Complex biology of constitutional ring chromosomes structure and (in)stability revealed by somatic cell reprogramming

Human ring chromosomes are often unstable during mitosis, and daughter cells can be partially or completely aneuploid. We studied the mitotic stability of four ring chromosomes, 8, 13, 18, and 22, in long-term cultures of skin fibroblasts and induced pluripotent stem cells (iPSCs) by GTG karyotyping and aCGH. Ring chromosome loss and secondary aberrations were observed in all fibroblast cultures except for r(18). We found monosomy, fragmentation, and translocation of indexed chromosomes. In iPSCs, aCGH revealed striking differences in mitotic stability both between iPSC lines with different rings and, in some cases, between cell lines with the same ring chromosome. We registered the spontaneous rescue of karyotype 46,XY,r(8) to 46,XY in all six iPSC lines through ring chromosome loss and intact homologue duplication with isoUPD(8)pat occurrence, as proven by SNP genotype distribution analysis. In iPSCs with other ring chromosomes, karyotype correction was not observed. Our results suggest that spontaneous correction of the karyotype with ring chromosomes in iPSCs is not universal and that pluripotency is compatible with a wide range of derivative karyotypes. We conclude that marked variability in the frequency of secondary rearrangements exists in both fibroblast and iPSC cultures, expanding the clinical significance of the constitutional ring chromosome.

Modeling of dynamic mosaicism on ring chromosomes in human fibroblasts and IPSCS

Митотическая нестабильность кольцевых хромосом может приводить к появлению клеточных клонов с различной генетической структурой. В качестве модели нестабильности кольцевых хромосом в митозе мы использовали фибробласты от пациентов с r(8), r(13), r(18) и r(22) и полученные из них индуцированные плюрипотентные стволовые клетки (ИПСК). Линии ИПСК с r(22) имели относительно стабильный кариотип на протяжении десятков (до 60) пассажей и сохраняли неизменную структуру кольцевой хромосомы. Кариотип линий ИПСК с r(8) и r(18) на ранних пассажах стабильный, планируется его изучение на поздних пассажах. Наибольшее разнообразие кариотипа выявлено в линиях ИПСК с r(13), в которых наблюдали различные перестройки и выраженную клеточную гетерогенность. Определение факторов, влияющих на митотическую стабильность кольцевых хромосом, может иметь значение для консультирования пациентов. Mitotic instability of ring chromosomes can lead to the appearance of cell clones with different genetic structure. IPSCs from fibroblasts of patients with r(8), r(13), r(18), and r(22) were used as a model of ring chromosomes mitotic behavior. Karyotypes of iPSC lines with r(8) and r(18) have so far been evaluated only in the early passages, lines with r(22) have maintained a relatively stable karyotype up to 60 passages. The occurrence of rearrangements and cellular heterogeneity was found characteristic for r(13) iPSCs. The determination of factors affecting the ring chromosomes mitotic stability would be beneficial for the patient’s prognosis.

Oct1/Pou2f1 is selectively required for gut regeneration and regulates gut malignancy

The transcription factor Oct1/Pou2f1 promotes poised gene expression states, mitotic stability, glycolytic metabolism and other characteristics of stem cell potency. To determine the effect of Oct1 loss on stem cell maintenance and malignancy, we deleted Oct1 in two different mouse gut stem cell compartments. Oct1 deletion preserved homeostasis in vivo and the ability to generate cultured organoids in vitro, but blocked the ability to regenerate after treatment with dextran sodium sulfate, and the ability to maintain organoids after passage. In a chemical model of colon cancer, loss of Oct1 in the colon severely restricted tumorigenicity. In contrast, loss of one or bothOct1alleles progressively increased tumor burden in a colon cancer model driven by loss of heterozygosity of the tumor suppressor geneApc.The different outcomes are consistent with prior findings that Oct1 promotes mitotic stability, and consistent with different gene expression signatures associated with the two models. These results reveal that Oct1 is selectively required for gut regeneration, and has potent effects in colon malignancy, with outcome (pro-oncogenic or tumor suppressive) dictated by tumor etiology.Author summaryColorectal cancer is the second leading cause of cancer death in the United States. Approximately 35% of diagnosed patients eventually succumb to disease. The high incidence and mortality due to colon cancer demand a better understanding of factors controlling the physiology and pathophysiology of the gastrointestinal tract. Previously, we and others showed that the widely expressed transcription factor is expressed at higher protein levels in stem cells, including intestinal stem cells. In this study we use a conditional mouseOct1(Pou2f1) allele deleted in two different intestinal stem cell compartments. The results indicate that Oct1 loss is dispensable for maintenance of the mouse gut, but required for regeneration. We also tested Oct1 loss in the context of two different mouse colon cancer models. We find that Oct1 loss has opposing effects in the two models, and further that the two models are associated with different gene expression signatures. The differentially expressed genes are enriched for previously identified Oct1 targets, suggesting that differential gene control by Oct1 is one mechanism underlying different outcomes.