An evolutionarily conserved DNA architecture determines target specificity of the TWIST family bHLH transcription factors

Chang, Andrew; Liu, Yuanjie; Ayyanathan, Kasirajan; Benner, Chris; Jiang, Yike; Prokop, Jeremy W.; Paz, Helicia; Wang, Dong; Li, Hairi; Fu, Xiang‐Dong; Rauscher, Frank J.; Yang, Jing

doi:10.1101/gad.242842.114

Cited by 70 publications

(78 citation statements)

References 36 publications

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“…While appearing paradoxical, this is necessary to maintain a pool of osteo-PCs that can support balanced rates of osteoblast differentiation. Interestingly, Twist1/E-protein heterodimers have high affinity for tandem e-box elements in the Bmp4 promoter (Chang et al, 2015), and we find that high levels of Twist1 coincide with low and/or undetectable levels of Bmp2/4 expression in osteo-PCs. This suggests that Twist1 maintains an osteoprogenitor state, in part, through the repression of BMP signaling.…”

Section: Discussionmentioning

confidence: 72%

Cerebral Vein Malformations Result from Loss of Twist1 Expression and BMP Signaling from Skull Progenitor Cells and Dura

et al. 2017

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Dural cerebral veins (CV) are required for cerebrospinal fluid reabsorption and brain homeostasis, but mechanisms that regulate their growth and remodeling are unknown. We report molecular and cellular processes that regulate dural CV development in mammals, and describe venous malformations in humans with craniosynostosis and TWIST1 mutations that are recapitulated in mouse models. Surprisingly, Twist1 is dispensable in endothelial cells but required for specification of osteoprogenitor cells that differentiate into preosteoblasts that produce Bone Morphogenetic Proteins. Inactivation of Bmp2 and Bmp4 in preosteoblasts and periosteal dura cause skull and CV malformations, similar to humans harboring TWIST1 mutations. Notably, arterial development appears normal, suggesting morphogens from the skull and dura establish optimal venous networks independent from arterial influences. Collectively, our work establishes a paradigm whereby CV malformations result from primary or secondary loss of paracrine BMP signaling from preosteoblasts and dura, highlighting unique cellular interactions that influence tissue-specific angiogenesis in mammals.

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Section: Discussionmentioning

confidence: 72%

Cerebral Vein Malformations Result from Loss of Twist1 Expression and BMP Signaling from Skull Progenitor Cells and Dura

et al. 2017

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“…4d). These findings suggest that GFP-WR is interacting with TWIST1 via WR-WR binding, an interaction illustrated by recent studies of higher order TWIST1 complexes [17]. In order to determine whether GFP-WR-mediated inhibition of TWIST1-RELA binding impacted downstream signaling, we again employed a dual luciferase assay to quantify IL-8 promoter activity.…”

Section: Resultsmentioning

confidence: 84%

Disruption of TWIST1-RELA binding by mutation and competitive inhibition to validate the TWIST1 WR domain as a therapeutic target

et al. 2017

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BackgroundMost cancer deaths result from tumor cells that have metastasized beyond their tissue of origin, or have developed drug resistance. Across many cancer types, patients with advanced stage disease would benefit from a novel therapy preventing or reversing these changes. To this end, we have investigated the unique WR domain of the transcription factor TWIST1, which has been shown to play a role in driving metastasis and drug resistance.MethodsIn this study, we identified evolutionarily well-conserved residues within the TWIST1 WR domain and used alanine substitution to determine their role in WR domain-mediated protein binding. Co-immunoprecipitation was used to assay binding affinity between TWIST1 and the NFκB subunit p65 (RELA). Biological activity of this complex was assayed using a dual luciferase assay system in which firefly luciferase was driven by the interleukin-8 (IL-8) promoter, which is upregulated by the TWIST1-RELA complex. Finally, in order to inhibit the TWIST1-RELA interaction, we created a fusion protein comprising GFP and the WR domain. Cell fractionation and proteasome inhibition experiments were utilized to elucidate the mechanism of action of the GFP-WR fusion.ResultsWe found that the central residues of the WR domain (W190, R191, E193) were important for TWIST1 binding to RELA, and for increased activation of the IL-8 promoter. We also found that the C-terminal 245 residues of RELA are important for TWIST1 binding and IL-8 promoter activation. Finally, we found the GFP-WR fusion protein antagonized TWIST1-RELA binding and downstream signaling. Co-expression of GFP-WR with TWIST1 and RELA led to proteasomal degradation of TWIST1, which could be inhibited by MG132 treatment.ConclusionsThese data provide evidence that mutation or inhibition of the WR domain reduces TWIST1 activity, and may represent a potential therapeutic modality.Electronic supplementary materialThe online version of this article (doi:10.1186/s12885-017-3169-9) contains supplementary material, which is available to authorized users.

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“…This domain is found in over one-third of Arabidopsis bHLH TF family members (Supplemental Data Set 3). The bHLH transcription factors usually function in dimer or tetramer form or by forming a complex with other TF family members (Ellenberger et al, 1994;Ma et al, 1994;Huffman et al, 2001;Li, 2014;Chang et al, 2015); often the bHLH motif is required for dimerization. In this study, we demonstrated that the plantspecific BIF domain is also important for the dimerization and the normal function of DYT1.…”

Section: The Bif Domain Is Required For Dyt1 Functionmentioning

confidence: 99%

Feedback Regulation of DYT1 by Interactions with Downstream bHLH Factors Promotes DYT1 Nuclear Localization and Anther Development

et al. 2016

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Transcriptional regulation is one of the most important mechanisms controlling development and cellular functions in plants and animals. The Arabidopsis thaliana bHLH transcription factor (TF) DYSFUNCTIONL TAPETUM1 (DYT1) is required for normal male fertility and anther development and activates the expression of the bHLH010/bHLH089/bHLH091 genes. Here, we showed that DYT1 is localized to both the cytoplasm and nucleus at anther stage 5 but specifically to the nucleus at anther stage 6 and onward. The bHLH010/bHLH089/bHLH091 proteins have strong nuclear localization signals, interact with DYT1, and facilitate the nuclear localization of DYT1. We further found that the conserved C-terminal BIF domain of DYT1 is required for its dimerization, nuclear localization, transcriptional activation activity, and function in anther development. Interestingly, when the BIF domain of DYT1 was replaced with that of bHLH010, the DYT1 N -bHLH010 BIF chimeric protein shows nuclearpreferential localization at anther stage 5 but could not fully rescue the dyt1-3 phenotype, suggesting that the normal spatiotemporal subcellular localization of DYT1 is important for DYT1 function and/or that the BIF domains from different bHLH members might be functionally distinct. Our results support an important positive feedback regulatory mechanism whereby downstream TFs increase the function of an upstream TF by enhancing its nucleus localization through the BIF domain.

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An evolutionarily conserved DNA architecture determines target specificity of the TWIST family bHLH transcription factors

Cited by 70 publications

References 36 publications

Cerebral Vein Malformations Result from Loss of Twist1 Expression and BMP Signaling from Skull Progenitor Cells and Dura

Cerebral Vein Malformations Result from Loss of Twist1 Expression and BMP Signaling from Skull Progenitor Cells and Dura

Disruption of TWIST1-RELA binding by mutation and competitive inhibition to validate the TWIST1 WR domain as a therapeutic target

Feedback Regulation of DYT1 by Interactions with Downstream bHLH Factors Promotes DYT1 Nuclear Localization and Anther Development

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