The Polycomb Group Mutant esc Leads to Augmented Levels of Paused Pol II in the Drosophila Embryo

Abstract Anteroposterior polarity of the Drosophila embryo is initiated by the localized activities of the maternal genes, bicoid and nanos, which establish a gradient of the hunchback (hb) morphogen. nanos determines the distribution of the maternal Hb protein by regulating its translation. To identify further components of this pathway we isolated suppressors of nanos. In the absence of nanos high levels of Hb protein repress the abdomen-specific genes knirps and giant. In suppressor-of-nanos mutants, knirps and giant are expressed in spite of high Hb levels. The suppressors are alleles of Enhancer of zeste (E(z)) a member of the Polycomb group (Pc-G) of genes. We show that E(z), and likely other Pc-G genes, are required for maintaining the expression domains of knirps and giant initiated by the maternal Hb protein gradient. We have identified a small region of the knirps promoter that mediates the regulation by E(z) and hb. Because Pc-G genes are thought to control gene expression by regulating chromatin, we propose that imprinting at the chromatin level underlies the determination of anteroposterior polarity in the early embryo.

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Transcriptional repression of Myc underlies AGO1’s tumour suppressor function

10.1101/2020.03.10.984906 ◽

2020 ◽

Author(s):

Olga Zaytseva ◽

Naomi C. Mitchell ◽

Linna Guo ◽

Owen J. Marshall ◽

Linda M. Parsons ◽

...

Keyword(s):

Transcriptional Repression ◽

Ribosome Biogenesis ◽

Rna Binding ◽

Tissue Growth ◽

Tumour Suppressor ◽

Polycomb Group ◽

Physical Interaction ◽

Suppressor Activity ◽

Rna Pol Ii ◽

Pol Ii

AbstractHere we report novel tumour suppressor activity for the Drosophila Argonaute family RNA binding protein AGO1, a component of the miRNA-dependent RNA-induced silencing complex (RISC). The mechanism for growth inhibition does not, however, involve canonical roles as part of the RISC; rather AGO1 controls cell and tissue growth by functioning as a direct transcriptional repressor of the master regulator of growth, Myc. AGO1 depletion in wing imaginal discs drives a significant increase in ribosome biogenesis, nucleolar expansion, and cell growth in a manner dependent on Myc abundance. Moreover, increased Myc promoter activity and elevated Myc mRNA in AGO1 depleted animals requires RNA Pol II transcription. Further support for transcriptional AGO1 functions is provided by physical interaction with the RNA Pol II transcriptional machinery (chromatin remodelling factors and Mediator Complex), punctate nuclear localisation in euchromatic regions and overlap with Polycomb Group transcriptional silencing loci. Moreover, significant AGO1 enrichment is observed on the Myc promoter and AGO1 interacts with the Myc transcriptional activator Psi. Together our data show AGO1 functions outside of the RISC to repress Myc transcription and inhibit developmental cell and tissue growth.

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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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Transcription and splicing dynamics during early Drosophila development

RNA ◽

10.1261/rna.078933.121 ◽

2021 ◽

pp. rna.078933.121

Author(s):

Pedro Prudencio ◽

Rosina Savisaar ◽

Kenny Rebelo ◽

Rui Goncalo Martinho ◽

Maria Carmo-Fonseca

Keyword(s):

Rna Polymerase Ii ◽

Developmental Stages ◽

Early Embryo ◽

Drosophila Embryo ◽

Dynamic Features ◽

Nascent Transcript ◽

Pol Ii ◽

Cleavage And Polyadenylation ◽

Dna Template ◽

Early Developmental

Widespread co-transcriptional splicing has been demonstrated from yeast to human. However, most studies to date addressing the kinetics of splicing relative to transcription used either Saccharomyces cerevisiae or metazoan cultured cell lines. Here, we adapted native elongating transcript sequencing technology (NET-seq) to measure co-transcriptional splicing dynamics during the early developmental stages of Drosophila melanogaster embryos. Our results reveal the position of RNA polymerase II (Pol II) when both canonical and recursive splicing occur. We found heterogeneity in splicing dynamics, with some RNAs spliced immediately after intron transcription, whereas for other transcripts no splicing was observed over the first 100 nucleotides of the downstream exon. Introns that show splicing completion before Pol II has reached the end of the downstream exon are necessarily intron-defined. We studied the splicing dynamics of both nascent pre-mRNAs transcribed in the early embryo, which have few and short introns, as well as pre-mRNAs transcribed later in embryonic development, which contain multiple long introns. As expected, we found a relationship between the proportion of spliced reads and intron size. However, intron definition was observed at all intron sizes. We further observed that genes transcribed in the early embryo tend to be isolated in the genome whereas genes transcribed later are often overlapped by a neighboring convergent gene. In isolated genes, transcription termination occurred soon after the polyadenylation site, while in overlapped genes Pol II persisted associated with the DNA template after cleavage and polyadenylation of the nascent transcript. Taken together, our data unravels novel dynamic features of Pol II transcription and splicing in the developing Drosophila embryo.

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Faculty Opinions recommendation of Paused Pol II coordinates tissue morphogenesis in the Drosophila embryo.

Faculty Opinions – Post-Publication Peer Review of the Biomedical Literature ◽

10.3410/f.718016240.793477801 ◽

2013 ◽

Author(s):

Christopher Gregg

Keyword(s):

Drosophila Embryo ◽

Tissue Morphogenesis ◽

Pol Ii

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Association of SARFH (sarcoma-associated RNA-binding fly homolog) with regions of chromatin transcribed by RNA polymerase II.

Molecular and Cellular Biology ◽

10.1128/mcb.15.8.4562 ◽

1995 ◽

Vol 15 (8) ◽

pp. 4562-4571 ◽

Cited By ~ 41

Author(s):

D Immanuel ◽

H Zinszner ◽

D Ron

Keyword(s):

Rna Polymerase ◽

Rna Polymerase Ii ◽

Mammalian Cells ◽

Rna Binding ◽

Rna Binding Proteins ◽

Polytene Chromosomes ◽

Drosophila Embryo ◽

Cdna Clones ◽

Rna Pol Ii ◽

Pol Ii

Many oncogenes associated with human sarcomas are composed of a fusion between transcription factors and the N-terminal portions of two similar RNA-binding proteins, TLS and EWS. Though the oncogenic fusion proteins lack the RNA-binding domain and do not bind RNA, the contribution from the N-terminal portion of the RNA-binding protein is essential for their transforming activity. TLS and EWS associate in vivo with RNA polymerase II (Pol II) transcripts. To learn more about the target gene specificity of this interaction, the localization of a Drosophila melanogaster protein that has extensive sequence identity to the C-terminal RNA-binding portions of TLS and EWS was studied in preparations of Drosophila polytene nuclei. cDNA clones encoding the full-length Drosophila TLS-EWS homolog, SARFH (stands for sarcoma-associated RNA-binding fly homolog), were isolated. Functional similarity to TLS and EWS was revealed by the association of SARFH with Pol II transcripts in mammalian cells and by the ability of SARFH to elicit homologous down-regulation of the levels of the mammalian proteins. The SARFH gene is expressed in the developing Drosophila embryo from the earliest stages of cellularization and is subsequently found in many cell types. In preparations of polytene chromosomes from salivary gland nuclei, SARFH antibodies recognize their target associated with the majority of active transcription units, revealed by colocalization with the phosphorylated form of RNA Pol II. We conclude that SARFH and, by homology, EWS and TLS participate in a function common to the expression of most genes transcribed by RNA Pol II.

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