Epigenetic modifications, such as DNA methylation and histone modifications, play important roles in gene expression and regulation, and are highly involved in cellular processes such as stem cell pluripotency/differentiation and tumorigenesis. Chromatin immunoprecipitation (ChIP) is the technique of choice for examining in vivo DNA-protein interactions and has been a great tool for studying epigenetic mechanisms. However, conventional ChIP assays require millions of cells for tests and are not practical for examination of samples from lab animals and patients. Automated microfluidic chips offer the advantage to handle small sample sizes and facilitate rapid reaction. They also eliminate cumbersome manual handling.
In this report, I will talk about three different projects that utilized microfluidic immunoprecipitation followed by next genereation sequencing technologies to enable low input and high through epigenomics profiling. First, I examined RNA polymerase II transcriptional regulation with microfluidic chromatin immunoprecipitation followed by next generation sequencing (ChIP-seq) assays. Second, I probed the temporal dynamics in the DNA methylome during cancer development using a transgenic mouse model with microfluidic methylated DNA immunoprecipitation followed by next generation sequencing (MeDIP-seq) assays. Third, I explored negative enrichment of circulating tumor cells (CTCs) followed by microfluidic ChIP-seq technology for studying temporal dynamic histone modification (H3K4me3) of patient-derived tumor xenograft on an immunodeficient mouse model during the course of cancer metastasis.
In the first study, I adapted microfluidic ChIP-seq devices to achieve ultrahigh sensitivity to study Pol2 transcriptional regulation from scarce cell samples. I dramatically increased the assay sensitivity to an unprecedented level (~50 K cells for pol2 ChIP-seq). Importantly, this is three orders of magnitude more sensitive than the prevailing pol2 ChIP-seq assays. I showed that MNase digestion provided better ChIP-seq signal than sonication, and two-steps fixation with MNase digestion provided the best ChIP-seq quality followed by one-step fixation with MNase digestion, and lastly, no fixation with MNase digestion.
In the second study, I probed dynamic epigenomic changes during tumorigenesis using mice often require profiling epigenomes using a tiny quantity of tissue samples. Conventional epigenomic tests do not support such analysis due to the large amount of materials required by these assays. In this study, I developed an ultrasensitive microfluidics-based methylated DNA immunoprecipitation followed by next-generation sequencing (MeDIP-seq) technology for profiling methylomes using as little as 0.5 ng DNA (or ~100 cells) with 1.5 h on-chip process for immunoprecipitation. This technology enabled me to examine genome-wide DNA methylation in a C3(1)/SV40 T-antigen transgenic mouse model during different stages of mammary cancer development. Using this data, I identified differentially methylated regions and their associated genes in different periods of cancer development. Interestingly, the results showed that methylomic features are dynamic and change with tumor developmental stage.
In the last study, I developed a negative enrichment of CTCs followed by ultrasensitive microfluidic ChIP-seq technology for profiling histone modification (H3K4Me3) of CTCs to resolve the technical challenges associated with CTC isolation and difficulties related with tools for profiling whole genome histone modification on tiny cell samples. / Ph. D. / The human genome has been sequenced and completed over a decade ago. The information provided by the genomic map inspired numerous studies on genetic variations and their roles in diseases. However, genomic information alone is not always sufficient to explain important biological processes. Gene activation and expression are not only associated with alteration in the DNA sequence, but also affected by other changes to DNA and histones. Epigenetics refers to the molecular mechanisms that affect gene expression and phenotypes without involving changes in the DNA sequence.
For example, the DNA can get methylated, the histone protein that is wrapped around by DNA can also get methylated or acetylatied, and transcription factors can bind to different part of DNA. All of these can affect gene expression without alter the DNA sequences. Epigenetic changes occur throughout all stages of cell development or in response to environmental cues. They change transcription patterns in a tissue/cell-specific fashion. For example, transcriptional silencing of tumor-suppressor genes by DNA methylation plays an important role in cancer development. Therefore, understanding of epigenetic regulations will help to improve various aspects of biomedicine. For instance, personalized medicine can be vi tailored based on epigenetic profile of certain patient to specifically control gene expression in the disease treatment. However, the technology for profiling epigenetic modifications, i.e. Chromatin Immunoprecipitation (ChIP), suffers from serious limitations. The key limitation is the sensitivity of the assay. Conventional assay requires a large number of cells (>10⁶ cells per ChIP). This is feasible when using cell lines. However, such requirement has become a major challenge when primary cells are used because very limited amounts of samples can be generated from lab animals or patients. Population heterogeneity information may also be lost when a large cell number is used.
In this project, we developed an automated ultrasensitive microfluidic chromatin/DNA immunoprecipitation followed by next-generation sequencing (ChIP/MeDIP-Seq) technology for profiling epigenetic modifications (e.g., histone modifications, transcriptional regulations, and DNA methylation). We extensively optimized design parameters for each and every step of ChIP/MeDIP (e.g. sonication/crosslinking time, antibody concentration, washing conditions) in order to reach highest sensitivity of 0.1 ng DNA (or ~50-100 cells) as starting material for IP, which is roughly 4-5 orders of magnitude higher than the prevailing protocol and 2-3 orders of magnitude higher than the-state-of-the-art(~50 ng). With such sensitivity, we were able to study temporal dynamics in the DNA methylomes during the various stages of mammary cancer development from a transgenic mouse mode. We were able to investigate transcriptional regulation of RNA polymerase II from scarce cell samples. We were also able to study histone modification (H3K4Me3) of circulating tumor cells during cancer metastasis.
| Identifer | oai:union.ndltd.org:VTETD/oai:vtechworks.lib.vt.edu:10919/83402 |
| Date | 25 May 2018 |
| Creators | Zhu, Yan |
| Contributors | Chemical Engineering, Lu, Chang-Tien, Ducker, William A., Li, Liwu, Goldstein, Aaron S. |
| Publisher | Virginia Tech |
| Source Sets | Virginia Tech Theses and Dissertation |
| Detected Language | English |
| Type | Dissertation |
| Format | ETD, application/pdf |
| Rights | In Copyright, http://rightsstatements.org/vocab/InC/1.0/ |
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