Higher 5-hydroxymethylcytosine identifies immortal DNA strand chromosomes in asymmetrically self-renewing distributed stem cells

Immortal strands are the targeted chromosomal DNA strands of nonrandom sister chromatid segregation, a mitotic chromosome segregation pattern unique to asymmetrically self-renewing distributed stem cells (DSCs). By nonrandom segregation, immortal DNA strands become the oldest DNA strands in asymmetrically self-renewing DSCs. Nonrandom segregation of immortal DNA strands may limit DSC mutagenesis, preserve DSC fate, and contribute to DSC aging. The mechanisms responsible for specification and maintenance of immortal DNA strands are unknown. To discover clues to these mechanisms, we investigated the 5-methylcytosine and 5-hydroxymethylcytosine (5hmC) content on chromosomes in mouse hair follicle DSCs during nonrandom segregation. Although 5-methylcytosine content did not differ significantly, the relative content of 5hmC was significantly higher in chromosomes containing immortal DNA strands than in opposed mitotic chromosomes containing younger mortal DNA strands. The difference in relative 5hmC content was caused by the loss of 5hmC from mortal chromosomes. These findings implicate higher 5hmC as a specific molecular determinant of immortal DNA strand chromosomes. Because 5hmC is an intermediate during DNA demethylation, we propose a ten-eleven translocase enzyme mechanism for both the specification and maintenance of nonrandomly segregated immortal DNA strands. The proposed mechanism reveals a means by which DSCs “know” the generational age of immortal DNA strands. The mechanism is supported by molecular expression data and accounts for the selection of newly replicated DNA strands when nonrandom segregation is initiated. These mechanistic insights also provide a possible basis for another characteristic property of immortal DNA strands, their guanine ribonucleotide dependency.Although distributed stem cells (DSCs) are universally essential for normal tissue function, health, and longevity (1–5), understanding of the cellular mechanisms responsible for their unique tissue functions is quite limited. In particular, the mechanisms responsible for the defining properties of DSCs—asymmetric self-renewal (ASR) and nonrandom sister chromatid segregation—are unknown.ASR is a special subclass of asymmetric cell division by which DSCs continuously renew mature differentiated tissue cells (6–8). During ASR, DSCs divide asymmetrically with retention of their own stem cell phenotype while simultaneously producing nonstem sisters that are precursors for short-lived tissue-specific differentiating cell lineages. This long-lived role of DSCs in the cell kinetics architecture of renewing tissues is the basis for the hypothesis that they are the predominant cells of origin for tumors induced by gene mutations (9, 10).Nonrandom sister chromatid segregation is tightly associated with ASR (11, 12). During mitosis, asymmetrically self-renewing DSCs (aDSCs) continuously cosegregate to themselves the set of mitotic chromosomes that contain the older of the two parental template DNA strands. By retaining the same set of template DNA strands over many successive ASR divisions, long-lived DSCs are proposed to reduce their rate of accrual of carcinogenic mutations by 100–1,000-fold compared with their shorter-lived differentiating progeny cells, which retain unrepaired and misrepaired replication errors as a consequence of random segregation (9, 13). Nonrandom segregation also may be an important factor contributing to tissue aging. Accrued chemical damage in the cosegregated template DNA strands, called “immortal DNA strands” (9), may compromise the function and viability of tissue DSCs (1). The immortal DNA strands themselves also might organize epigenomic regulators that are responsible for maintaining the DSC fate (3).Nonrandom segregation of mitotic sister chromatids was discovered in experiments with cultured mouse fetal fibroblasts (14) and the root tips of legumes and wheats (15, 16). More recently, nonrandom segregation has been described in a diverse range of mammalian species and normal (3, 4, 11, 12, 17–27) and cancerous (28–30) tissue types. Thus, far, these studies have shed only limited light on the nature of the responsible molecular mechanisms. In particular, the molecular basis for the specification and maintenance of immortal DNA strands during nonrandom segregation remains unknown nearly a half century after their discovery (16).As proposed initially (9), we have confirmed that nonrandom segregation occurs specifically when DSCs adopt an ASR program (4, 11, 12). Five specific cellular proteins—p53, inosine 5′ monophosphate dehydrogenase type II (EC 1.2.1.14) (12), left-right dynein (25), histone H2A.Z (3), and PIM-1 kinase (26)—have been identified as playing a role in nonrandom chromosome segregation mechanisms. In addition, we have shown that guanine ribonucleotide precursors and high cell density are physiological regulators of ASR and nonrandom segregation (4, 12, 31, 32). However, so far, none of these factors has led to an understanding of how DSCs achieve nonrandom segregation.Two recently discovered properties of the histone H2A variant H2A.Z motivated us to investigate the 5-methylcytosine (5mC) and 5-hydroxymethylcytosine (5hmC) content of immortal and mortal chromosomes in mouse hair follicle DSCs undergoing nonrandom segregation. H2A.Z chromosomal detection shows a reciprocal relationship with 5mC sites (33), and H2A.Z is molecularly masked on the mortal set of chromosomes in nonrandomly segregating DSCs (3). These analyses reveal that immortal chromosomes, which contain immortal DNA strands, have a significantly higher level of 5hmC modification. The identification of this chromosomal 5hmC asymmetry suggests a molecular mechanism that can account for immortal DNA strand specification and maintenance as well as their long-enigmatic guanine ribonucleotide dependency (12, 32).