Figure 8–65 Using cluster analysis to identify sets of genes that are coordinately regulated. Genes that have the same expression pattern are likely to be involved in common pathways or processes. To perform a cluster analysis, RNA-seq or microarray data are obtained from cell samples exposed to a variety of different conditions, and genes that show coordinate changes in their expression pattern are grouped together. In this experiment, human fibroblasts were deprived of serum for 48 hours; serum was then added back to the cultures at time 0 and the cells were harvested for microarray analysis at different time points. Of the 8600 genes depicted here (each represented by a thin, vertical line), just over 300 showed threefold or greater variation in their expression patterns in response to serum reintroduction. Here, red indicates an increase in expression; green is a decrease in expression. On the basis of the results of many other experiments, the 8600 genes have been grouped in clusters based on similar patterns of expression. The results of this analysis show that genes involved in wound healing are turned on in response to serum, while genes involved in regulating cell-cycle progression and cholesterol biosynthesis are shut down. (From M.B. Eisen et al., Proc. Natl Acad. Sci. USA 94:14863–14868, 1998. With permission from National Academy of Sciences.)
Figure 8–66 Chromatin immunoprecipitation. This method allows the identification of all the sites in a genome that a transcription regulator occupies in vivo. The identities of the precipitated, amplified DNA fragments are determined by DNA sequencing.
Genome-wide Chromatin Immunoprecipitation Identifies Sites on the Genome Occupied by Transcription Regulators
We have discussed several strategies to measure the levels of individual RNAs in a cell and to monitor changes in their levels in response to external signals. But this information does not tell us how such changes are brought about. We saw in Chapter 7 that transcription regulators, by binding to cis-regulatory sequences in DNA, are responsible for establishing and changing patterns of transcription. Typically, these proteins do not occupy all of their potential cis-regulatory sequences in the genome under all conditions. For example, in some cell types, the regulatory protein may not be expressed, or it may be present but lack an obligatory partner protein, or it may be excluded from the nucleus until an appropriate signal is received from the cell’s environment. Even if the protein is present in the nucleus and is competent to bind DNA, other transcription regulators or components of chromatin can occupy overlapping DNA sequences and thereby occlude some of its cis-regulatory sequences in the genome.
Chromatin immunoprecipitation provides a way to experimentally determine all the cis-regulatory sequences in a genome that are occupied by a given transcription regulator under a particular set of conditions (Figure 8–66). In this approach, proteins are covalently cross-linked to DNA in living cells, the cells are broken open, and the DNA is mechanically sheared into small fragments. Antibodies directed against a given transcription regulator are then used to purify the DNA that became covalently cross-linked to that protein in the cell. This DNA is then sequenced using the rapid methods discussed earlier; the precise location of each precipitated DNA fragment along the genome is determined by comparing its DNA sequence to that of the whole genome sequence (Figure 8–67). In this way, all of the sites occupied by the transcription regulator in the cell sample can be mapped across the cell’s genome (see Figure 7–37). In combination with microarray or RNA-seq information, chromatin immunoprecipitation can identify the key transcriptional regulator responsible for specifying a particular pattern of gene expression.