Ecdysone-Induced 3D Chromatin Reorganization Involves Active Enhancers Bound by Pipsqueak and Polycomb

Gutiérrez-Pérez, Irene; Rowley, M. Jordan; Lyu, Xiaowen; Valadéz-Graham, Viviana; Vallejo, Diana M.; Ballesta-Illán, Esther; López‐Atalaya, José P.; Kremsky, Isaac; Caparrós, Esther; Corcés, Victor G.; Domı́nguez, Marı́a

doi:10.1016/j.celrep.2019.07.096

Cited by 38 publications

(44 citation statements)

References 61 publications

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“…We found a complete absence of CP190 binding at the STARR-Seq peaks demonstrating that observed CP190-bound EcR sites are not the 20E-dependent enhancers. This result was in agreement with a recent study in Drosophila Kc cells, where CP190 binding was found at EcR sites which are not enriched with enhancer markers H3K9me1, H3K27Ac, and CBP 31 .…”

Section: Cp190 Is Present At Only a Portion Of Ecr-bound Sites In Thesupporting

confidence: 93%

“…This is well correlated with a previous time-course analysis of CP190 binding in Kc cells (3-hour treatment with 20E), where it was shown that CP190 participates in stabilization of a basic regulatory hub at 20E-dependent genes in a hormone-independent manner 29 . The mode of CP190 action during 20E-dependent induction is likely to maintain a complex 3D structure at the loci enabling proper interactions between enhancers and promoters of genes, which appears to be transient and signal-dependent 31 . To illustrate all the possible ways of CP190 action at the 20E-dependent loci we provide a model of an average 20E-dependent gene (Fig.…”

Section: Discussionmentioning

confidence: 99%

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Proximity-dependent biotin labelling reveals CP190 as an EcR/Usp molecular partner

Mazina

Ziganshin

Magnitov

et al. 2020

Sci Rep

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proximity-dependent biotin labelling revealed undescribed participants of the ecdysone response in Drosophila. Two labelling enzymes (BioID2 and APEX2) were fused to EcR or Usp to biotin label the surrounding proteins. the ecR/Usp heterodimer was found to collaborate with nuclear pore subunits, chromatin remodelers, and architectural proteins. Many proteins identified through proximity-dependent labelling with ecR/Usp were described previously as functional components of an ecdysone response, corroborating the potency of this labelling method. A link to ecdysone response was confirmed for some newly discovered regulators by immunoprecipitation of prepupal nuclear extract with anti-ecR antibodies and functional experiments in Drosophila S2 cells. A more in-depth study was conducted to clarify the association of EcR/Usp with one of the detected proteins, CP190, a well-described cofactor of Drosophila insulators. CP190 was found to co-immunoprecipitate with the EcR subunit of EcR/Usp in a 20E-independent manner. ChIP-Seq experiments revealed only partial overlapping between CP190 and EcR bound sites in the Drosophila genome and complete absence of CP190 binding at 20E-dependent enhancers. Analysis of Hi-C data demonstrated an existence of remote interactions between 20E-dependent enhancers and CP190 sites which suggests formation of a protein complex between EcR/Usp and CP190 through the space. Our results support the previous concept that CP190 has a role in stabilization of specific chromatin loops for proper activation of transcription of genes regulated by 20E hormone.www.nature.com/scientificreports www.nature.com/scientificreports/ rate (e.g., promoter-bound transcriptional complexes). Recently, an additional directed mutagenesis procedure of BirA has allowed for the development of the fast TurboID enzyme 7 .An alternative to the BioID2 technology is a combination of APEX2, a plant ascorbate peroxidase, and a biotin-phenol substrate. In the presence of hydrogen peroxide, the APEX2 enzyme can catalyse the substrate conversion (biotin-phenol) into active and short-lived radicals which can modify the surrounding proteins 3 . Although the method is less common than the BioID technique, APEX2 labelling requires significantly less time to biotinylate surrounding proteins, making it more suited for researching unstable complexes 8,9 . For example, it was recently integrated with the CRISPR/Cas9 system to investigate DNA-protein interactions, as an alternative to ChIP-Seq 10 .The main research interest of our group is to accurately describe the molecular mechanisms of transcriptional activation in eukaryotes. The ultimate goal is to understand the general mechanism for most genes, including the cooperative function of hundreds of transcriptional factors. As an experimental approach we use Drosophila melanogaster developmental genes (e.g., 20-hydroxyecdysone (20E)-dependent), which are a well-regulated physiological system that is convenient to study 11,12 . The main molecular sensor of an ecdysone system is the EcR/Us...

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Section: Cp190 Is Present At Only a Portion Of Ecr-bound Sites In Thesupporting

confidence: 93%

Section: Discussionmentioning

confidence: 99%

Proximity-dependent biotin labelling reveals CP190 as an EcR/Usp molecular partner

Mazina

Ziganshin

Magnitov

et al. 2020

Sci Rep

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“…S2C). We then tested if anchors of loops identified by SIP were enriched in proteins previously found to be important for looping in Drosophila (Eagen et al 2017;Ogiyama et al 2018;Gutierrez-Perez et al 2019). Indeed, Polycomb (Pc) and pipsqueak (Psq) are enriched on SIP loop anchors (Supplemental Fig.…”

Section: Sip and Sipmeta Allow Identification And Visualization Of Lomentioning

confidence: 99%

Analysis of Hi-C data using SIP effectively identifies loops in organisms from C. elegans to mammals

et al. 2020

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Chromatin loops are a major component of 3D nuclear organization, visually apparent as intense point-to-point interactions in Hi-C maps. Identification of these loops is a critical part of most Hi-C analyses. However, current methods often miss visually evident CTCF loops in Hi-C data sets from mammals, and they completely fail to identify high intensity loops in other organisms. We present SIP, Significant Interaction Peak caller, and SIPMeta, which are platform independent programs to identify and characterize these loops in a time- and memory-efficient manner. We show that SIP is resistant to noise and sequencing depth, and can be used to detect loops that were previously missed in human cells as well as loops in other organisms. SIPMeta corrects for a common visualization artifact by accounting for Manhattan distance to create average plots of Hi-C and HiChIP data. We then demonstrate that the use of SIP and SIPMeta can lead to biological insights by characterizing the contribution of several transcription factors to CTCF loop stability in human cells. We also annotate loops associated with the SMC component of the dosage compensation complex (DCC) in Caenorhabditis elegans and demonstrate that loop anchors represent bidirectional blocks for symmetrical loop extrusion. This is in contrast to the asymmetrical extrusion until unidirectional blockage by CTCF that is presumed to occur in mammals. Using HiChIP and multiway ligation events, we then show that DCC loops form a network of strong interactions that may contribute to X Chromosome–wide condensation in C. elegans hermaphrodites.

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“…Psq is essential for oogenesis (Horowitz and Berg 1996), and it has been suggested to have a role as a repressor and in chromatin looping (Gutierrez-Perez et al 2019;Huang et al 2002).…”

Section: Introductionmentioning

confidence: 99%

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

Gaskill

Gibson

Larson

et al. 2021

eLife

101

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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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Ecdysone-Induced 3D Chromatin Reorganization Involves Active Enhancers Bound by Pipsqueak and Polycomb

Cited by 38 publications

References 61 publications

Proximity-dependent biotin labelling reveals CP190 as an EcR/Usp molecular partner

Proximity-dependent biotin labelling reveals CP190 as an EcR/Usp molecular partner

Analysis of Hi-C data using SIP effectively identifies loops in organisms from C. elegans to mammals

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

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