GAF is essential for zygotic genome activation and chromatin accessibility in the early Drosophila embryo

Following fertilization, the genomes of the germ cells are reprogrammed to form the totipotent embryo. Pioneer transcription factors are essential for remodeling the chromatin and driving the initial wave of zygotic gene expression. In Drosophila melanogaster, the pioneer factor Zelda is essential for development through this dramatic period of reprogramming, known as the maternal-to-zygotic transition (MZT). However, it was unknown whether additional pioneer factors were required for this transition. We identified an additional maternally encoded factor required for development through the MZT, GAGA Factor (GAF). GAF is necessary to activate widespread zygotic transcription and to remodel the chromatin accessibility landscape. We demonstrated that Zelda preferentially controls expression of the earliest transcribed genes, while genes expressed during widespread activation are predominantly dependent on GAF. Thus, progression through the MZT requires coordination of multiple pioneer-like factors, and we propose that as development proceeds control is gradually transferred from Zelda to GAF.

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The pioneer factor GAF is essential for zygotic genome activation and chromatin accessibility in the early Drosophila embryo

10.1101/2020.07.15.204248 ◽

2020 ◽

Author(s):

Marissa M. Gaskill ◽

Tyler J. Gibson ◽

Elizabeth D. Larson ◽

Melissa M. Harrison

Keyword(s):

Transcriptional Control ◽

Chromatin Accessibility ◽

Drosophila Embryo ◽

Gaga Factor ◽

Zygotic Transcription ◽

Early Drosophila Embryo ◽

Zygotic Gene ◽

Pioneer Factor ◽

Pioneer Factors ◽

Genome Activation

AbstractFollowing fertilization, the genomes of the germ cells are reprogrammed to form the totipotent embryo. Pioneer transcription factors are required for remodeling the chromatin and driving the initial wave of zygotic gene expression. In Drosophila melanogaster, the pioneer factor Zelda is essential for development through this dramatic period of reprogramming, known as the maternal- to-zygotic transition (MZT). However, it was unknown whether additional pioneer factors were necessary for this transition. We identified an additional maternally encoded factor required for development through the MZT, GAGA Factor (GAF). GAF is needed to activate widespread zygotic transcription and to remodel the chromatin accessibility landscape. We demonstrated that Zelda preferentially controls expression of the earliest transcribed genes, while genes expressed during widespread activation are predominantly dependent on GAF. Thus, progression through the MZT requires coordination of multiple pioneer factors, and we propose that as development proceeds transcriptional control is gradually transferred from Zelda to GAF.

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Odd-paired is a pioneer-like factor that coordinates with Zelda to control gene expression in embryos

eLife ◽

10.7554/elife.59610 ◽

2020 ◽

Vol 9 ◽

Author(s):

Theodora Koromila ◽

Fan Gao ◽

Yasuno Iwasaki ◽

Peng He ◽

Lior Pachter ◽

...

Keyword(s):

Gene Expression ◽

Chromatin Accessibility ◽

Secondary Wave ◽

Genome Wide ◽

Zygotic Transcription ◽

Early Embryos ◽

Zygotic Gene ◽

Pioneer Factor ◽

Pioneer Factors

Pioneer factors such as Zelda (Zld) help initiate zygotic transcription in Drosophila early embryos, but whether other factors support this dynamic process is unclear. Odd-paired (Opa), a zinc-finger transcription factor expressed at cellularization, controls the transition of genes from pair-rule to segmental patterns along the anterior-posterior axis. Finding that Opa also regulates expression through enhancer sog_Distal along the dorso-ventral axis, we hypothesized Opa’s role is more general. Chromatin-immunoprecipitation (ChIP-seq) confirmed its in vivo binding to sog_Distal but also identified widespread binding throughout the genome, comparable to Zld. Furthermore, chromatin assays (ATAC-seq) demonstrate that Opa, like Zld, influences chromatin accessibility genome-wide at cellularization, suggesting both are pioneer factors with common as well as distinct targets. Lastly, embryos lacking opa exhibit widespread, late patterning defects spanning both axes. Collectively, these data suggest Opa is a general timing factor and likely late-acting pioneer factor that drives a secondary wave of zygotic gene expression.

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Odd-paired is a late-acting pioneer factor coordinating with Zelda to broadly regulate gene expression in early embryos

10.1101/853028 ◽

2019 ◽

Cited By ~ 2

Author(s):

Theodora Koromila ◽

Fan Gao ◽

Yasuno Iwasaki ◽

Peng He ◽

Lior Pachter ◽

...

Keyword(s):

Gene Expression ◽

Chromatin Accessibility ◽

Secondary Wave ◽

Genome Wide ◽

Zygotic Transcription ◽

Early Embryos ◽

Zygotic Gene ◽

Pioneer Factor ◽

Pioneer Factors

ABSTRACTPioneer factors such as Zelda help initiate zygotic transcription in Drosophila early embryos, but whether other factors support this dynamic process is unclear. Odd-paired (Opa), a zinc-finger transcription factor expressed at cellularization, controls transition of genes from pair-rule to segmental patterns along the anterior-posterior axis. Finding that Opa also regulates late expression through enhancer sog_Distal, along the dorso-ventral axis, we hypothesized that Opa acts as a general timing factor. Chromatin-immunoprecipitation (ChIP-seq) confirmed Opa in vivo binding to sog_Distal but also identified widespread binding throughout the genome, comparable to Zelda. Furthermore, chromatin assays (ATAC-seq) demonstrate that Opa, like Zelda, influences chromatin accessibility genome-wide, suggesting both are pioneer factors with common as well as distinct targets. Lastly, embryos lacking opa exhibit widespread, late patterning defects spanning both axes. Collectively, these data suggest Opa, a general timing factor and likely a late-acting pioneer factor, heralds in a secondary wave of zygotic gene expression.

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Spatially uniform establishment of chromatin accessibility in the early Drosophila embryo

10.1101/195073 ◽

2017 ◽

Author(s):

Jenna E. Haines ◽

Michael B. Eisen

Keyword(s):

Chromatin Accessibility ◽

Drosophila Embryo ◽

Direct Result ◽

Open Chromatin ◽

Relative Contribution ◽

Genome Wide ◽

Zygotic Transcription ◽

Early Drosophila Embryo ◽

Nucleosome Density ◽

Anterior Posterior

AbstractAs the Drosophila embryo transitions from the use of maternal RNAs to zygotic transcription, domains of open chromatin, with relatively low nucleosome density and specific histone marks, are established at promoters and enhancers involved in patterned embryonic transcription. However, it remains unclear whether open chromatin is a product of activity - transcription at promoters and patterning transcription factor binding at enhancers - or whether it is established by independent mechanisms. Recent work has implicated the ubiquitously expressed, maternal factor Zelda in this process. To assess the relative contribution of activity in the establishment of chromatin accessibility, we have probed chromatin accessibility across the anterior-posterior axis of early Drosophila melanogaster embryos by applying a transposon based assay for chromatin accessibility (ATAC-seq) to anterior and posterior halves of hand-dissected, cellular blastoderm embryos. We find that genome-wide chromatin accessibility is remarkably similar between the two halves. Promoters and enhancers that are active in exclusively one half of the embryo have open chromatin in the other half, demonstrating that chromatin accessibility is not a direct result of activity. However, there is a small skew at enhancers that drive transcription exclusively in either the anterior or posterior half of the embryo, with greater accessibility in the region of activity. Taken together these data support a model in which regions of chromatin accessibility are defined and established by ubiquitous factors, and fine-tuned subsequently by activity.

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CLAMP and Zelda function together to promote Drosophila zygotic genome activation

eLife ◽

10.7554/elife.69937 ◽

2021 ◽

Vol 10 ◽

Author(s):

Jingyue Duan ◽

Leila Rieder ◽

Megan M Colonnetta ◽

Annie Huang ◽

Mary Mckenney ◽

...

Keyword(s):

Transcription Factor ◽

Embryonic Development ◽

Essential Role ◽

Chromatin Accessibility ◽

Zygotic Genome Activation ◽

Zygotic Transcription ◽

Pioneer Factor ◽

Pioneer Transcription Factor ◽

Genome Activation ◽

Zygotic Genome

During the essential and conserved process of zygotic genome activation (ZGA), chromatin accessibility must increase to promote transcription. Drosophila is a well-established model for defining mechanisms that drive ZGA. Zelda (ZLD) is a key pioneer transcription factor (TF) that promotes ZGA in the Drosophila embryo. However, many genomic loci that contain GA-rich motifs become accessible during ZGA independent of ZLD. Therefore, we hypothesized that other early TFs that function with ZLD have not yet been identified, especially those that are capable of binding to GA-rich motifs such as CLAMP. Here, we demonstrate that Drosophila embryonic development requires maternal CLAMP to: 1) activate zygotic transcription; 2) increase chromatin accessibility at promoters of specific genes that often encode other essential TFs; 3) enhance chromatin accessibility and facilitate ZLD occupancy at a subset of key embryonic promoters. Thus, CLAMP functions as a pioneer factor which plays a targeted yet essential role in ZGA.

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Sex-specific transcript diversity is regulated by a maternal pioneer factor in early Drosophila embryos

10.1101/2021.03.18.436074 ◽

2021 ◽

Author(s):

Mukulika Ray ◽

Ashley Mae Conard ◽

Jennifer Urban ◽

Erica Larschan

Keyword(s):

Chromatin Accessibility ◽

Early Embryo ◽

Specific Transcript ◽

Genome Wide ◽

Zygotic Transcription ◽

Pioneer Factor ◽

Alternatively Spliced ◽

Transcript Diversity ◽

Changes Over Time ◽

Genome Activation

Maternally deposited RNAs and proteins play a crucial role in initiating zygotic transcription during early embryonic development. However, the mechanisms by which maternal factors regulate zygotic transcript diversity early in development remain poorly understood. Furthermore, how early in development sex-specific transcript diversity occurs is not known genome-wide in any organism. Here, we determine that sex-specific transcript diversity occurs much earlier in development than previously thought in Drosophila, concurrent with Zygotic genome activation (ZGA). We use genetic, biochemical, and genomic approaches to demonstrate that the essential maternally-deposited pioneer factor CLAMP (Chromatin linked adapter for MSL proteins) is a key regulator of sex-specific transcript diversity in the early embryo via the following mechanisms: 1) In both sexes, CLAMP directly binds to the gene bodies of female and male sex-specifically spliced genes. 2) In females, CLAMP modulates chromatin accessibility of an alternatively-spliced exon within Sex-lethal, the master regulator of sex determination, to promote protein production. 3) In males, CLAMP regulates Maleless (MLE) distribution, a spliceosome component to prevent aberrant sex-specific splicing. Thus, we demonstrate for the first time how a maternal factor regulates early zygotic transcriptome diversity sex-specifically. We also developed a new tool to measure how splicing changes over time called time2splice.

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CLAMP regulates Zygotic Genome Activation in Drosophila embryos

Genetics ◽

10.1093/genetics/iyab107 ◽

2021 ◽

Author(s):

Megan M Colonnetta ◽

Juan E Abrahante ◽

Paul Schedl ◽

Daryl M Gohl ◽

Girish Deshpande

Keyword(s):

Temporal Dynamics ◽

Embryonic Patterning ◽

Zygotic Genome Activation ◽

Promoter Sequences ◽

Genome Wide ◽

Zygotic Transcription ◽

Pioneer Factor ◽

Genome Activation ◽

Drosophila Embryos ◽

Zygotic Genome

Abstract Embryonic patterning is critically dependent on zygotic genome activation (ZGA). In Drosophila melanogaster embryos, the pioneer factor Zelda directs ZGA, possibly in conjunction with other factors. Here we have explored novel involvement of Chromatin-Linked Adapter for MSL Proteins (CLAMP) during ZGA. CLAMP binds thousands of sites genome-wide throughout early embryogenesis. Interestingly, CLAMP relocates to target promoter sequences across the genome when ZGA is initiated. Although there is a considerable overlap between CLAMP and Zelda binding sites, the proteins display distinct temporal dynamics. To assess whether CLAMP occupancy affects gene expression, we analyzed transcriptomes of embryos zygotically compromised for either clamp or zelda and found that transcript levels of many zygotically-activated genes are similarly affected. Importantly, compromising either clamp or zelda disrupted the expression of critical segmentation and sex determination genes bound by CLAMP (and Zelda). Furthermore, clamp knockdown embryos recapitulate other phenotypes observed in Zelda-depleted embryos, including nuclear division defects, centrosome aberrations, and a disorganized actomyosin network. Based on these data, we propose that CLAMP acts in concert with Zelda to regulate early zygotic transcription.

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The GAGA factor is required in the early Drosophila embryo not only for transcriptional regulation but also for nuclear division

Development ◽

10.1242/dev.122.4.1113 ◽

1996 ◽

Vol 122 (4) ◽

pp. 1113-1124 ◽

Cited By ~ 1

Author(s):

K.M. Bhat ◽

G. Farkas ◽

F. Karch ◽

H. Gyurkovics ◽

J. Gausz ◽

...

Keyword(s):

Gene Expression ◽

Chromatin Remodeling ◽

In Vitro Transcription ◽

Drosophila Embryo ◽

Gaga Factor ◽

Early Drosophila Embryo ◽

Hypersensitive Sites ◽

And Function ◽

Stimulatory Factor

The GAGA protein of Drosophila was first identified as a stimulatory factor in in vitro transcription assays using the engrailed and Ultrabithorax promoters. Subsequent studies have suggested that the GAGA factor promotes transcription by blocking the repressive effects of histones; moreover, it has been shown to function in chromatin remodeling, acting together with other factors in the formation of nuclease hypersensitive sites in vitro. The GAGA factor is encoded by the Trithorax-like locus and in the studies reported here we have used the maternal effect allele Trl13C to examine the functions of the protein during embryogenesis. We find that GAGA is required for the proper expression of a variety of developmental loci that contain GAGA binding sites in their upstream regulatory regions. The observed disruptions in gene expression are consistent with those expected for a factor involved in chromatin remodeling. In addition to facilitating gene expression, the GAGA factor appears to have a more global role in chromosome structure and function. This is suggested by the spectrum of nuclear cleavage cycle defects observed in Trl13C embryos. These defects include asynchrony in the cleavage cycles, failure in chromosome condensation, abnormal chromosome segregation and chromosome fragmentation. These defects are likely to be related to the association of the GAGA protein with heterochromatic satellite sequences which is observed throughout the cell cycle.

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Establishment of chromatin accessibility by the conserved transcription factor Grainy head is developmentally regulated

10.1101/798454 ◽

2019 ◽

Cited By ~ 1

Author(s):

Markus Nevil ◽

Tyler J. Gibson ◽

Constantine Bartolutti ◽

Anusha Iyengar ◽

Melissa M Harrison

Keyword(s):

Transcription Factor ◽

Transcription Factors ◽

Chromatin Accessibility ◽

Mitotic Chromosomes ◽

Developmentally Regulated ◽

Regulatory Modules ◽

Pioneer Factor ◽

Pioneer Factors ◽

Coordinate Changes ◽

Accessible Chromatin

AbstractThe dramatic changes in gene expression required for development necessitate the establishment of cis-regulatory modules defined by regions of accessible chromatin. Pioneer transcription factors have the unique property of binding closed chromatin and facilitating the establishment of these accessible regions. Nonetheless, much of how pioneer transcription factors coordinate changes in chromatin accessibility during development remains unknown. To determine whether pioneer-factor function is intrinsic to the protein or whether pioneering activity is developmentally modulated, we studied the highly conserved, essential transcription factor, Grainy head (Grh). Grh is expressed throughout Drosophila development and functions as a pioneer factor in the larvae. We demonstrated that Grh remains bound to condensed mitotic chromosomes, a property shared with other pioneer factors. By assaying chromatin accessibility in embryos lacking either maternal or zygotic Grh at three stages of development, we discovered that Grh is not required for chromatin accessibility in early embryogenesis, in contrast to its essential functions later in development. Our data reveal that the pioneering activity of Grh is temporally regulated and is likely influenced by additional factors expressed at a given developmental stage.

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An "individualist" model of an active genome in a developing embryo

10.1101/2021.01.08.425929 ◽

2021 ◽

Author(s):

Shao-Kuei Huang ◽

Sayantan Dutta ◽

Peter H Whitney ◽

Stanislav Shvartsman ◽

Christine Rushlow

Keyword(s):

Rna Polymerase Ii ◽

Physical Distance ◽

Drosophila Embryo ◽

Drosophila Genome ◽

Pol Ii ◽

Individual Gene ◽

Early Drosophila Embryo ◽

Primary Driver ◽

Genome Activation ◽

Active Genes

The early Drosophila embryo provides unique experimental advantages for addressing fundamental questions of gene regulation at multiple levels of organization, from individual gene loci to the whole genome. Using Drosophila embryos undergoing the first wave of genome activation, we detected discrete "speckles" of RNA Polymerase II (Pol II), and showed that they overlap with transcribing loci. We characterized the spatial distribution of Pol II speckles and quantified how this distribution changes in the absence of the primary driver of Drosophila genome activation, the pioneer factor Zelda. Although the number and size of Pol II speckles were reduced, indicating that Zelda promotes Pol II speckle formation, we observed a uniform distribution of distances between active genes in the nuclei of both wildtype and zelda mutant embryos. This suggests that the topologically associated domains identified by Hi-C studies do little to spatially constrain groups of transcribed genes at this time. We provide evidence that linear genomic distance between transcribed genes is the primary determinant of measured physical distance between the active loci. Furthermore, we show active genes can have distinct Pol II pools even if the active loci are in close proximity. In contrast to the emerging model whereby active genes are clustered to facilitate co-regulation and sharing of transcriptional resources, our data support an "individualist" model of gene control at early genome activation in Drosophila. This model is in contrast to a "collectivist" model where active genes are spatially clustered and share transcriptional resources, motivating rigorous tests of both models in other experimental systems.

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