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The zinc-finger protein Zelda is a key activator of the early zygotic genome in Drosophila.

Liang HL, Nien CY, Liu HY, Metzstein MM, Kirov N, Rushlow C - Nature (2008)

Bottom Line: However, the transcription factor(s) that activate the zygotic genome remain elusive.Mutant embryos lacking zld are defective in cellular blastoderm formation, and fail to activate many genes essential for cellularization, sex determination and pattern formation.Global expression profiling confirmed that Zld has an important role in the activation of the early zygotic genome and suggests that Zld may also regulate maternal RNA degradation during the maternal-to-zygotic transition.

View Article: PubMed Central - PubMed

Affiliation: Department of Biology, New York University, 100 Washington Square East, New York, New York 10003, USA.

ABSTRACT
In all animals, the initial events of embryogenesis are controlled by maternal gene products that are deposited into the developing oocyte. At some point after fertilization, control of embryogenesis is transferred to the zygotic genome in a process called the maternal-to-zygotic transition. During this time, many maternal RNAs are degraded and transcription of zygotic RNAs ensues. There is a long-standing question as to which factors regulate these events. The recent findings that microRNAs and Smaug mediate maternal transcript degradation have shed new light on this aspect of the problem. However, the transcription factor(s) that activate the zygotic genome remain elusive. The discovery that many of the early transcribed genes in Drosophila share a cis-regulatory heptamer motif, CAGGTAG and related sequences, collectively referred to as TAGteam sites raised the possibility that a dedicated transcription factor could interact with these sites to activate transcription. Here we report that the zinc-finger protein Zelda (Zld; Zinc-finger early Drosophila activator) binds specifically to these sites and is capable of activating transcription in transient transfection assays. Mutant embryos lacking zld are defective in cellular blastoderm formation, and fail to activate many genes essential for cellularization, sex determination and pattern formation. Global expression profiling confirmed that Zld has an important role in the activation of the early zygotic genome and suggests that Zld may also regulate maternal RNA degradation during the maternal-to-zygotic transition.

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TAGteam sites bind Zld and mediate transcriptional activation(a) DNA sequence of the 91 bp zen enhancer (uppercase) plus surrounding sequences (lowercase). Base substitutions are in purple. (b) Schematic organization of the zld locus (CG12701; Flybase) with the two predicted transcription start sites, RB and RA. The P{RS3}UM8171-3 insertion site is between -661 and -660. The nucleotides deleted in zld294 and zld681 are indicated as blank space between solid lines. (c) Zld binding to oligonucleotides containing different TAGteam sites (denoted beneath each section of the gel). The first lane in each section contains free probe. The second lane contains probe plus 10ng GST-ZldC, the third 30 ng GST-ZldC. (d) S2 cells were transfected with 0 ng (blue bar), 50 ng (red bar), or 100 ng (yellow bar) of plasmid expressing zld under control of the inducible metallothionein promoter, the zen91-lacZ or zen91m-lacZ reporter plasmids, and the luciferase control. Error bars, s.e.m.; n=3.
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Figure 1: TAGteam sites bind Zld and mediate transcriptional activation(a) DNA sequence of the 91 bp zen enhancer (uppercase) plus surrounding sequences (lowercase). Base substitutions are in purple. (b) Schematic organization of the zld locus (CG12701; Flybase) with the two predicted transcription start sites, RB and RA. The P{RS3}UM8171-3 insertion site is between -661 and -660. The nucleotides deleted in zld294 and zld681 are indicated as blank space between solid lines. (c) Zld binding to oligonucleotides containing different TAGteam sites (denoted beneath each section of the gel). The first lane in each section contains free probe. The second lane contains probe plus 10ng GST-ZldC, the third 30 ng GST-ZldC. (d) S2 cells were transfected with 0 ng (blue bar), 50 ng (red bar), or 100 ng (yellow bar) of plasmid expressing zld under control of the inducible metallothionein promoter, the zen91-lacZ or zen91m-lacZ reporter plasmids, and the luciferase control. Error bars, s.e.m.; n=3.

Mentions: In Drosophila, an initial wave of zygotic gene transcription occurs between 1-2 hours of development during mitotic cleavage cycles 8-13. This is followed by a major burst of activity between 2-3 hours (cycle 14) when the embryo is undergoing cellular blastoderm formation. Many pre-cellular genes contain TAGteam sites in their upstream regulatory regions including several direct targets of Bicoid, Dorsal, and other key regulators of patterning5-7. ten Bosch et. al.5 demonstrated that TAGteam sites are required for the early expression of the dorsoventral (DV) gene zen, and the sex determination genes sisB and Sxl. To isolate the TAGteam binding factor, we performed a yeast one-hybrid screen with a 91 bp fragment (Fig. 1a, sequences in uppercase) from the zen cis-regulatory region8,9, which contains four TAGteam sites5 (Fig. 1a in red, the first two are the reverse complement). zld (CG12701 on the X-chromosome) was selected as the only candidate of the 11 recovered that had the potential to bind specific DNA sequences since it encoded a protein with six C2H2 zinc fingers (represented as green boxes in Fig. 1b). Oligonucleotides (Fig. 1a, underlined sequences) with different TAGteam sites were tested in gel shift assays with the 357 amino-acid C-terminal region of Zld fused to GST (GST-ZldC; Fig. 1b, stippled region). They all formed complexes with GST-ZldC, though with different affinities (Fig. 1c lanes 1-9), while mutations (Fig. 1a, in purple) in the heptanucleotide sequence abolished binding (Fig. 1c, lanes 10-12). Interestingly, the site with the strongest affinity, CAGGTAG, is the site most over represented in regulatory elements of pre-blastoderm genes versus postblastoderm genes5. A plasmid expressing full-length Zld protein promoted transcriptional activation of a zen91-lacZ reporter but not a mutated zen91m-lacZ reporter after co-transfection in Drosophila S2 cells (Fig. 1d). Taken together, these data strongly suggest that Zld activates transcription of zen, and likely other TAGteam containing genes.


The zinc-finger protein Zelda is a key activator of the early zygotic genome in Drosophila.

Liang HL, Nien CY, Liu HY, Metzstein MM, Kirov N, Rushlow C - Nature (2008)

TAGteam sites bind Zld and mediate transcriptional activation(a) DNA sequence of the 91 bp zen enhancer (uppercase) plus surrounding sequences (lowercase). Base substitutions are in purple. (b) Schematic organization of the zld locus (CG12701; Flybase) with the two predicted transcription start sites, RB and RA. The P{RS3}UM8171-3 insertion site is between -661 and -660. The nucleotides deleted in zld294 and zld681 are indicated as blank space between solid lines. (c) Zld binding to oligonucleotides containing different TAGteam sites (denoted beneath each section of the gel). The first lane in each section contains free probe. The second lane contains probe plus 10ng GST-ZldC, the third 30 ng GST-ZldC. (d) S2 cells were transfected with 0 ng (blue bar), 50 ng (red bar), or 100 ng (yellow bar) of plasmid expressing zld under control of the inducible metallothionein promoter, the zen91-lacZ or zen91m-lacZ reporter plasmids, and the luciferase control. Error bars, s.e.m.; n=3.
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Figure 1: TAGteam sites bind Zld and mediate transcriptional activation(a) DNA sequence of the 91 bp zen enhancer (uppercase) plus surrounding sequences (lowercase). Base substitutions are in purple. (b) Schematic organization of the zld locus (CG12701; Flybase) with the two predicted transcription start sites, RB and RA. The P{RS3}UM8171-3 insertion site is between -661 and -660. The nucleotides deleted in zld294 and zld681 are indicated as blank space between solid lines. (c) Zld binding to oligonucleotides containing different TAGteam sites (denoted beneath each section of the gel). The first lane in each section contains free probe. The second lane contains probe plus 10ng GST-ZldC, the third 30 ng GST-ZldC. (d) S2 cells were transfected with 0 ng (blue bar), 50 ng (red bar), or 100 ng (yellow bar) of plasmid expressing zld under control of the inducible metallothionein promoter, the zen91-lacZ or zen91m-lacZ reporter plasmids, and the luciferase control. Error bars, s.e.m.; n=3.
Mentions: In Drosophila, an initial wave of zygotic gene transcription occurs between 1-2 hours of development during mitotic cleavage cycles 8-13. This is followed by a major burst of activity between 2-3 hours (cycle 14) when the embryo is undergoing cellular blastoderm formation. Many pre-cellular genes contain TAGteam sites in their upstream regulatory regions including several direct targets of Bicoid, Dorsal, and other key regulators of patterning5-7. ten Bosch et. al.5 demonstrated that TAGteam sites are required for the early expression of the dorsoventral (DV) gene zen, and the sex determination genes sisB and Sxl. To isolate the TAGteam binding factor, we performed a yeast one-hybrid screen with a 91 bp fragment (Fig. 1a, sequences in uppercase) from the zen cis-regulatory region8,9, which contains four TAGteam sites5 (Fig. 1a in red, the first two are the reverse complement). zld (CG12701 on the X-chromosome) was selected as the only candidate of the 11 recovered that had the potential to bind specific DNA sequences since it encoded a protein with six C2H2 zinc fingers (represented as green boxes in Fig. 1b). Oligonucleotides (Fig. 1a, underlined sequences) with different TAGteam sites were tested in gel shift assays with the 357 amino-acid C-terminal region of Zld fused to GST (GST-ZldC; Fig. 1b, stippled region). They all formed complexes with GST-ZldC, though with different affinities (Fig. 1c lanes 1-9), while mutations (Fig. 1a, in purple) in the heptanucleotide sequence abolished binding (Fig. 1c, lanes 10-12). Interestingly, the site with the strongest affinity, CAGGTAG, is the site most over represented in regulatory elements of pre-blastoderm genes versus postblastoderm genes5. A plasmid expressing full-length Zld protein promoted transcriptional activation of a zen91-lacZ reporter but not a mutated zen91m-lacZ reporter after co-transfection in Drosophila S2 cells (Fig. 1d). Taken together, these data strongly suggest that Zld activates transcription of zen, and likely other TAGteam containing genes.

Bottom Line: However, the transcription factor(s) that activate the zygotic genome remain elusive.Mutant embryos lacking zld are defective in cellular blastoderm formation, and fail to activate many genes essential for cellularization, sex determination and pattern formation.Global expression profiling confirmed that Zld has an important role in the activation of the early zygotic genome and suggests that Zld may also regulate maternal RNA degradation during the maternal-to-zygotic transition.

View Article: PubMed Central - PubMed

Affiliation: Department of Biology, New York University, 100 Washington Square East, New York, New York 10003, USA.

ABSTRACT
In all animals, the initial events of embryogenesis are controlled by maternal gene products that are deposited into the developing oocyte. At some point after fertilization, control of embryogenesis is transferred to the zygotic genome in a process called the maternal-to-zygotic transition. During this time, many maternal RNAs are degraded and transcription of zygotic RNAs ensues. There is a long-standing question as to which factors regulate these events. The recent findings that microRNAs and Smaug mediate maternal transcript degradation have shed new light on this aspect of the problem. However, the transcription factor(s) that activate the zygotic genome remain elusive. The discovery that many of the early transcribed genes in Drosophila share a cis-regulatory heptamer motif, CAGGTAG and related sequences, collectively referred to as TAGteam sites raised the possibility that a dedicated transcription factor could interact with these sites to activate transcription. Here we report that the zinc-finger protein Zelda (Zld; Zinc-finger early Drosophila activator) binds specifically to these sites and is capable of activating transcription in transient transfection assays. Mutant embryos lacking zld are defective in cellular blastoderm formation, and fail to activate many genes essential for cellularization, sex determination and pattern formation. Global expression profiling confirmed that Zld has an important role in the activation of the early zygotic genome and suggests that Zld may also regulate maternal RNA degradation during the maternal-to-zygotic transition.

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