Epigenetic Characteristics of Human Subtelomeres Vary in Cells Utilizing the Alternative Lengthening of Telomeres (ALT) Pathway

Most human cancers circumvent senescence by activating a telomere length maintenance mechanism, most commonly involving telomerase activation. A minority of cancers utilize the recombination-based alternative lengthening of telomeres (ALT) pathway. The exact requirements for unleashing normally repressed recombination at telomeres are yet unclear. Epigenetic modifications at telomeric regions were suggested to be pivotal for activating ALT; however, conflicting data exist regarding their exact nature and necessity. To uncover common ALT-positive epigenetic characteristics, we performed a comprehensive analysis of subtelomeric DNA methylation, histone modifications, and TERRA expression in several ALT-positive and ALT-negative cell lines. We found that subtelomeric DNA methylation does not differentiate between the ALT-positive and ALT-negative groups, and most of the analyzed subtelomeres within each group do not share common DNA methylation patterns. Additionally, similar TERRA levels were measured in the ALT-positive and ALT-negative groups, and TERRA levels varied significantly among the members of the ALT-positive group. Subtelomeric H3K4 and H3K9 trimethylation also differed significantly between samples in the ALT-positive group. Our findings do not support a common route by which epigenetic modifications activate telomeric recombination in ALT-positive cells, and thus, different therapeutic approaches will be necessary to overcome ALT-dependent cellular immortalization.

Cell-to-cell expression dispersion of B-cell surface proteins is linked to genetic variants in humans

Variability in gene expression across a population of homogeneous cells is known to influence various biological processes. In model organisms, natural genetic variants were found that modify expression dispersion (variability at a fixed mean) but very few studies have detected such effects in humans. Here, we analyzed single-cell expression of four proteins (CD23, CD55, CD63 and CD86) across cell lines derived from individuals of the Yoruba population. Using data from over 30 million cells, we found substantial inter-individual variation of dispersion. We demonstrate, via de novo cell line generation and subcloning experiments, that this variation exceeds the variation associated with cellular immortalization. We detected a genetic association between the expression dispersion of CD63 and the rs971 SNP. Our results show that human DNA variants can have inherently-probabilistic effects on gene expression. Such subtle genetic effects may participate to phenotypic variation and disease outcome.

Cell-to-cell expression dispersion of B-cell surface proteins displays genetic variation among humans

ABSTRACTVariability in gene expression across a population of homogeneous cells is known to influence various biological processes. In model organisms, natural genetic variants were found that modify expression dispersion (variability at a fixed mean) but whether such effects exist in humans has not been fully demonstrated. Here, we analyzed single-cell expression of four proteins (CD23, CD55, CD63 and CD86) across cell lines derived from individuals of the Yoruba population. Using data from over 30 million cells, we found substantial inter-individual variation of dispersion. We demonstrate, via de novo cell line generation and subcloning experiments, that this variation exceeds the variation associated with cellular immortalization. By association mapping, we linked the expression dispersion of CD63 to the rs971 SNP. Our results show that human DNA variants can have inherently-probabilistic effects on gene expression. Such subtle genetic effects may participate to phenotypic variation and disease predisposition.

Potential for Isolation of Immortalized Hepatocyte Cell Lines by Liver-DirectedIn VivoGene Delivery of Transposons in Mice

Isolation of hepatocytes and their culturein vitrorepresent important avenues to explore the function of such cells. However, these studies are often difficult to perform because of the inability of hepatocytes to proliferatein vitro. Immortalization of isolated hepatocytes is thus an important step toward continuousin vitroculture. For cellular immortalization, integration of relevant genes into the host chromosomes is a prerequisite. Transposons, which are mobile genetic elements, are known to facilitate integration of genes of interest (GOI) into chromosomesin vitroandin vivo. Here, we proposed that a combination of transposon- and liver-directed introduction of nucleic acids may confer acquisition of unlimited cellular proliferative potential on hepatocytes, enabling the possible isolation of immortalized hepatocyte cell lines, which has often failed using more traditional immortalization methods.