Transcriptome analysis of cynomolgus macaques throughout their lifespan reveals age-related immune patterns.

Despite the different perspectives by diverse research sectors spanning several decades, aging research remains uncharted territory for human beings. Therefore, we investigated the transcriptomic characteristics of eight male healthy cynomolgus macaques, and the annual sampling was designed with two individuals in four age groups. As a laboratory animal, the macaques were meticulously shielded from all environmental factors except aging. The results showed recent findings of certain immune response and the age-associated network of primate immunity. Three important aging patterns were identified and each gene clusters represented a different immune response. The increased expression pattern was predominantly associated with innate immune cells, such as Neutrophils and NK cells, causing chronic inflammation with aging whereas the other two decreased patterns were associated with adaptive immunity, especially "B cell activation" affecting antibody diversity of aging. Furthermore, the hub gene network of the patterns reflected transcriptomic age and correlated with human illness status, aiding in future human disease prediction. Our macaque transcriptome profiling results offer systematic insights into the age-related immunological features of primates.

Utilization of nicking properties of CRISPR-Cas12a effector for genome editing.

The CRISPR-Cas nickase system for genome editing has attracted considerable attention owing to its safety, efficiency, and versatility. Although alternative effectors to Cas9 have the potential to expand the scope of genome editing, their application has not been optimized. Herein, we used an enhanced CRISPR-Cas12a nickase system to induce mutations by targeting genes in a human-derived cell line. The optimized CRISPR-Cas12a nickase system effectively introduced mutations into target genes under a specific directionality and distance between nickases. In particular, the single-mode Cas12a nickase system can induce the target-specific mutations with less DNA double-strand breaks. By inducing mutations in the Thymine-rich target genes in single- or dual-mode, Cas12a nickase compensates the limitations of Cas9 nickase and is expected to contribute to the development of future genome editing technologies.

Identification of microRNAs Derived from Transposable Elements in the Macaca mulatta (Rhesus Monkey) Genome.

Transposable elements (TEs) are mobile DNA entities that can move within the host genome. Over long periods of evolutionary time, TEs are typically silenced via the accumulation of mutations in the genome, ultimately resulting in their immobilization. However, they still play an important role in the host genome by acting as regulatory elements. They influence host transcription in various ways, one of which as the origin of the generation of microRNAs (miRNAs), which are so-called miRNAs derived from TEs (MDTEs). miRNAs are small non-coding RNAs that are involved in many biological processes by regulating gene expression at the post-transcriptional level. Here, we identified MDTEs in the Macaca mulatta (rhesus monkey) genome, which is phylogenetically close species to humans, based on the genome coordinates of miRNAs and TEs. The expression of 5 out of 17 MDTEs that were exclusively registered in M. mulatta from the miRBase database (v22) was examined via quantitative polymerase chain reaction (qPCR). Moreover, Gene Ontology analysis was performed to examine the functional implications of the putative target genes of the five MDTEs.

Generation of induced pluripotent stem cells (cmESF-iPS-C5) derived from cynomolgus monkey ear skin fibroblasts (cmESF).

Cynomolgus monkeys, due to their close anatomical, genetic and physiological similarity to humans, have been employed as a popular laboratory non-human primate model over rodents. Primate animal induced pluripotent stem cell (iPSC) have been used to aid on the investigation of autologous regenerative therapies. Here, we reprogrammed cynomolgus monkey ear skin fibroblasts (cmESFs) into iPSCs as a starting material for autologous based study. The resulting cmESF-iPSCs with canonical features of PSCs will advance the development of autologous transplantation.

Generation and characterization of cynomolgus monkey kidney fibroblasts (cmKF)-derived induced pluripotent stem cells (cmKF-iPS-C5).

Cynomolgus monkeys, a non-human primate species, are genetically and physiologically similar to humans; hence, they have been employed as an ideal developmental and biomedical model. Non-human primate animals and their induced pluripotent stem cell (iPSC) derivatives have been used as a research tool to investigate autologous regenerative medicine. Here, we reprogrammed cynomolgus monkey kidney fibroblasts (cmKFs) as a control for animal iPSCs and to study autologous transplant. The resulting cmKF-iPSCs, which displayed canonical characteristics of PSCs, could be used as a unique model for autologous cell-based therapy.

Hsa-miR-422a Originated from Short Interspersed Nuclear Element Increases ARID5B Expression by Collaborating with NF-E2.

MicroRNAs (miRNAs) are a class of small non-coding RNAs that regulate the expression of target messenger RNA (mRNA) complementary to the 3' untranslated region (UTR) at the post-transcriptional level. Hsa-miR-422a, which is commonly known as miRNA derived from transposable element (MDTE), was derived from short interspersed nuclear element (SINE). Through expression analysis, hsa-miR-422a was found to be highly expressed in both the small intestine and liver of crab-eating monkey. AT-Rich Interaction Domain 5 B (ARID5B) was selected as the target gene of hsa-miR-422a, which has two binding sites in both the exon and 3'UTR of ARID5B. To identify the interaction between hsa-miR-422a and ARID5B, a dual luciferase assay was conducted in HepG2 cell line. The luciferase activity of cells treated with the hsa-miR-422a mimic was upregulated and inversely downregulated when both the hsa-miR-422a mimic and inhibitor were administered. Nuclear factor erythroid-2 (NF-E2) was selected as the core transcription factor (TF) via feed forward loop analysis. The luciferase expression was downregulated when both the hsa-miR-422a mimic and siRNA of NF-E2 were treated, compared to the treatment of the hsa-miR-422a mimic alone. The present study suggests that hsa-miR-422a derived from SINE could bind to the exon region as well as the 3'UTR of ARID5B. Additionally, hsa-miR-422a was found to share binding sites in ARID5Bwith several TFs, including NF-E2. The hsa-miR-422a might thus interact with TF to regulate the expression of ARID5B, as demonstrated experimentally. Altogether, hsa-miR-422a acts as a super enhancer miRNA of ARID5Bby collaborating with TF and NF-E2.

Anti-Obesity Effect of Pine Needle Extract on High-Fat Diet-Induced Obese Mice.

BACKGROUND: Obesity due to an excessive intake of nutrient disturbs the hypothalamus-mediated energy metabolism subsequently develops metabolic disorders. In this study, we investigated the effect of pine needle extract (PNE) on the hypothalamic proopiomelanocortin (POMC) neurons involved in the regulation of energy balance via melanocortin system and fat tissue metabolism. METHODS: We performed electrophysiological and immunohistochemical analyses to determine the effect of PNE on POMC neurons. Mice were fed a normal or high-fat diet for 12 weeks, then received PNE for the last 2 weeks to measure the following physiological indices: Body weight, food intake, fat/lean mass, glucose metabolism, and plasma leptin levels. In addition, changes of thermogenic, lipolytic, and lipogenetic markers were evaluated in brown adipose tissue (BAT) and white adipose tissue (WAT) by western blotting, respectively. RESULTS: PNE increased hypothalamic POMC neuronal activity, and the effect was abolished by blockade of melanocortin 3/4 receptors (MC3/4Rs). PNE decreased body weight, fat mass, plasma leptin levels, and improved glucose metabolism after high-fat-induced obesity. However, PNE did not change the expression of thermogenic markers of the BAT in HFD fed groups, but decreased only the lipogenetic markers of WAT. This study suggests that PNE has a potent anti-obesity effect, inhibiting lipogenesis in WAT, even though HFD-induced leptin resistance-mediated disruption of POMC neuronal activity.

The enhancer activity of long interspersed nuclear element derived microRNA 625 induced by NF-κB.

Transposable elements (TEs) are DNA sequences that cut or introduced into the genome, and they represent a massive portion of the human genome. TEs generate a considerable number of microRNAs (miRNAs) are derived from TEs (MDTEs). Numerous miRNAs are related to cancer, and hsa-miRNA-625 is a well-known oncomiR derived from long interspersed nuclear elements (LINEs). The relative expression of hsa-miRNA-625-5p differs in humans, chimpanzees, crab-eating monkeys, and mice, and four primers were designed against the 3'UTR of GATAD2B to analyze the different quantities of canonical binding sites and the location of miRNA binding sites. Luciferase assay was performed to score for the interaction between hsa-miRNA-625 and the 3'UTR of GATAD2B, while blocking NF-κB. In summary, the different numbers of canonical binding sites and the locations of miRNA binding sites affect gene expression, and NF-κB induces the enhancer activity of hsa-miRNA-625-5p by sharing the binding sites.

Sense-oriented AluYRa1 elements provide a lineage-specific transcription environment for polyadenylation.

Transposable elements cause alternative splicing (AS) in different ways, contributing to transcript diversification. Alternative polyadenylation (APA), one of the AS events, is related to the generation of mRNA isoforms in 70% of human genes. In this study, we tried to investigate AluYRa1s located at the terminal region of cynomolgus monkey genes, utilizing both computational analysis and molecular experimentation. We found that ten genes had AluYRa1 at their 3' end, and nine of these AluYRa1s were sense-oriented. Furthermore, in seven genes, AluYRa1s were expected to have a similar consensus sequence for polyadenylation cleavage. Additional computational analysis using the annotation files from the UCSC database showed that AluYRa1 was more involved in polyadenylation than in open reading frame exon splicing. To examine the extent of AluYRa1 involvement in polyadenylation, RNA-seq data from 30 normal cynomolgus monkeys were analyzed using TAPAS, a recently devised software that detects all the promising polyadenylation sites including APA sites. We observed that approximately 74% of possible polyadenylation sites in the analyzed genes were provided by sense-oriented AluYRa1. In conclusion, AluYRa1 is an Old-World monkey-specific TE, and its sense-oriented insertion at the 3'UTR region tends to provide a favorable environment for polyadenylation, diversifying gene transcripts.

Longitudinal profiling of the blood transcriptome in an African green monkey aging model.

African green monkeys (AGMs, Chlorocebus aethiops) are Old World monkeys which are used as experimental models in biomedical research. Recent technological advances in next generation sequencing are useful for unraveling the genetic mechanisms underlying senescence, aging, and age-related disease. To elucidate the normal aging mechanisms in older age, the blood transcriptomes of nine healthy, aged AGMs (15‒23 years old), were analyzed over two years. We identified 910‒1399 accumulated differentially expressed genes (DEGs) in each individual, which increased with age. Aging-related DEGs were sorted across the three time points. A major proportion of the aging-related DEGs belonged to gene ontology (GO) categories involved in translation and rRNA metabolic processes. Next, we sorted common aging-related DEGs across three time points over two years. Common aging-related DEGs belonged to GO categories involved in translation, cellular component biogenesis, rRNA metabolic processes, cellular component organization, biogenesis, and RNA metabolic processes. Furthermore, we identified 29 candidate aging genes that were upregulated across the time series analysis. These candidate aging genes were linked to protein synthesis. This study describes a changing gene expression pattern in AGMs during aging using longitudinal transcriptome sequencing. The candidate aging genes identified here may be potential targets for the treatment of aging.

Enhancer Function of MicroRNA-3681 Derived from Long Terminal Repeats Represses the Activity of Variable Number Tandem Repeats in the 3' UTR of SHISA7.

microRNAs (miRNAs) are non-coding RNA molecules involved in the regulation of gene expression. miRNAs inhibit gene expression by binding to the 3' untranslated region (UTR) of their target gene. miRNAs can originate from transposable elements (TEs), which comprise approximately half of the eukaryotic genome and one type of TE, called the long terminal repeat (LTR) is found in class of retrotransposons. Amongst the miRNAs derived from LTR, hsa-miR-3681 was chosen and analyzed using bioinformatics tools and experimental analysis. Studies on hsa-miR-3681 have been scarce and this study provides the relative expression analysis of hsa-miR-3681-5p from humans, chimpanzees, crab-eating monkeys, and mice. Luciferase assay for hsa-miR-3681-5p and its target gene SHISA7 supports our hypothesis that the number of miRNA binding sites affects target gene expression. Especially, the variable number tandem repeat (VNTR) and hsa-miR-3681-5p share the binding sites in the 3' UTR of SHISA7, which leads the enhancer function of hsa-miR-3681-5p to inhibit the activity of VNTR. In conclusion, hsa-miR-3681-5p acts as a super-enhancer and the enhancer function of hsa-miR-3681-5p acts as a repressor of VNTR activity in the 3' UTR of SHISA7.

A single mutation in the ACTR8 gene associated with lineage-specific expression in primates.

BACKGROUND: Alternative splicing (AS) generates various transcripts from a single gene and thus plays a significant role in transcriptomic diversity and proteomic complexity. Alu elements are primate-specific transposable elements (TEs) and can provide a donor or acceptor site for AS. In a study on TE-mediated AS, we recently identified a novel AluSz6-exonized ACTR8 transcript of the crab-eating monkey (Macaca fascicularis). In the present study, we sought to determine the molecular mechanism of AluSz6 exonization of the ACTR8 gene and investigate its evolutionary and functional consequences in the crab-eating monkey. RESULTS: We performed RT-PCR and genomic PCR to analyze AluSz6 exonization in the ACTR8 gene and the expression of the AluSz6-exonized transcript in nine primate samples, including prosimians, New world monkeys, Old world monkeys, and hominoids. AluSz6 integration was estimated to have occurred before the divergence of simians and prosimians. The Alu-exonized transcript obtained by AS was lineage-specific and expressed only in Old world monkeys and apes, and humans. This lineage-specific expression was caused by a single G duplication in AluSz6, which provides a new canonical 5' splicing site. We further identified other alternative transcripts that were unaffected by the AluSz6 insertion. Finally, we observed that the alternative transcripts were transcribed into new isoforms with C-terminus deletion, and in silico analysis showed that these isoforms do not have a destructive function. CONCLUSIONS: The single G duplication in the TE sequence is the source of TE exonization and AS, and this mutation may suffer a different fate of ACTR8 gene expression during primate evolution.

Chronic Infiltration of T Lymphocytes into the Brain in a Non-human Primate Model of Parkinson's Disease.

Study of interactions between the nervous system and immunity offers insights into the pathogenesis of Parkinson's disease (PD) and potential therapeutic strategies for neurodegenerative diseases. Studies on rodents have revealed regulatory mechanisms of microglial activation and T lymphocyte recruitment in PD. However, the mechanisms underlying chronic T lymphocyte infiltration into the brain after 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) injection into a non-human primate (NHP) model of PD remain unknown. This study aimed to investigate changes in serum RANTES (regulated on activation, normal T cell expression and secretion) and analyze the chronic infiltration of T lymphocytes into the brain and microglia activation in NHPs at 48 weeks post-MPTP administration. We found selective and local chronic infiltration of CD4+ and CD8+ T lymphocytes, loss of dopaminergic neurons, dopamine transporter expression, chronic normalization of RANTES in the peripheral blood, and altered microglial morphology at 48 weeks after MPTP injection. This study confirms the involvement of CD4+ and CD8+ T lymphocyte infiltration in MPTP-induced NHP models of PD. Additionally, we corroborated previous findings regarding the mechanisms of T lymphocyte-induced neurodegeneration. The findings of chronic infiltration of T lymphocytes in our NHP model of PD provide novel insights into PD pathogenesis and the development of preventive and therapeutic agents.

Cooperative evolution of two different TEs results in lineage-specific novel transcripts in the BLOC1S2 gene.

BACKGROUND: The BLOC1S2 gene encodes the multifunctional protein BLOS2, a shared subunit of two lysosomal trafficking complexes: i) biogenesis of lysosome-related organelles complex-1 and i) BLOC-1-related complex. In our previous study, we identified an intriguing unreported transcript of the BLOC1S2 gene that has a novel exon derived from two transposable elements (TEs), MIR and AluSp. To investigate the evolutionary footprint and molecular mechanism of action of this transcript, we performed PCR and RT-PCR experiments and sequencing analyses using genomic DNA and RNA samples from humans and various non-human primates. RESULTS: The results showed that the MIR element had integrated into the genome of our common ancestor, specifically in the BLOC1S2 gene region, before the radiation of all primate lineages and that the AluSp element had integrated into the genome of our common ancestor, fortunately in the middle of the MIR sequences, after the divergence of Old World monkeys and New World monkeys. The combined MIR and AluSp sequences provide a 3' splice site (AG) and 5' splice site (GT), respectively, and generate the Old World monkey-specific transcripts. Moreover, branch point sequences for the intron removal process are provided by the MIR and AluSp combination. CONCLUSIONS: We show for the first time that sequential integration into the same location and sequence divergence events of two different TEs generated lineage-specific transcripts through sequence collaboration during primate evolution.

Real-time PCR quantification of spliced X-box binding protein 1 (XBP1) using a universal primer method.

X-box binding protein 1 (XBP1) mRNA processing plays a crucial role in the unfolded protein response (UPR), which is activated in response to endoplasmic reticulum (ER) stress. Upon accumulation of the UPR-converted XBP1 mRNA splicing from an unspliced (u) XBP1 (inactive) isoform to the spliced (s) XBP1 (active) isoform, inositol-requiring enzyme 1 α (IRE1α) removes a 26-nucleotide intron from uXBP1 mRNA. Recent studies have reported the assessment of ER stress by examining the ratio of sXBP1 to uXBP1 mRNA (s/uXBP1 ratio) via densitometric analysis of PCR bands relative to increased levels of sXBP1 to uXBP1 using a housekeeping gene for normalization. However, this measurement is visualized by gel electrophoresis, making it very difficult to quantify differences between the two XBP1 bands and complicating data interpretation. Moreover, most commonly used housekeeping genes display an unacceptably high variable expression pattern of the s/uXBP1 ratio under different experimental conditions, such as various phases of development and different cell types, limiting their use as internal controls. For a more quantitative determination of XBP1 splicing activity, we measured the expression levels of total XBP1 (tXBP1: common region of s/uXBP1) and sXBP1 via real-time PCR using specific primer sets. We also designed universal real-time PCR primer sets capable of amplifying a portion of each u/s/tXBP1 mRNA that is highly conserved in eukaryotes, including humans, monkeys, cows, pigs, and mice. Therefore, we provide a more convenient and easily approachable quantitative real-time PCR method that can be used in various research fields to assess ER stress.

Abnormal Mitochondria in a Non-human Primate Model of MPTP-induced Parkinson's Disease: Drp1 and CDK5/p25 Signaling.

Mitochondria continuously fuse and divide to maintain homeostasis. An impairment in the balance between the fusion and fission processes can trigger mitochondrial dysfunction. Accumulating evidence suggests that mitochondrial dysfunction is related to neurodegenerative diseases such as Parkinson's disease (PD), with excessive mitochondrial fission in dopaminergic neurons being one of the pathological mechanisms of PD. Here, we investigated the balance between mitochondrial fusion and fission in the substantia nigra of a non-human primate model of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced PD. We found that MPTP induced shorter and abnormally distributed mitochondria. This phenomenon was accompanied by the activation of dynamin-related protein 1 (Drp1), a mitochondrial fission protein, through increased phosphorylation at S616. Thereafter, we assessed for activation of the components of the cyclin-dependent kinase 5 (CDK5) and extracellular signal-regulated kinase (ERK) signaling cascades, which are known regulators of Drp1(S616) phosphorylation. MPTP induced an increase in p25 and p35, which are required for CDK5 activation. Together, these findings suggest that the phosphorylation of Drp1(S616) by CDK5 is involved in mitochondrial fission in the substantia nigra of a non-human primate model of MPTP-induced PD.

Evaluation of fecal microbiomes associated with obesity in captive cynomolgus monkeys (Macaca fascicularis).

Microorganisms play important roles in obesity; however, the role of the gut microbiomes in obesity is controversial because of the inconsistent findings. This study investigated the gut microbiome communities in obese and lean groups of captive healthy cynomolgus monkeys reared under strict identical environmental conditions, including their diet. No significant differences in the relative abundance of Firmicutes, Bacteroidetes and Prevotella were observed between the obese and lean groups, but a significant difference in Spirochetes (p < 0.05) was noted. Microbial diversity and richness were similar, but highly variable results in microbial composition, diversity, and richness were observed in individuals, irrespective of their state of obesity. Distinct clustering between the groups was not observed by principal coordinate analysis using an unweighted pair group method. Higher sharedness values (95.81% ± 2.28% at the genus level, and 79.54% ± 5.88% at the species level) were identified among individual monkeys. This paper reports the association between the gut microbiome and obesity in captive non-human primate models reared under controlled environments. The relative proportion of Firmicutes and Bacteroidetes as well as the microbial diversity known to affect obesity were similar in the obese and lean groups of monkeys reared under identical conditions. Therefore, obesity-associated microbial changes reported previously appear to be associated directly with environmental factors, particularly diet, rather than obesity.

Reference values of hematological and biochemical parameters in young-adult cynomolgus monkey (Macaca fascicularis) and rhesus monkey (Macaca mulatta) anesthetized with ketamine hydrochloride.

Nonhuman primate models are valuable in biomedical research. However, reference data for clinical pathology parameters in cynomolgus and rhesus monkeys are limited. In the present study, we established hematologic and biochemical reference intervals for healthy cynomolgus and rhesus monkeys anesthetized with ketamine hydrochloride. A total of 142 cynomolgus monkeys (28 males and 114 females) and 42 rhesus monkeys (22 males and 20 females) were selected and analyzed in order to examine reference intervals of 20 hematological and 16 biochemical parameters. The effects of sex were also investigated. Reference intervals for hematological and biochemical parameters were separately established by species (cynomolgus and rhesus) and sex (male and female). No sex-related differences were determined in erythrocyte-related parameters for cynomolgus and rhesus monkey housed in indoor laboratory conditions. Alkaline phosphatase and gamma glutamyltransferase were significantly lower in females than males in both cynomolgus and rhesus monkeys aged 48-96 months. The reference values for hematological and biochemical parameters established herein might provide valuable information for researchers using cynomolgus and rhesus monkeys in experimental conditions for biomedical studies.

A non-human primate model for stable chronic Parkinson's disease induced by MPTP administration based on individual behavioral quantification.

BACKGROUND: The guidelines for applying individual adjustments to macaques according to the severity of behavioral symptoms during 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) treatment were provided to reproduce stable chronic Parkinsonism in a recent study (Potts et al., 2014). But, since there are insufficient guidelines regarding objective severity criteria of individual symptoms for adjustments of MPTP treatment, it is difficult to develop MPTP-induced chronic non-human primate (NHP) models with stable symptoms. NEW METHOD: The individual adjustments of MPTP administration based on results of automatic quantification of global activity (GA) using a video-based tracking system were applied to develop MPTP-PD model. Low-dose (0.2 mg/kg) intramuscular injection was repeated continuously until GA was lower than 8% of baseline Parkinsonian behavior scores. The positron emission tomography imaging were used to follow the longitudinal course of Parkinson's disease (PD). RESULTS: Significant reductions in GA and dopamine transporter activity, along with significant increases in Parkinsonian behavior scores were found from 4 to 48 weeks following the first administration. GA was correlated with the Parkinsonian behavior score. The dopamine transporter activity was correlated with GA and the Parkinsonian behavior score. However, it was not correlated with the total dose of MPTP. Damage of dopaminergic neuronal systems in the basal ganglia was confirmed by immunohistochemistry and Western blot. COMPARISON WITH EXISTING METHOD: This study reinforces previous guidelines regarding production of NHP models with stable Parkinsonian symptoms. CONCLUSIONS: This novel strategy of MPTP administration based on global activity evaluations provides an important conceptual advance for the development of chronic NHP Parkinsonian models.

Successful application of human-based methyl capture sequencing for methylome analysis in non-human primate models.

BACKGROUND: The characterization of genomic or epigenomic variation in human and animal models could provide important insight into pathophysiological mechanisms of various diseases, and lead to new developments in disease diagnosis and clinical intervention. The African green monkey (AGM; Chlorocebus aethiops) and cynomolgus monkey (CM; Macaca fascicularis) have long been considered important animal models in biomedical research. However, non-human primate-specific methods applicable to epigenomic analyses in AGM and CM are lacking. The recent development of methyl-capture sequencing (MC-seq) has an unprecedented advantage of cost-effectiveness, and further allows for extending the methylome coverage compared to conventional sequencing approaches. RESULTS: Here, we used a human probe-designed MC-seq method to assay DNA methylation in DNA obtained from 13 CM and three AGM blood samples. To effectively adapt the human probe-designed target region for methylome analysis in non-human primates, we redefined the target regions, focusing on regulatory regions and intragenic regions with consideration of interspecific sequence homology and promoter region variation. Methyl-capture efficiency was controlled by the sequence identity between the captured probes based on the human reference genome and the AGM and CM genome sequences, respectively. Using reasonable guidelines, 56 and 62% of the human-based capture probes could be effectively mapped for DNA methylome profiling in the AGM and CM genome, respectively, according to numeric global statistics. In particular, our method could cover up to 89 and 87% of the regulatory regions of the AGM and CM genome, respectively. CONCLUSIONS: Use of human-based MC-seq methods provides an attractive, cost-effective approach for the methylome profiling of non-human primates at the single-base resolution level.