Zebrafish globin switching occurs in two developmental stages and is controlled by the LCR

Abstract

Globin gene switching is a complex, highly regulated process allowing expression of distinct globin genes at specific developmental stages. Here, for the first time, we have characterized all of the zebrafish globins based on the completed genomic sequence. Two distinct chromosomal loci, termed major (chromosome 3) and minor (chromosome 12), harbor the globin genes containing α/β pairs in a 5'-3' to 3'-5' orientation. Both these loci share synteny with the mammalian α-globin locus. Zebrafish globin expression was assayed during development and demonstrated two globin switches, similar to human development. A conserved regulatory element, the locus control region (LCR), was revealed by analyzing DNase I hypersensitive sites, H3K4 trimethylation marks and GATA1 binding sites. Surprisingly, the position of these sites with relation to the globin genes is evolutionarily conserved, despite a lack of overall sequence conservation. Motifs within the zebrafish LCR include CACCC, GATA, and NFE2 sites, suggesting functional interactions with known transcription factors but not the same LCR architecture. Functional homology to the mammalian α-LCR MCS-R2 region was confirmed by robust and specific reporter expression in erythrocytes of transgenic zebrafish. Our studies provide a comprehensive characterization of the zebrafish globin loci and clarify the regulation of globin switching.

Figure 1. Major and minor zebrafish chromosomal loci and protein sequences

(A) The major and minor zebrafish globin loci are located on chromosome 3 and 12 respectively. The major locus was assembled by aligning bacterial artificial chromosomes (BAC) and phage artificial chromosomes (PAC), AC103581, AL953863, BX004811 and CU464181, and contains 13 globin genes. The minor locus was assembled by aligning 2 BACs, CR352324 and BX572076, and contains 4 globin genes. The timing of expression is denoted by “embryonic,” “embryonic/larval,” and “adult.”The surrounding regions included are syntenic with other teleost and mammalian species (“Results”). The scale indicated the distance of the region from the beginning of rhbdf1 on both loci. (B) The human α-globin locus, adapted from Higgs & Wood (2008), is syntenic with both the major and minor zebrafish globin loci. (C) Analysis of the similarity of the various globins, broken into α- and β-globins, by protein sequence. Phylogenic analysis was performed using the Clustal W method (MegAlign; DNASTAR). Shades of gray indicate level of conservation at the amino acid level.

Figure 2. Relative globin expression levels throughout development

The relative expression level changes of the α-globin (A) genes and the β-globin genes (B) in the zebrafish are shown. In both A and B, the approximate embryonic (emb), larval and adult stages of globin expression are denoted by shades of gray. The first time point depicted is the 16 somite stage. Relative expression levels were determined by quantitative real-time PCR and normalized to band3. The globin genes α_e4 and α_a2 are not depicted as no significant expression was detected at any of these time points.

Figure 3

globin expression by in situ hybridization through 5 dpf. Expression patterns of the α- (A) and β-globin (B) genes. For detected genes, expression is seen in bilateral stripes at the 16ss, in the ICM at the 25ss and 24 hpf stage. With the onset of circulation at 24 hpf, globin positive cells can be seen throughout the vasculature with probes for expressed globin genes, particularly in the vascular plexus of the caudal hematopoietic tissue.

Figure 4

Analysis of the chromosomal state of the major and minor globin loci. All tracks were mapped to Zv9 on the UCSU genome browser (http://genome.ucsc.edu/). In some cases annotated genes were renamed in order to adhere to the naming convention, and in cases where a globin gene was not annotated, the UCSC BLAT tool was used to locate the ORFs included in the figure. The shaded gray areas indicate the confirmed and putative regulatory regions in the major and minor loci respectively.

Figure 5

Generation and characterization of LCR-GFP transgenic zebrafish line. (A) Sequence and location of the HS-26 DNase I hypersensitivity site (HS-26; yellow rectangle) and locus control region (LCR; red rectangle) within the major globin locus. The proximal globin promoter used is indicated with a red star. Shaded boxes indicate binding motifs for canonical erythrocyte transcription factors. (B) Representation of the vectors assembled from genomic fragments and the resulting GFP expression patterns. Fluorescent images of LCR-GFP transgenic zebrafish embryos at 16ss, 22 hpf and 36 hpf respectively (C-E). (F) Fluorescent image of the flank of an adult LCR-GFP transgenic zebrafish. (G) Red blood cell gate as determined by forward and side scatter for peripheral blood, and the analysis of the percent of GFP positive red blood cells in an LCR-GFP transgenic adult (green) versus in a wild-type adult (red).