Early-life DNA methylation profiles are indicative of age-related transcriptome changes.

BACKGROUND: Alterations to cellular and molecular programs with brain aging result in cognitive impairment and susceptibility to neurodegenerative disease. Changes in DNA methylation patterns, an epigenetic modification required for various CNS functions are observed with brain aging and can be prevented by anti-aging interventions, but the relationship of altered methylation to gene expression is poorly understood. RESULTS: Paired analysis of the hippocampal methylome and transcriptome with aging of male and female mice demonstrates that age-related differences in methylation and gene expression are anti-correlated within gene bodies and enhancers. Altered promoter methylation with aging was found to be generally un-related to altered gene expression. A more striking relationship was found between methylation levels at young age and differential gene expression with aging. Highly methylated gene bodies and promoters in early life were associated with age-related increases in gene expression even in the absence of significant methylation changes with aging. As well, low levels of methylation in early life were correlated to decreased expression with aging. This relationship was also observed in genes altered in two mouse Alzheimer\u27s models. CONCLUSION: DNA methylation patterns established in youth, in combination with other epigenetic marks, were able to accurately predict changes in transcript trajectories with aging. These findings are consistent with the developmental origins of disease hypothesis and indicate that epigenetic variability in early life may explain differences in aging trajectories and age-related disease

Epigenetics

Epigenetic changes are heritable and reversible modifications that significantly affect gene expression without any change in DNA sequence. The epigenetic signature is remodelled during the lifespan as a direct consequence of both environment and lifestyle. Therefore, health or disease status strongly depends on epigenetic marks. This book summarizes the current knowledge in the field and includes chapters on epigenetics in plants and epigenetics in health and disease. It is written for a wide audience of basic and clinical scientists, teachers and students interested in gaining a better understanding of epigenetics

Regulation of fetal brain development in short versus long lived mice

How fetal brain development is regulated in mice with reduced life span is a primary objective of this research. Three studies were performed to investigate the fetal brain development in short and lived mice strains. The scientific premise and background of brain development and aging are provided review of literature (Chapter 1). In the first study (Chapter 2), experiments were performed to test early-life origin of brain aging. Mouse epigenetic clock (epiclock) database which represents specific genomic sites that are methylated in an age-correlated manner was profiled in three life stages: fetal (gestation day 15), postnatal (day 5), and adult (week 70) brains of male and female C57BL/6J inbred mice. Data analysis showed that the female adult brain was epigenetically younger than the male adult brain even when the chronological age (time from birth) was the same (week 70). Specific methylations in the developing brain predictive of epigenetic differences in the aging brain between sexes were identified by predictive modeling by neural network. This study also showed that gene expression of epiclock genes were similar between placenta and fetal brain. However, genes unrelated to epiclock did not show this pattern. Whole-genome bisulfite sequencing identified sites that were methylated in a coordinated manner in the placenta and in a sex-specific manner in the fetal brain. These sites showed that methylation level in the epiclock genes and genes associated with gonadotropin-releasing hormone (GnRH) signaling pathway genes changes in a fetal-sex dependent manner both in the placenta and fetal brain. Furthermore, these methylations were maintained in the brain in the adult life stages. These findings suggested the fetal origin of sex differences in brain aging is epigenetically linked to the placenta. In the second study (Chapter 3), experiments were performed to test if reducing life span of mouse by ablating Caveolin 1 (Cav-1), a prolongevity gene that codes for an abundant structural protein of plasma membrane in endothelial cells, dysregulate brain development at the fetal stage. Further relevance of studying this specific gene is that mice lacking Cav-1 show neurodegeneration and multiple hallmarks of Alzheimer's disease (AD) at an early age. As a result, most Cav-1- null mice die within a year. Gene expression in bulk brain tissue as well as single cells were analyzed in the Cav-1-null fetal brain compared to the wildtype (WT). The results of this study showed that lack of Cav-1 leads to extensive dysregulation of genes of fetal brain at specific gestation time (day 15). Several epigenetic clock genes were differentially methylated in Cav-1-KO compared to WT mouse fetal brain. Single nuclei RNA sequencing identified specific glial and neuronal cells being dysregulated in the fetal brain due to the absence of Cav-1. In addition, methylation analysis was performed to investigate effect of Cav-1 on epiclock genes. Based on these results, a model was proposed for fetal links of Alzheimer's symptoms in mice lacking Cav-1. Lastly, in the third study (Chapter 4), experiments were performed to test if reduced lifespan in mice due to murine leukemia virus induced cancer influences fetal brain development. In this experiment, gene expression pattern of fetal brain and placenta of AKR/J mice, which mostly survive for a year due to onset of cancer, was compared with C57BL/6J mice to understand molecular and cellular links between aging and leukemia. The C57BL/6J has longer life span ([greater than] 2 years) and is refractory to AK virus that causes leukemia in AKR/J mice. The gene expression studies showed that genes related to aging and neurodegenerative diseases are differentially regulated in the fetal brain and placenta of AKR/J mice compared to that in C57BL/6J. Targeted methylation profiling of a total of 2,045 single bases of mouse genome, which are associated with mouse epigenetic clock data, showed that brain of AKR/J mice ages faster than C57BL/6J mice suggesting a link between leukemia and neuronal aging. By generating a F2 mapping population from AKR/J x C57BL/6J crosses, Bulk Segregant Analysis (BSA) was performed with the pooled DNA of F2 progenies by whole-genome sequencing to identify genetic variants associated with accelerated brain aging in AKR/J mice. Single-cell ATAC-seq (Assay for Transposase-Accessible Chromatin by sequencing) analysis further predicted that specific transcription factors are involved in the differential gene regulation of fetal brain in AKR/J mice compared to C57BL/6J mice. Together, the results of these study provide foundational knowledge to establish molecular and cellular links between reproduction and aging.Includes bibliographical references

Genome-Wide methylation and transcriptome analyses of bone marrow mesenchymal stem cells from osteoporotic patients

RESUMEN: La diferenciación de las células madre mesenquimales (MSCs) es esencial para el mantenimiento de la masa ósea. Nuestro objetivo fue caracterizar las marcas de metilación, la expresión génica (codificante y no codificante de proteína) y la capacidad de diferenciación de las MSC de médula ósea (BMSCs) de pacientes con fracturas de cadera osteoporóticas. Las BMSCs de pacientes con fracturas mostraron una mayor proliferación y una capacidad de diferenciación alterada. Los análisis de metilación de ADN revelaron que la mayoría los sitios diferencialmente metilados se dan en regiones genómicas con actividad potenciadora que a su vez se asociaron con genes expresados diferencialmente enriquecidos en vías relacionadas con la diferenciación osteogénica. Nuestros resultados sugieren que los mecanismos epigenéticos estudiados juegan un papel importante en la determinación del patrón de expresión génica de BMSCs derivadas de pacientes con osteoporosis. Y un mejor conocimiento de estas vías nos permitirá mejorar el metabolismo óseo en la osteoporosis.ABSTRACT: Mesenchymal stem cells (MSCs) osteogenic differentiation is essential for the maintenance of bone mass. The aim of this study was to characterize the DNA methylation marks, gene expression (coding and nonprotein-coding) and the ability to differentiate bone marrow stem cells (BMSCs) from patients with osteoporotic hip fractures. The BMSCs of patients with fractures showed greater proliferation and an altered differentiation capacity. DNA methylation analysis revealed that most differentially methylated sites are in genomic regions with enhancer activity. These enhancer regions were associated with differentially expressed genes, and these genes were enriched in bone related pathways, such as, osteogenic differentiation. Our results suggest that epigenetic mechanisms play an important role in the regulation of gene expression of BMSCs derived from patients with osteoporosis. A better knowledge of these pathways will permit us to improve bone metabolism in osteoporosis.Research carried out in this thesis was mainly developed in the Department of Medicine and Psychiatry of the Faculty of Medicine, University of Cantabria/ Mineral and lipid metabolism group of IDIVAL. The research was funded with grants from the Instituto de Salud Carlos III (PI12/615 and PI16/915). I have been funded by a predoctoral fellowship from the University of Cantabria and the Research Institute of Marques de Valdecilla Hospital (IDIVAL) (CVE-2016-11669)

Multi-tissue DNA methylation age predictor in mouse.

BACKGROUND: DNA methylation changes at a discrete set of sites in the human genome are predictive of chronological and biological age. However, it is not known whether these changes are causative or a consequence of an underlying ageing process. It has also not been shown whether this epigenetic clock is unique to humans or conserved in the more experimentally tractable mouse. RESULTS: We have generated a comprehensive set of genome-scale base-resolution methylation maps from multiple mouse tissues spanning a wide range of ages. Many CpG sites show significant tissue-independent correlations with age which allowed us to develop a multi-tissue predictor of age in the mouse. Our model, which estimates age based on DNA methylation at 329 unique CpG sites, has a median absolute error of 3.33 weeks and has similar properties to the recently described human epigenetic clock. Using publicly available datasets, we find that the mouse clock is accurate enough to measure effects on biological age, including in the context of interventions. While females and males show no significant differences in predicted DNA methylation age, ovariectomy results in significant age acceleration in females. Furthermore, we identify significant differences in age-acceleration dependent on the lipid content of the diet. CONCLUSIONS: Here we identify and characterise an epigenetic predictor of age in mice, the mouse epigenetic clock. This clock will be instrumental for understanding the biology of ageing and will allow modulation of its ticking rate and resetting the clock in vivo to study the impact on biological age

Dnmt1 in Intestinal Development and Cancer

Patterns of DNA methylation are established and maintained by DNA methyltransferases (Dnmts), which have traditionally been subdivided into the ‘de novo’ methyltransferases, Dnmt3a and Dnmt3b, and the ‘maintenance’ methyltransferase, Dnmt1. Dnmt1 maintains DNA methylation patterns and genomic stability in several in vitro cell systems, but its function in tissue-specific development, homeostasis, and disease in vivo is only beginning to be investigated. Recently, the Kaestner lab demonstrated that loss of Dnmt1 in the adult intestinal epithelium causes a two-fold expansion of the proliferative crypt zone, indicating that Dnmt1 and DNA methylation regulate proliferative processes in the intestine. I hypothesized that loss of Dnmt1 may impart similar effects during intestinal development and tumorigenesis, and employed distinct Cre-loxP mouse models to ablate Dnmt1 in progenitor cells during intestinal development and in the mature intestinal epithelium of cancer-prone ApcMin/+ mice. In the first part of my thesis, I show that loss of Dnmt1 in intervillus progenitor cells in the developing intestine causes global hypomethylation, DNA damage, premature differentiation, and apoptosis. I confirm this novel role for Dnmt1 during crypt development using the in vitro organoid culture system, and illustrate a differential requirement for Dnmt1 in immature versus mature organoids. These results demonstrate an essential role for Dnmt1 in maintaining genomic stability during intestinal development and the establishment of intestinal crypts. DNA methylation is thought to drive CRC progression by the repression of tumor suppressor genes via promoter methylation. In the second part of my thesis, I utilize inducible intestinal epithelial-specific gene ablation to determine the requirement of Dnmt1 in intestinal tumorigenesis. Surprisingly, I find that loss of Dnmt1 in cancer-prone ApcMin/+ mice results in accelerated, not decreased, intestinal tumor development. Dnmt1 deletion precipitates an acute response in mature intestinal epithelium characterized by hypomethylation of repetitive elements, genomic instability, and apoptosis, which is followed by remethylation with time. This recovery is entirely dependent on the activity of the de novo methyltransferase Dnmt3b. In light of these data, the current dogma regarding the role of DNA methylation in colon cancer needs to be revisited

DNA methylation dynamics in aging: How far are we from understanding the mechanisms?

DNA methylation is currently the most promising molecular marker for monitoring aging and predicting life expectancy. However, the mechanisms underlying age-related DNA methylation changes remain mostly undiscovered.Here we discuss the current knowledge of the dynamic nature of DNA epigenome landscape in mammals, and propose putative molecular mechanisms for aging-associated DNA epigenetic changes. Specifically, we describe age-related variations of methylcytosine and its oxidative derivatives in relation to the dynamics of chromatin structure, histone post-translational modifications and their modulators.Finally, we are proposing a conceptual framework that could explain the complex nature of the effects of age on DNA methylation patterns. This combines the accumulation of DNA methylation noise and also all of the predictable, site-specific DNA methylation changes.Gathering information in this area would pave the way for future investigation aimed at establishing a possible causative role of epigenetic mechanisms in aging

Methylation profiling and validation of candidate tDMRs for identification of human blood, saliva, semen and vaginal fluid and its application in forensics.

Masters Degree. University of KwaZulu-Natal, Durban.Identification of body fluids and tissues is an essential step in forensic investigation because it can be used as strong evidence in identifying suspects and victims. Currently in forensic investigations, catalytic, enzymatic and immunological techniques are used to identify body fluids, however, are limited due to lack of sensitivity and specificity. Hence, researchers are always on the lookout for novel methods that can be used to identify and analyse body fluids. Recently, DNA methylation-based markers have proven to be more sensitive and specific than conventional methods for body fluid identification. Genome-wide methylation studies have demonstrated that tissue specific differentially methylated regions (tDMRs) vary in methylation profiles in various cell types and tissues. The differences in methylation profiles of tDMRs can be targeted to be used as biomarkers to differentiate between body fluids and tissues. To date, only a few DNA methylation-based markers have been reported to identify body fluids. To enhance the specificity and robustness of DNA methylation-based identification, novel markers are required. Additionally, methylation-based markers require further interrogation, to evaluate the stability of their methylation profiles under simulated forensics conditions such as UV light, temperature, rain and microbes, which could cause DNA degradation and affect DNA recovery as well as the methylation status of body fluids. In a previous study, based on differential gene expression in blood, saliva, semen and vaginal fluid, gene body CpG islands were selected, in genes Zinc finger protein 282 (ZNF282), Protein tyrosine phosphatase, receptor S (PTPRS) and Hippocalcin like 1 (HPCAL1), that have potential tDMRs to differentiate between, blood, saliva, semen and vaginal fluid. It was proposed that differential gene expression could be possibly due to differences in methylation patterns. The present study was undertaken to establish the methylation status of potential tDMRs in target body fluids by using methylation specific PCR (MSP) and bisulfite sequencing (BS). In both MSP and BS, the methylation status of 3 genes ZNF282, PTPRS and HPCAL1 were analysed in 10 samples of each body fluid. With MSP analysis the ZNF282 and PTPRS1 tDMR displayed semen-specific hypomethylation while HPCAL1 tDMR showed saliva-specific hypomethylation. The PTPRS 2 tDMR did not differentiate between any body fluids due to presence of methylation and unmethylation for all body fluids. With quantitative analysis by BS the ZNF282 tDMR showed statistically significant difference in overall methylation status between semen and all other body fluids as well as at individual CpG sites (p 0.05). The BS study showed that the tDMR for the HPCAL1 gene displayed non-specific amplification therefore was not further analysed. Furthermore, a sensitivity and forensic simulation study was conducted to determine the stability of methylation profiles. To determine the lowest DNA concentration that can be evaluated with MSP, a sensitivity study was conducted using five-fold serial dilution (25, 20, 15, 10, 5, 1 ng) of blood DNA samples. Each DNA dilution was subjected to bisulfite modification, followed by amplification with ZNF282, PTPRS 1, PTPRS 2, and HPCAL1 primers. The results showed that the detection limits were 10 ng for ZNF282 tDMR, 5 ng for PTPRS 1, 15 ng for PTPRS 2, and 5 ng for HPCAL1 tDMR. Thus, it was concluded that a DNA concentration greater than 10 ng would yield successful results with MSP analyses. To evaluate whether environmental conditions has an effect on the stability of methylation profiles of the ZNF282 tDMR, five samples of each body fluid were subjected to five different forensic simulated conditions (dry at room temperature, wet in an exsiccator, outside on the ground, sprayed with alcohol and sprayed with bleach) for 50 days. Following the 50 days, vaginal fluid showed highest DNA recovery under all conditions while semen had least DNA quantity. Under outside on the ground condition, all body fluids except semen showed decrease in methylation level, however, significant decrease in methylation level was observed for saliva. A statistical significant difference was observed for saliva and semen (p < 0.05) in the outside on the ground condition. No differences in methylation level were observed for the ZNF282 tDMR under all conditions for vaginal fluid samples. Thus, ZNF282 tDMR is stable under environmental insults and can be used as reliable semen-specific hypomethylated marker. The analysis of tDMRs represents a unique, efficient and reliable technique that can be used to differentiate between human body fluids. In the future, identification and validation of new tDMRs based markers as well as determining methylation differences in other forensically relevant body fluids will be beneficial for forensics applications.Supervisor on university system as Joshi, Meenu