Evolutionarily conserved sequences on human chromosome 21

Abstract

Comparison of human sequences with the DNA of other mammals is an excellent means of identifying functional elements in the human genome. Here we describe the utility of high-density oligonucleotide arrays as a rapid approach for comparing human sequences with the DNA of multiple species whose sequences are not presently available. High-density arrays representing approximately 22.5 Mb of nonrepetitive human chromosome 21 sequence were synthesized and then hybridized with mouse and dog DNA to identify sequences conserved between humans and mice (human-mouse elements) and between humans and dogs (human-dog elements). Our data show that sequence comparison of multiple species provides a powerful empiric method for identifying actively conserved elements in the human genome. A large fraction of these evolutionarily conserved elements are present in regions on chromosome 21 that do not encode known genes.

Figure 1

Detection and analysis of evolutionarily conserved sequences on human chromosome 21 by cross-species comparisons using high-density arrays. (A) The chromosome 21 arrays were designed using nonrepetitive sequences and hybridized with syntenic mouse and dog BACs (horizontal lines). A low-magnification view of a fluorescence hybridization image of an array is shown. (B) Chromosome 21 reference sequence was tiled as 25-mer oligonucleotides (probes). Each nucleotide of the reference sequence was interrogated by four probes: one probe complementary to the sequence and three probes noncomplementary at the central position. When the fluorescent intensities (white squares) of the complementary probes are greater than that of the noncomplementary probes, similarities between the tiled human 21q sequences and the hybridized animal DNA exist. (C) (Top) Enlarged view of a 21q array hybridized with syntenic dog BAC DNA. Two 30-nt intervals, one with high conformance between human and dog sequences (97% conformance, red rectangle) and one with low conformance between human and dog sequences (60% conformance, blue rectangle), are shown. (Bottom) For the conserved sequence with high conformance, the 29 conforming nucleotides are indicated in red. For the conserved sequence with low conformance, the 18 conforming nucleotides are indicated in blue. (D) CONSEQ plots showing the conserved sequences (red peaks that are highlighted yellow) detected on the above 21q array relative to their position in the human reference sequence (horizontal axis) and their percent conformances (vertical axis). The high-conformance (97%) conserved sequence has been merged with neighboring conserved sequences to form a 190-nt element. The low-conformance (60%) conserved sequence is an isolated 30-nt element. (Small tan rectangles) Positions of interspersed repeats, which compose ∼33.5% of chromosome 21 sequence and were not tiled on the arrays (Supplemental Table 4, http://www.genome.org); therefore, conformance information is absent.

Figure 1

Detection and analysis of evolutionarily conserved sequences on human chromosome 21 by cross-species comparisons using high-density arrays. (A) The chromosome 21 arrays were designed using nonrepetitive sequences and hybridized with syntenic mouse and dog BACs (horizontal lines). A low-magnification view of a fluorescence hybridization image of an array is shown. (B) Chromosome 21 reference sequence was tiled as 25-mer oligonucleotides (probes). Each nucleotide of the reference sequence was interrogated by four probes: one probe complementary to the sequence and three probes noncomplementary at the central position. When the fluorescent intensities (white squares) of the complementary probes are greater than that of the noncomplementary probes, similarities between the tiled human 21q sequences and the hybridized animal DNA exist. (C) (Top) Enlarged view of a 21q array hybridized with syntenic dog BAC DNA. Two 30-nt intervals, one with high conformance between human and dog sequences (97% conformance, red rectangle) and one with low conformance between human and dog sequences (60% conformance, blue rectangle), are shown. (Bottom) For the conserved sequence with high conformance, the 29 conforming nucleotides are indicated in red. For the conserved sequence with low conformance, the 18 conforming nucleotides are indicated in blue. (D) CONSEQ plots showing the conserved sequences (red peaks that are highlighted yellow) detected on the above 21q array relative to their position in the human reference sequence (horizontal axis) and their percent conformances (vertical axis). The high-conformance (97%) conserved sequence has been merged with neighboring conserved sequences to form a 190-nt element. The low-conformance (60%) conserved sequence is an isolated 30-nt element. (Small tan rectangles) Positions of interspersed repeats, which compose ∼33.5% of chromosome 21 sequence and were not tiled on the arrays (Supplemental Table 4, http://www.genome.org); therefore, conformance information is absent.

Figure 1

Detection and analysis of evolutionarily conserved sequences on human chromosome 21 by cross-species comparisons using high-density arrays. (A) The chromosome 21 arrays were designed using nonrepetitive sequences and hybridized with syntenic mouse and dog BACs (horizontal lines). A low-magnification view of a fluorescence hybridization image of an array is shown. (B) Chromosome 21 reference sequence was tiled as 25-mer oligonucleotides (probes). Each nucleotide of the reference sequence was interrogated by four probes: one probe complementary to the sequence and three probes noncomplementary at the central position. When the fluorescent intensities (white squares) of the complementary probes are greater than that of the noncomplementary probes, similarities between the tiled human 21q sequences and the hybridized animal DNA exist. (C) (Top) Enlarged view of a 21q array hybridized with syntenic dog BAC DNA. Two 30-nt intervals, one with high conformance between human and dog sequences (97% conformance, red rectangle) and one with low conformance between human and dog sequences (60% conformance, blue rectangle), are shown. (Bottom) For the conserved sequence with high conformance, the 29 conforming nucleotides are indicated in red. For the conserved sequence with low conformance, the 18 conforming nucleotides are indicated in blue. (D) CONSEQ plots showing the conserved sequences (red peaks that are highlighted yellow) detected on the above 21q array relative to their position in the human reference sequence (horizontal axis) and their percent conformances (vertical axis). The high-conformance (97%) conserved sequence has been merged with neighboring conserved sequences to form a 190-nt element. The low-conformance (60%) conserved sequence is an isolated 30-nt element. (Small tan rectangles) Positions of interspersed repeats, which compose ∼33.5% of chromosome 21 sequence and were not tiled on the arrays (Supplemental Table 4, http://www.genome.org); therefore, conformance information is absent.

Figure 1

Detection and analysis of evolutionarily conserved sequences on human chromosome 21 by cross-species comparisons using high-density arrays. (A) The chromosome 21 arrays were designed using nonrepetitive sequences and hybridized with syntenic mouse and dog BACs (horizontal lines). A low-magnification view of a fluorescence hybridization image of an array is shown. (B) Chromosome 21 reference sequence was tiled as 25-mer oligonucleotides (probes). Each nucleotide of the reference sequence was interrogated by four probes: one probe complementary to the sequence and three probes noncomplementary at the central position. When the fluorescent intensities (white squares) of the complementary probes are greater than that of the noncomplementary probes, similarities between the tiled human 21q sequences and the hybridized animal DNA exist. (C) (Top) Enlarged view of a 21q array hybridized with syntenic dog BAC DNA. Two 30-nt intervals, one with high conformance between human and dog sequences (97% conformance, red rectangle) and one with low conformance between human and dog sequences (60% conformance, blue rectangle), are shown. (Bottom) For the conserved sequence with high conformance, the 29 conforming nucleotides are indicated in red. For the conserved sequence with low conformance, the 18 conforming nucleotides are indicated in blue. (D) CONSEQ plots showing the conserved sequences (red peaks that are highlighted yellow) detected on the above 21q array relative to their position in the human reference sequence (horizontal axis) and their percent conformances (vertical axis). The high-conformance (97%) conserved sequence has been merged with neighboring conserved sequences to form a 190-nt element. The low-conformance (60%) conserved sequence is an isolated 30-nt element. (Small tan rectangles) Positions of interspersed repeats, which compose ∼33.5% of chromosome 21 sequence and were not tiled on the arrays (Supplemental Table 4, http://www.genome.org); therefore, conformance information is absent.

Figure 2

Comparison of the conserved human–mouse and human–dog elements. (A) CONSEQ plots showing the conserved elements identified by hybridizing a 100-kb interval (upstream and encoding the 5′ end of the single-minded [SIM2] gene) with dog and mouse DNA. The conserved elements are shown relative to their position in the human reference sequence (horizontal axis) and their percent conformances (vertical axis). (Top) Conserved elements identified in both the human–dog and human–mouse comparisons (yellow rectangles), identified in only the human–dog comparison (blue rectangles), identified in only the human–mouse comparison (green rectangles), are indicated. In this 100-kb region, 3 of the conserved elements were identified only in human–mouse comparison, 17 of the conserved elements were identified in both comparisons, and 35 of the conserved elements were identified in only the human–dog comparison. (B) Analysis of the percent of human–mouse elements that are also conserved in the dog based on length. (C) Venn diagrams showing that 77% of the human–mouse IE elements and 51% of the human–mouse NIE elements were also identified as conserved elements in the human–dog comparison. In contrast, only 62% of the conserved human–dog IE elements and 13% of the conserved human–dog NIE elements were also identified as conserved elements in the human–mouse comparison.

Figure 2

Comparison of the conserved human–mouse and human–dog elements. (A) CONSEQ plots showing the conserved elements identified by hybridizing a 100-kb interval (upstream and encoding the 5′ end of the single-minded [SIM2] gene) with dog and mouse DNA. The conserved elements are shown relative to their position in the human reference sequence (horizontal axis) and their percent conformances (vertical axis). (Top) Conserved elements identified in both the human–dog and human–mouse comparisons (yellow rectangles), identified in only the human–dog comparison (blue rectangles), identified in only the human–mouse comparison (green rectangles), are indicated. In this 100-kb region, 3 of the conserved elements were identified only in human–mouse comparison, 17 of the conserved elements were identified in both comparisons, and 35 of the conserved elements were identified in only the human–dog comparison. (B) Analysis of the percent of human–mouse elements that are also conserved in the dog based on length. (C) Venn diagrams showing that 77% of the human–mouse IE elements and 51% of the human–mouse NIE elements were also identified as conserved elements in the human–dog comparison. In contrast, only 62% of the conserved human–dog IE elements and 13% of the conserved human–dog NIE elements were also identified as conserved elements in the human–mouse comparison.

Figure 2

Comparison of the conserved human–mouse and human–dog elements. (A) CONSEQ plots showing the conserved elements identified by hybridizing a 100-kb interval (upstream and encoding the 5′ end of the single-minded [SIM2] gene) with dog and mouse DNA. The conserved elements are shown relative to their position in the human reference sequence (horizontal axis) and their percent conformances (vertical axis). (Top) Conserved elements identified in both the human–dog and human–mouse comparisons (yellow rectangles), identified in only the human–dog comparison (blue rectangles), identified in only the human–mouse comparison (green rectangles), are indicated. In this 100-kb region, 3 of the conserved elements were identified only in human–mouse comparison, 17 of the conserved elements were identified in both comparisons, and 35 of the conserved elements were identified in only the human–dog comparison. (B) Analysis of the percent of human–mouse elements that are also conserved in the dog based on length. (C) Venn diagrams showing that 77% of the human–mouse IE elements and 51% of the human–mouse NIE elements were also identified as conserved elements in the human–dog comparison. In contrast, only 62% of the conserved human–dog IE elements and 13% of the conserved human–dog NIE elements were also identified as conserved elements in the human–mouse comparison.

Figure 3

Distribution analysis of conserved human–mouse elements in genic and nongenic intervals on chromosome 21. (A) Diagram illustrating the definition of genic regions as all sequences ± 10 kb of an annotated chromosome 21 gene. (B) The percent of the base pairs identified as conserved in the human–mouse comparison that are located in nongenic and genic intervals.

Figure 3

Distribution analysis of conserved human–mouse elements in genic and nongenic intervals on chromosome 21. (A) Diagram illustrating the definition of genic regions as all sequences ± 10 kb of an annotated chromosome 21 gene. (B) The percent of the base pairs identified as conserved in the human–mouse comparison that are located in nongenic and genic intervals.