UTX mediates demethylation of H3K27me3 at muscle-specific genes during myogenesis.

Polycomb (PcG) and Trithorax (TrxG) group proteins act antagonistically to establish tissue-specific patterns of gene expression. The PcG protein Ezh2 facilitates repression by catalysing histone H3-Lys27 trimethylation (H3K27me3). For expression, H3K27me3 marks are removed and replaced by TrxG protein catalysed histone H3-Lys4 trimethylation (H3K4me3). Although H3K27 demethylases have been identified, the mechanism by which these enzymes are targeted to specific genomic regions to remove H3K27me3 marks has not been established. Here, we demonstrate a two-step mechanism for UTX-mediated demethylation at muscle-specific genes during myogenesis. Although the transactivator Six4 initially recruits UTX to the regulatory region of muscle genes, the resulting loss of H3K27me3 marks is limited to the region upstream of the transcriptional start site. Removal of the repressive H3K27me3 mark within the coding region then requires RNA Polymerase II (Pol II) elongation. Interestingly, blocking Pol II elongation on transcribed genes leads to increased H3K27me3 within the coding region, and formation of bivalent (H3K27me3/H3K4me3) chromatin domains. Thus, removal of repressive H3K27me3 marks by UTX occurs through targeted recruitment followed by spreading across the gene.

Human health effects of traffic-related air pollution (TRAP): a scoping review protocol.

BACKGROUND: Traffic-related air pollution (TRAP) is one of the major sources of exposure in urban areas and has been associated with a wide range of adverse human health effects. Much of the Canadian population is regularly exposed to TRAP as a result of daily activities (e.g., commuting) and a significant portion of the population resides in close proximity to major roadways. The objective of this scoping review is to develop an evidence map of the epidemiological literature of the human health effects of exposure to TRAP, to support future reviews and assessments by Health Canada. METHODS: Literature searches will be conducted in Ovid EMBASE and Ovid MEDLINE database. DistillerSR will be used to manage the review process. Two reviewers will independently screen the studies in a two-part process (title and abstract; full text) for eligibility. Epidemiological studies and reviews will be included if they report on the human health effects of exposure to TRAP. Data collection will include study design parameters and human health outcomes evaluated in the study. A descriptive analysis will be used to provide a high-level summary of the number of studies evaluating the different types of health effects and cross-tabulations by study design parameters. DISCUSSION: The scoping review will be used to identify subject areas for more detailed review and evaluation of the human health effects of TRAP by the Air Health Effects Assessment Division of Health Canada.

Time-dependent rescue of gene expression by androgens in the mouse proximal caput epididymidis-1 cell line after androgen withdrawal.

Androgens are the primary regulators of epididymal structure and functions. Principal cells are the major cell type of the tissue and are particularly sensitive to androgen removal. To distinguish the direct effects of androgens on this cell type, DNA microarrays were used to identify androgen-regulated genes in the mouse proximal caput epididymidis (PC-1) cell line. PC-1 cells display tissue- and caput-specific gene expression. We examined changes in gene expression occurring 2, 4, and 6 d after androgen deprivation and 2 d after androgen supplementation after being deprived of androgen for 2 or 4 d. Changes in transcript levels were investigated for mediators of androgen action; selected genes were analyzed by real-time RT-PCR, and changes at the protein levels were examined. Four distinct patterns of gene expression were activated after androgen withdrawal; the vast majority of genes displayed an early or late transient increase in expression levels. A differential ability of rescue was seen among androgen-regulated genes, depending on time of androgen supplementation. Many of the genes that were rescued at 4 d were functionally linked by direct interactions and converged on IGF-I. The ability for rescue after 4 d of androgen deprivation was severely compromised in many genes belonging to specific functional gene families (cell adhesion, cell growth, apoptosis, and cell cycle) and may be mediated in part by changes in androgen receptor coregulator expression. These results provide novel insights into the mechanisms of androgen regulation in epididymal principal cells.

The promoter of the rat 5alpha-reductase type 1 gene is bidirectional and Sp1-dependent.

In many androgen target tissues, testosterone is reduced to the more potent androgen, dihydrotestosterone, by steroid 5alpha-reductase. Two isoforms of 5alpha-reductase, type 1 and type 2, have been cloned. They are differentially expressed and regulated. To determine the mechanisms of regulation of 5alpha-reductase type 1 expression, we have cloned its 5'upstream region and defined its promoter. The proximal 5'upstream region of 5alpha-reductase type 1 displays all the features of a CpG island and has numerous Sp1 binding sites. By transient transfection assays, we have identified a bidirectional promoter activity in this region; this activity was highest in the negative orientation, in the direction of the methyltransferase Nsun2 (predicted) gene. Promoter activity, in either orientation, was lost in Sp1 deficient cells but was rescued following co-transfection with a Sp1 expression vector. Thus, the 5'upstream region of rat 5alpha-reductase type 1 contains a bidirectional promoter with an activity that is Sp1-dependent.

Androgenic regulation of novel genes in the epididymis.

The epididymis is critically dependent on the presence of the testis. Although several hormones, such as retinoids and progestins, and factors secreted directly into the epididymal lumen, such as androgen binding protein and fibroblast growth factor, might play regulatory roles in epididymal function, testosterone (T) and its metabolites, dihydrotestosterone (DHT) and estradiol (E2), are accepted as the primary regulators of epididymal structure and functions, with the former playing the greater role. To ascertain the molecular action of androgens on the epididymis, three complementary approaches were pursued to monitor changes in gene expression in response to different hormonal milieux. The first was to establish changes in gene expression along the epididymis as androgenic support is withdrawn. The second was to determine the sequence of responses that occur in an androgen deprived tissue upon re-administration of the two metabolites of T, DHT and E2. The third was to study the effects of androgen withdrawal and re-administration on gene expression in immortalized murine caput epididymidal principal cells. Specific responses were observed under each of these conditions, with an expected major difference in the panoply of genes expressed upon hormone withdrawal and re-administration; however, some key common features were the common roles of genes in insulin like growth factor/epidermal growth factor and the relatively minor and specific effects of E2 as compared to DHT. Together, these results provide novel insights into the mechanisms of androgen regulation in epididymal principal cells.

Cloning and characterization of the 5alpha-reductase type 2 promoter in the rat epididymis.

Steroid 5alpha-reductase converts testosterone to the more potent androgen, dihydrotestosterone. The molecular mechanisms responsible for maintaining high concentrations of the 5alpha-reductase type 2 mRNA in the caput epididymidis and for regulating its region-specific expression are unknown. To gain insight into its transcriptional regulation, the cloning and characterization of the 5' upstream region of 5alpha-reductase type 2 were undertaken. Sequential deletion analysis was done to map the 2243-base pair (bp) cloned 5' upstream region, and the constructs were transfected into epididymal PC1 cells and prostatic PC3 cells. In both cell lines, regulatory elements and the minimal promoter were mapped to the 485-bp region upstream of the start codon. Primer extension and 5' RACE identified one transcriptional start site at 33-bp upstream of the start codon. Using electrophoretic mobility shift assay, a specific band was observed in the -68- to -32-bp region in the presence of nuclear extracts. Supershift and mutational studies confirmed the binding of SP1 and, to a lesser extent, SP3 to the two potential SP1 binding sites and the preference of these proteins to one binding site over the other. SP1 and SP3 were both predominantly immunolocalized to the principal cells of the epididymis and follow distinct distribution patterns in this tissue. These results provide a framework crucial in the further investigation of the transcriptional regulation of 5alpha-reductase type 2 in the rat epididymis.