Sarah Kimmins

Disruption of histone methylation in developing sperm impairs offspring health transgenerationally

Generations affected by histone changes Parent and even grandparent environmental exposure can transmit adverse health effects to offspring. The mechanism of transmission is unclear, but some studies have implicated variations in DNA methylation. In a mouse model, Siklenka et al. found that alterations in histone methylation during sperm formation in one generation leads to reduced survival and developmental abnormalities in three subsequent generations (see the Perspective by McCarrey). Although changes in DNA methylation were not observed, altered sperm RNA content and abnormal gene expression in offspring were measured. Thus, chromatin may act as a mediator of molecular memory in transgenerational inheritance. Science, this issue p. 10.1126/science.aab2006; see also p. 634 Overexpression of a histone demethylase in the mouse germ line reveals a mode of transgenerational epigenetic inheritance. [Also see Perspective by McCarrey] INTRODUCTION Despite the father transmitting half of the heritable information to the embryo, the focus of preconception health has been the mother. Paternal effects have been linked to complex diseases such as cancer, diabetes, and obesity. These diseases are increasing in prevalence at rates that cannot be explained by genetics alone and highlight the potential for disease transmission via nongenetic inheritance, through epigenetic mechanisms. Epigenetic mechanisms include DNA methylation, posttranslational modifications of histones, and noncoding RNA. Studies in humans and animals suggest that epigenetic mechanisms may serve in the transmission of environmentally induced phenotypic traits from the father to the offspring. Such traits have been associated with altered gene expression and tissue function in first and second offspring generations, a phenomenon known as intergenerational or transgenerational inheritance, respectively. The mechanisms underlying such paternal epigenetic transmission are unclear. RATIONALE Sperm formation involves rapid cell division and distinctive transcription programs, resulting in a motile cell with highly condensed chromatin. Within the highly compacted sperm nucleus, few histones are retained in a manner that suggests an influential role in development. Despite being the major focus of studies in epigenetic inheritance, the role of DNA methylation in paternal epigenetic inheritance is unresolved, as only minor changes in DNA methylation in sperm at CpG-enriched regions have been associated with transmission of environmentally induced traits. Instead, there may be a combination of molecular mechanisms underlying paternal transgenerational epigenetic inheritance involving changes in histone states and/or RNA in sperm. The function of sperm histones and their modifications in embryonic development, offspring health, and epigenetic inheritance is unknown. By overexpressing the human KDM1A histone lysine 4 demethylase during mouse spermatogenesis, we generated a mouse model producing spermatozoa with reduced H3K4me2 within the CpG islands of genes implicated in development, and we studied the development and fitness of the offspring. RESULTS Male transgenic offspring were bred with C57BL/6 females, generating the experimental heterozygous transgenic (TG) and nontransgenic (nonTG) brothers. Each generation from TG and nonTG animals (F1 to F3 in our transgenerational studies) was bred with C57BL/6 females, and the offspring (pups from generations F1 to F4) were analyzed for intergenerational and transgenerational effects. We found that KDM1A overexpression in one generation severely impaired development and survivability of offspring. These defects lasted for two subsequent generations in the absence of KDM1A germline expression. We characterized histone and DNA methylation states in the sperm of TG and nonTG sires. Overexpression of KDM1A was associated with a specific loss of H3K4me2 at more than 2300 genes, including many developmental regulatory genes. Unlike in other examples of paternal transgenerational inheritance, we observed no changes in sperm DNA methylation associated with primarily CpG-enriched regions. Instead, we measured robust and analogous changes in sperm RNA content of TG and nonTG descendants, as well as in their offspring, at the two-cell stage. These changes in expression and the phenotypic abnormalities observed in offspring correlated with altered histone methylation levels at genes in sperm. This study demonstrates that KDM1A activity during sperm development has major developmental consequences for offspring and implicates histone methylation and sperm RNA as potential mediators of transgenerational inheritance. Our data emphasize the complexity of transgenerational epigenetic inheritance likely involving multiple molecular factors, including the establishment of chromatin states in spermatogenesis and sperm-borne RNA. CONCLUSION Correct histone methylation during spermatogenesis is critical for offspring development and survival over multiple generations. These findings demonstrate the potential of histone methylation as a molecular mechanism underlying paternal epigenetic inheritance. Its modification by environmental influences may alter embryo development and complex disease transmission across generations. An essential next step is to establish functional links between environmental exposures, the composition of the sperm epigenome, and consequent altered gene expression and metabolic processes in offspring. Considering the mounting evidence, it may soon be reasonable to suggest that future fathers protect their sperm epigenome. Disruption of histone methylation in developing sperm by exposure to the KDM1A transgene in one generation severely impaired development and survivability of offspring. These defects were transgenerational and occurred in nonTG descendants in the absence of KDM1A germline expression. Developmental defects in offspring and embryos were associated with altered RNA expression in sperm and embryos. A father’s lifetime experiences can be transmitted to his offspring to affect health and development. However, the mechanisms underlying paternal epigenetic transmission are unclear. Unlike in somatic cells, there are few nucleosomes in sperm, and their function in epigenetic inheritance is unknown. We generated transgenic mice in which overexpression of the histone H3 lysine 4 (H3K4) demethylase KDM1A (also known as LSD1) during spermatogenesis reduced H3K4 dimethylation in sperm. KDM1A overexpression in one generation severely impaired development and survivability of offspring. These defects persisted transgenerationally in the absence of KDM1A germline expression and were associated with altered RNA profiles in sperm and offspring. We show that epigenetic inheritance of aberrant development can be initiated by histone demethylase activity in developing sperm, without changes to DNA methylation at CpG-rich regions.

Dynamic Regulation of Histone H3 Methylation at Lysine 4 in Mammalian Spermatogenesis

Abstract Spermatogenesis is a highly complex cell differentiation process that is governed by unique transcriptional regulation and massive chromatin alterations, which are required for meiosis and postmeiotic maturation. The underlying mechanisms involve alterations to the epigenetic layer, including histone modifications and incorporation of testis-specific nuclear proteins, such as histone variants and protamines. Histones can undergo methylation, acetylation, and phosphorylation among other modifications at their N-terminus, and these modifications can signal changes in chromatin structure. We have identified the temporal and spatial distributions of histone H3 mono-, di-, and trimethylation at lysine 4 (K4), and the lysine-specific histone demethylase AOF2 (amine oxidase flavin-containing domain 2, previously known as LSD1) during mammalian spermatogenesis. Our results reveal tightly regulated distributions of H3-K4 methylation and AOF2, and that H3-K4 methylation is very similar between the mouse and the marmoset. The AOF2 protein levels were found to be higher in the testes than in the somatic tissues. The distribution of AOF2 matched the cell- and stage-specific patterns of H3-K4 methylation. Interaction studies revealed unique epigenetic regulatory complexes associated with H3-K4 methylation in the testis, including the association of AOF2 and methyl-CpG-binding domain protein 2 (MBD2a/b) in a complex with histone deacetylase 1 (HDAC1). These studies enhance our understanding of epigenetic modifications and their roles in chromatin organization during male germ cell differentiation in both normal and pathologic states.

The dynamic epigenetic program in male germ cells: Its role in spermatogenesis, testis cancer, and its response to the environment.

Spermatogenesis is a truly remarkable process that requires exquisite control and synchronization of germ cell development. It is prone to frequent error, as paternal infertility contributes to 30–50% of all infertility cases; yet, in many cases, the mechanisms underlying its causes are unknown. Strikingly, aberrant epigenetic profiles, in the form of anomalous DNA and histone modifications, are characteristic of cancerous testis cells. Germ cell development is a critical period during which epigenetic patterns are established and maintained. The progression from diploid spermatogonia to haploid spermatozoa involves stage‐ and testis‐specific gene expression, mitotic and meiotic division, and the histone–protamine transition. All are postulated to engender unique epigenetic controls. In support of this idea are the findings that mouse models with gene deletions for epigenetic modifiers have severely compromised fertility. Underscoring the importance of understanding how epigenetic marks are set and interpreted is evidence that abnormal epigenetic programming of gametes and embryos contributes to heritable instabilities in subsequent generations. Numerous studies have documented the existence of transgenerational consequences of maternal nutrition, or other environmental exposures, but it is only now recognized that there are sex‐specific male‐line transgenerational responses in humans and other species. Epigenetic events in the testis have just begun to be studied. New work on the function of specific histone modifications, chromatin modifiers, DNA methylation, and the impact of the environment on developing sperm suggests that the correct setting of the epigenome is required for male reproductive health and the prevention of paternal disease transmission. Microsc. Res. Tech., 2009.

miRNet - dissecting miRNA-target interactions and functional associations through network-based visual analysis

MicroRNAs (miRNAs) can regulate nearly all biological processes and their dysregulation is implicated in various complex diseases and pathological conditions. Recent years have seen a growing number of functional studies of miRNAs using high-throughput experimental technologies, which have produced a large amount of high-quality data regarding miRNA target genes and their interactions with small molecules, long non-coding RNAs, epigenetic modifiers, disease associations, etc. These rich sets of information have enabled the creation of comprehensive networks linking miRNAs with various biologically important entities to shed light on their collective functions and regulatory mechanisms. Here, we introduce miRNet, an easy-to-use web-based tool that offers statistical, visual and network-based approaches to help researchers understand miRNAs functions and regulatory mechanisms. The key features of miRNet include: (i) a comprehensive knowledge base integrating high-quality miRNA-target interaction data from 11 databases; (ii) support for differential expression analysis of data from microarray, RNA-seq and quantitative PCR; (iii) implementation of a flexible interface for data filtering, refinement and customization during network creation; (iv) a powerful fully featured network visualization system coupled with enrichment analysis. miRNet offers a comprehensive tool suite to enable statistical analysis and functional interpretation of various data generated from current miRNA studies. miRNet is freely available at http://www.mirnet.ca.

Impaired Fertility and FSH Synthesis in Gonadotrope-Specific Foxl2 Knockout Mice

Impairments in pituitary FSH synthesis or action cause infertility. However, causes of FSH dysregulation are poorly described, in part because of our incomplete understanding of mechanisms controlling FSH synthesis. Previously, we discovered a critical role for forkhead protein L2 (FOXL2) in activin-stimulated FSH β-subunit (Fshb) transcription in immortalized cells in vitro. Here, we tested the hypothesis that FOXL2 is required for FSH synthesis in vivo. Using a Cre/lox approach, we selectively ablated Foxl2 in murine anterior pituitary gonadotrope cells. Conditional knockout (cKO) mice developed overtly normally but were subfertile in adulthood. Testis size and spermatogenesis were significantly impaired in cKO males. cKO females exhibited reduced ovarian weight and ovulated fewer oocytes in natural estrous cycles compared with controls. In contrast, ovaries of juvenile cKO females showed normal responses to exogenous gonadotropin stimulation. Both male and female cKO mice were FSH deficient, secondary to diminished pituitary Fshb mRNA production. Basal and activin-stimulated Fshb expression was similarly impaired in Foxl2 depleted primary pituitary cultures. Collectively, these data definitively establish FOXL2 as the first identified gonadotrope-restricted transcription factor required for selective FSH synthesis in vivo.

Diet exposure to technical hexabromocyclododecane (HBCD) affects testes and circulating testosterone and thyroxine levels in American kestrels (Falco sparverius).

Hexabromocyclododecane (HBCD) is a high-production-volume, brominated flame-retardant that is used in items such as polystyrene foams. HBCD has been detected in the environment, wildlife tissues and in humans globally with some of the highest recorded levels in predatory birds. This study examined the effects of exposure to environmentally relevant levels of HBCD on the reproductive physiology of captive male American kestrels (Falco sparverius), a predatory bird. Two sets of males were used: one group not housed with females (unpaired: nc=12, nHBCD=10) and the second group housed with females (breeding: nc=10, nHBCD=20). All treatment birds were exposed to 0.51 μg HBCD/g kestrel/day technical HBCD, and controls to safflower oil only, injected into their food during seasonal testicular development. Unpaired males were exposed for 3 weeks and euthanized for testicular analysis. Breeding males were exposed for 3 weeks prior to pairing and throughout the courtship period. The HBCD-exposed unpaired males had heavier testes (p≤0.017) and a trend towards more seminiferous tubules containing elongated spermatids (p=0.052). There was also a moderate increase in plasma testosterone concentrations (p=0.056) compared to controls. In breeding males, testosterone levels increased during courtship to culminate in higher levels than controls by the time the first egg was laid (p=0.010) and circulating free and total T4 was reduced throughout. The number of sperm cells reaching the perivitelline layer of the first egg for breeding males did not differ between the two groups. This study is the first report that HBCD exposure at environmentally relevant levels alters reproductive physiology in male birds and suggests that birds may be more sensitive to HBCD than mammals.

The flame retardant β-1,2-dibromo-4-(1,2-dibromoethyl)cyclohexane: fate, fertility, and reproductive success in American kestrels (Falco sparverius).

Captive American kestrels (Falco sparverius) were exposed via diet during reproduction to an environmentally relevant concentration of β-1,2-dibromo-4-(1,2-dibromoethyl)cyclohexane (β-TBECH). The β-TBECH isomer was injected into the food source at a daily dosing concentration of 0.239 ng/g kestrel/day (22 pairs); control birds were exposed via diet to the safflower oil vehicle only (24 pairs). Eight pairs in each group were exposed for four weeks and sacrificed for tissue analysis; the remaining pairs completed their breeding cycle, with exposure ceasing at the end of incubation (82 days). α- and β-TBECH appeared to be rapidly metabolized and/or eliminated from fat, liver, and plasma; both isomers and potential hydroxylated metabolites of β-TBECH (plasma) were undetected. Notwithstanding, compared to controls, pairs exposed to β-TBECH laid fewer eggs (p = 0.019) and laid lighter eggs (successful eggs: p = 0.009). Exposed pairs also demonstrated poorer egg fertility (p = 0.035) although testis mass and histology were similar among males. Reductions in egg production and fertility resulted in decreased hatchling success (p = 0.023). The β-TBECH-exposed pairs also produced fewer males overall (p = 0.009), which occurred concurrently with increased estradiols maternally deposited in eggs (p = 0.039). These findings demonstrate that β-TBECH may be detrimental for breeding in wild birds receiving similar exposure levels.

Histone H3 Tails Containing Dimethylated Lysine and Adjacent Phosphorylated Serine Modifications Adopt a Specific Conformation during Mitosis and Meiosis

ABSTRACT Condensation of chromatin, mediated in part by posttranslational modifications of histones, is essential for cell division during mitosis. Histone H3 tails are dimethylated on lysine (Kme2) and become phosphorylated on serine (Sp) residues during mitosis. We have explored the possibility that these double modifications are involved in the establishment of H3 tail conformations during the cell cycle. Here we describe a specific chromatin conformation occurring at Kme2 and adjacently phosphorylated S of H3 tails upon formation of a hydrogen bond. This conformation appears exclusively between early prophase and early anaphase of the mitosis, when chromatin condensation is highest. Moreover, we observed that the conformed H3Kme2Sp tail is present at the diplotene and metaphase stages in spermatocytes and oocytes. Our data together with results obtained by cryoelectron microscopy suggest that the conformation of Kme2Sp-modified H3 tails changes during mitosis and meiosis. This is supported by biostructural modeling of a modified histone H3 tail bound by an antibody, indicating that Kme2Sp-modified H3 tails can adopt at least two different conformations. Thus, the H3K9me2S10p and the H3K27me2S28p sites are involved in the acquisition of specific chromatin conformations during chromatin condensation for cell division.

Embryonic Exposure to the Polybrominated Diphenyl Ether Mixture, DE-71, Affects Testes and Circulating Testosterone Concentrations in Adult American Kestrels (Falco sparverius)

Polybrominated diphenyl ethers (PBDEs) are additive flame retardants that are environmentally persistent and bioaccumulative. The developmental effects of in ovo exposure to environmentally relevant levels of the PBDE technical mixture, DE-71, on male reproductive physiology in captive American kestrels (Falco sparverius) was determined. Males were exposed in ovo by direct maternal transfer to DE-71 at three mean concentrations of 289 ng/g ww (low exposure), 1131 ng/g ww (high-exposure), or background levels of 3 ng/g ww (control). As adults, males were paired with unexposed females for breeding and, 1 year later, sacrificed for testes evaluation. While breeding, high-exposure males demonstrated a trend of reduced circulating testosterone levels when their female mate commenced egg laying when compared with controls (p = 0.056). No differences in circulating free T₄ or T₃ were detected. Sperm numbers were elevated on the perivitelline layer of the first egg of both high- and low-exposure males when compared with controls (p = 0.021). High-exposure males had a higher gonadosomatic index (p = 0.046) and heavier right testis than controls (p = 0.034) with a similar trend for their left testis (p = 0.055). High-exposure males had more seminiferous tubules containing lumen than controls (p = 0.030), and in proportion to the total number of tubules, low-exposure males had more tubules containing lumen than did controls (p = 0.016). Only high-exposure males had fewer than half of tubules containing final spermatids (43%). The results of the present study demonstrate that embryonic exposure to technical DE-71 affects the reproductive tract of adult male kestrels.

Epigenetic mechanisms regulate stem cell expressed genes Pou5f1 and Gfra1 in a male germ cell line.

Male fertility is declining and an underlying cause may be due to environment-epigenetic interactions in developing sperm, yet nothing is known of how the epigenome controls gene expression in sperm development. Histone methylation and acetylation are dynamically regulated in spermatogenesis and are sensitive to the environment. Our objectives were to determine how histone H3 methylation and acetylation contribute to the regulation of key genes in spermatogenesis. A germ cell line, GC-1, was exposed to either the control, or the chromatin modifying drugs tranylcypromine (T), an inhibitor of the histone H3 demethylase KDM1 (lysine specific demethylase 1), or trichostatin (TSA), an inhibitor of histone deacetylases, (HDAC). Quantitative PCR (qPCR) was used to identify genes that were sensitive to treatment. As a control for specificity the Myod1 (myogenic differentiation 1) gene was analyzed. Chromatin immunoprecipitation (ChIP) followed by qPCR was used to measure histone H3 methylation and acetylation at the promoters of target genes and the control, Myod1. Remarkably, the chromatin modifying treatment specifically induced the expression of spermatogonia expressed genes Pou5f1 and Gfra1. ChIP-qPCR revealed that induction of gene expression was associated with a gain in gene activating histone H3 methylation and acetylation in Pou5f1 and Gfra1 promoters, whereas CpG DNA methylation was not affected. Our data implicate a critical role for histone H3 methylation and acetylation in the regulation of genes expressed by spermatogonia – here, predominantly mediated by HDAC-containing protein complexes.