Structural and Functional Studies of the RBPJ-SHARP Complex Reveal Conserved Corepressor Binding Site

Yuan, Zhenyu; VanderWielen, Bradley D.; Giaimo, Benedetto Daniele; Pan, Leiling; Collins, Courtney; Turkiewicz, Aleksandra; Hein, Kerstin; Oswald, Franz; Borggrefe, Tilman; Kovall, Rhett A.

doi:10.2139/ssrn.3195335

Cited by 11 publications

(18 citation statements)

References 48 publications

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“…Here we also show that doxorubicin treatment of cardiomyocytes alters the activity of RBPJ, an important transcriptional regulator of NOTCH 79 . Additionally, since TWIST, HEY, and HES proteins all belong to the basic helix-loop-helix (bHLH) transcription factors family 80,81 , it seems reasonable to hypothesize that the co-regulation of these TFs with NRF2 may be related to the 60 amino acid region which contains two highly conserved domains, considered as involved in interactions with TFs 82 .…”

Section: Discussionsupporting

confidence: 54%

NRF2-related transcriptomic alterations as a long-term effect of doxorubicin treatment for childhood acute lymphoblastic leukemia

Totoń‐Żurańska¹,

Sulicka-Grodzicka²,

Seweryn³

et al. 2021

Preprint

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The use of doxorubicin is associated with an increased risk of acute and long-term cardiomyopathy. Despite that the number of cancer survivors is growing constantly, little is known about the transcriptional mechanisms which progress in time leading to severe cardiac outcome. It is also unclear whether long-term transcriptomic alterations are related to an acute response to doxorubicin. We have sequenced miRNA from total plasma and extracellular vesicles (EVs) from 66 acute lymphoblastic leukemia (ALL) survivors treated with doxorubicin and 61 healthy controls (254 samples in total). We identified 94 and 33 TFs regulating differentially expressed miRNA in plasma and EVs compartments, respectively. For total plasma we found: HEY1, NRF2, HIF1A, NOTCH1, and for EVs: TGFB, ZEB1, ASCL2, PELP1, SIP1, TWIST1. Analysis of the data from patients with dilated and idiopathic cardiomyopathy revealed similarities with our data from EVs, especially in the activity of TFs related to epithelial-to-mesenchymal transition (EMT). To verify if similarities exist between acute and long-term response to doxorubicin, we performed experiments on cultured as well as we studied the role of NRF2, previously considered as an important player in acute response, in transcriptomic network upon doxorubicin treatment). KEGG analysis of miRNA targets for EVs indicates development of cardiomyopathy, whereas among GO process we found terms related to cardiac septum. In vitro experiments revealed that NRF2 is co-regulated with NOTCH effectors from HEY family, with known role in muscle regeneration. NRF2 and doxorubicin treatment contribute also to the dysregulation of TWIST family, important for the process of EMT.

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Section: Discussionsupporting

confidence: 54%

NRF2-related transcriptomic alterations as a long-term effect of doxorubicin treatment for childhood acute lymphoblastic leukemia

Totoń‐Żurańska¹,

Sulicka-Grodzicka²,

Seweryn³

et al. 2021

Preprint

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“…Furthermore, the technique of MBP-mediated crystallization enabled us to simply solve the crystallographic phase problem by the MR method using the MBP structure as a template and even to determine the high-resolution structure. Other than AtBIL1/BZR1, four structures of nucleic acid-bound proteins have been revealed by MBP fusion crystallographic systems [9][10][11]13 . Moreover, there are no successful structural analyses of nucleic acid-bound proteins with other fusion crystallographic systems including thioredoxin (Trx)-or glutathione S-transferase (GST)-fusion.…”

Section: Discussionmentioning

confidence: 99%

Highlighting the potential utility of MBP crystallization chaperone for Arabidopsis BIL1/BZR1 transcription factor-DNA complex

Nosaki

Terada

Nakamura

et al. 2021

Sci Rep

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The maltose-binding protein (MBP) fusion tag is one of the most commonly utilized crystallization chaperones for proteins of interest. Recently, this MBP-mediated crystallization technique was adapted to Arabidopsis thaliana (At) BRZ-INSENSITIVE-LONG (BIL1)/BRASSINAZOLE-RESISTANT (BZR1), a member of the plant-specific BZR TFs, and revealed the first structure of AtBIL1/BZR1 in complex with target DNA. However, it is unclear how the fused MBP affects the structural features of the AtBIL1/BZR1-DNA complex. In the present study, we highlight the potential utility of the MBP crystallization chaperone by comparing it with the crystallization of unfused AtBIL1/BZR1 in complex with DNA. Furthermore, we assessed the validity of the MBP-fused AtBIL1/BZR1-DNA structure by performing detailed dissection of crystal packings and molecular dynamics (MD) simulations with the removal of the MBP chaperone. Our MD simulations define the structural basis underlying the AtBIL1/BZR1-DNA assembly and DNA binding specificity by AtBIL1/BZR1. The methodology employed in this study, the combination of MBP-mediated crystallization and MD simulation, demonstrates promising capabilities in deciphering the protein-DNA recognition code.

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“…S1c), suggesting that L3MBTL2 interacts exclusively with N-terminal part of RBPJ. This finding was unexpected since most of the interactions of RBPJ with other corepressors like KyoT2 or RITA are within BTD, or like SHARP, with both BTD, and CTD of RBPJ (29,(31)(32)(33)(34). Together, RBPJ and L3MBTL2 strongly interact confirming the unbiased initial mass spectrometric results.…”

Section: Dna-bound Rbpj Is Associated With Several Prc16 Componentsmentioning

confidence: 99%

SUMOylated Non-canonical Polycomb PRC1.6 Complex as a Prerequisite for Recruitment of Transcription Factor RBPJ

Sotomska

Liefke

Ferrante

et al. 2021

Preprint

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BackgroundNotch signaling controls cell fate decisions in many contexts during development and adult stem cell homeostasis and, when dysregulated, leads to carcinogenesis. The central transcription factor RBPJ assembles the Notch coactivator complex in the presence of Notch signalling, and represses Notch target gene expression in its absence.ResultsWe identified L3MBTL2 and additional members of the non-canonical polycomb repressive PRC1.6 complex in DNA-bound RBPJ associated complexes and demonstrate that L3MBTL2 directly interacts with RBPJ. Depletion of RBPJ does not affect occupancy of PRC1.6 components at Notch target genes. Conversely, absence of L3MBTL2 reduces RBPJ occupancy at enhancers of Notch target genes. Since L3MBTL2 and additional members of the PRC1.6 are known to be SUMOylated, we investigated whether RBPJ uses SUMO-moieties as contact points. Indeed, we found that RBPJ binds to SUMO2/3 and that this interaction depends on a defined SUMO-interaction motif. Furthermore, we show that pharmacological inhibition of SUMOylation reduces RBPJ occupancy at Notch target genes.ConclusionsWe propose that the PRC1.6 complex and its conjugated SUMO-modifications provide a scaffold that is recognized by RBPJ and promotes its recruitment to Notch target genes.

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Structural and Functional Studies of the RBPJ-SHARP Complex Reveal Conserved Corepressor Binding Site

Cited by 11 publications

References 48 publications

NRF2-related transcriptomic alterations as a long-term effect of doxorubicin treatment for childhood acute lymphoblastic leukemia

NRF2-related transcriptomic alterations as a long-term effect of doxorubicin treatment for childhood acute lymphoblastic leukemia

Highlighting the potential utility of MBP crystallization chaperone for Arabidopsis BIL1/BZR1 transcription factor-DNA complex

SUMOylated Non-canonical Polycomb PRC1.6 Complex as a Prerequisite for Recruitment of Transcription Factor RBPJ

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