Structure and regulation of the chicken erythroid δ-aminolevulinate synthase gene

Abstract

Erythroid cells regulate heme biosynthesis in a manner that is distinct from all other cell types. While heme negatively regulates the synthesis of the housekeeping delta-aminolevulinate synthase (ALAS-N) in all non-erythroid cells, the expression of an erythroid-specific isozyme (ALAS-E) is developmentally regulated in red blood cells. As a first step towards understanding the molecular basis for the transcriptional regulation of ALAS-E during erythropoiesis, we cloned and characterized the chicken ALAS-E locus. This gene spans 18 kbp and is composed of eleven exons. The intron/exon structure of erythroid ALAS was found to be conserved among several vertebrate species. Direct RNA sequencing identified a 5' untranslated region that is derived from two continuous exons and is predicted to form a very stable stem-loop structure that bears resemblance to the ferritin iron-responsive element. Tissue-specific expression of the ALAS-E gene was analyzed by transient transfection assays in hematopoietic cells of both erythroid and non-erythroid origins. These experiments identified distal (-784 to -505 bp) and proximal (-155 to +21 bp) promoter elements which are required for high level, erythroid-specific transcription.

Transcriptional Regulation of the Human Erythroid 5-Aminolevulinate Synthase Gene

“…Binding sites for GATA-1, NF-E2, and CACCC box-binding proteins have also been identified in the human ferrochelatase promoter (72). In contrast, the chicken ALAS2 promoter contains multiple binding sites for Sp1 (12). These studies, together with the information on globin gene expression (19), confirm that there is likely to be only a small number of erythroid cell-specific factors that act in a combinatorial fashion to ensure the coordinated regulation of heme and globin synthesis during erythropoiesis.…”

Bach Proteins Belong to a Novel Family of BTB-Basic Leucine Zipper Transcription Factors That Interact with MafK and Regulate Transcription through the NF-E2 Site

“…QT6 cells were transfected with the reporter and effector plasmids by the calcium phosphate precipitation method as previously described (60). The chicken erythroid cell line HD3 (5) was maintained and transfected by the DEAE-dextran method as previously described (41). Luciferase assays were performed by the luciferase assay system (Promega) by following the supplier's protocol and with a Biolumat luminometer (Berthold).…”

Structure and regulation of vertebrate δ‐aminolevulinate synthases

“…The first exon of the gene was identified to be a small discrete exon coding for a 5' UTR, and the gene consists of eleven exons which encompass about 14 kb in the rat genome. As shown in Figure 1, comparison of the structure and organization of the rat ALAS-N gene to those of chicken ALAS-N [31] and the chicken [33], mouse [25] and human [34] ALAS-E genes revealed that the organization of these genes is highly conserved. ALAS-N genes appeared to be organized more compactly than ALAS-E genes.…”

“…This observation suggested that the genes encoding ALAS-N and ALAS-E have diverged from a common ancestor, and the first exon in the chicken ALAS-N gene fused during molecular evolution. The structure of ALAS-E first exons is highly conserved among the chicken, human and mouse ALAS-E genes [15,33,35,361. When processed to m R N 4 the calculated free energy of the predicted stem-loop secondary structure formed by the first and a portion of the second exons of chicken ALAS-E is -16.8 kCal/mol, suggesting that this RNA structure may be of functional significance.…”