Abstract
In many cases, the DNA-binding activity of a transcription factor does not change, while its transcriptional activity is greatly influenced by the make-up of bound proteins. In this study, we assessed the protein composition and DNA-binding ability of the E2F transcription factor complex to provide insight into cell cycle control in an anoxia tolerant turtle through the use of a modified ELISA protocol. This modification also permits the use of custom DNA probes that are tailored to a specific DNA binding region, introducing the ability to design capture probes for non-model organisms. Through the use of EMSA and ELISA DNA binding assays, we have successfully determined the in vitro DNA binding activity and complex dynamics of the Rb/E2F cell cycle regulatory mechanisms in an anoxic turtle, Trachemys scripta elegans. Repressive cell cycle proteins (E2F4, Rb, HDAC4 and Suv39H1) were found to significantly increase at E2F DNA-binding sites upon anoxic exposure in anoxic turtle liver. The lack of p130 involvement in the E2F DNA-bound complex indicates that anoxic turtle liver may maintain G1 arrest for the duration of stress survival.
Highlights
Heterochromatin is composed of genomic DNA tightly packed by histones and nonhistone proteins (Lu & Gilbert, 2007; Ferreira et al, 2001)
We developed a method to identify protein binding partners in transcription factors that are actively binding to DNA in vitro
In order to determine whether E2F could bind the DNA probes utilized in the enzyme-linked immunosorbent assay (ELISA) experiments, the relative qualitative level of E2F binding to DNA was visualized in extracts from control and anoxic liver of T. scripta elegans
Summary
Heterochromatin is composed of genomic DNA tightly packed by histones and nonhistone proteins (Lu & Gilbert, 2007; Ferreira et al, 2001). Dynamic changes in chromatin structure prevent the access of transcription factors, such as E2F, to nucleosomal DNA. At least two primary mechanisms can be used by the cell to remodel chromatin structure. The second mechanism involves covalent modifications of histone N-terminal tails that protrude from the chromatin structure (Ferreira et al, 2001; Trojer et al, 2007; Wu, Connelly & Biggar, 2017). The particular associations between Rb and chromatin remodeling factors have been found to be dependent on the type of cell cycle exit (Lu & Gilbert, 2007)
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