scholarly journals Quantitative imaging of transcription in living Drosophila embryos reveals the impact of core promoter motifs on promoter state dynamics

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Virginia L. Pimmett ◽  
Matthieu Dejean ◽  
Carola Fernandez ◽  
Antonio Trullo ◽  
Edouard Bertrand ◽  
...  
Keyword(s):  
Core Promoter ◽  
Quantitative Imaging ◽  
Tata Box ◽  
Polymerase Pausing ◽  
Multicellular Organisms ◽  
Genome Activation ◽  
The Impact ◽  
Rate Limiting ◽  
Drosophila Embryos ◽  
Zygotic Genome

AbstractGenes are expressed in stochastic transcriptional bursts linked to alternating active and inactive promoter states. A major challenge in transcription is understanding how promoter composition dictates bursting, particularly in multicellular organisms. We investigate two key Drosophila developmental promoter motifs, the TATA box (TATA) and the Initiator (INR). Using live imaging in Drosophila embryos and new computational methods, we demonstrate that bursting occurs on multiple timescales ranging from seconds to minutes. TATA-containing promoters and INR-containing promoters exhibit distinct dynamics, with one or two separate rate-limiting steps respectively. A TATA box is associated with long active states, high rates of polymerase initiation, and short-lived, infrequent inactive states. In contrast, the INR motif leads to two inactive states, one of which relates to promoter-proximal polymerase pausing. Surprisingly, the model suggests pausing is not obligatory, but occurs stochastically for a subset of polymerases. Overall, our results provide a rationale for promoter switching during zygotic genome activation.

scholarly journals Quantitative imaging of transcription in living Drosophila embryos reveals the impact of core promoter motifs on promoter state dynamics

2021 ◽  
Author(s):  
Virginia L Pimmett ◽  
Matthieu Dejean ◽  
Carola Fernandez ◽  
Antonio Trullo ◽  
Edouard Bertrand ◽  
...  
Keyword(s):  
Core Promoter ◽  
Quantitative Imaging ◽  
Tata Box ◽  
Polymerase Pausing ◽  
Multicellular Organisms ◽  
Genome Activation ◽  
The Impact ◽  
Rate Limiting ◽  
Drosophila Embryos ◽  
Zygotic Genome

AbstractGenes are expressed in stochastic transcriptional bursts linked to alternating active and inactive promoter states. A major challenge in transcription is understanding how promoter composition dictates bursting, particularly in multicellular organisms. We investigate two key Drosophila developmental promoter motifs, the TATA box (TATA) and the Initiator (INR). Using live imaging in Drosophila embryos and new computational methods, we demonstrate that bursting occurs on multiple timescales ranging from seconds to minutes. TATA-containing promoters and INR-containing promoters exhibit distinct dynamics, with one or two separate rate-limiting steps respectively. A TATA box is associated with long active states, high rates of polymerase initiation, and short-lived, infrequent inactive states. In contrast, the INR motif leads to two inactive states, one of which relates to promoter-proximal polymerase pausing. Surprisingly, the model suggests pausing is not obligatory, but occurs stochastically for a subset of polymerases. Overall, our results provide a rationale for promoter switching during zygotic genome activation.

CLAMP regulates Zygotic Genome Activation in Drosophila embryos

Genetics ◽  
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.

scholarly journals The miR-430 locus with extreme promoter density is a transcription body organizer, which facilitates long range regulation in zygotic genome activation

2021 ◽  
Author(s):  
Yavor Hadzhiev ◽  
Lucy Wheatley ◽  
Ledean Cooper ◽  
Federico Ansaloni ◽  
Celina Whalley ◽  
...  
Keyword(s):  
Rna Polymerase Ii ◽  
Gene Cluster ◽  
Core Promoter ◽  
Single Copy ◽  
Early Embryo ◽  
Zygotic Genome Activation ◽  
Pol Ii ◽  
A Minor ◽  
Genome Activation ◽  
Zygotic Genome

In anamniote embryos the major wave of zygotic genome activation (ZGA) starts during the mid-blastula transition. This major wave of ZGA is facilitated by several mechanisms, including dilution of repressive maternal factors and accumulation of activating transcription factors during the fast cell division cycles preceding the mid-blastula transition. However, a set of genes escape global genome repression and are activated substantially earlier, during what is called, the minor wave of genome activation. While the mechanisms underlying the major wave of genome activation have been studied extensively, the minor wave of genome activation is little understood. In zebrafish the earliest expressed RNA polymerase II (Pol II) transcribed genes are activated in a pair of large transcription bodies depleted of chromatin, abundant in elongating Pol II and nascent RNAs (Hadzhiev et al., 2019; Hilbert et al., 2021). This transcription body includes the miR-430 gene cluster required for maternal mRNA clearance. Here we explored the genomic, chromatin organisation and cis-regulatory mechanisms of the minor wave of genome activation occurring in the transcription body. By long read genome sequencing we identified a remarkable cluster of miR-430 genes with over 300 promoters and spanning 0.6 Mb, which represent the highest promoter density of the genome. We demonstrate that the miR-430 gene cluster is required for the formation of the transcription body and acts as a transcription organiser for minor wave activation of a set of zinc finger genes scattered on the same chromosome arm, which share promoter features with the miR-430 cluster. These promoter features are shared among minor wave genes overall and include the TATA-box and sharp transcription start site profile. Single copy miR-430 promoter transgene reporter experiments indicate the importance of promoter-autonomous mechanisms regulating escape from global repression of the early embryo. These results together suggest that formation of the transcription body in the early embryo is the result of high promoter density coupled to a minor wave-specific core promoter code for transcribing key minor wave ZGA genes, which are required for the overhaul of the transcriptome during early embryonic development.

scholarly journals The genome-wide multi-layered architecture of chromosome pairing in early Drosophila embryos

2019 ◽  
Vol 10 (1) ◽  
Author(s):  
Jelena Erceg ◽  
Jumana AlHaj Abed ◽  
Anton Goloborodko ◽  
Bryan R. Lajoie ◽  
Geoffrey Fudenberg ◽  
...  
Keyword(s):  
Computational Approach ◽  
Homologous Chromosomes ◽  
Layered Architecture ◽  
Homolog Pairing ◽  
Genome Wide ◽  
Mammalian Embryos ◽  
Pioneer Factor ◽  
Genome Activation ◽  
Drosophila Embryos ◽  
Zygotic Genome

Abstract Genome organization involves cis and trans chromosomal interactions, both implicated in gene regulation, development, and disease. Here, we focus on trans interactions in Drosophila, where homologous chromosomes are paired in somatic cells from embryogenesis through adulthood. We first address long-standing questions regarding the structure of embryonic homolog pairing and, to this end, develop a haplotype-resolved Hi-C approach to minimize homolog misassignment and thus robustly distinguish trans-homolog from cis contacts. This computational approach, which we call Ohm, reveals pairing to be surprisingly structured genome-wide, with trans-homolog domains, compartments, and interaction peaks, many coinciding with analogous cis features. We also find a significant genome-wide correlation between pairing, transcription during zygotic genome activation, and binding of the pioneer factor Zelda. Our findings reveal a complex, highly structured organization underlying homolog pairing, first discovered a century ago in Drosophila. Finally, we demonstrate the versatility of our haplotype-resolved approach by applying it to mammalian embryos.

scholarly journals Chiffon triggers global histone H3 acetylation and expression of developmental genes in Drosophila embryos

2021 ◽  
Author(s):  
Eliana F. Torres-Zelada ◽  
Smitha George ◽  
Hannah R. Blum ◽  
Vikki M. Weake
Keyword(s):  
Gene Expression ◽  
Histone Acetylation ◽  
Potential Role ◽  
Developmental Genes ◽  
Histone Acetyltransferase Gcn5 ◽  
Genome Activation ◽  
H3 Acetylation ◽  
Drosophila Embryos ◽  
Cell Cycle Kinase ◽  
Zygotic Genome

The histone acetyltransferase Gcn5 is critical for gene expression and development. In Drosophila, Gcn5 is part of four complexes (SAGA, ATAC, CHAT, and ADA) that are essential for fly viability and have key roles in regulating gene expression. Here, we show that while the SAGA, ADA, and CHAT complexes play redundant roles in embryonic gene expression, the insect-specific CHAT complex uniquely regulates expression of a subset of developmental genes. We also identify a substantial decrease in histone acetylation in chiffon mutant embryos that exceeds that observed in ada2b, suggesting broader roles for Chiffon in regulating histone acetylation outside of the Gcn5 complexes. The chiffon gene encodes two independent polypeptides that nucleate formation of either the CHAT or Dbf4-dependent kinase (DDK) complexes. DDK includes the cell cycle kinase Cdc7, which is necessary for maternally-driven DNA replication in the embryo. We identify a temporal switch between the expression of these chiffon gene products during a short window during the early nuclear cycles in embryos that correlates with the onset of zygotic genome activation, suggesting a potential role for CHAT in this process.

Fluorescent potentiometric dyes for membrane-potential imaging

1994 ◽  
Vol 52 ◽  
pp. 166-167
Author(s):  
Leslie M. Loew
Keyword(s):  
Membrane Potential ◽  
Single Cells ◽  
Quantitative Imaging ◽  
Optical Recording ◽  
Biological Processes ◽  
Major Focus ◽  
The Past ◽  
Excitable Systems ◽  
Major Application ◽  
The Impact

A major application of potentiometric dyes has been the multisite optical recording of electrical activity in excitable systems. After being championed by L.B. Cohen and his colleagues for the past 20 years, the impact of this technology is rapidly being felt and is spreading to an increasing number of neuroscience laboratories. A second class of experiments involves using dyes to image membrane potential distributions in single cells by digital imaging microscopy - a major focus of this lab. These studies usually do not require the temporal resolution of multisite optical recording, being primarily focussed on slow cell biological processes, and therefore can achieve much higher spatial resolution. We have developed 2 methods for quantitative imaging of membrane potential. One method uses dual wavelength imaging of membrane-staining dyes and the other uses quantitative 3D imaging of a fluorescent lipophilic cation; the dyes used in each case were synthesized for this purpose in this laboratory.

scholarly journals Role of PFKFB3 and PFKFB4 in Cancer: Genetic Basis, Impact on Disease Development/Progression, and Potential as Therapeutic Targets

Cancers ◽  
2021 ◽  
Vol 13 (4) ◽  
pp. 909
Author(s):  
Krzysztof Kotowski ◽  
Jakub Rosik ◽  
Filip Machaj ◽  
Stanisław Supplitt ◽  
Daniel Wiczew ◽  
...  
Keyword(s):  
Current Knowledge ◽  
Treatment Options ◽  
Protein Structures ◽  
New Drugs ◽  
Proliferating Cells ◽  
Cancer Tissues ◽  
The Impact ◽  
Rate Limiting ◽  
Synthesis And Degradation

Glycolysis is a crucial metabolic process in rapidly proliferating cells such as cancer cells. Phosphofructokinase-1 (PFK-1) is a key rate-limiting enzyme of glycolysis. Its efficiency is allosterically regulated by numerous substances occurring in the cytoplasm. However, the most potent regulator of PFK-1 is fructose-2,6-bisphosphate (F-2,6-BP), the level of which is strongly associated with 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase activity (PFK-2/FBPase-2, PFKFB). PFK-2/FBPase-2 is a bifunctional enzyme responsible for F-2,6-BP synthesis and degradation. Four isozymes of PFKFB (PFKFB1, PFKFB2, PFKFB3, and PFKFB4) have been identified. Alterations in the levels of all PFK-2/FBPase-2 isozymes have been reported in different diseases. However, most recent studies have focused on an increased expression of PFKFB3 and PFKFB4 in cancer tissues and their role in carcinogenesis. In this review, we summarize our current knowledge on all PFKFB genes and protein structures, and emphasize important differences between the isoenzymes, which likely affect their kinase/phosphatase activities. The main focus is on the latest reports in this field of cancer research, and in particular the impact of PFKFB3 and PFKFB4 on tumor progression, metastasis, angiogenesis, and autophagy. We also present the most recent achievements in the development of new drugs targeting these isozymes. Finally, we discuss potential combination therapies using PFKFB3 inhibitors, which may represent important future cancer treatment options.

scholarly journals Glycosphingolipids are required for sorting melanosomal proteins in the Golgi complex

2001 ◽  
Vol 155 (3) ◽  
pp. 369-380 ◽  
Author(s):  
Hein Sprong ◽  
Sophie Degroote ◽  
Tijs Claessens ◽  
Judith van Drunen ◽  
Viola Oorschot ◽  
...  
Keyword(s):  
Golgi Complex ◽  
Protein Transport ◽  
Transmembrane Domain ◽  
Direct Pathway ◽  
Mouse Melanoma Cell ◽  
Multicellular Organisms ◽  
Rate Limiting ◽  
Tyrosinase Related Protein ◽  
Mouse Melanoma Cell Line

A;lthough glycosphingolipids are ubiquitously expressed and essential for multicellular organisms, surprisingly little is known about their intracellular functions. To explore the role of glycosphingolipids in membrane transport, we used the glycosphingolipid-deficient GM95 mouse melanoma cell line. We found that GM95 cells do not make melanin pigment because tyrosinase, the first and rate-limiting enzyme in melanin synthesis, was not targeted to melanosomes but accumulated in the Golgi complex. However, tyrosinase-related protein 1 still reached melanosomal structures via the plasma membrane instead of the direct pathway from the Golgi. Delivery of lysosomal enzymes from the Golgi complex to endosomes was normal, suggesting that this pathway is not affected by the absence of glycosphingolipids. Loss of pigmentation was due to tyrosinase mislocalization, since transfection of tyrosinase with an extended transmembrane domain, which bypassed the transport block, restored pigmentation. Transfection of ceramide glucosyltransferase or addition of glucosylsphingosine restored tyrosinase transport and pigmentation. We conclude that protein transport from Golgi to melanosomes via the direct pathway requires glycosphingolipids.

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