Genomewide analysis of Drosophila GAGA factor target genes reveals context-dependent DNA binding - PubMed (original) (raw)
Genomewide analysis of Drosophila GAGA factor target genes reveals context-dependent DNA binding
Bas van Steensel et al. Proc Natl Acad Sci U S A. 2003.
Abstract
The association of sequence-specific DNA-binding factors with their cognate target sequences in vivo depends on the local molecular context, yet this context is poorly understood. To address this issue, we have performed genomewide mapping of in vivo target genes of Drosophila GAGA factor (GAF). The resulting list of approximately 250 target genes indicates that GAF regulates many cellular pathways. We applied unbiased motif-based regression analysis to identify the sequence context that determines GAF binding. Our results confirm that GAF selectively associates with (GA)(n) repeat elements in vivo. GAF binding occurs in upstream regulatory regions, but less in downstream regions. Surprisingly, GAF binds abundantly to introns but is virtually absent from exons, even though the density of (GA)(n) is roughly the same. Intron binding occurs equally frequently in last introns compared with first introns, suggesting that GAF may not only regulate transcription initiation, but possibly also elongation. We provide evidence for cooperative binding of GAF to closely spaced (GA)(n) elements and explain the lack of GAF binding to exons by the absence of such closely spaced GA repeats. Our approach for revealing determinants of context-dependent DNA binding will be applicable to many other transcription factors.
Figures
Figure 1
Distribution of GAF-Dam/Dam methylation ratios (log2 values; average of three independent experiments). To illustrate the skewed distribution toward positive values, a Gaussian distribution (drawn curve) was fitted to values below the mode of the histogram (gray bars). Black bars show counts of statistically significant GAF target loci (see Materials and Methods).
Figure 2
(A) Sequences used for
reduce
analysis. For a given gene (filled bars), a corresponding cDNA probe (open bars) detects only methylation levels of matching exon sequences. Because targeted methylation spreads in cis, we included introns and a variable amount of 5′ and 3′ flanking sequences (dashed lines) in the
reduce
analysis (gray bar labeled “Probed locus”). In Table 2 and Fig. 3 the amount of flanking sequence was set to 2 kb. (B and C) Determination of optimal length of flanking sequences. Density of GAGAG motifs (B) and t value corresponding to the Pearson correlation between GAF binding and the number of occurrences of the GAGAG motif (C) for the probed loci plus 0, 1, 2, 4, or 8 kb of flanking sequence on each side.
Figure 3
Binding of GAF to GAGAG elements in subregions of target genes. (Left) t value calculated as in Fig. 2_C_; for each bar, the statistical significance is indicated by value of −log10(P). (Right) GAGAG density. GAGAG occurrences were counted only in specific subregions of the probed loci as indicated in Fig. 2_A_.
Figure 4
Spacing distribution of GAGAG elements in 500 loci with strongest GAF binding (A, average methylation log ratio = 0.96) and 500 loci with weakest GAF binding (B, average methylation log ratio = −0.44), for intergenic regions (filled bars), introns (shaded bars), and exons (open bars). Lines indicate the spacing distribution after randomization of the positions of all GAGAG elements in each locus (average of >1,000 random simulations is shown).
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