Tracing ancestry with methylation patterns: most crypts appear distantly related in normal adult human colon

Abstract

Background: The ability to discern ancestral relationships between individual human colon crypts is limited. Widely separated crypts likely trace their common ancestors to a time around birth, but closely spaced adult crypts may share more recent common ancestors if they frequently divide by fission to form clonal patches. Alternatively, adult crypts may be long-lived structures that infrequently divide or die.

Methods: Methylation patterns (the 5' to 3' order of methylation) at CpG sites that exhibit random changes with aging were measured from isolated crypts by bisulfite genomic sequencing. This epigenetic drift may be used to infer ancestry because recently related crypts should have similar methylation patterns.

Results: Methylation patterns were different between widely separated ("unrelated") crypts greater than 15 cm apart. Evidence for a more recent relationship between directly adjacent or branched crypts could not be found because their methylation pattern distances were not significantly different than widely separated crypt pairs. Methylation patterns are essentially equally different between two adult human crypts regardless of their relative locations.

Conclusions: Methylation patterns appear to record somatic cell trees. Starting from a single cell at conception, sequences replicate and may drift apart. Most adult human colon crypts appear to be long-lived structures that become mosaic with respect to methylation during aging.

Figure 1

Colon somatic cell ancestral trees resemble a "big bang", originating from a zygote and progressively growing with aging. All cells eventually relate to each other, and therefore "Y" shaped trees characterize the relationship between any two cells. The last possible common ancestor between widely spaced crypts is around birth, and their ancestral trees must have short trunks and essentially life-long branches. In contrast, more closely spaced crypts may be related by recent crypt fission, yielding ancestral trees with relatively longer trunks and shorter branches. Tree branch lengths may be inferred from methylation pattern drift – methylation at some CpG sites appears to randomly change with aging. Methylation patterns were not significantly different between widely or closely spaced (within the same 1–2 cm² patch or directly adjacent) crypts, consistent with stable, long-lived adult crypts.

Figure 2

Methylation tags. A) The bisulfite converted sequence of the BGN methylation tag, which contains 9 CpG sites (underlined). A capital "T" represents bisulfite conversion of a non-CpG site "C". B) Intracrypt and intercrypt distances between methylation tags. Average distances (line) change with aging according to our model of human crypt niches [11]. 95% simulation intervals (dotted lines) reflect that drift and therefore distances are stochastic and not deterministic. The lower simulation bound for intracrypt distances is zero. Intracrypt distances remain relatively constant through life because stem cell niche turnover results in the periodic loss of all stem cell lineages except one. These "bottlenecks" ensure that all cells within a crypt are relatively closely related to each other. In contrast, intercrypt distances increase with age, reflecting that methylation tags randomly drift apart in unrelated crypts. C) Examples of BGN methylation tags (5' to 3') in adjacent and branched crypts. Filled circles are methylated CpG sites and tags from individual crypts are grouped. Intracrypt and intercrypt distances are labeled.

Figure 3

Distances between BGN tags. Intracrypt distances are significant less than intercrypt distances. There are no significant differences between intercrypt distances of adjacent crypts, crypts randomly sampled from the same 1–2 cm² patch, or crypts from widely separated patches. Intracrypt distances of branched crypts were significantly greater than individual crypts and not significantly different from intercrypt distances. These distance relationships are consistent with long-lived individual or branched crypts. Shaded in yellow are adjacent crypts with the smallest intercrypt distances. These adjacent crypt pairs appeared to be distantly related when examined at another locus (see Table 2).

Figure 4

Example of a branched crypt analyzed for methylation patterns.