Trisomy 21 causes Down syndrome (DS), but the mechanisms by which the extra chromosome leads to deficient intellectual and immune function are not well understood. Here, we profile CpG methylation in DS and control cerebral and cerebellar cortex of adults and cerebrum of fetuses. We purify neuronal and non-neuronal nuclei and T lymphocytes and find biologically relevant genes with DS-specific methylation (DS-DM) in each of these cell types. Some genes show brain-specific DS-DM, while others show stronger DS-DM in T cells. Both 5-methyl-cytosine and 5-hydroxy-methyl-cytosine contribute to the DS-DM. Thirty percent of genes with DS-DM in adult brain cells also show DS-DM in fetal brains, indicating early onset of these epigenetic changes, and we find early maturation of methylation patterns in DS brain and lymphocytes. Some, but not all, of the DS-DM genes show differential expression. DS-DM preferentially affected CpGs in or near specific transcription factor binding sites (TFBSs), implicating a mechanism involving altered TFBS occupancy. Methyl-seq of brain DNA from mouse models with sub-chromosomal duplications mimicking DS reveals partial but significant overlaps with human DS-DM and shows that multiple chromosome 21 genes contribute to the downstream epigenetic effects. These data point to novel biological mechanisms in DS and have general implications for trans effects of chromosomal duplications and aneuploidies on epigenetic patterning.

Down syndrome (DS), caused by trisomy 21, is the most common chromosomal disorder associated with developmental cognitive deficits. Despite intensive efforts, the genetic mechanisms underlying developmental cognitive deficits remain poorly understood, and no treatment has been proven effective. The previous mouse-based experiments suggest that the so-called Down syndrome critical region of human chromosome 21 is an important region for this phenotype, which is demarcated by Setd4/Cbr1 and Fam3b/Mx2. We first confirmed the importance of the Cbr1-Fam3b region using compound mutant mice, which carry a duplication spanning the entire human chromosome 21 orthologous region on mouse chromosome 16 [Dp(16)1Yey] and Ms1Rhr. By dividing the Setd4-Mx2 region into complementary Setd4-Kcnj6 and Kcnj15-Mx2 intervals, we started an unbiased dissection through generating and analyzing Dp(16)1Yey/Df(16Setd4-Kcnj6)Yey and Dp(16)1Yey/Df(16Kcnj15-Mx2)Yey mice. Surprisingly, the Dp(16)1Yey-associated cognitive phenotypes were not rescued by either deletion in the compound mutants, suggesting the possible presence of at least one causative gene in each of the two regions. The partial rescue by a Dyrk1a mutation in a compound mutant carrying Dp(16)1Yey and the Dyrk1a mutation confirmed the causative role of Dyrk1a, whereas the absence of a similar rescue by Df(16Dyrk1a-Kcnj6)Yey in Dp(16)1Yey/Df(16Dyrk1a-Kcnj6)Yey mice demonstrated the importance of Kcnj6. Our results revealed the high levels of complexities of gene actions and interactions associated with the Setd4/Cbr1-Fam3b/Mx2 region as well as their relationship with developmental cognitive deficits in DS.

Testosterone deficiency is associated with increased serum cholesterol levels. However, how testosterone deficiency precisely affects cholesterol metabolism remains unclear. Therefore, in the current study, we examined the effect of testosterone deficiency on cholesterol metabolism and liver gene expression in pigs fed a high-fat and high-cholesterol (HFC) diet. Sexually mature male miniature pigs (6–7 months old) were randomly divided into 3 groups as follows: intact male pigs fed an HFC diet (IM + HFC), castrated male pigs fed an HFC diet (CM + HFC), and castrated pigs with testosterone replacement fed an HFC diet (CM + HFC + T). Serum testosterone levels and lipid profiles were measured, and gene expression levels associated with hepatic cholesterol metabolism were determined. Furthermore, total hepatic cholesterol contents and the activities of enzymes mediating hepatic cholesterol metabolism were measured. Serum testosterone levels were significantly decreased in CM + HFC pigs, and testosterone replacement attenuated castration-induced testosterone deficiency. Castration significantly increased the serum levels of total cholesterol, low-density lipoprotein cholesterol and triglycerides, as well as hepatic lipid contents in pigs fed an HFC diet. Compared with IM + HFC and CM + HFC + T pigs, low-density lipoprotein receptor (LDLR) mRNA expression and protein levels were significantly decreased in the livers of CM + HFC pigs. In contrast, we found that compared with IM + HFC pigs, hepatic proprotein convertase subtilisin/kexin type 9 (PCSK9) mRNA and serum PCSK9 protein levels were significantly increased in CM + HFC pigs. Moreover, testosterone treatment reversed the increase in PCSK9 expression in CM + HFC pigs. However, neither castration nor testosterone replacement affected the expression of the other hepatic genes that were tested. This study demonstrated that castration-induced testosterone deficiency caused severe hypercholesterolemia in pigs fed an HFC diet; furthermore, these effects could be reversed by testosterone replacement therapy. Altered hepatic PCSK9 and LDLR expression, resulting in reduced LDL-cholesterol clearance, may contribute to the increased serum cholesterol levels induced by testosterone deficiency and an HFC diet. These results deepen our understanding of the underlying molecular mechanisms that mediate the effects of testosterone deficiency on cholesterol metabolism.

Long interspersed elements (LINEs), through both self-mobilization and trans-mobilization of short interspersed elements and processed pseudogenes, have made an indelible impact on the structure and function of the human genome. One consequence is the creation of new CpG islands (CGIs). In fact, more than half of all CGIs in the genome are associated with repetitive DNA, three-quarters of which are derived from retrotransposons. However, little is known about the epigenetic impact of newly inserted CGIs. We utilized a transgenic LINE-1 mouse model and tracked DNA methylation dynamics of individual germline insertions during mouse development. The retrotransposed GFP marker sequence, a strong CGI, is hypomethylated in male germ cells but hypermethylated in somatic tissues, regardless of genomic location. The GFP marker is similarly methylated when delivered into the genome via the Sleeping Beauty DNA transposon, suggesting that the observed methylation pattern may be independent of the mode of insertion. Comparative analyses between insertion- and non-insertion-containing alleles further reveal a graded influence of the retrotransposed CGI on flanking CpG sites, a phenomenon that we described as “sloping shores.” Computational analyses of human and mouse methylomic data at single-base resolution confirm that sloping shores are universal for hypomethylated CGIs in sperm and somatic tissues. Additionally, the slope of a hypomethylated CGI can be affected by closely positioned CGI neighbors. Finally, by tracing sloping shore dynamics through embryonic and germ cell reprogramming, we found evidence of bookmarking, a mechanism that likely determines which CGIs will be eventually hyper- or hypomethylated.

Trisomy 21 (Down syndrome, DS) is the most common genetic cause of developmental cognitive deficits, and the so-called Down syndrome critical region (DSCR) has been proposed as a major determinant of this phenotype. The regions on human chromosome 21 (Hsa21) are syntenically conserved on mouse chromosome 10 (Mmu10), Mmu16 and Mmu17. DSCR is conserved between the Cbr1 and Fam3b genes on Mmu16. Ts65Dn mice carry three copies of ∼100 Hsa21 gene orthologs on Mmu16 and exhibited impairments in the Morris water maze and hippocampal long-term potentiation (LTP). Converting the Cbr1-Fam3b region back to two copies in Ts65Dn mice rescued these phenotypes. In this study, we performed similar conversion of the Cbr1-Fam3b region in Dp(16)1Yey/+ mice that is triplicated for all ∼115 Hsa21 gene orthologs on Mmu16, which also resulted in the restoration of the wild-type phenotypes in the Morris water maze and hippocampal LTP. However, converting the Cbr1-Fam3b region back to two copies in a complete model, Dp(10)1Yey/+;Dp(16)1Yey/+;Dp(17)1Yey/+, failed to yield the similar phenotypic restorations. But, surprisingly, converting both the Cbr1-Fam3b region and the Hsa21 orthologous region on Mmu17 back to two copies in the complete model did completely restore these phenotypes to the wild-type levels. Our results demonstrated that the Hsa21 orthologous region on Mmu17 is a major determinant of DS-related developmental cognitive deficits. Therefore, the inclusion of the three copies of this Hsa21 orthologous region in mouse models is necessary for unraveling the mechanism underlying DS-associated developmental cognitive deficits and for developing effective interventions for this clinical manifestation.