Technique / Molecular Biology / RNA transcriptional post-transcriptional regulation / DNase I hypersensitivity assay

The study of eukaryotic gene transcription depends on methods to discover distal cis-acting control sequences. Comparative bioinformatics is one powerful strategy to reveal these domains, but still requires conventional wet-bench techniques to elucidate their specificity and function. The DNase I hypersensitivity assay (DHA) is also a method to identify regulatory domains, but can also suggest their function. Technically however, the classical DHA is constrained to mapping gene loci in small increments of approximately 20 kb. This limitation hinders efficient and comprehensive analysis of distal gene regions. Here, we report an improved method termed mega-DHA that extends the range of existing DHAs to facilitate assaying intervals that approach 100 kb. We demonstrate its feasibility for efficient analysis of single-copy genes within a large and complex genome by assaying 230 kb of the human ADAMTS14-perforin-paladin gene cluster in four experiments. The results identify distinct networks of regulatory domains specific to expression of perforin and its two neighboring genes.

Human C/EBPepsilon is a recently cloned member of the C/EBP family of transcriptional factors. Previous studies demonstrated that the expression of this gene is tightly regulated in a tissue-specific manner; it is expressed almost exclusively in myeloid cells. To understand the mechanism by which the expression of C/EBPepsilon gene is controlled, we cloned a large genomic region surrounding the C/EBPepsilon gene and performed a DNase I hypersensitivity analysis of this locus. These sites probably represent areas of binding of proteins modulating gene transcription. Hypersensitive (HS) regions in 30 kb of DNA surrounding the C/EBPepsilon gene were examined in C/EBPepsilon high-expressing (NB4, HL-60), low-expressing (Jurkat), very-low-expressing (KG-1), and non-expressing (K562) hematopoietic cells as well as in non-hematopoietic-non-expressing cells (MCF-7, DU 145, PC-3). Three HS sites were detected near the first exon of C/EBPepsilon gene. They were found only in hematopoietic cells and were especially prominent in C/EBPepsilon expressing cells, suggesting that these sites play an important role in transcribing the gene. These hypersensitive bands did not change when the cells were cultured with retinoids. Gel-shift assays using 200 bp of nucleotide sequences that encompassed the hypersensitive sites and nuclear extracts from NB4 and Jurkat cells (C/EBPepsilon expressing) as well as K562 and MCF-7 cells (non-expressing) showed different retarded bands on gel electrophoresis. A fourth HS site, located about 11 kb upstream of exon 1, was found only in cells highly expressing C/EBPepsilon. Two sites, one about 4.5 kb upstream of exon 1 and another about 8.5 kb downstream of exon 2, were positive only in non-expressing cell lines, suggesting that repressors may bind in these areas. Taken together, we have found six specific DNase I hypersensitive sites in the region of C/EBPepsilon that may be involved in regulating transcription of this gene.

Interleukin-4 (IL-4) induces germline C epsilon transcripts prior to C epsilon switch recombination in human B-lymphocytes. In chromatin, nucleosome-free regions known as nuclease hypersensitive sites represent the "open windows" that allow enhanced access of crucial resident cis-acting DNA sequences to transacting factors. In this study, lymphoblastoid cell lines (LCLs) were established from surface IgE negative B-cells of healthy children and patients with high levels of serum IgE, using Epstein-Barr virus. The germline C epsilon transcript was amplified from cDNA of the patients' LCLs cultured with low concentrations of recombinant interleukin-4 (rIL-4, 10 IU/ml), while it was not amplified from the cDNA of the healthy LCLs with the low concentration of rIL-4. The germline C epsilon transcript was strongly amplified from cDNA of the patients' LCLs with high concentrations of rIL-4 (100 IU/ml), compared with that of the healthy LCLs with the high concentrations of rIL-4. Moreover, the DNase I hypersensitive site of the I epsilon region was abnormally induced in the patients' LCLs even by a low concentration of rIL-4, compared with that of healthy LCLs. Our results indicate that DNase I hypersensitive sites in the I epsilon region of immunoglobulin-heavy chain genes are abnormally induced by IL-4 in the B-cells of atopic patients with high levels of serum IgE, and, as a result, the germline C epsilon transcript is abnormally expressed.