EPEN-24. YAP1 FUSION PROTEINS MEDIATE ONCOGENIC ACTIVITY IN EPENDYMOMA VIA INTERACTION WITH TEAD TRANSCRIPTION FACTORS

Abstract The transcription factors Oct4 and Sox2 are two of the main regulators of pluripotency in embryonic stem cells. Since the importance of non-genetic modification is continually increasing, particularly for therapeutic application of manipulated cells, the aim of the present study was to generate cell-permeant Oct4 and Sox2 proteins for the direct cellular delivery of active proteins. Protein transduction allowing cellular manipulation to circumvent genetic modification of target cells has recently been developed. We present a new expression vector system, pSESAME, that facilitates the generation of transducible proteins. Using pSESAME, both Oct4 and Sox2 were genetically fused with a TAT protein transduction domain that promotes cellular penetration. The recombinant purified Oct4 and Sox2 fusion proteins display DNA-binding properties comparable to their endogenous counterparts, and exhibit cellular entry and the ability to modulate the transcriptional machinery maintaining pluripotency of mouse embryonic stem cells. In a rescue assay we demonstrate that transducible Oct4 and Sox2 fusion proteins can compensate knockdown of Pou5f1 and Sox2, respectively. This study provides powerful tools for the modulation of stem cell properties without genetic interference.

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TAZ-CAMTA1 and YAP-TFE3 alter the TAZ/YAP transcriptome by recruiting the ATAC histone acetyltransferase complex

eLife ◽

10.7554/elife.62857 ◽

2021 ◽

Vol 10 ◽

Author(s):

Nicole Merritt ◽

Keith Garcia ◽

Dushyandi Rajendran ◽

Zhen-Yuan Lin ◽

Xiaomeng Zhang ◽

...

Keyword(s):

Transcription Factors ◽

Cell Lines ◽

Fusion Proteins ◽

Histone Acetyltransferase ◽

Clinical Course ◽

Epithelioid Hemangioendothelioma ◽

Complex Interaction ◽

Transcriptional Program ◽

Oncogenic Driver ◽

Generation Sequencing

Epithelioid hemangioendothelioma (EHE) is a vascular sarcoma that metastasizes early in its clinical course and lacks an effective medical therapy. The TAZ-CAMTA1 and YAP-TFE3 fusion proteins are chimeric transcription factors and initiating oncogenic drivers of EHE. A combined proteomic/genetic screen in human cell lines identified YEATS2 and ZZZ3, components of the Ada2a-containing histone acetyltransferase (ATAC) complex, as key interactors of both fusion proteins despite the dissimilarity of the C terminal fusion partners CAMTA1 and TFE3. Integrative next generation sequencing approaches in human and murine cell lines showed that the fusion proteins drive a unique transcriptome by simultaneously hyperactivating a TEAD-based transcriptional program and modulating the chromatin environment via interaction with the ATAC complex. Interaction of the ATAC complex with both fusion proteins indicates that it is a key oncogenic driver and unifying enzymatic therapeutic target for this sarcoma. This study presents an approach to mechanistically dissect how chimeric transcription factors drive the formation of human cancers.

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Hoxa9 and Meis1 Are Key Targets for MLL-ENL-Mediated Cellular Immortalization

Molecular and Cellular Biology ◽

10.1128/mcb.24.2.617-628.2004 ◽

2004 ◽

Vol 24 (2) ◽

pp. 617-628 ◽

Cited By ~ 229

Author(s):

Bernd B. Zeisig ◽

Tom Milne ◽

María-Paz García-Cuéllar ◽

Silke Schreiner ◽

Mary-Ellen Martin ◽

...

Keyword(s):

Transcription Factors ◽

Microarray Analysis ◽

Fusion Proteins ◽

Transformed Cells ◽

Myeloid Differentiation ◽

Continuous Growth ◽

Cellular Immortalization ◽

Proliferation And Differentiation ◽

Myeloid Leukemias ◽

Transforming Activity

ABSTRACT MLL fusion proteins are oncogenic transcription factors that are associated with aggressive lymphoid and myeloid leukemias. We constructed an inducible MLL fusion, MLL-ENL-ERtm, that rendered the transcriptional and transforming properties of MLL-ENL strictly dependent on the presence of 4-hydroxy-tamoxifen. MLL-ENL-ERtm-immortalized hematopoietic cells required 4-hydroxy-tamoxifen for continuous growth and differentiated terminally upon tamoxifen withdrawal. Microarray analysis performed on these conditionally transformed cells revealed Hoxa9 and Hoxa7 as well as the Hox coregulators Meis1 and Pbx3 among the targets upregulated by MLL-ENL-ERtm. Overexpression of the Hox repressor Bmi-1 inhibited the growth-transforming activity of MLL-ENL. Moreover, the enforced expression of Hoxa9 in combination with Meis1 was sufficient to substitute for MLL-ENL-ERtm function and to maintain a state of continuous proliferation and differentiation arrest. These results suggest that MLL fusion proteins impose a reversible block on myeloid differentiation through aberrant activation of a limited set of homeobox genes and Hox coregulators that are consistently expressed in MLL-associated leukemias.

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Inhibition of CRM1-Mediated Nuclear Export of Transcription FACTORS by Leukemogenic NUP98 Fusion Proteins.

Blood ◽

10.1182/blood.v114.22.1959.1959 ◽

2009 ◽

Vol 114 (22) ◽

pp. 1959-1959

Author(s):

Akiko Takeda ◽

Anmaar M Abdul-Nabi ◽

Nabeel R. Yaseen

Keyword(s):

Transcription Factors ◽

Nuclear Export ◽

Fusion Proteins ◽

Conflicts Of Interest ◽

Nuclear Accumulation ◽

Human Leukemia ◽

Dependent Manner ◽

Nucleocytoplasmic Trafficking ◽

Nuclear Retention

Abstract Abstract 1959 Poster Board I-982 NUP98 is a nucleoporin that plays complex roles in the nucleocytoplasmic trafficking of macromolecules. Rearrangements of the NUP98 gene in human leukemia result in the expression of numerous fusion oncoproteins whose effect on nucleocytoplasmic trafficking is poorly understood. The present study was undertaken to determine the effects of leukemogenic NUP98 fusion proteins on CRM1-mediated nuclear export. NUP98-HOXA9, a prototypic NUP98 fusion, inhibited the nuclear export of two known CRM1 substrates: mutated cytoplasmic nucleophosmin (NPMc) and HIV-1 Rev. In-vitro binding assays revealed that NUP98-HOXA9 binds CRM1 through the FG-repeat motif in a Ran-GTP dependent manner similar to but stronger than the interaction between CRM1 and its export substrates. Two NUP98 fusions, NUP98-HOXA9 and NUP98-DDX10, whose fusion partners are structurally and functionally unrelated, interacted with endogenous CRM1 in cells as shown by co-immunoprecipitation. These leukemogenic NUP98 fusion proteins interacted with CRM1, Ran, and the nucleoporin NUP214 in a manner fundamentally different from that of wild-type NUP98. NUP98-HOXA9 and NUP98-DDX10 formed characteristic aggregates within the nuclei of myeloid cells and caused aberrant localization of CRM1 to these aggregates. These NUP98 fusions caused nuclear accumulation of two transcription factors, NFAT and NFΚB, that are regulated by CRM1-mediated export. The nuclear entrapment of NFAT and NFΚB correlated with enhanced transcription from promoters responsive to these transcription factors. Taken together, the results suggest a new mechanism by which NUP98 fusions dysregulate transcription and cause leukemia, namely, inhibition of CRM1-mediated nuclear export with aberrant nuclear retention of transcriptional regulators. Disclosures: No relevant conflicts of interest to declare.

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The Acute Promyelocytic Leukemia (APL)-Associated Fusion Proteins PML/RAR and PLZF/RAR Directly Bind to and Inhibit GATA-1.

Blood ◽

10.1182/blood.v108.11.1929.1929 ◽

2006 ◽

Vol 108 (11) ◽

pp. 1929-1929

Author(s):

Anita Seshire ◽

Claudia Oancea ◽

Dieter Hoelzer ◽

Martin Ruthardt ◽

Elena Puccetti

Keyword(s):

Transcription Factors ◽

Fusion Proteins ◽

Binding Capacity ◽

Globin Gene ◽

Direct Interaction ◽

Erythroid Differentiation ◽

Beta Globin ◽

Hematopoietic Stem ◽

All Trans Retinoic Acid

Abstract The pathogenesis of acute myeloid leukemia (AML) is strictly related to a block of terminal differentiation. The APL is a well characterized subtype of AML, which is related in the 95% of the cases by the presence of the t(15;17) and in 2% by the presence of t(11;17). In several cell models the resulting PML/RAR and PLZF/RAR fusion proteins (X-RAR) recapitulate the leukemic phenotype by inducing a state of refractoriness to various inducers of myeloid differentiation. Accordingly, expression in animal models of both PML/RAR and PLZF/RAR leads to the development of leukemia. The treatment with all trans retinoic acid (t-RA) is able to overcome the block of differentiation of PML/RARα- but not that of PLZF/RAR-positive-blasts. These fusion proteins block differentiation through several mechanisms such as aberrant chromatin modeling by aberrant recruitment of histone deacetylase activity or the deregulation of differentiation-relevant transcription factors such as PU.1, VDR or C/EBPalpha. The deregulated function of these transcription factors can be due to their transcriptional down-regulation or to a sequester by direct interaction. Nothing is known about how the X-RAR block erythroid and megacaryocytic differentiation. Therefore we investigated whether and how the X-RAR interfere with the functionality of the differentiation-relevant transcription factor GATA-1. It has been recently reported that the lack of GATA-1 severely impairs erythroid differentiation and contributes to the accumulation of immature megakaryocytic blasts. He we report thatGATA-1 directly interacts with X-RAR in vivo as revealed by co-immunoprecipitation and mammalian two hybrid assays;GATA-1 expression was not transcriptionally deregulated by the X-RAR;the GATA-1 binding capacity to the H2S beta-globin locus was severely inhibited by the presence of the X-RAR as revealed by ChIP experiments in K562, whereas the transactivation of the GATA-target promotor alpha-IIb was not impaired by the the X-RAR in classical transient promoter studies;treatment with t-RA restored GATA-1 binding to the H2S locus of the beta-globin gene;the overexpression of GATA-1 in the presence of EPO reduced the the colony forming units of PLZF/RAR-positive Sca1+/lin− hematopoietic stem cells (HSC) and diminished the replating efficiency of PML/RAR-positive HSC, but did not increase erythroid differentiation monitored by TER 119 expression. Taken together our data demonstrate that the X-RAR interfere with the functionality of GATA-1 by direct interaction with GATA-1. It remains to definitively clarify whether the X-RAR inhibit the access of GATA-1 to its target promoters or whether they interfere with the accessibility of the GATA-1 for transcriptional co-activators. The fact that the overexpression of GATA-1 did not increase differentiation of HSC and the X-RAR were unable to inhibit the transactivation of a GATA-1 target promoter strongly suggests that the X-RAR interfere with the GATA-1 functionality in the context of the chromatin.

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FUS Oncofusion Protein Condensates Recruit mSWI/SNF Chromatin Remodelers via Heterotypic Interactions Between Prion-like Domains

10.1101/2021.04.23.440992 ◽

2021 ◽

Author(s):

Richoo B. Davis ◽

Taranpreet Kaur ◽

Mahdi Muhammad Moosa ◽

Priya R. Banerjee

Keyword(s):

Transcription Factors ◽

Chromatin Remodeling ◽

Single Molecule ◽

Fusion Proteins ◽

Dna Binding Domain ◽

Chromatin Remodelers ◽

Transcriptional Reprogramming ◽

Essential Components ◽

Heterotypic Interactions ◽

Transcription Regulators

AbstractFusion transcription factors generated by genomic translocations are common drivers of several types of cancers including sarcomas and leukemias. Oncofusions of the FET (FUS, EWSR1, and TAF15) family of proteins result from fusion of the prion-like domain (PLD) of FET proteins to the DNA-binding domain (DBD) of certain transcription regulators and are implicated in aberrant transcriptional programs through interactions with chromatin remodelers. Here, we show that FUS-DDIT3, a FET oncofusion protein, undergoes PLD-mediated phase separation into liquid-like condensates. Nuclear FUS-DDIT3 condensates can recruit essential components of the global transcriptional machinery such as the chromatin remodeler SWI/SNF. The recruitment of mammalian SWI/SNF is driven by heterotypic PLD-PLD interactions between FUS-DDIT3 and core subunits of SWI/SNF, such as the catalytic component BRG1. Further experiments with single-molecule correlative force-fluorescence microscopy support a model wherein the fusion protein forms condensates on DNA surface and enrich BRG1 to activate transcription by ectopic chromatin remodeling. Similar PLD-driven co-condensation of mSWI/SNF with transcription factors can be employed by other oncogenic fusion proteins with a generic PLD-DBD domain architecture for global transcriptional reprogramming.

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Recurrent evolution of vertebrate transcription factors by transposase capture

10.1101/2020.05.07.082677 ◽

2020 ◽

Author(s):

Rachel L. Cosby ◽

Julius Judd ◽

Ruiling Zhang ◽

Alan Zhong ◽

Nathaniel Garry ◽

...

Keyword(s):

Transcription Factors ◽

Fusion Proteins ◽

Fusion Gene ◽

Regulatory Elements ◽

Exon Shuffling ◽

Protein Coding ◽

Regulatory Domains ◽

Dna Binding Domains ◽

Binding Domains ◽

Cellular Genes

AbstractHow genes with novel cellular functions evolve is a central biological question. Exon shuffling is one mechanism to assemble new protein architectures. Here we show that DNA transposons, which are mobile and pervasive in genomes, have provided a recurrent supply of exons and splice sites to assemble protein-coding genes in vertebrates via exon-shuffling. We find that transposase domains have been captured, primarily via alternative splicing, to form new fusion proteins at least 94 times independently over ∼350 million years of tetrapod evolution. Evolution favors fusion of transposase DNA-binding domains to host regulatory domains, especially the Krüppel-associated Box (KRAB), suggesting transposase capture frequently yields new transcriptional repressors. We show that four independently evolved KRAB-transposase fusion proteins repress gene expression in a sequence-specific fashion. Genetic knockout and rescue of the bat-specific KRABINER fusion gene in cells demonstrates that it binds its cognate transposons genome-wide and controls a vast network of genes and cis-regulatory elements. These results illustrate a powerful mechanism by which a transcription factor and its dispersed binding sites emerge at once from a transposon family.One Sentence SummaryHost-transposase fusion generates novel cellular genes, including deeply conserved and lineage specific transcription factors.

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