Aaron D. Clark

Hairpin-bisulfite PCR: Assessing epigenetic methylation patterns on complementary strands of individual DNA molecules

Epigenetic inheritance, the transmission of gene expression states from parent to daughter cells, often involves methylation of DNA. In eukaryotes, cytosine methylation is a frequent component of epigenetic mechanisms. Failure to transmit faithfully a methylated or an unmethylated state of cytosine can lead to altered phenotypes in plants and animals. A central unresolved question in epigenetics concerns the mechanisms by which a locus maintains, or changes, its state of cytosine methylation. We developed “hairpin-bisulfite PCR” to analyze these mechanisms. This method reveals the extent of methylation symmetry between the complementary strands of individual DNA molecules. Using hairpin-bisulfite PCR, we determined the fidelity of methylation transmission in the CpG island of the FMR1 gene in human lymphocytes. For the hypermethylated CpG island of this gene, characteristic of inactive-X alleles, we estimate a maintenance methylation efficiency of ≈0.96 per site per cell division. For de novo methylation efficiency (Ed), remarkably different estimates were obtained for the hypermethylated CpG island (Ed = 0.17), compared with the hypomethylated island on the active-X chromosome (Ed < 0.01). These results clarify the mechanisms by which the alternative hypomethylated and hypermethylated states of CpG islands are stably maintained through many cell divisions. We also analyzed a region of human L1 transposable elements. These L1 data provide accurate methylation patterns for the complementary strand of each repeat sequence analyzed. Hairpin-bisulfite PCR will be a powerful tool in studying other processes for which genetic or epigenetic information differs on the two complementary strands of DNA.

Deficits in human trisomy 21 iPSCs and neurons

Down syndrome (trisomy 21) is the most common genetic cause of intellectual disability, but the precise molecular mechanisms underlying impaired cognition remain unclear. Elucidation of these mechanisms has been hindered by the lack of a model system that contains full trisomy of chromosome 21 (Ts21) in a human genome that enables normal gene regulation. To overcome this limitation, we created Ts21-induced pluripotent stem cells (iPSCs) from two sets of Ts21 human fibroblasts. One of the fibroblast lines had low level mosaicism for Ts21 and yielded Ts21 iPSCs and an isogenic control that is disomic for human chromosome 21 (HSA21). Differentiation of all Ts21 iPSCs yielded similar numbers of neurons expressing markers characteristic of dorsal forebrain neurons that were functionally similar to controls. Expression profiling of Ts21 iPSCs and their neuronal derivatives revealed changes in HSA21 genes consistent with the presence of 50% more genetic material as well as changes in non-HSA21 genes that suggested compensatory responses to oxidative stress. Ts21 neurons displayed reduced synaptic activity, affecting excitatory and inhibitory synapses equally. Thus, Ts21 iPSCs and neurons display unique developmental defects that are consistent with cognitive deficits in individuals with Down syndrome and may enable discovery of the underlying causes of and treatments for this disorder.

Hemimethylation and Non-CpG Methylation Levels in a Promoter Region of Human LINE-1 (L1) Repeated Elements

DNA methylation within the promoter region of human LINE1 (L1) transposable elements is important for maintaining transcriptional inactivation and for inhibiting L1 transposition. Determining methylation patterns on the complementary strands of repeated sequences is difficult using standard bisulfite methylation analysis. Evolutionary changes in each repeat and the variations between cells or alleles of the same repeat lead to a heterogeneous population of sequences. Potential sequence biases can arise during analyses that are different for the converted sense and antisense strands. These problems can be avoided with hairpin-bisulfite PCR, a double-stranded PCR method in which complementary strands of individual molecules are attached by a hairpin linker ligated to genomic DNA. Using human L1 elements to study methylation of repeated sequences, (i) we distinguish valid L1 sequences from redundant and contaminant sequences by applying the powerful new method of molecular barcodes, (ii) we resolve a controversy on the level of hemimethylation of L1 sequences in fetal fibroblasts in favor of relatively little hemimethylation, (iii) we report that human L1 sequences in different cell types also have primarily concordant CpG methylation patterns on complementary strands, and (iv) we provide evidence that non-CpG cytosines within the regions analyzed are rarely methylated.

Seasonal and social modulation of testosterone in Costa Rican rufous-collared sparrows (Zonotrichia capensis costaricensis)

Previous work shows that most birds breeding in northern temperate regions adjust production of testosterone in response to stage of the breeding cycle and in some cases following social interactions. In contrast, prior research suggests that tropical breeding birds are less likely to modulate testosterone in response to social interactions (the propensity to increase testosterone in response to social instability is known as the challenge hypothesis). To further test the challenge hypothesis in tropical birds, we investigated whether variation in season affects reproductive condition, aggressive behavior, and social modulation of testosterone in two populations of Costa Rican rufous-collared sparrow, Zonotrichia capensis costaricensis. We conducted our study at three distinct times of year: the dry season (March and May); the veranillo, a hiatus in the rainy season (July); and the late rainy season (November). Significantly more birds were in breeding condition in the dry season than in the rainy season or veranillo. In each time period, we collected baseline testosterone samples and conducted simulated territorial intrusions (STIs). Our study shows that testosterone is modulated with season independent of breeding condition, as testosterone levels were affected by season, breeding condition, and an interaction of the two factors. Males breeding in the dry season had higher plasma testosterone levels than non-breeding males in the dry season and both breeding and non-breeding males in the veranillo and rainy season. Males did not socially modulate testosterone in any season. Aggressive behaviors expressed during STIs did not differ among seasons with the exception that males sang fewer songs during the rainy season.

From the Cover: Hairpin-bisulfite PCR: Assessing epigenetic methylation patterns on complementary strands of individual DNA molecules

Epigenetic inheritance, the transmission of gene expression states from parent to daughter cells, often involves methylation of DNA. In eukaryotes, cytosine methylation is a frequent component of epigenetic mechanisms. Failure to transmit faithfully a methylated or an unmethylated state of cytosine can lead to altered phenotypes in plants and animals. A central unresolved question in epigenetics concerns the mechanisms by which a locus maintains, or changes, its state of cytosine methylation. We developed “hairpin-bisulfite PCR” to analyze these mechanisms. This method reveals the extent of methylation symmetry between the complementary strands of individual DNA molecules. Using hairpin-bisulfite PCR, we determined the fidelity of methylation transmission in the CpG island of the FMR1 gene in human lymphocytes. For the hypermethylated CpG island of this gene, characteristic of inactive-X alleles, we estimate a maintenance methylation efficiency of ≈0.96 per site per cell division. For de novo methylation efficiency (Ed), remarkably different estimates were obtained for the hypermethylated CpG island (Ed = 0.17), compared with the hypomethylated island on the active-X chromosome (Ed < 0.01). These results clarify the mechanisms by which the alternative hypomethylated and hypermethylated states of CpG islands are stably maintained through many cell divisions. We also analyzed a region of human L1 transposable elements. These L1 data provide accurate methylation patterns for the complementary strand of each repeat sequence analyzed. Hairpin-bisulfite PCR will be a powerful tool in studying other processes for which genetic or epigenetic information differs on the two complementary strands of DNA.

Endocrine responsiveness to social challenges in northern and southern hemisphere populations of Zonotrichia

Northern populations of the White-crowned Sparrow, Zonotrichia leucophrys, show varying degrees of social modulation of testosterone. In general, males of mid-latitude breeding and multiple-brooded populations show transient increases in secretions of luteinizing hormone and testosterone when challenged by another male for a territory, or when exposed to sexually receptive females. These surges of testosterone do not appear to activate aggression associated with territory defense or mate-guarding, but appear to enhance persistence of aggression during and after the behavioral interaction. Males may continue to sing spontaneously and patrol the territory for many hours even after the behavioral interaction is over. However, males of high latitude and altitude populations do not socially modulate testosterone levels. Several hypotheses, not necessarily mutually exclusive, have been proposed to explain why males in some populations do socially modulate testosterone and others do not. Males will not socially modulate testosterone if: (1) the breeding season is so restricted in time (e.g., high latitude and altitude) there are few social interactions; (2) populations in which males are essential for parental care; and (3) populations in which extra-pair copulations are low and male-male interactions are minimal. Tropical populations of the Rufous-collared Sparrow, Z. capensis costaricensis, have extended breeding seasons and they are multiple-brooded, but do not socially modulate testosterone. This is unlike Z. leucophrys. Whether austral populations of the Rufous-collared Sparrow socially modulate testosterone under presumably similar constraints of mid- to high-latitude seasonality are currently under investigation.

Disentangling the Effects of Environment and Life‐History Stage on Corticosterone Modulation in Costa Rican Rufous‐Collared Sparrows, Zonotrichia capensis costaricensis

This study explores the factors that influence modulation of baseline corticosterone levels and the hypothalamo‐pituitary‐adrenal cortex (HPA) axis response to stress in Costa Rican rufous‐collared sparrows (Zonotrichia capensis costaricensis). Individuals in our study population vary in their timing of breeding and molt. Thus, at multiple times of the year, we were able to investigate how the HPA axis changes with life‐history stage (breeding, molt) and the interaction of season and life‐history stage. Season explained most of the variation in corticosterone data. During the dry season, males had higher baseline corticosterone levels and both females and males had a higher stress response, regardless of breeding or molting condition. Breeding did not significantly affect corticosterone levels in either males or females. There was no direct effect of molt on corticosterone levels in males or in females, but there was a significant interaction between season and molt for male baseline corticosterone levels and HPA axis responsiveness. Baseline corticosterone levels were not correlated with body condition, suggesting that body condition is not an explanatory factor for HPA axis change. Our data indicate that environmental conditions may affect how the HPA axis in birds is modulated during different life‐history stages. They also emphasize the importance of studying species with diverse life cycles and seasonality.

Modulation of androgens in southern hemisphere temperate breeding sparrows (Zonotrichia capensis): an altitudinal comparison.

Most work investigating modulation of testosterone (T) levels in birds has focused on northern temperate and Arctic species, and to a lesser degree, tropical species. Studies exploring modulation of T in birds in temperate regions of the Southern Hemisphere are lacking. Here we explore patterns of T secretion across the breeding season in two populations of temperate Zonotrichia capensis in Chile, located only 130 km apart, but separated by 2000 m in elevation. We then compared these T profiles to those of conspecifcs in the tropics and congeners in northern zones. We measured baseline T levels during pre-breeding in lowland Z. c. chilensis, early breeding in highland Z. c. chilensis and mid-breeding in both populations. We also tested for social stimulation of T secretion during mid-breeding in both populations. Lastly, we challenged the hypothalamic-pituitary-gonadal (HPG) axis of the lowland population with gonadotropin-releasing hormone (GnRH) to determine maximum possible T production. We found that the highland population adjusted T secretion across the breeding season like northern species. Neither Z. c. chilensis population modulated T in response to social stimuli, nor the HPG axis of the lowland population was not maximally active during either pre- or mid-breeding. These results suggest that patterns of circulating T in the highland population of Z. c. chilensis in the Southern Hemisphere are similar to congeners in the temperate Northern Hemisphere, but those of the lowland population of Z. c. chilensis are not, and are more similar to conspecifics breeding in the tropics.

Examination of nocturnal activity and behaviour in resident white-crowned sparrows (Zonotrichia leucophrys nuttalli)

Observations of nocturnal activity in resident species held in captivity are often attributed to migratory restlessness (MR). Previous publications investigating migratory white-crowned sparrows (Zonotrichia leucophrys gambelii) provided a distinct set of traits characteristic of MR, which we used to test the expression of activity and behaviour in the resident subspecies (Z. l. nuttalli). Under a winter photocycle, the 24-h activity profile of Z. l. nuttalli closely resembled that of migrant relatives. Following photostimulation, most birds expressed some activity during the dark phase; however, the diel pattern differed greatly from that of migrants. Unlike Z. l. gambelii, peak activity levels during the light phase remained greater than those expressed during the dark phase. Furthermore, birds did not express a quiescent phase prior to the initiation of the dark phase, nor did photostimulation result in increases in body mass or fat deposits. However, two birds did exhibit migration-specific behaviours (beak-up and beak-up flight) coupled with intense dark phase locomotor activity, which is consistent with MR expression. The suite of contrasts between conspecifics however, may suggest a different context of nocturnal behaviour, perhaps representing an underlying ancestral migratory phenotype that may now be associated with territorial or reproductive activities.

Seasonal Modulation of Testosterone during Breeding of the Rufous-Collared Sparrow (Zonotrichia capensis australis) in Southern Patagonia

The roles of testosterone (T) during reproduction are often complex and vary among and within vertebrate taxa and locations, making general hypotheses relating T to breeding behavior and success difficult to integrate. In birds, T is thought to influence degrees of territoriality and associated aggression in males to maximize breeding success. Importantly, most work supporting these ideas has been conducted in the Northern Hemisphere. However, accumulating work on tropical species has shown divergent patterns of T in association with breeding behavior. The compilation of work from northern temperate and tropical species suggests that the function of T in relation to breeding behavior varies across latitude and environmental conditions. We investigate the patterns of T in relation to breeding behavior in a subspecies of the rufous-collared sparrow Zonotrichia capensis australis breeding at high latitude in the Southern Hemisphere (55°S). We then compare the T profiles and breeding behaviors of male Z. c. australis to conspecifics breeding in the tropics and congeners in North America to test the hypothesis that environments with breeding seasons of similar lengths will drive similar patterns of T in relation to breeding behavior. We found that Z. c. australis have high levels of T during the early-breeding periods when territories are being established and low levels of T during the parental phase of breeding, similar to temperate and Arctic birds in the Northern Hemisphere but unlike tropical Zonotrichia capensis costaricensis. In contrast, we found that Z. c. australis also exhibit similar aggressive behaviors in early breeding and midbreeding, unlike many birds in the Northern Hemisphere.