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Transcription factors bind thousands of active and inactive regions in the Drosophila blastoderm.

Li XY, MacArthur S, Bourgon R, Nix D, Pollard DA, Iyer VN, Hechmer A, Simirenko L, Stapleton M, Luengo Hendriks CL, Chu HC, Ogawa N, Inwood W, Sementchenko V, Beaton A, Weiszmann R, Celniker SE, Knowles DW, Gingeras T, Speed TP, Eisen MB, Biggin MD - PLoS Biol. (2008)

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Genomics Division, Lawrence Berkeley National Laboratory, Berkeley, California, United States of America.

ABSTRACT
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.

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Patterns of mRNA Expression of the Six Maternal and Gap Genes Controlling Trunk Segmentation along the Anterior–Posterior AxisExpression (protein for BCD and mRNA for the other factors) is shown in orthographic projections using PointCloudXplore [100] to display data from the BDTNP's VirtualEmbryo [11] (BDTNP, unpublished data). The embryos are shown with anterior to the left, posterior right, dorsal at the top, and ventral at the bottom.
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pbio-0060027-g001: Patterns of mRNA Expression of the Six Maternal and Gap Genes Controlling Trunk Segmentation along the Anterior–Posterior AxisExpression (protein for BCD and mRNA for the other factors) is shown in orthographic projections using PointCloudXplore [100] to display data from the BDTNP's VirtualEmbryo [11] (BDTNP, unpublished data). The embryos are shown with anterior to the left, posterior right, dorsal at the top, and ventral at the bottom.

Mentions: In this system, A-P patterning is initially established by maternally controlled activity gradients of two transcription factors: Bicoid (BCD), which has its highest activity in the anterior portion of the embryo and decays more posteriorly, and Caudal (CAD), which has its highest activity in the posterior portion of the embryo and decays anteriorly (Figure 1). Amongst the earliest zygotically transcribed genes are four targets of BCD and CAD—hunchback (hb), Krüppel (Kr), knirps (kni), and giant (gt)—the “gap” genes (Figure 1). These six genes encode transcription factors that work together to segment the A-P axis of the embryonic trunk (a collection of additional regulatory factors are involved in patterning the head and tail) [14–16]. For example, the second stripe of the pair-rule gene even-skipped (eve) is produced by the action of BCD, HB, KR, and GT on a CRM located approximately 1.5 kb upstream of the coding gene. In this case, BCD and HB act coordinately as activators in the same cells, whereas KR and GT each acts to repress expression in different parts of the embryo, restricting CRM output to a narrow stripe lying between the single band of KR expression and the anterior expression domain of GT [17,18]. This same collection of factors when bound to other CRMs produces different patterns of gene expression. The different combinations of recognition sequences in each CRM dictate the binding of factors in CRM-specific numbers and orientations. This binding, in turn, modulates the activity of each factor (in some cases changing activators into repressors, in others leading to binding-site competition or cooperative interactions) and produces a distinct transcriptional response [19–22]. Thus it is essential to study and model the action of these proteins in their native context.


Transcription factors bind thousands of active and inactive regions in the Drosophila blastoderm.

Li XY, MacArthur S, Bourgon R, Nix D, Pollard DA, Iyer VN, Hechmer A, Simirenko L, Stapleton M, Luengo Hendriks CL, Chu HC, Ogawa N, Inwood W, Sementchenko V, Beaton A, Weiszmann R, Celniker SE, Knowles DW, Gingeras T, Speed TP, Eisen MB, Biggin MD - PLoS Biol. (2008)

Patterns of mRNA Expression of the Six Maternal and Gap Genes Controlling Trunk Segmentation along the Anterior–Posterior AxisExpression (protein for BCD and mRNA for the other factors) is shown in orthographic projections using PointCloudXplore [100] to display data from the BDTNP's VirtualEmbryo [11] (BDTNP, unpublished data). The embryos are shown with anterior to the left, posterior right, dorsal at the top, and ventral at the bottom.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2235902&req=5

pbio-0060027-g001: Patterns of mRNA Expression of the Six Maternal and Gap Genes Controlling Trunk Segmentation along the Anterior–Posterior AxisExpression (protein for BCD and mRNA for the other factors) is shown in orthographic projections using PointCloudXplore [100] to display data from the BDTNP's VirtualEmbryo [11] (BDTNP, unpublished data). The embryos are shown with anterior to the left, posterior right, dorsal at the top, and ventral at the bottom.
Mentions: In this system, A-P patterning is initially established by maternally controlled activity gradients of two transcription factors: Bicoid (BCD), which has its highest activity in the anterior portion of the embryo and decays more posteriorly, and Caudal (CAD), which has its highest activity in the posterior portion of the embryo and decays anteriorly (Figure 1). Amongst the earliest zygotically transcribed genes are four targets of BCD and CAD—hunchback (hb), Krüppel (Kr), knirps (kni), and giant (gt)—the “gap” genes (Figure 1). These six genes encode transcription factors that work together to segment the A-P axis of the embryonic trunk (a collection of additional regulatory factors are involved in patterning the head and tail) [14–16]. For example, the second stripe of the pair-rule gene even-skipped (eve) is produced by the action of BCD, HB, KR, and GT on a CRM located approximately 1.5 kb upstream of the coding gene. In this case, BCD and HB act coordinately as activators in the same cells, whereas KR and GT each acts to repress expression in different parts of the embryo, restricting CRM output to a narrow stripe lying between the single band of KR expression and the anterior expression domain of GT [17,18]. This same collection of factors when bound to other CRMs produces different patterns of gene expression. The different combinations of recognition sequences in each CRM dictate the binding of factors in CRM-specific numbers and orientations. This binding, in turn, modulates the activity of each factor (in some cases changing activators into repressors, in others leading to binding-site competition or cooperative interactions) and produces a distinct transcriptional response [19–22]. Thus it is essential to study and model the action of these proteins in their native context.

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Genomics Division, Lawrence Berkeley National Laboratory, Berkeley, California, United States of America.

ABSTRACT
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.

Show MeSH