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At the frontier of epigenetics of brain sex differences.

McCarthy MM, Nugent BM - Front Behav Neurosci (2015)

Bottom Line: The notion that epigenetics may play an important role in the establishment and maintenance of sex differences in the brain has garnered great enthusiasm but the reality in terms of actual advances has been slow.The majority of emphasis has been on candidate genes such as steroid receptors.Only recently have more generalized survey type approaches been achieved and these promise to open new vistas and accelerate discovery of important roles for DNA methylation, histone modification, genomic imprinting and microRNAs (miRs).

View Article: PubMed Central - PubMed

Affiliation: Department of Pharmacology, University of Maryland School of Medicine Baltimore, MD, USA.

ABSTRACT
The notion that epigenetics may play an important role in the establishment and maintenance of sex differences in the brain has garnered great enthusiasm but the reality in terms of actual advances has been slow. Two general approaches include the comparison of a particular epigenetic mark in males vs. females and the inhibition of key epigenetic enzymes or co-factors to determine if this eliminates a particular sex difference in brain or behavior. The majority of emphasis has been on candidate genes such as steroid receptors. Only recently have more generalized survey type approaches been achieved and these promise to open new vistas and accelerate discovery of important roles for DNA methylation, histone modification, genomic imprinting and microRNAs (miRs). Technical challenges abound and, while not unique to this field, will require novel thinking and new approaches by behavioral neuroendocrinologists.

No MeSH data available.


Related in: MedlinePlus

CpG methylation is modulated by hormones. Cytosines are methylated at the 5′ carbon by a class of enzymes called DNA Methyl Transferases (DNMTs). The number of fully methylated CpGs was quantified in DNA extracted from the preoptic area (POA) of 3 newborn females and 3 females treated with a masculinizing dose of estradiol for the previous 2 days. Estradiol treatment significantly reduced the number of fully methylated sites and analyses of where in the genome these sex differences reside indicated they are predominantly in the intragenic region, where DNA methylation is highest.
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Figure 3: CpG methylation is modulated by hormones. Cytosines are methylated at the 5′ carbon by a class of enzymes called DNA Methyl Transferases (DNMTs). The number of fully methylated CpGs was quantified in DNA extracted from the preoptic area (POA) of 3 newborn females and 3 females treated with a masculinizing dose of estradiol for the previous 2 days. Estradiol treatment significantly reduced the number of fully methylated sites and analyses of where in the genome these sex differences reside indicated they are predominantly in the intragenic region, where DNA methylation is highest.

Mentions: Similar to the discovery based approaches described above, we also took a “big picture” view of the epigenome and sexual differentiation by quantifying the overall level of DNA methylation in the POA of male, female and masculinized female rat pups (Nugent et al., 2015). We found females had significantly higher overall DNA methylation compared to either males or masculinized females. Whole-genome-bisulfite-sequencing (WGBS) revealed this was due to more highly methylated CpG sites in females and that most of this was not in CpG islands or gene promoters but was instead in the intergenic regions, where most DNA methylation is found. Moreover, the sex difference in methylation was broadly distributed across the chromosomes (Figure 3). In order to establish if the sex difference in DNA methylation was functionally important we used two approaches. First was to determine if reducing the DNA methylation in females during the critical window would masculinize the POA and adult reproductive behavior, and the second was to identify what genes were being repressed in females as a result of elevated DNA methylation. Both aims were achieved with the use of DNMT inhibitors, specifically zebularine and RG108, which have slightly different mechanisms but are similar in that they block all DNMT activity and result in de-methylation of the DNA (Yoo et al., 2004). After confirming this was also true in the developing POA, newborn males and females were treated with either RG108 or zebularine, raised to adulthood and tested for male sexual behavior by being placed with a sexually receptive females (all test subjects also underwent gonadectomy and testosterone replacement to both simulate adult male circulating hormone levels and equalize hormone levels across animals). Both the brain and behavior of females treated with DNMT inhibitor as neonates were fully masculinized in adulthood, meaning they exhibited a male-like synaptic pattern and showed normal male sexual behavior (Figure 4). This was not true of females treated with vehicle as neonates, they showed very little male-like behavior despite having equal circulating testosterone levels as the time of testing as the other animals. Thus the demethylation that had occurred some 60 days prior resulted in a gene expression pattern that masculinized the brain. What those genes might be was assessed by conducting RNA-Seq on mRNA from the POA of males and females with and without DNMT inhibitor treatment. To our surprise the number of genes expressed at significantly different levels in male and female POA was relatively small, ~70, but consistent with the low number observed by Vilain and colleagues using RRBS (Ghahramani et al., 2014). About half of the genes were expressed at higher levels in males and half at higher levels in females. Candidate genes for masculinization are those that increased in females following DNMT inhibition and were also higher in control males than females. Of the 34 genes expressed at higher levels in males, 25 of them increased in females treated with a DNMT inhibitor (Nugent et al., 2015). It is logical to predict that in females these genes would have increased DNA methylation in the promoter region compared to males or masculinized females, but exhaustive sequencing analysis found no evidence that was the case, suggesting the change in expression of these genes was secondary to other events or are located outside of promoters.


At the frontier of epigenetics of brain sex differences.

McCarthy MM, Nugent BM - Front Behav Neurosci (2015)

CpG methylation is modulated by hormones. Cytosines are methylated at the 5′ carbon by a class of enzymes called DNA Methyl Transferases (DNMTs). The number of fully methylated CpGs was quantified in DNA extracted from the preoptic area (POA) of 3 newborn females and 3 females treated with a masculinizing dose of estradiol for the previous 2 days. Estradiol treatment significantly reduced the number of fully methylated sites and analyses of where in the genome these sex differences reside indicated they are predominantly in the intragenic region, where DNA methylation is highest.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4543874&req=5

Figure 3: CpG methylation is modulated by hormones. Cytosines are methylated at the 5′ carbon by a class of enzymes called DNA Methyl Transferases (DNMTs). The number of fully methylated CpGs was quantified in DNA extracted from the preoptic area (POA) of 3 newborn females and 3 females treated with a masculinizing dose of estradiol for the previous 2 days. Estradiol treatment significantly reduced the number of fully methylated sites and analyses of where in the genome these sex differences reside indicated they are predominantly in the intragenic region, where DNA methylation is highest.
Mentions: Similar to the discovery based approaches described above, we also took a “big picture” view of the epigenome and sexual differentiation by quantifying the overall level of DNA methylation in the POA of male, female and masculinized female rat pups (Nugent et al., 2015). We found females had significantly higher overall DNA methylation compared to either males or masculinized females. Whole-genome-bisulfite-sequencing (WGBS) revealed this was due to more highly methylated CpG sites in females and that most of this was not in CpG islands or gene promoters but was instead in the intergenic regions, where most DNA methylation is found. Moreover, the sex difference in methylation was broadly distributed across the chromosomes (Figure 3). In order to establish if the sex difference in DNA methylation was functionally important we used two approaches. First was to determine if reducing the DNA methylation in females during the critical window would masculinize the POA and adult reproductive behavior, and the second was to identify what genes were being repressed in females as a result of elevated DNA methylation. Both aims were achieved with the use of DNMT inhibitors, specifically zebularine and RG108, which have slightly different mechanisms but are similar in that they block all DNMT activity and result in de-methylation of the DNA (Yoo et al., 2004). After confirming this was also true in the developing POA, newborn males and females were treated with either RG108 or zebularine, raised to adulthood and tested for male sexual behavior by being placed with a sexually receptive females (all test subjects also underwent gonadectomy and testosterone replacement to both simulate adult male circulating hormone levels and equalize hormone levels across animals). Both the brain and behavior of females treated with DNMT inhibitor as neonates were fully masculinized in adulthood, meaning they exhibited a male-like synaptic pattern and showed normal male sexual behavior (Figure 4). This was not true of females treated with vehicle as neonates, they showed very little male-like behavior despite having equal circulating testosterone levels as the time of testing as the other animals. Thus the demethylation that had occurred some 60 days prior resulted in a gene expression pattern that masculinized the brain. What those genes might be was assessed by conducting RNA-Seq on mRNA from the POA of males and females with and without DNMT inhibitor treatment. To our surprise the number of genes expressed at significantly different levels in male and female POA was relatively small, ~70, but consistent with the low number observed by Vilain and colleagues using RRBS (Ghahramani et al., 2014). About half of the genes were expressed at higher levels in males and half at higher levels in females. Candidate genes for masculinization are those that increased in females following DNMT inhibition and were also higher in control males than females. Of the 34 genes expressed at higher levels in males, 25 of them increased in females treated with a DNMT inhibitor (Nugent et al., 2015). It is logical to predict that in females these genes would have increased DNA methylation in the promoter region compared to males or masculinized females, but exhaustive sequencing analysis found no evidence that was the case, suggesting the change in expression of these genes was secondary to other events or are located outside of promoters.

Bottom Line: The notion that epigenetics may play an important role in the establishment and maintenance of sex differences in the brain has garnered great enthusiasm but the reality in terms of actual advances has been slow.The majority of emphasis has been on candidate genes such as steroid receptors.Only recently have more generalized survey type approaches been achieved and these promise to open new vistas and accelerate discovery of important roles for DNA methylation, histone modification, genomic imprinting and microRNAs (miRs).

View Article: PubMed Central - PubMed

Affiliation: Department of Pharmacology, University of Maryland School of Medicine Baltimore, MD, USA.

ABSTRACT
The notion that epigenetics may play an important role in the establishment and maintenance of sex differences in the brain has garnered great enthusiasm but the reality in terms of actual advances has been slow. Two general approaches include the comparison of a particular epigenetic mark in males vs. females and the inhibition of key epigenetic enzymes or co-factors to determine if this eliminates a particular sex difference in brain or behavior. The majority of emphasis has been on candidate genes such as steroid receptors. Only recently have more generalized survey type approaches been achieved and these promise to open new vistas and accelerate discovery of important roles for DNA methylation, histone modification, genomic imprinting and microRNAs (miRs). Technical challenges abound and, while not unique to this field, will require novel thinking and new approaches by behavioral neuroendocrinologists.

No MeSH data available.


Related in: MedlinePlus