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  • About
  • The Global ETD Search service is a free service for researchers to find electronic theses and dissertations. This service is provided by the Networked Digital Library of Theses and Dissertations.
    Our metadata is collected from universities around the world. If you manage a university/consortium/country archive and want to be added, details can be found on the NDLTD website.
1

Developing DamID-seq to investigate transcription factor binding in mammalian cells

Tosti, Luca January 2017 (has links)
In order to understand gene regulatory networks (GRNs) in mammalian cells, it is pivotal to assess the interaction between proteins and DNA. In particular, the specific DNA binding activity of transcription factors (TFs) determines the expression of target genes and in general the overall connectivity of the GRN. However, the genomic location of TF binding cannot be predicted just from the DNA sequence, and functional assays are required to detect this interaction. The investigation of the binding of TF to DNA is usually accomplished by chromatin immunoprecipitation followed by next-generation sequencing (ChIP-seq). While in the last 10 years this method enabled a better understanding of how transcription is regulated in living cells, it does have some drawbacks. In particular, the need for very highly specific antibodies and the large amount of starting material limit the ability of ChIP-seq to address biological questions when dealing with samples of small quantity. A technique called DNA Adenine Methyltransferase Identification (DamID) was developed in Drosophila as an alternative method for the detection of protein- DNA interactions and it is based on the fusion of a protein of interest (POI) with the DNA adenine methyltransferase (Dam). This fusion causes DNA methylation of adenines surrounding the sites where POI binds and the subsequent identification of the methylation sites allows mapping of the binding event without antibodies and using less cells as starting material. While this technology was successful in detecting the interaction between nuclear lamina and DNA in mammalian cells, to date little reports are present in the literature about TF DamID. This is mainly due to the different nature of TF binding compared to Lamin (punctuated instead of broad) and to the elevated intrinsic activity of Dam that makes the detection of real signal above the noise challenging. I here demonstrate a step-by-step optimization of the DamID technology coupled to next-generation sequencing (DamID-seq) that I used to map the binding of the mouse embryonic stem cell master regulator Oct4 in as few as 1,000 cells. This new technology paves the way for exciting new experiments where the number of cells is scarce such as in vitro cell state change or in vivo processes.
2

Methods for Global Characterization of Chromatin Regulators in Human Cells

Zhou, Vicky 17 August 2012 (has links)
Chromatin is a multi-layered structure composed of DNA, nucleosomes, histone modifications, and associated proteins that critically affects genome function. Recently developed sequencing technologies enable genomewide characterization of certain aspects of chromatin structure, including nucleosome positioning and histone modifications. However, chromatin proteins present several challenges due to their dynamic nature and variable association with DNA. Chromatin proteins such as Polycomb regulators and heterochromatic factors play critical and global roles in epigenetic repression and hence new approaches are needed for their study. We first sought to identify sequences that recruit Polycomb repressive complex 2 (PRC2) in mammalian cells. We combined chromatin immunoprecipitation with sequencing (ChIP-seq) to map the candidate transcription factor YY1, and found that it does not correlate with PRC2 localization, suggesting that YY1 is not directly involved in PRC2 recruitment. We also identified GC-rich sequences that are necessary and sufficient for PRC2 recruitment. Yet attempts to map additional Polycomb proteins and other repressors using ChIP-seq proved difficult. Since chromatin proteins are often broadly, secondarily or transiently bound to DNA, they are difficult to crosslink. Antibody quality also varies, further hampering ChIP-seq technology. Here, we adapt DamID, a method for mapping chromatin regulators that uses a fusion enzyme and that does not rely on crosslinking or antibodies, for high-throughput sequencing. We show that DamID-seq can be used to globally characterize chromatin repressors in human cells. We used DamID-seq to map the binding of 12 chromodomain-containing and related proteins in K562 cells. We found that these proteins cluster into two modules: 1) Polycombrelated and 2) heterochromatin-related. Polycomb proteins bind developmental genes, while heterochromatin proteins bind broad olfactory receptor (OR) and zinc finger (ZNF) domains. Surprisingly, unlike other Polycomb proteins, CBX2 uniquely binds genes involved with modifying proteins. Our findings advance the model that the genome is compartmentalized into domains, and identify the distinct protein components that associate respectively with Polycomb and heterochromatin domains in human cells. We expect that DamID-seq, along with further advancements in characterizing the three-dimensional organization of chromatin, will bring us towards a better understanding of the role of chromatin in differentiation, development, and disease.

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