Transcription Factors Bind Thousands of Active and Inactive Regions in the Drosophila Blastoderm

By Xiaoyong Li, Stewart MacArthur, Richard Bourgon, D. Nix, Daniel A. Pollard, Venky N. Iyer, A. Hechmer, L. Simirenko, M. Stapleton, C. L. Hendriks and 12 more. 2008

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Identifying the genomic regions bound by sequence-specific regulatory factors is central both to deciphering the complex DNA cis-regulatory code that controls transcription in metazoans and to determining the range of genes that shape animal morphogenesis. We used whole-genome tiling arrays to map sequences bound in Drosophila melanogaster embryos by the six maternal and gap transcription factors that initiate anterior–posterior patterning. We find that these sequence-specific DNA binding proteins bind with quantitatively different specificities to highly overlapping sets of several thousand genomic regions in blastoderm embryos. Specific high- and moderate-affinity in vitro recognition sequences for each factor are enriched in bound regions. This enrichment, however, is not sufficient to explain the pattern of binding in vivo and varies in a context-dependent manner, demonstrating that higher-order rules must govern targeting of transcription factors. The more highly bound regions include all of the over 40 well-characterized enhancers known to respond to these factors as well as several hundred putative new cis-regulatory modules clustered near developmental regulators and other genes with patterned expression at this stage of embryogenesis. The new targets include most of the microRNAs (miRNAs) transcribed in the blastoderm, as well as all major zygotically transcribed dorsal–ventral patterning genes, whose expression we show to be quantitatively modulated by anterior–posterior factors. In addition to these highly bound regions, there are several thousand regions that are reproducibly bound at lower levels. However, these poorly bound regions are, collectively, far more distant from genes transcribed in the blastoderm than highly bound regions; are preferentially found in protein-coding sequences; and are less conserved than highly bound regions. Together these observations suggest that many of these poorly bound regions are not involved in early-embryonic transcriptional regulation, and a significant proportion may be nonfunctional. Surprisingly, for five of the six factors, their recognition sites are not unambiguously more constrained evolutionarily than the immediate flanking DNA, even in more highly bound and presumably functional regions, indicating that comparative DNA sequence analysis is limited in its ability to identify functional transcription factor targets.

Subfields: Molecular Biology, Plant Science

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Citations in the corpus

Variation in homeodomain DNA binding revealed by high-resolution analysis of sequence preferences. (2008, methodology): “Chromatin Immunoprecipitation Analyses We obtained 1331 bound sequences in the Caudal data set by selecting those in the 1% false discovery rate set where a peak was also reported (Li et al., 2008).”
Systematic discovery and characterization of regulatory motifs in ENCODE TF binding experiments (2013, background): “Moreover, TF binding frequently occurs in the absence of any discernible motif instance (7,8) or to ‘hot-spots’ where several factors are simultaneously found (9).”
DNA-binding specificities of plant transcription factors and their potential to define target genes (2014, background): “ChIP-based techniques revealed that TFs may bind to thousands of genomic fragments, suggesting that the TF is interacting indirectly with DNA or that the binding requires additional cooperative factors (5, 6).”
Impacts of the ubiquitous factor Zelda on Bicoid-dependent DNA binding and transcription in Drosophila (2014, methodology): “Previous work suggested that Zld and Bcd each binds to thousands of sites during early development (Li et al. 2008; Harrison et al. 2011; Nien et al. 2011).”
Dynamic reprogramming of chromatin accessibility during Drosophila embryo development (2011, methodology): “To map the occupancy patterns of specific regulators, chromatin immunoprecipitation (ChIP) has been applied to over 20 developmental transcription factors and RNA polymerase in the blastoderm embryo and, for several factors, at later stages of embryogenesis [30-36].”
Temporal Coordination of Gene Networks by Zelda in the Early Drosophila Embryo (2011, background): “However, there was a significant difference (Fisher’s exact, p,0.0001) between the percentage of genes bound by Zld within 2 kb of their TSS for genes considered expressed (78.5% in blue) versus those considered not expressed (45.3% in red).”
Zelda is differentially required for chromatin accessibility, transcription factor binding, and gene expression in the early Drosophila embryo (2015, background): “…are bound by a large number of different transcription factors, have been identified in multiple organisms, including worms, flies, and humans (Moorman et al. 2006; The ENCODE Project Consortium 2007; Li et al. 2008; MacArthur et al. 2009; Gerstein et al. 2010; Roy et al. 2010; Negre et al. 2011).”
Establishment of regions of genomic activity during the Drosophila maternal to zygotic transition (2014, background): “, 2007; Combs and Eisen, 2013), a result of the differential binding by around 50 spatially patterned transcription factors to several thousands known and putative patterning transcriptional enhancers (Li et al., 2008; MacArthur et al., 2009).”
Incremental causal effects (2019, background): “More specificaly, we consider the activity of p = 36 transcription factors in Drosophila embryos on n = 7809 segments of the genome (Li et al., 2008; MacArthur et al., 2009).”
Non-coding RNA: what is functional and what is junk? (2015, unknown): “…that most TF binding sites which are occupied by TF proteins are not conserved between highly related species (Paris et al., 2013) and that many TF binding events have little to no impact on the expression of nearby genes (Li et al., 2008; Biggin, 2011; Lickwar et al., 2012; Paris et al., 2013).”
ChIP-Seq of transcription factors predicts absolute and differential gene expression in embryonic stem cells (2009, background): “This is consistent with the finding that many genomic loci are bound by a large number of different TFs (33, 34).”
DNA regions bound at low occupancy by transcription factors do not drive patterned reporter gene expression in Drosophila (2012, methodology): “We used two well-validated antibodies that each recognize nonoverlapping regions of the KR protein to generate two separate microarray based in vivo crosslinking (ChIP-chip) datasets from blastoderm (stage 5) embryos (9).”
Comprehensive Genome-wide Protein-DNA Interactions Detected at Single Nucleotide Resolution (2011, background): “Importantly, low-occupancy binding is widespread in genomes (Li et al., 2008), but its physiological importance and distinction from noise have been difficult to discern by any assay thus far.”