Long-range comparison of human and mouse SCL loci: localized regions of sensitivity to restriction endonucleases correspond precisely with peaks of conserved noncoding sequences

Abstract

Long-range comparative sequence analysis provides a powerful strategy for identifying conserved regulatory elements. The stem cell leukemia (SCL) gene encodes a bHLH transcription factor with a pivotal role in hemopoiesis and vasculogenesis, and it displays a highly conserved expression pattern. We present here a detailed sequence comparison of 193 kb of the human SCL locus to 234 kb of the mouse SCL locus. Four new genes have been identified together with an ancient mitochondrial insertion in the human locus. The SCL gene is flanked upstream by the SIL gene and downstream by the MAP17 gene in both species, but the gene order is not collinear downstream from MAP17. To facilitate rapid identification of candidate regulatory elements, we have developed a new sequence analysis tool (SynPlot) that automates the graphical display of large-scale sequence alignments. Unlike existing programs, SynPlot can display the locus features of more than one sequence, thereby indicating the position of homology peaks relative to the structure of all sequences in the alignment. In addition, high-resolution analysis of the chromatin structure of the mouse SCL gene permitted the accurate positioning of localized zones accessible to restriction endonucleases. Zones known to be associated with functional regulatory regions were found to correspond precisely with peaks of human/mouse homology, thus demonstrating that long-range human/mouse sequence comparisons allow accurate prediction of the extent of accessible DNA associated with active regulatory regions.

Figure 1

Structure of the human and murine SCL loci. The gene structure of the human and mouse SCL loci is shown above a profile displaying the respective G/C content. Arrows indicate the direction of genes. M1 and M2 refer to the sequences homologous to mitochondrial DNA, and (pCYP1) and (pCYP2) refer to partial segments of CYP4 genes present in the mouse locus.

Figure 2

A transposition of mitochondrial DNA 3′ of the human MAP17 gene. (A) Diagram showing the position of the two mitochondrial homology regions relative to the MAP17 and CYP4A11 genes. Boxes labeled M correspond to the mitochondrial homology regions shown in (B), and boxes labeled R show the position of repeat elements. (B) Alignment of mitochondrial homology regions 1 and 2 to mitochondrial sequence of cow and human.

Figure 2

A transposition of mitochondrial DNA 3′ of the human MAP17 gene. (A) Diagram showing the position of the two mitochondrial homology regions relative to the MAP17 and CYP4A11 genes. Boxes labeled M correspond to the mitochondrial homology regions shown in (B), and boxes labeled R show the position of repeat elements. (B) Alignment of mitochondrial homology regions 1 and 2 to mitochondrial sequence of cow and human.

Figure 3

SynPlot analysis of the human and mouse SCL loci. Human and mouse clones starting with the last five exons of SIL, and ranging to beyond the CYP4A11/Cyp4a21 genes, were aligned using Dialign. The alignment, together with locus features, was displayed using SynPlot. Numbers on the horizontal axis represent distance (nucleotides) from the beginning of the aligned file. Numbers on the vertical axis represent the proportion of identical nucleotides within a 49 nt window, moved by 25 nt increments across the entire alignment. Hence, regions with gaps of >50 bp show 0% identity. The horizontal lines above the profile represent the human and mouse sequences and illustrate the position of gaps introduced to permit optimum alignment. Red boxes show exon positions, and the smaller boxes represent repeats as follows: (dark blue) LINEs,(light blue) SINEs, (magenta) tandem repeats. Green arrowheads indicate the positions of previously mapped DNaseI hypersensitive sites, and the yellow bar delimits the portion of the profile shown in Figure 4. Gray shading indicates background similarity of ≤25%.

Figure 4

Localized regions of sensitivity to restriction endonucleases correspond to peaks of human/mouse homology. (A) Restriction endonuclease accessibility assay showing the mapping of the hypersensitive sites at promoter 1b, +1 (+1HS), and +3 (+3HS) (numbering corresponds to distances in kb from the start of exon 1a) in the 416B and M1 primitive myeloid cell lines. Nuclei were incubated with HaeIII. DNA was subsequently extracted, digested with SacI, and hybridized with probes indicated in B. The absence of the +3 hypersensitive site in M1 is consistent with previous DNaseI hypersensitive site analysis (Göttgens et al. 1997). (B) Summary of restriction endonuclease data for the 5′ region of the mouse SCL gene. The top part of the diagram shows the approximate locations of previously mapped DNaseI hypersensitive sites (gray arrowheads labeled P1a, P1b, +1HS, and +3HS) followed by the positions of mouse SCL exons 1a to 3 and the SacI sites used for the Southern blots shown in part A. This is followed by restriction maps for the three enzymes (AvaII, HhaI, and HaeIII) used to determine endonuclease sensitivity, and a summary of the endonuclease sensitivity experiments in which black and gray boxes represent the minimum and maximum regions of endonuclease accessibility in 416B and/or M1 cells. The profile of the mouse/human alignment underneath is a 6250 nucleotide section of the alignment from Fig. 3 (see yellow bar in Fig. 3) and shows the concordance of endonuclease accessibility and sequence conservation.