DNMT3B PHD Proposal - Adnan

Dynamics of DNMT3B
Methyltransferase Expression and

localization at CpG Islands
Adnan Tahir
Student ID: 3820170072
Supervisor: Prof. Hong Qing
PhD student of Biomedical Engineering
2/9/2022 1
Outline of Presentation
Item 1 Introduction and Background
Item 2 Problem Statement
Item 3 Aims and Objectives
Item 4 Methodology
Item 5 Possible Outcomes
Item 6 Estimated Plan and Budget
Item 7 References
2/9/2022 2
Dementia and Alzheimer’s Disease
• Dementia, an age-related neurodegenerative disorder characterized by

progressive cognitive decline, affects 35.6 million people worldwide and is
becoming an increasingly relevant concern as the population ages.
• Alzheimer’s Disease (AD) is the most common type of dementia, accounting for
50–80% of all dementia cases.
• Many report have reported that epigenetics play a

important role in neurological disorders such as AD,
Parkinson Disease (PD), Huntington Disease (HD) and
Amyotrophic lateral sclerosis (ALS).
2/9/2022 3
Epigenetics
• Epigenetics is the study of heritable changes in gene function that do not involve
changes in the DNA sequence.
2/9/2022 4
DNA Methyltransferases
• DNA methyltransferase (DNMT) enzymes, of which DNMT1, DNMT3A and

DNMT3B are best characterized, catalyze the transfer of a methyl group to
DNA.
2/9/2022 5
Cytosine Methylation
• An elevation in plasma homocysteine, is associated with a greater risk for developing dementia and AD, thus
implicating DNA methylation and alterations in one-carbon metabolism with AD pathogenesis.
2/9/2022 6
Domain Structure of Human DNMTs
2/9/2022 7
Domain Structure of Human DNMTs
DNMT3B
2/9/2022 8
DNMT3B
• During the embryogenesis the DNMT3 family establishes the initial CpG
methylation pattern.
• Knockout of DNMT3A or DNMT3B is lethal, being both essential for embryonic
development in mice. Mouse DNMT3B knockout embryos die in utero, whereas
the DNMT3A knockout animals die shortly after birth.
• The protein localizes primarily to the nucleus and its expression is
developmentally regulated. Mutations in this gene cause the immunodeficiency-
centromeric instability-facial anomalies (ICF) syndrome. Eight alternatively spliced
transcript variants have been described.
2/9/2022 9
Problem Statement
• While the genome-wide distribution of this Epigenetics has been

studied to great detail, the mechanisms responsible for its correct
deposition, as well as the cause for its aberrant localization in
neurodegenerative disorders, have not been fully elucidated.
2/9/2022 10
Aims and Objectives
• To identify the DNMT3B variations in genomic binding and DNA methylation

activity at regulatory sites.
• To explore the role of DNMT3B in DNA methylation patterns in mouse ES and

neuronal progenitor cells at these sites.
• To analyze the expression of DNMT3B
• To reveal the significance of DNMT3B in sustaining methylcytosine at bivalent

CpG islands.
• Comparative analysis with other de novo methyltransferase i.e. DNMT3A
2/9/2022 11
Methodology Proposed
Cell Culture
Cell Line Generation
Transfection
Nuclear protein extraction Co-immunoprecipitation

Mass Spectrometry (MS) (CoIP)
Chromatin Extraction
Chromatin
DNA purification
immunoprecipitation (chIP)
Libraries Preparations
High-throughput
Sequencing
Sequencing
Reads Processing CpG Islands Detection
Methylation percentage, CpG and m-CpG
Bioinformatics Analyses
2/9/2022
Identification of DNMT enriched regions
12
Techniques
• Cell Culturing
• Mass Spectrometry
• Co-Immunoprecipitation
• Chromatin Immunoprecipitation
• High-throughput Sequencing
• Bioinformatics Analyses
• Western Blotting
• Statistical Analysis
• Comparative analysis
2/9/2022 13
Expected Results
• DNMT3B will preferentially localized to the methylated shores of bivalent CpG

island promoters.
• This may will coincide with elevated or depleted the hydroxymethylcytosine (5-
hmC) deposition, suggesting an involvement of in mediating turnover of DNA
methylation at these sites.
• Through experiments, we will be able to demonstrate the specific recruitment of

DNMT3B in de novo DNA methylation at CpG island shores, with potential
implications on histones mediated regulation of developmental genes.
2/9/2022 14
Estimated Plan
Phases Activity Time Frame /(Duration)

1 Literature Review & Instruments Purchasing Jul, 2018-Sep, 2018 (3 months)
2 Cell Cultures and Cell Lines Generations Oct, 2018-Feb, 2019 (6 months)
3 Proteins extraction and Mass Spectrometry Mar, 2019-May, 2019 (3 months)
4 Chromatin Extraction and DNA Purification Jun, 2019-Aug 2019 (3 months
5 Libraries preparations and Sequencing Sep, 2019-Dec, 2019 (4 months)
6 Bioinformatics Analyses and DNA Methylation Jan, 2020-Apr, 2020 (4 months)
7 Methylated Enrichment and Western Blotting May, 2020- Oct, 2020 (6 Months)
8 Results Interpretation and Manuscript Nov, 2020- Apr, 2021 (6 months)
preparation
2/9/2022 15
Estimated Budget
No. Items Estimated
Costs
1 Mouse Embryonic stem Cell 20,000 RMB
2 Reagents 100,000 RMB
3 Instrumentation 200,000 RMB
4 High-throughput Sequencing 100,000 RMB
5 Computational Machine 50,000 RMB
6 Publication Costs 15,000 RMB
7 Miscellaneous 50,000 RMB
Estimated Total Costs: 530,000 RMB
2/9/2022 16
References
• Manzo, M., Wirz, J., Ambrosi, C., Villaseñor, R., Roschitzki, B., & Baubec, T. (2017). Isoform‐specific localization of
DNMT3A regulates DNA methylation fidelity at bivalent CpG islands. The EMBO journal, e201797038.
• Bayraktar, G., & Kreutz, M. R. (2017). Neuronal DNA Methyltransferases: Epigenetic Mediators between Synaptic
Activity and Gene Expression?. The Neuroscientist, 1073858417707457.
• Yokoyama, A. S., Rutledge, J. C., & Medici, V. (2017). DNA methylation alterations in Alzheimer’s disease.
Environmental Epigenetics, 3(2), dvx008.
• Saini, S. K., Mangalhara, K. C., Prakasam, G., & Bamezai, R. N. K. (2017). DNA Methyltransferase1 (DNMT1)
Isoform3 methylates mitochondrial genome and modulates its biology. Scientific reports, 7(1), 1525.
• Baubec, T., Colombo, D. F., Wirbelauer, C., Schmidt, J., Burger, L., Krebs, A. R., ... & Schübeler, D. (2015). Genomic
profiling of DNA methyltransferases reveals a role for DNMT3B in genic methylation. Nature, 520(7546), 243.
• Liao, J., Karnik, R., Gu, H., Ziller, M. J., Clement, K., Tsankov, A. M., ... & Pop, R. (2015). Targeted disruption of
DNMT1, DNMT3A and DNMT3B in human embryonic stem cells. Nature genetics, 47(5), 469.
• Duymich, C. E., Charlet, J., Yang, X., Jones, P. A., & Liang, G. (2016). DNMT3B isoforms without catalytic activity
stimulate gene body methylation as accessory proteins in somatic cells. Nature communications, 7, 11453.
• Irier, H. A., & Jin, P. (2012). Dynamics of DNA methylation in aging and Alzheimer's disease. DNA and cell
biology, 31(S1), S-42.
2/9/2022 17
Thank You!
2/9/2022 18

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Dynamics of DNMT3B

Methyltransferase Expression and

Item 2 Problem Statement

Item 3 Aims and Objectives

Item 5 Possible Outcomes

Item 6 Estimated Plan and Budget

• Dementia, an age-related neurodegenerative disorder characterized by

• Many report have reported that epigenetics play a

• DNA methyltransferase (DNMT) enzymes, of which DNMT1, DNMT3A and

• While the genome-wide distribution of this Epigenetics has been

• To identify the DNMT3B variations in genomic binding and DNA methylation

• To explore the role of DNMT3B in DNA methylation patterns in mouse ES and

• To analyze the expression of DNMT3B

• To reveal the significance of DNMT3B in sustaining methylcytosine at bivalent

Nuclear protein extraction Co-immunoprecipitation

• DNMT3B will preferentially localized to the methylated shores of bivalent CpG

• Through experiments, we will be able to demonstrate the specific recruitment of

Phases Activity Time Frame /(Duration)

2 Reagents 100,000 RMB

3 Instrumentation 200,000 RMB

4 High-throughput Sequencing 100,000 RMB

5 Computational Machine 50,000 RMB

6 Publication Costs 15,000 RMB

7 Miscellaneous 50,000 RMB

Estimated Total Costs: 530,000 RMB

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