, with bare skin lying between the tracts, is termed the macropattern, while the smaller scale periodic spacing between individual feathers is referred to as the micropattern. The degree of integration between the patterning mechanisms that operate on these two scales during development and the mechanisms underlying the remarkable evolvability of skin macropatterns are unknown. A striking example of macropattern variation is the convergent loss of neck feathering in multiple species, a trait associated with heat tolerance in both wild and domestic birds. In chicken, a mutation called Naked neck is characterized by a reduction of body feathering and completely bare neck. Here we perform genetic fine mapping of the causative region and identify a large insertion associated with the Naked neck trait. A strong candidate gene in the critical interval, BMP12/GDF7, displays markedly elevated expression in Naked neck embryonic skin due to a cis-regulatory effect of the causative mutation. BMP family members inhibit embryonic feather formation by acting in a reaction-diffusion mechanism, and we find that selective production of retinoic acid by neck skin potentiates BMP signaling, making neck skin more sensitive than body skin to suppression of feather development. This selective production of retinoic acid by neck skin constitutes a cryptic pattern as its effects on feathering are not revealed until gross BMP levels are altered. This developmental modularity of neck and body skin allows simple quantitative changes in BMP levels to produce a sparsely feathered or bare neck while maintaining robust feather patterning on the body