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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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Highly Bound Regions Are Associated with Genes Transcribed and Patterned in the BlastodermAnalyzed are the percentage of primary peaks that are within 10 kb of the 5' end of a gene. Genes are divided into three categories, all genes (from genome release 4.3, March 2006), genes with known patterned expression (hand annotated based on Berkeley Drosophila Genome Project [BDGP] in situ images [35]) and transcribed genes (defined by our RNA Polymerase II ChIP/chip binding, see Materials and Methods). Percentages are calculated in nonoverlapping windows of 100 peaks down the rank list to the 80% FDR threshold. The position of the 1% and 25% FDR cutoffs are indicated with vertical dotted lines. (A) shows the results for the BCD1 antisera and (B) the results for the KR1 antisera.
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pbio-0060027-g007: Highly Bound Regions Are Associated with Genes Transcribed and Patterned in the BlastodermAnalyzed are the percentage of primary peaks that are within 10 kb of the 5' end of a gene. Genes are divided into three categories, all genes (from genome release 4.3, March 2006), genes with known patterned expression (hand annotated based on Berkeley Drosophila Genome Project [BDGP] in situ images [35]) and transcribed genes (defined by our RNA Polymerase II ChIP/chip binding, see Materials and Methods). Percentages are calculated in nonoverlapping windows of 100 peaks down the rank list to the 80% FDR threshold. The position of the 1% and 25% FDR cutoffs are indicated with vertical dotted lines. (A) shows the results for the BCD1 antisera and (B) the results for the KR1 antisera.

Mentions: The most highly bound regions for each factor were preferentially found near genes that are transcribed in the blastoderm (Figures 7 and S5). For example, 49% of the 100 regions most highly bound by KR are within 10 kb of a transcribed gene, but at the 1% FDR threshold, a point at which there are insufficient false positives to significantly affect the analysis, only 22% of regions are within that distance of a transcribed gene (Figure 7B). There is also a strongly nonrandom association of highly bound regions with genes that show patterned expression in the blastoderm (Figures 7A, 7B, and S5) [35].


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)

Highly Bound Regions Are Associated with Genes Transcribed and Patterned in the BlastodermAnalyzed are the percentage of primary peaks that are within 10 kb of the 5' end of a gene. Genes are divided into three categories, all genes (from genome release 4.3, March 2006), genes with known patterned expression (hand annotated based on Berkeley Drosophila Genome Project [BDGP] in situ images [35]) and transcribed genes (defined by our RNA Polymerase II ChIP/chip binding, see Materials and Methods). Percentages are calculated in nonoverlapping windows of 100 peaks down the rank list to the 80% FDR threshold. The position of the 1% and 25% FDR cutoffs are indicated with vertical dotted lines. (A) shows the results for the BCD1 antisera and (B) the results for the KR1 antisera.
© Copyright Policy
Related In: Results  -  Collection

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

pbio-0060027-g007: Highly Bound Regions Are Associated with Genes Transcribed and Patterned in the BlastodermAnalyzed are the percentage of primary peaks that are within 10 kb of the 5' end of a gene. Genes are divided into three categories, all genes (from genome release 4.3, March 2006), genes with known patterned expression (hand annotated based on Berkeley Drosophila Genome Project [BDGP] in situ images [35]) and transcribed genes (defined by our RNA Polymerase II ChIP/chip binding, see Materials and Methods). Percentages are calculated in nonoverlapping windows of 100 peaks down the rank list to the 80% FDR threshold. The position of the 1% and 25% FDR cutoffs are indicated with vertical dotted lines. (A) shows the results for the BCD1 antisera and (B) the results for the KR1 antisera.
Mentions: The most highly bound regions for each factor were preferentially found near genes that are transcribed in the blastoderm (Figures 7 and S5). For example, 49% of the 100 regions most highly bound by KR are within 10 kb of a transcribed gene, but at the 1% FDR threshold, a point at which there are insufficient false positives to significantly affect the analysis, only 22% of regions are within that distance of a transcribed gene (Figure 7B). There is also a strongly nonrandom association of highly bound regions with genes that show patterned expression in the blastoderm (Figures 7A, 7B, and S5) [35].

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