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Evidence of pioneer factor activity of an oncogenic fusion transcription factor.

ABSTRACT: Recent characterizations of pioneer transcription factors provide insights into their structures and patterns of chromatin recognition associated with their roles in cell fate commitment and transformation. Intersecting with these basic science concepts, identification of pioneer factors (PFs) fused together as driver translocations in childhood cancers raises questions of whether these fusions retain the fundamental ability to invade repressed chromatin, consistent with their monomeric PF constituents. This study defines the cellular and chromatin localization of PAX3-FOXO1, an oncogenic driver of childhood rhabdomyosarcoma (RMS), derived from a fusion of PFs. To quantitatively define its chromatin-targeting functions and capacity to drive epigenetic reprogramming, we developed a ChIP-seq workflow with per-cell normalization (pc-ChIP-seq). Our quantitative localization studies address structural variation in RMS genomes and reveal insights into inactive chromatin localization of PAX3-FOXO1. Taken together, our studies are consistent with pioneer function for a driver oncoprotein in RMS, with repressed chromatin binding and nucleosome-motif targeting.

SUBMITTER: Sunkel BD

PROVIDER: S-EPMC8346656 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Pioneer activity of an oncogenic fusion transcription factor at inaccessible chromatin

Project description:We developed a new method for ChIP-seq with per-cell normalization (pc-ChIP-seq) to define PAX3-FOXO1 localization in rhabdomyosarcoma (RMS). We report novel pioneer function for the major driver oncogene in RMS, with nucleosome-motif targeting and kinetic displacement of nucleosomes in human cells.

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Nucleosome allostery in pioneer transcription factor binding.

Project description:While recent experiments revealed that some pioneer transcription factors (TFs) can bind to their target DNA sequences inside a nucleosome, the binding dynamics of their target recognitions are poorly understood. Here we used the latest coarse-grained models and molecular dynamics simulations to study the nucleosome-binding procedure of the two pioneer TFs, Sox2 and Oct4. In the simulations for a strongly positioning nucleosome, Sox2 selected its target DNA sequence only when the target was exposed. Otherwise, Sox2 entropically bound to the dyad region nonspecifically. In contrast, Oct4 plastically bound on the nucleosome mainly in two ways. First, the two POU domains of Oct4 separately bound to the two parallel gyres of the nucleosomal DNA, supporting the previous experimental results of the partial motif recognition. Second, the POUS domain of Oct4 favored binding on the acidic patch of histones. Then, simulating the TFs binding to a genomic nucleosome, the LIN28B nucleosome, we found that the recognition of a pseudo motif by Sox2 induced the local DNA bending and shifted the population of the rotational position of the nucleosomal DNA. The redistributed DNA phase, in turn, changed the accessibility of a distant TF binding site, which consequently affected the binding probability of a second Sox2 or Oct4. These results revealed a nucleosomal DNA-mediated allosteric mechanism, through which one TF binding event can change the global conformation, and effectively regulate the binding of another TF at distant sites. Our simulations provide insights into the binding mechanism of single and multiple TFs on the nucleosome.

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Histone modifications regulate pioneer transcription factor cooperativity.

Project description:Pioneer transcription factors have the ability to access DNA in compacted chromatin1. Multiple transcription factors can bind together to a regulatory element in a cooperative way, and cooperation between the pioneer transcription factors OCT4 (also known as POU5F1) and SOX2 is important for pluripotency and reprogramming2-4. However, the molecular mechanisms by which pioneer transcription factors function and cooperate on chromatin remain unclear. Here we present cryo-electron microscopy structures of human OCT4 bound to a nucleosome containing human LIN28B or nMATN1 DNA sequences, both of which bear multiple binding sites for OCT4. Our structural and biochemistry data reveal that binding of OCT4 induces changes to the nucleosome structure, repositions the nucleosomal DNA and facilitates cooperative binding of additional OCT4 and of SOX2 to their internal binding sites. The flexible activation domain of OCT4 contacts the N-terminal tail of histone H4, altering its conformation and thus promoting chromatin decompaction. Moreover, the DNA-binding domain of OCT4 engages with the N-terminal tail of histone H3, and post-translational modifications at H3K27 modulate DNA positioning and affect transcription factor cooperativity. Thus, our findings suggest that the epigenetic landscape could regulate OCT4 activity to ensure proper cell programming.

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Stepwise modifications of transcriptional hubs link pioneer factor activity to a burst of transcription.

Project description:Binding of transcription factors (TFs) promotes the subsequent recruitment of coactivators and preinitiation complexes to initiate eukaryotic transcription, but this time course is usually not visualized. It is commonly assumed that recruited factors eventually co-reside in a higher-order structure, allowing distantly bound TFs to activate transcription at core promoters. We use live imaging of endogenously tagged proteins, including the pioneer TF Zelda, the coactivator dBrd4, and RNA polymerase II (RNAPII), to define a cascade of events upstream of transcriptional initiation in early Drosophila embryos. These factors are sequentially and transiently recruited to discrete clusters during activation of non-histone genes. Zelda and the acetyltransferase dCBP nucleate dBrd4 clusters, which then trigger pre-transcriptional clustering of RNAPII. Subsequent transcriptional elongation disperses clusters of dBrd4 and RNAPII. Our results suggest that activation of transcription by eukaryotic TFs involves a succession of distinct biomolecular condensates that culminates in a self-limiting burst of transcription.

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Nucleosome binding by the pioneer transcription factor OCT4.

Project description:Transcription factor binding to genomic DNA is generally prevented by nucleosome formation, in which the DNA is tightly wrapped around the histone octamer. In contrast, pioneer transcription factors efficiently bind their target DNA sequences within the nucleosome. OCT4 has been identified as a pioneer transcription factor required for stem cell pluripotency. To study the nucleosome binding by OCT4, we prepared human OCT4 as a recombinant protein, and biochemically analyzed its interactions with the nucleosome containing a natural OCT4 target, the LIN28B distal enhancer DNA sequence, which contains three potential OCT4 target sequences. By a combination of chemical mapping and cryo-electron microscopy single-particle analysis, we mapped the positions of the three target sequences within the nucleosome. A mutational analysis revealed that OCT4 preferentially binds its target DNA sequence located near the entry/exit site of the nucleosome. Crosslinking mass spectrometry consistently showed that OCT4 binds the nucleosome in the proximity of the histone H3 N-terminal region, which is close to the entry/exit site of the nucleosome. We also found that the linker histone H1 competes with OCT4 for the nucleosome binding. These findings provide important information for understanding the molecular mechanism by which OCT4 binds its target DNA in chromatin.

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CLOCK:BMAL1 is a pioneer-like transcription factor.

Project description:The mammalian circadian clock relies on the master genes CLOCK and BMAL1 to drive rhythmic gene expression and regulate biological functions under circadian control. Here we show that rhythmic CLOCK:BMAL1 DNA binding promotes rhythmic chromatin opening. Mechanisms include CLOCK:BMAL1 binding to nucleosomes and rhythmic chromatin modification; e.g., incorporation of the histone variant H2A.Z. This rhythmic chromatin remodeling mediates the rhythmic binding of other transcription factors adjacent to CLOCK:BMAL1, suggesting that the activity of these other transcription factors contributes to the genome-wide CLOCK:BMAL1 heterogeneous transcriptional output. These data therefore indicate that the clock regulation of transcription relies on the rhythmic regulation of chromatin accessibility and suggest that the concept of pioneer function extends to acute gene regulation.

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Histone modifications regulate pioneer transcription factor binding and cooperativity.

Project description:Pioneer transcription factors have the ability to access DNA in compacted chromatin. Multiple transcription factors can bind together to a regulatory element in a cooperative way and cooperation between pioneer transcription factors Oct4 and Sox2 is important for pluripotency and reprogramming. However, the molecular mechanisms by which pioneer transcription factors function and cooperate remain unclear. Here we present cryo-EM structures of human Oct4 bound to a nucleosome containing human Lin28B and nMatn1 DNA sequences, which bear multiple binding sites for Oct4. Our structural and biochemistry data reveal that Oct4 binding induces changes to the nucleosome structure, repositions the nucleosomal DNA and facilitates cooperative binding of additional Oct4 and of Sox2 to their internal binding sites. The flexible activation domain of Oct4 contacts the histone H4 N-terminal tail, altering its conformation and thus promoting chromatin decompaction. Moreover, the DNA binding domain of Oct4 engages with histone H3 N-terminal tail, and posttranslational modifications at H3K27 modulate DNA positioning and affect transcription factor cooperativity. Thus, our data show that the epigenetic landscape can regulate Oct4 activity to ensure proper cell reprogramming.

| S-EPMC10055048 | biostudies-literature

Interaction of the pioneer transcription factor GATA3 with nucleosomes.

Project description:During cellular reprogramming, the pioneer transcription factor GATA3 binds chromatin, and in a context-dependent manner directs local chromatin remodeling and enhancer formation. Here, we use high-resolution nucleosome mapping in human cells to explore the impact of the position of GATA motifs on the surface of nucleosomes on productive enhancer formation, finding productivity correlates with binding sites located near the nucleosomal dyad axis. Biochemical experiments with model nucleosomes demonstrate sufficiently stable transcription factor-nucleosome interaction to empower cryo-electron microscopy structure determination of the complex at 3.15 Å resolution. The GATA3 zinc fingers efficiently bind their target 5'-GAT-3' sequences in the nucleosome when they are located in solvent accessible, consecutive major grooves without significant changes in nucleosome structure. Analysis of genomic loci bound by GATA3 during reprogramming suggests a correlation of recognition motif sequence and spacing that may distinguish productivity of new enhancer formation.

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RNA helicase A activity is inhibited by oncogenic transcription factor EWS-FLI1.

Project description:RNA helicases impact RNA structure and metabolism from transcription through translation, in part through protein interactions with transcription factors. However, there is limited knowledge on the role of transcription factor influence upon helicase activity. RNA helicase A (RHA) is a DExH-box RNA helicase that plays multiple roles in cellular biology, some functions requiring its activity as a helicase while others as a protein scaffold. The oncogenic transcription factor EWS-FLI1 requires RHA to enable Ewing sarcoma (ES) oncogenesis and growth; a small molecule, YK-4-279 disrupts this complex in cells. Our current study investigates the effect of EWS-FLI1 upon RHA helicase activity. We found that EWS-FLI1 reduces RHA helicase activity in a dose-dependent manner without affecting intrinsic ATPase activity; however, the RHA kinetics indicated a complex model. Using separated enantiomers, only (S)-YK-4-279 reverses the EWS-FLI1 inhibition of RHA helicase activity. We report a novel RNA binding property of EWS-FLI1 leading us to discover that YK-4-279 inhibition of RHA binding to EWS-FLI1 altered the RNA binding profile of both proteins. We conclude that EWS-FLI1 modulates RHA helicase activity causing changes in overall transcriptome processing. These findings could lead to both enhanced understanding of oncogenesis and provide targets for therapy.

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