Reprogramming of mammalian genome methylation is critically important but poorly understood. Klf4, a transcription factor directing reprogramming, contains a DNA binding domain with three consecutive C2H2 zinc fingers. Klf4 recognizes CpG or TpG within a specific sequence. Mouse Klf4 DNA binding domain has roughly equal affinity for methylated CpG or TpG, and slightly lower affinity for unmodified CpG. The structural basis for this key preference is unclear, though the side chain of Glu446 is known to contact the methyl group of 5-methylcytosine (5mC) or thymine (5-methyluracil). We examined the role of Glu446 by mutagenesis. Substituting Glu446 with aspartate (E446D) resulted in preference for unmodified cytosine, due to decreased affinity for 5mC. In contrast, substituting Glu446 with proline (E446P) increased affinity for 5mC by two orders of magnitude. Structural analysis revealed hydrophobic interaction between the proline's aliphatic cyclic structure and the 5-methyl group of the pyrimidine (5mC or T). As in wild-type Klf4 (E446), the proline at position 446 does not interact directly with either the 5mC N4 nitrogen or the thymine O4 oxygen. In contrast, the unmethylated cytosine's exocyclic N4 amino group (NH2) and its ring carbon C5 atom hydrogen bond directly with the aspartate carboxylate of the E446D variant. Both of these interactions would provide a preference for cytosine over thymine, and the latter one could explain the E446D preference for unmethylated cytosine. Finally, we evaluated the ability of these Klf4 mutants to regulate transcription of methylated and unmethylated promoters in a luciferase reporter assay.

CDC: Centers for Disease Control; DMSO: dimethyl sulfoxide; GHR: growth hormone receptor; hESCs: human embryonic stem cells; PFASs: per- and polyfluoroalkyl substances; PFCs: perfluorinated compounds; PFNA: perfluorononanoic acid; PFOS: perfluorooctanesulfonic acid; PFOA: perfluorooctanoic acid; PLZF: promyelocytic leukemia zinc finger; ROS: reactive oxygen species; HILI: RNA-mediated gene silencing 2; SSC: spermatogonial stem cell.

In 1973, the Velsicol Chemical Company, which manufactured FireMaster, a brominated flame retardant, and NutriMaster, a nutritional supplement, mistakenly shipped hundreds of pounds of FireMaster to grain mills around Michigan where it was incorporated into animal feed and then into the food chain across the state. An estimated 6.5 million Michigan residents consumed polybrominated biphenyl (PBB)-laced animal products leading to one of the largest agricultural accidents in U.S. history. To date, there have been no studies investigating the effects of PBB on epigenetic regulation in sperm, which could explain some of the endocrine-related health effects observed among children of PBB-exposed parents. Fusing epidemiological approaches with a novel in vitro model of human spermatogenesis, we demonstrate that exposure to PBB153, the primary component of FireMaster, alters the epigenome in human spermatogenic cells. Using our novel stem cell-based spermatogenesis model, we show that PBB153 exposure decreases DNA methylation at regulatory elements controlling imprinted genes. Furthermore, PBB153 affects DNA methylation by reducing de novo DNA methyltransferase activity at increasing PBB153 concentrations as well as reducing maintenance DNA methyltransferase activity at the lowest tested PBB153 concentration. Additionally, PBB153 exposure alters the expression of genes critical to proper human development. Taken together, these results suggest that PBB153 exposure alters the epigenome by disrupting methyltransferase activity leading to defects in imprint establishment causing altered gene expression, which could contribute to health concerns in the children of men exposed to PBB153. While this chemical is toxic to those directly exposed, the results from this study indicate that the epigenetic repercussions may be detrimental to future generations. Above all, this model may be expanded to model a multitude of environmental exposures to elucidate the effect of various chemicals on germline epigenetics and how paternal exposure may impact the health of future generations. In recent years, a major focus of research has been the Developmental Origins of Health and Disease (DOHaD) 1. A component of DOHaD research examines the impact of pre-and post-conception risk factors on long-term health and disease progression in adulthood. In terms of pre-conception risk factors, many researchers report that toxicants and environmental exposures can potentially alter the epigenome in gametes, giving these exposures the potential to ultimately impact the health of the next generation 2-5. In 1973, farms across Michigan mistakenly received cattle feed mixed with FireMaster BP-6, a chemical flame retardant composed of a mixture of polybrominated biphenyl compounds (PBBs), rather than NutriMaster, a food additive that increased milk production in farm animals 6-9. PBB-laced animal products were consumed by millions of residents, and the decline in animal

SUMMARY Sperm counts have rapidly declined in Western males over the past four decades. This rapid decline remains largely unexplained, but exposure to environmental toxicants provides one potential explanation for this decline. Flame retardants are highly prevalent and persistent in the environment, but many have not been assessed for their effects on human spermatogenesis. Using a human stem cell-based model of spermatogenesis, we evaluated two major flame retardants, hexabromocyclododecane (HBCDD) and tetrabromobisphenol A (TBBPA), under acute conditions simulating occupational-level exposures. Here we show that HBCDD and TBBPA are human male reproductive toxicants in vitro. Although these toxicants do not specifically affect the survival of haploid spermatids, they affect spermatogonia and primary spermatocytes through mitochondrial membrane potential perturbation and reactive oxygen species generation, ultimately causing apoptosis. Taken together, these results show that HBCDD and TBBPA affect human spermatogenesis in vitro and potentially implicate this highly prevalent class of toxicants in the decline of Western males’ sperm counts.

The Eph receptors and their cognate ephrin ligands play key roles in many aspects of nervous system development. These interactions typically occur within an individual tissue type, serving either to guide axons to their terminal targets or to define boundaries between the rhombomeres of the hindbrain. We have identified a novel role for the Caenorhabditis elegans ephrin EFN-4 in promoting primary neurite outgrowth in AIY interneurons and D-class motor neurons. Rescue experiments reveal that EFN-4 functions non-cell autonomously in the epidermis to promote primary neurite outgrowth. We also find that EFN-4 plays a role in promoting ectopic axon branching in a C. elegans model of X-linked Kallmann syndrome. In this context, EFN-4 functions non-cell autonomously in the body-wall muscle and in parallel with HS modification genes and HSPG core proteins. This is the first report of an epidermal ephrin providing a developmental cue to the nervous system. KEYWORDS ephrin; Eph receptor; heparan sulfate proteoglycan; axon outgrowth; axon branching A CCURATE development of the central nervous system requires contributions both from the extracellular environment in the form of guidepost cues and secreted guidance molecules and from contact with adjacent neurons or other tissues in the form of cell-surface receptors that can detect and transduce navigation cues. Guideposts typically take the form of specific tissue types such as the ventral midline of Caenorhabditis elegans or Drosophila, which provides a permissive environment for neurons to migrate along, while secreted guidance molecules provide spatial information and navigation instructions in the form of repulsive or attractive cues (Chilton 2006;Killeen and Sybingco 2008). Many studies have identified how individual neurons and guidance cues act to provide spatial and navigation information, but how does a single cell that possesses multiple axon guidance receptors find its target when exposed to multiple guidance cues? The nervous system of the nematode C. elegans provides a simple and defined model to examine the interplay between multiple neuronal guidance systems. The morphology and connectivity of C. elegans neurons have been established by serial-section electron microscopy, and the C. elegans genome possesses orthologs of most vertebrate axon guidance and guidepost genes (White et al. 1986;Bargmann 1998;Chisholm and Jin 2005;Ackley 2014). One of the most important classes of axon guidance molecules is the Eph receptor tyrosine kinases and their cognate ligands, the ephrins (Flanagan 2006;Lisabeth et al. 2013;Cayuso et al. 2015). Eph receptors and ephrins are required for the accurate connectivity of many parts of the vertebrate brain and also have roles in cell adhesion and embryonic morphogenesis (George et al. 1998;Chin-Sang et al. 1999;Chin-Sang et al. 2002;Klein 2012). We previously showed that the C. elegans ephrin EFN-4 functions in concert with the KAL-1/anosmin-heparan sulfate proteoglycan (HSPG) pathway to regulate neuroblast migration during...