Discovery of 33mer in chromosome 21 - the largest alpha satellite higher order repeat unit among all human somatic chromosomes

Abstract

The centromere is important for segregation of chromosomes during cell division in eukaryotes. Its destabilization results in chromosomal missegregation, aneuploidy, hallmarks of cancers and birth defects. In primate genomes centromeres contain tandem repeats of ~171 bp alpha satellite DNA, commonly organized into higher order repeats (HORs). In spite of crucial importance, satellites have been understudied because of gaps in sequencing - genomic "black holes". Bioinformatical studies of genomic sequences open possibilities to revolutionize understanding of repetitive DNA datasets. Here, using robust (Global Repeat Map) algorithm we identified in hg38 sequence of human chromosome 21 complete ensemble of alpha satellite HORs with six long repeat units (≥20 mers), five of them novel. Novel 33mer HOR has the longest HOR unit identified so far among all somatic chromosomes and novel 23mer reverse HOR is distant far from the centromere. Also, we discovered that for hg38 assembly the 33mer sequences in chromosomes 21, 13, 14, and 22 are 100% identical but nearby gaps are present; that seems to require an additional more precise sequencing. Chromosome 21 is of significant interest for deciphering the molecular base of Down syndrome and of aneuploidies in general. Since the chromosome identifier probes are largely based on the detection of higher order alpha satellite repeats, distinctions between alpha satellite HORs in chromosomes 21 and 13 here identified might lead to a unique chromosome 21 probe in molecular cytogenetics, which would find utility in diagnostics. It is expected that its complete sequence analysis will have profound implications for understanding pathogenesis of diseases and development of new therapeutic approaches.

Figure 1

Alpha satellite HOR ideogram for linear positioning of alpha satellite HOR arrays with long repeat units (n ≥ 8) obtained by applying GRM algorithm to the hg38 assembly sequence of human chromosome 21. CEN21 denotes location of the centromere. Only a segment of chromosome 21 containing alpha satellite HOR arrays is displayed. Ten HOR arrays are located within the centromere. The 23mer HOR with reverse monomers in the long arm of chromosome 21 is removed far from the centromere. Closer description of HOR arrays is given in Table 1.

Figure 2

GRM diagrams for the whole human chromosome 21 and for the contig NT_187321 which contains 33mer HOR array. (a) GRM diagram for the whole chromosome 21. Pronounced peaks that correspond to alpha satellite HORs are denoted by number of monomers in nmer HOR repeat unit. Inserts give magnified presentation of weak peaks for 22mer and 33mer, which are sizably screened by a noise of different other repeats in the whole chromosome 21. (b) GRM diagram for contig NT_187321 in which the 33mer HOR array is located. The pronounced GRM peak at 5,639 bp is a signature of 33mer HOR (5,639: 171 ≈ 33).

Figure 3

Schematic presentation of aligned monomer structure of 33mer HOR and 23mer HOR (reverse) arrays in human chromosome 21. (a) 33mer (4 complete HOR copies). Top: enumeration of columns corresponding to 33 constituent consensus monomers (Nos. 1 to 33, enumeration of every fifth monomer is displayed). Each of the four 33mer HOR copies (denoted h_i, i = 1, 2, 3, 4) is presented by 33 bars in the ith row. Each monomer of the same type (from consensus HOR) in different HOR copies is presented by a bar in the same column, corresponding to monomer enumeration at the top. (b) 23mer with reverse monomers (20 HOR copies). Each of the twenty 23mer HOR copies (denoted h_i, i = 1, 2, … 20) is presented by 23 bars in the ith row.

Figure 4

Dot-matrix plots of 33mer HORs in four acrocentric chromosomes. (a) chromosome 21; (b) chromosome 13; (c) chromosome 14; (d) chromosome 22. Dot-matrix analyses determined the presence or absence of HOR structure using a window size of monomer length and mismatch limits ranging at ~7%. Monomers are labeled in order of appearance, displayed in matrix along the upper horizontal axis (from left to right) and along the left vertical axis (from up to down): at both axes label 1 corresponds to the first monomer in alpha satellite ensemble, label 2 to the second monomer, etc. In this way, the alpha satellite ensemble is compared with itself, giving pairwise comparisons of divergence between constituting alpha satellite monomers. Each cell in dot-matrix which represents divergence between monomers located at identical positions in different HOR copies (e.g. the second monomer in the third HOR copy on the horizontal axis and the second monomer in the fourth HOR copy on the vertical axis, etc.) correspond to relatively small divergence between monomers (here chosen below 7%) and is shown as colored dot. The other cells in dot-matrix correspond to higher divergence (above 7%) are blank. In this way, for each HOR array the dot-matrix diagram is obtained as a set of equidistant diagonal lines at spacing equal to the number of monomers in HOR unit (n = 33), parallel to the self-diagonal.