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Fig. 3. All three cis-regulatory elements of the Eklf locus contain
highly conserved Smad binding motifs adjacent to Gata sites. (A)
Alignment of five mammalian Eklf genomic loci encompassing 17 kb
(
30 kb global alignment length) surrounding the Eklf
transcription unit in between the two nearest neighboring genes, Gcdh
and Dnase2a. Each sequence is represented as a black line (breaks
demarcate gaps in the alignment), repeats or low complexity DNA are
represented as blue bars, untranslated regions (UTR) as light tan bars and
exons as brown bars. The degree of sequence conservation between species is
expressed as an alignment score (y-axis) per nucleotide position
(x-axis; global length). Peaks of conservation above 0.6 (red line)
within non-coding regions are shaded in orange. The 950 bp region upstream of
Eklf exon 1, exon 1 itself, and intron 1 are highlighted in light
yellow. (B) Enlarged view of the aligned mouse Eklf sequence
from -950 bp to the beginning of exon 2. The three conserved Eklf
cis-regulatory elements are indicated (upstream enhancer, proximal promoter,
intronic enhancer) together with blocks of four or more nucleotides of perfect
homology (green bars), erythroid hypersensitive sites (EHS1 and EHS2, dark
yellow bars) and previously known Gata and Cp1 sites (small red bars). Arrows
indicate positions of newly identified Smad binding motifs (SBM) and WGATAR
motifs. (C) Detail of seven-species alignment of all three
Eklf cis-regulatory regions (upstream enhancer, proximal promoter,
intronic enhancer) at the single nucleotide level. Newly identified Smad
binding motifs (SBM1 through SBM10) are highlighted (blue), as are unassigned
conserved blocks of perfect homology (green), WGATAR motifs (red), E-box
motifs (yellow), Cp1 (pink) and putative Sp1 (brown) sites. The EHS1/GEG
region within the upstream enhancer is underlined.
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