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Cruciform-forming inverted repeats appear to have mediated many of the microinversions that distinguish the human and chimpanzee genomes

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Abstract

Submicroscopic inversions have contributed significantly to the genomic divergence between humans and chimpanzees over evolutionary time. Those microinversions which are flanked by segmental duplications (SDs) are presumed to have originated via non-allelic homologous recombination between SDs arranged in inverted orientation. However, the nature of the mechanisms underlying those inversions which are not flanked by SDs remains unclear. We have investigated 35 such inversions, ranging in size from 51-nt to 22056-nt, with the goal of characterizing the DNA sequences in the breakpoint-flanking regions. Using the macaque genome as an outgroup, we determined the lineage specificity of these inversions and noted that the majority (N = 31; 89%) were associated with deletions (of length beween 1-nt and 6754-nt) immediately adjacent to one or both inversion breakpoints. Overrepresentations of both direct and inverted repeats, ≥ 6-nt in length and capable of non-B DNA structure formation, were noted in the vicinity of breakpoint junctions suggesting that these repeats could have contributed to double strand breakage. Inverted repeats capable of cruciform structure formation were also found to be a common feature of the inversion breakpoint-flanking regions, consistent with these inversions having originated through the resolution of Holliday junction-like cruciforms. Sequences capable of non-B DNA structure formation have previously been implicated in promoting gross deletions and translocations causing human genetic disease. We conclude that non-B DNA forming sequences may also have promoted the occurrence of mutations in an evolutionary context, giving rise to at least some of the inversion/deletions which now serve to distinguish the human and chimpanzee genomes.

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Abbreviations

aCGH:

array-based comparative genomic hybridization

bp:

base pairs

DSBs:

double strand breaks

DR:

direct repeat

HJ:

Holliday junction

IR:

inverted repeat

LINEs:

long interspersed nuclear elements

LTR:

long terminal repeat

MIR:

mammalian-wide interspersed repeat

nt:

nucleotides

SD:

segmental duplication

SE:

symmetric element

SINEs:

small interspersed nuclear elements

SSA-type:

single-strand annealing-type

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Authors and Affiliations

  1. Institute of Human Genetics, University of Ulm, Albert-Einstein-Allee 11, 89081, Ulm, Germany

    Jessica Kolb, Josef Högel & Hildegard Kehrer-Sawatzki

  2. School of Computing, Engineering and Physical Sciences, University of Central Lancashire, Preston, PR1 2HE, UK

    Nadia A. Chuzhanova

  3. Science Park-Research Division, Department of Carcinogenesis, The University of Texas M.D. Anderson Cancer Center, Smithville, TX, 78957, USA

    Karen M. Vasquez & Albino Bacolla

  4. Institute of Medical Genetics, School of Medicine, Cardiff University, Cardiff, CF14 4XN, UK

    David N. Cooper

Authors
  1. Jessica Kolb
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  2. Nadia A. Chuzhanova
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  3. Josef Högel
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  4. Karen M. Vasquez
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  5. David N. Cooper
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  6. Albino Bacolla
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  7. Hildegard Kehrer-Sawatzki
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Corresponding author

Correspondence to Hildegard Kehrer-Sawatzki.

Additional information

Responsible Editor: Fengtang Yang.

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Fig. S1

Alignment dot-plots. The panels depict the alignments of the 35 inversions between the human genomic plus strands and the chimpanzee genomic minus strands, including ~10 nt of non-inverted DNA sequences at both ends of each sequence. The genomic coordinates for the human sequences are given in Table 1. The genomic coordinates for the orthologous chimpanzee sequences following BLAT searches were as follows: panel 1, chr10:12136276-12136329; panel 2, chr10:116844843-116844954; panel 3, chr10:53922309-53922470; panel 4, chr10:126100936-126101213; panel 5, chr7:95515505-95515791; panel 6, chr11:113304150-113304491; panel 7, chr10:11718879-11719507; panel 8, chr6:72091037-72091659; panel 9, chr5:35330988-35331976; panel 10, chr16:85430135-85431243; panel 11, chr6:12644332-12645386; panel 12, chr7:137167855-137169253; panel 13, chr5:20180206-20181756; panel 14, chr5:119533003-119534741; panel 15, chr11:87128690-87129600; panel 16, chr7:124887989-124892425; panel 17, chr10:36549197-36554419; panel 18, chr5:136839424-136840615; panel 19, chr5:147021847-147035189; panel 20, chr4:43620932-43622318; panel 21, chr2a:8128093-8132020; panel 22, chr13:82801968-82807302; panel 23, chr4:44508164-44511072; panel 24, chr2b:160340903-160341245; panel 25, chr8:136861733-136862113; panel 26, chr11:131738359-131760399; panel 27, chr2b:180789656-180789856; panel 28, chr4:98469220-98469364; panel 29, chr13:115695710-115696114; panel 30, chr14:48396349-48396784; panel 31, chr17:73655852-73656289; panel 32, chr18:18030460-18030973; panel 33, chr13:113714126-113714652; panel 34, chr14:94557537-94558775; panel 35, chr6:168834882-168837248 (PDF 626 kb)

Fig. S2

Localization of the sequences included in the datasets (D) used to determine the repeat element frequency in regions flanking the breakpoints of the inversions >1000-nt. Vertical black lines indicate the positions of the breakpoints. Dataset 1 (D1) included 500-nt from the non-inverted region whereas dataset 2 (D2) included 500-nt directly flanking the inversion breakpoints within the inverted segments. Dataset (D3) included the sequences within the deleted regions in the ancestral sequence. The frequency of SINE and LINE elements in these datasets was investigated in both human and chimpanzee (PPT 119 kb)

Fig. S3

Case 32 comprises a 594 bp inversion in the human genome (bold and ^). In the chimpanzee genome, it is characterized by the presence of a 146 bp IR in which the left hand repeat copy is located almost entirely outside of the proximal breakpoint (with the exception of the last 8 bp, broken black arrows), whereas the right hand repeat copy is located mostly (118/146) within the inverted region. In human, the entire left arm of the IR has been lost along with an additional 71 bp (lower case), whereas a 14 bp sequence (blue) has been inserted de novo just distal to the inversion breakpoint. The 14 nt is exactly complementary to the proximally inverted region (red arrows). We speculate that the 146 bp IRs mediated the inversion through the formation of a cruciform structure and that following deletion of the left hand repeat copy, a novel cruciform structure was created during the resolution of the secondary structure intermediate. In case 35, a human-specific inversion of 2352 bp occurred (bold and ^). The inversion occurred with stark precision within the last bases of a 669 bp IR (black arrows), with no gain or loss of nts with respect to the ancestral chimpanzee genomic sequence. Sequence alignment analyses support the conclusion that the IR sequences became inverted between the two species. The inversion is readily explicable by the parsimony model presented in Fig. 3, whereby the basal HJ was resolved through the non-intersecting strands whereas the apical HJ was resolved through the intersecting strands. Case 4 represents an inversion of 258 bp in human (bold and ^). In addition, a 330 bp segment flanked by a TGA microhomology (yellow highlighting) has been deleted from the chimpanzee genome downstream of the distal inversion breakpoint (lower case). By contrast, a 173 bp insertion (blue) is evident at the proximal breakpoint, representing an inverted copy of the last 173 bp of the inverted segment (broken red arrows). This inversion is explicable in terms of the model presented in Fig. 3, Structures V and VI. In case 23, the human-specific inversion involves 2885 nt in human and 2889 nt in chimpanzee (bold and ^). However, because part of the inversion is present as an inverted repeat in human, the precise position of the upstream breakpoint is ambiguous. Thus, the inversion could have occurred over a 2252 nt interval in human and 2225 nt in the chimpanzee (left broken red arrow). Interestingly, the distal breakpoint, which is unambiguous, occurs over a repeating (AC)5 tract, a sequence known to be capable of adopting a left-handed Z-DNA conformation under conditions of torsional stress (negative supercoiling). In the human derivative chromosome, an inverted duplication of 634 bp has occurred, thereby creating a large IR (broken red arrows). In addition, a 193 bp segment has been transposed from downstream of the inversion breakpoint to upstream of the proximal inversion breakpoint (grey highlighting), whereas its last 12 nt have become duplicated distally to the inversion (blue arrows). [The transposition is independent of the inversion size]. Most (10 nt) of the Z-DNA-forming tract was deleted in the process (lower case). We conclude that the large IR was likely to have been critical both to the inversion process and to the direct transposition event. Finally, we consider case 5 in which a chimpanzee-specific inversion of 267 bp occurred (bold and ^). In the ancestral human genome, the inversion breakpoints occurred at a homologous GA dinucleotide (yellow highlighting). In addition, Mfold analyses predicted several folded branched hairpin structures with moderate stability at both ends (red, black and green arrows). Upon inversion in the chimpanzee genome, a larger hairpin structure was predicted at the proximal breakpoint (red arrows). We postulate that these short IRs played a crucial role in this inversion, as outlined in Fig. 3 for the simpler cases (DOC 41.5 kb)

Fig. S4

Mfold modeling of the inversion #34 cruciform structure. The two 178-nt inverted repeat sequences flanking the breakpoints of human inversion #34 (GTTAG---ATATTTA and TAAATAT---CTAATA, Fig. 2) were joined by a 4-nt loop (TTTT) and used as the query. The Figure illustrates the only result returned by the analysis (DOC 55.5 kb)

Table S1

Sequences of PCR primers used to amplify across the inversion breakpoints in the species in which the inversion occurred (DOC 49 kb)

Table S2

Coordinates of the inversions investigated by dotplot analysis and genes affected by these inversions (XLS 41.5 kb)

Table S3

Number of SINE/LINE elements in the human genome sequence flanking the 24 human-specific inversion breakpoints (DOC 48 kb)

Table S4

Number of SINE/LINE elements in the chimpanzee genome sequence flanking the sites of the 24 human-specific inversion breakpoints (DOC 47.5 kb)

Table S5

Number of SINE/LINE elements in the chimpanzee genome sequence flanking the 11 chimpanzee-specific inversion breakpoints (DOC 34 kb)

Table S6

Number of SINE/LINE elements in the human genome sequence flanking the sites of the 11 chimpanzee-specific inversion breakpoints (DOC 34 kb)

Table S7

Total numbers of SINE or LINE elements in the human and chimpanzee genomes according to Gibbs et al. (2007). (DOC 28.5 kb)

Table S8

Motifs associated with site-specific cleavage/recombination, high frequency mutation and gene rearrangement or recombination ≥ 5 bp of length (DOC 66.5 kb)

Table S9

SINE/LINE frequency in the breakpoint-flanking regions of the 24 human-specific inversions in human as compared to chimpanzee (DOC 59 kb)

Table S10

SINE/LINE frequencies in the breakpoint-flanking regions of the 11 chimpanzee-specific inversions in human as compared to chimpanzee (DOC 44.5 kb)

Text S1 (DOC 31 kb)

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Kolb, J., Chuzhanova, N.A., Högel, J. et al. Cruciform-forming inverted repeats appear to have mediated many of the microinversions that distinguish the human and chimpanzee genomes. Chromosome Res 17, 469–483 (2009). https://doi.org/10.1007/s10577-009-9039-9

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