Lack of cell cycle checkpoints and uninterrupted passage through S-phase continuously renew the embryonic stem (ES) cell population and maintain pluripotency. Here, we show that to regulate mitotic progression and pluripotency ES cells must keep the aryl hydrocarbon receptor (AHR), an environmental sensor and transcriptional regulator, in a persistent state of repression. This repression, however, is not always absolute, causing the AHR to fluctuate between reversible states of expression and repression, with a fraction of the cells escaping repression at any one time. Cells that escape AHR repression exhibit reduced levels of the pluripotency factors OCT4 and SOX2 and show an extended mitotic traverse time due to AHR-dependent MID1 repression and the subsequent disruption of the MID1-PP2A-CDC25B-CDK1 signaling pathway that regulates mitosis. Unlike the bulk of the cell population that differentiates into cardiomyocytes upon stimulation, AHR-expressing ES cells restrict cardiogenesis and commit to a neuroglia cell fate. It appears that the untimely expression of the Ahr gene needs to be repressed to maintain ES cell mitotic progression and prevent premature loss of pluripotency. Stem Cells 2016;34:2825-2839.

The unique DNA packaging of spermatozoa renders them resistant to DNA isolation techniques used for somatic cells, requiring alternative methods that are slow and labor intensive. Here we present a rapid method for isolating high-quality sperm DNA. Isolated human sperm cells were homogenized with 0.2 mm steel beads for 5 min at room temperature in the presence of guanidine thiocyanate lysis buffer supplemented with 50 mM tris(2-carboxyethyl)phosphine (TCEP). Our method yielded >90% high-quality DNA using 3 different commercially available silica-based spin columns. DNA yields did not differ between immediate isolation (2.84 ± 0.04 pg/cell) and isolation after 2 weeks of homogenate storage at room temperature (2.91 ± 0.13 pg/cell). DNA methylation analyses revealed similar methylation levels at both time points for three imprinted loci. Our protocol has many advantages: it is conducted at room temperature; lengthy proteinase K (ProK) digestions are eliminated; the reducing agent, TCEP, is odorless and stable at room temperature; nucleic acids are stabilized, allowing storage of homogenate; and it is adaptable for other mammalian species. Taken together, the benefits of our improved method have important implications for settings where sample processing constraints exist.

Congenital heart disease (CHD), the leading birth defect worldwide, has a largely unknown etiology, likely to result from complex interactions between genetic and environmental factors during heart development, at a time when the heart adapts to diverse physiological and pathophysiological conditions. Crucial among these is the regulation of cardiomyocyte development and postnatal maturation, governed by dynamic changes in DNA methylation. Previous work from our laboratory has shown that exposure to the environmental toxicant tetrachlorodibenzo-p-dioxin (TCDD) disrupts several molecular networks responsible for heart development and function. To test the hypothesis that the disruption caused by TCDD in the heart results from changes in DNA methylation and gene expression patterns of cardiomyocytes, we established a stable mouse embryonic stem cell line expressing a puromycin resistance selectable marker under control of the cardiomyocyte-specific Nkx2-5 promoter. Differentiation of these cells in the presence of puromycin induces the expression of a large suite of cardiomyocyte specific markers. To assess the consequences of TCDD treatment on gene expression and DNA methylation in these cardiomyocytes, we subjected them to transcriptome and methylome analyses in the presence of TCDD. Unlike control cardiomyocytes maintained in vehicle, the TCDD-treated cardiomyocytes showed extensive gene expression changes, with a significant correlation between differential RNA expression and DNA methylation in 111 genes, many of which are key elements of pathways that regulate cardiovascular development and function. Our findings provide an important clue towards the elucidation of the complex interactions between genetic and epigenetic mechanisms after developmental TCDD exposure that may contribute to CHD.

Congenital heart disease (CHD) is the most common congenital abnormality. A precise etiology for CHD remains elusive, but likely results from interactions between genetic and environmental factors during development, when the heart adapts to physiological and pathophysiological conditions. Further, it has become clearer that early exposure to toxins that do not result in overt CHD may be associated with adverse cardiac outcomes that are not manifested until later life. Previously, interference with endogenous developmental functions of the aryl hydrocarbon receptor (AHR), either by gene ablation or by in utero exposure to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), a potent AHR ligand, was shown to cause structural, molecular and functional cardiac abnormalities and altered heart physiology in mouse embryos. Here, we show that continuous exposure to TCDD from fertilization throughout adulthood caused male mice to underperform at exercise tolerance tests compared to their control and female counterparts, confirming previous observations of a sexually dimorphic phenotype. Renin-angiotensin stimulation by angiotensin II (Ang II) caused measurable increases in blood pressure and left ventricle mass, along with decreased end diastolic volume and preserved ejection fraction. Interestingly, TCDD exposure caused measurable reductions in the myocardial hypertrophic effects of Ang II, suggesting that endogenous AHR signaling present in adulthood may play a role in the pathogenesis of hypertrophy. Overall, the findings reported in this pilot study highlight the complex systems underlying TCDD exposure in the development of cardiac dysfunction in later life.