ZFP57 maintains the parent-of-origin-specific expression of the imprinted genes and differentially affects non-imprinted targets in mouse embryonic stem cells.

ZFP57 is necessary for maintaining repressive epigenetic modifications at Imprinting control regions (ICRs). In mouse embryonic stem cells (ESCs), ZFP57 binds ICRs (ICRBS) and many other loci (non-ICRBS). To address the role of ZFP57 on all its target sites, we performed high-throughput and multi-locus analyses of inbred and hybrid mouse ESC lines carrying different gene knockouts. By using an allele-specific RNA-seq approach, we demonstrate that ZFP57 loss results in derepression of the imprinted allele of multiple genes in the imprinted clusters. We also find marked epigenetic differences between ICRBS and non-ICRBS suggesting that different cis-acting regulatory functions are repressed by ZFP57 at these two classes of target loci. Overall, these data demonstrate that ZFP57 is pivotal to maintain the allele-specific epigenetic modifications of ICRs that in turn are necessary for maintaining the imprinted expression over long distances. At non-ICRBS, ZFP57 inactivation results in acquisition of epigenetic features that are characteristic of poised enhancers, suggesting that another function of ZFP57 in early embryogenesis is to repress cis-acting regulatory elements whose activity is not yet required.

ZFP57 recognizes multiple and closely spaced sequence motif variants to maintain repressive epigenetic marks in mouse embryonic stem cells.

Imprinting Control Regions (ICRs) need to maintain their parental allele-specific DNA methylation during early embryogenesis despite genome-wide demethylation and subsequent de novo methylation. ZFP57 and KAP1 are both required for maintaining the repressive DNA methylation and H3-lysine-9-trimethylation (H3K9me3) at ICRs. In vitro, ZFP57 binds a specific hexanucleotide motif that is enriched at its genomic binding sites. We now demonstrate in mouse embryonic stem cells (ESCs) that SNPs disrupting closely-spaced hexanucleotide motifs are associated with lack of ZFP57 binding and H3K9me3 enrichment. Through a transgenic approach in mouse ESCs, we further demonstrate that an ICR fragment containing three ZFP57 motif sequences recapitulates the original methylated or unmethylated status when integrated into the genome at an ectopic position. Mutation of Zfp57 or the hexanucleotide motifs led to loss of ZFP57 binding and DNA methylation of the transgene. Finally, we identified a sequence variant of the hexanucleotide motif that interacts with ZFP57 both in vivo and in vitro. The presence of multiple and closely located copies of ZFP57 motif variants emerges as a distinct characteristic that is required for the faithful maintenance of repressive epigenetic marks at ICRs and other ZFP57 binding sites.

Determinants of prakriti, the human constitution types of Indian traditional medicine and its correlation with contemporary science.

BACKGROUND: Constitutional type of an individual or prakriti is the basic clinical denominator in Ayurveda, which defines physical, physiological, and psychological traits of an individual and is the template for individualized diet, lifestyle counseling, and treatment. The large number of phenotype description by prakriti determination is based on the knowledge and experience of the assessor, and hence subject to inherent variations and interpretations. OBJECTIVE: In this study we have attempted to relate dominant prakriti attribute to body mass index (BMI) of individuals by assessing an acceptable tool to provide the quantitative measure to the currently qualitative ayurvedic prakriti determination. MATERIALS AND METHODS: The study is cross sectional, multicentered, and prakriti assessment of a total of 3416 subjects was undertaken. Healthy male, nonsmoking, nonalcoholic volunteers between the age group of 20-30 were screened for their prakriti after obtaining written consent to participate in the study. The prakriti was determined on the phenotype description of ayurvedic texts and simultaneously by the use of a computer-aided prakriti assessment tool. Kappa statistical analysis was employed to validate the prakriti assessment and Chi-square, Cramer's V test to determine the relatedness in the dominant prakriti to various attributes. RESULTS: We found 80% concordance between ayurvedic physician and software in predicting the prakriti of an individual. The kappa value of 0.77 showed moderate agreement in prakriti assessment. We observed a significant correlations of dominant prakriti to place of birth and BMI with Chi-square, P < 0.01 (Cramer's V-value of 0.156 and 0.368, respectively). CONCLUSION: The present study attempts to integrate knowledge of traditional ayurvedic concepts with the contemporary science. We have demonstrated analysis of prakriti classification and its association with BMI and place of birth with the implications to one of the ways for human classification.

Immunophenotyping of normal individuals classified on the basis of human dosha prakriti.

BACKGROUND: Human variations related to immune response and disease susceptibility is well-documented in Ayurveda. Prakriti (body constitution) is the basic constitution of an individual established at the time of birth and distinguishes variations, into three broad phenotype categories such as vata, pitta and kapha. Variation in immune response is often attributed to and measured from the difference in cluster differentiation (CD) markers expressed in lymphocytes. Currently, there are no reports available on the expression of CD markers related to prakriti. OBJECTIVE: This is a pilot study performed to evaluate a panel of lymphocyte subset CD markers in dominant prakriti individuals. MATERIALS AND METHODS: Immunophenotyping was carried out using whole blood from a total of healthy 222 subjects, who are grouped into kapha (n = 95), pitta (n = 57) and vata (n = 70) prakritis. CD markers such as CD3, CD4, CD8, CD14, CD25, CD56, CD69, CD71 and HLA-DR were analyzed using flow cytometry method. Differences between groups were analyzed using one-way ANOVA or Kruskal-Wallis analysis of variance (ANOVA) and multiple comparisons between groups were performed by Bonferroni or Mann-Whitney U test with corrections for type I error respectively. Significance was evaluated by ANOVA and Pearson's correlation. RESULTS: We observed a significant difference (P < 0.05) in the expression of CD markers such as CD14 (monocytes), CD25 (activated B cells) and CD56 (Natural killer cells) between different prakriti groups. CD25 and CD56 expression was significantly higher in kapha prakriti samples than other prakriti groups. Similarly, slightly higher levels of CD14 were observed in pitta prakriti samples. CONCLUSION: Significant difference in the expression of CD14, CD25 and CD56 markers between three different prakriti is demonstrated. The increased level of CD25 and CD56 in kapha prakriti may indicate ability to elicit better immune response, which is in conformity with textual references in Ayurveda.