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ChIP to Chip The
Special Case of DNA-Binding Proteins
Figure 1. To investigate the physiological effects of the neuropeptide hormone orexin in brain cells, we will compare ChIP to chip results using antibodies raised against a variety of transcription factors in samples that either have or have not been treated with orexin. The goal is to identify transcription factors that play a key role in orexin signaling. These data will also be supplemented with traditional expression microarray data. The long-term goal is to understand in detail how orexin stimulates wakefulness.
Sug1 is found throughout the activated GAL1 gene
Figure 2. An example of a ChIP assay done in yeast using an antibodies raised against the gene-specific transcription factor Gal4 and the general transcription factor and proteasome constituent Sug1. Different PCR primers (amplifying DNA segments A-E) were employed to probe for the presence of the two proteins along the GAL1 gene under non-inducing (raffinose) or inducing (galactose) conditions. Gal4 is known to be localized to the promoter. Therefore, the ChIP signals in regions B and C for this protein reflect the presence of longer DNAs in the sample which are co-IP’d with Gal4 and amplify using the B and C region primers. This reflects the relatively low resolution of the ChIP assay. Sug1 is an elongation factor and is therefore found throughput the gene. The "ChIP to Chip" Assay
Figure 4. In the so-called ChIP to chip assay, all of the DNA fragments co-immunoprecipitated with the protein of interest are amplified. This is done by ligating common linkers to the ends of the (repaired) DNA fragments. The amplified material is then annealed to a DNA microarray containing the appropriate probes. While the figure shows a one color experiment, this is often done with two colors, the other being the total DNA as a control. Any DNA enriched in the immunoprecipitation above the level of the total is scored as a site of protein binding. The Orexin Neuropeptide Hormone
Figure 5. To investigate the physiological effects of the neuropeptide hormone orexin in brain cells, we will compare ChIP to chip results using antibodies raised against a variety of transcription factors in samples that either have or have not been treated with orexin. The goal is to identify transcription factors that play a key role in orexin signaling. These data will also be supplemented with traditional expression microarray data. The long-term goal is to understand in detail how orexin stimulates wakefulness.
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