Mapping Transcription Network

In the early 1960s, bacterial genetics laid the concept of regulatory circuits controlling expression of genes. In its simplest form, circuits are turned "on" or "off' by the binding of transcription factors or repressors to the upstream regulatory sequences of genes respectively. Expression analysis by microarrays described in the earlier section revealed the co-regulated expression and repression of sometimes hundreds of genes during specific cellular

Figure 6.14 A schematic representation of a microarray experiment. Labeled cDNA prepared from untreated and treated cells was mixed and hybridized on a microarray slide spotted with 6200 yeast open reading frames (DNA sequences with initiation and termination codons). The red spots represent higher abundance of cyanin5-labeled genes and green spots represent higher abundance of cyanin3-labeled genes. Yellow spots are genes present in equal abundance (blending of red and green color in equal proportion). (See color insert following page 140.)

events. Richard A. Young used the microarray technique to identify regulatory sequences bound by all transcription factors encoded by the yeast genome (Lee et al., 2002). Based on the expectation that the promoter elements are located within the non-coding regions upstream of every gene, the group amplified the non-coding sequences between genes (intergenic regions) and spotted them on slides to create microarrays of all promoter regions of yeast. Next, they identified 141 genes in the yeast genome that are predicted to code for transcription factors and tagged them with an epitope (myc-tag) at the c-terminus. Each epitope-tagged transcription factor was inserted into its native genomic locus by homologous recombination and analyzed for the level of expression by Western blot using an antibody specific to the myc-tag. To identify the DNA-binding sequences of the transcription

106 strain«, each ChranaQn IP In enrich Mtctwrcy to Idenlfy Wtmateaged promoter» bound prtcnotors bourtd regulator try regulator In vfw try regulator In vtvo

Figure 6.15 Genome-wide location analyses of yeast transcription regulators. Tagged transcription regulators are expressed in yeast. Bound regulators are crosslinked to DNA and DNA-protein complex is digested with DNase to obtain fragments of DNA complexed with regulators. Bound regulators are immunoprecipitated using antibodies specific to the tag. The DNA associated with the regulator protein is isolated and hybridized to microar-rays to identify the sequence. The binding of the transcription regulator is mapped to the sequence. (Lee et al. (2002), Transcriptional regulatory networks in S. cerevisiae, Science 298, Figure 1, AAAS. With permission.) (See color insert following page 140.)

106 strain«, each ChranaQn IP In enrich Mtctwrcy to Idenlfy Wtmateaged promoter» bound prtcnotors bourtd regulator try regulator In vfw try regulator In vtvo

Figure 6.15 Genome-wide location analyses of yeast transcription regulators. Tagged transcription regulators are expressed in yeast. Bound regulators are crosslinked to DNA and DNA-protein complex is digested with DNase to obtain fragments of DNA complexed with regulators. Bound regulators are immunoprecipitated using antibodies specific to the tag. The DNA associated with the regulator protein is isolated and hybridized to microar-rays to identify the sequence. The binding of the transcription regulator is mapped to the sequence. (Lee et al. (2002), Transcriptional regulatory networks in S. cerevisiae, Science 298, Figure 1, AAAS. With permission.) (See color insert following page 140.)

factors, a genome-wide location analysis called ChIP (crosslinking chromatin immunopre-cipitation) was performed (Ren et al., 2000). The strategy is shown in Figure 6.15. Each yeast strain expressing a unique myc-tagged transcription factor was grown in rich medium and the DNA cross-linked to bound proteins in vivo. The protein-DNA complex was digested with the enzyme DNAse to cleave DNA into smaller fragments. The transcription factor-bound DNA fragments were enriched by immunoprecipitation using an antibody against the tag. After isolation of bound DNA from the protein-DNA complex, it was labeled and hybridized to a microarray slide spotted with the intergenic DNA. A positive hybridization signal identified specific intergenic regions to which the transcription factor was bound. Sequence comparison among the intergenic regions that lighted up with each transcription factor identified conserved motifs recognized by the transcription factor. A genome-wide search for the presence of motifs in upstream sequences of genes resulted in the discovery of genes that are co-regulated under the control of the transcription factor. Using this approach it was revealed that about 200 genes are regulated by SCB binding factor (SBF) and MluI cell cycle box (MCB) binding factor (MBF) transcription factors during G1-S phase transition of the cell cycle (Iyer et al., 2001; Simon et al., 2001).

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