An Extended DNA Binding Domain of the Estrogen Receptor Alpha Directly Interacts with RNAs <i>in Vitro</i>

Steiner, Halley R.; Lammer, Nickolaus C.; Batey, Robert T.; Wuttke, Deborah S.

doi:10.1021/acs.biochem.2c00536

Cited by 16 publications

(30 citation statements)

References 35 publications

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“…Accumulating evidence suggests that RNA-binding activity is common among chromatin-associated proteins (35–39). As discussed above, our findings provide mechanistic evidence for a robust RNA-mediated regulatory function of one chromatin-modifying protein, PRC2.…”

Section: Discussionmentioning

confidence: 99%

PRC2 direct transfer from G-quadruplex RNA to dsDNA: Implications for RNA-binding chromatin modifiers

Hemphill

Fenske

Gooding

et al. 2022

Preprint

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The chromatin-modifying enzyme, Polycomb Repressive Complex 2 (PRC2), deposits the H3K27me3 epigenetic mark to negatively regulate expression at numerous target genes, and this activity has been implicated in embryonic development, cell differentiation, and various cancers. A biological role for RNA binding in regulating PRC2 histone methyltransferase activity is generally accepted, but the nature and mechanism of this relationship remains an area of active investigation. Notably, manyin vitrostudies demonstrate that RNA inhibits PRC2 activity on nucleosomes through mutually antagonistic binding, while somein vivostudies indicate that PRC2’s RNA-binding activity is critical for facilitating its biological function(s). Here we use biochemical, biophysical, and computational approaches to interrogate PRC2’s RNA and DNA binding kinetics. Our findings demonstrate that PRC2-polynucleotide dissociation rates are dependent on the concentration of free ligand, indicating the potential for direct transfer between ligands without a free-enzyme intermediate. Direct transfer explains the variation in dissociation kinetics reported previously, allows reconciliation of priorin vitroandin vivostudies, and expands the potential mechanisms of RNA-mediated PRC2 regulation. Moreover, simulations indicate that such a direct transfer mechanism could be obligatory for RNA to recruit proteins to chromatin.SignificanceStudies of PRC2in vitroindicate that RNA inhibits its histone methyltransferase (HMTase) activity through mutually antagonistic binding with nucleosomes, but somein vivostudies paradoxically suggest that RNA binding is necessary to facilitate chromatin occupancy and HMTase activity. Our findings unveil a protein-intrinsic mechanism for directly exchanging RNA and DNA/nucleosome in PRC2’s binding site(s), which reconciles these prior findings by allowing antagonistic or synergistic RNA-mediated regulation dependent on RNA-nucleosome proximity. Furthermore, there is an increasing awareness that multiple chromatin-associated proteins exhibit regulatory RNA binding activity, and our findings indicate this “direct transfer” mechanism may be generally required for such regulation.

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Section: Discussionmentioning

confidence: 99%

PRC2 direct transfer from G-quadruplex RNA to dsDNA: Implications for RNA-binding chromatin modifiers

Hemphill

Fenske

Gooding

et al. 2022

Preprint

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“…Among the several RNA molecules that ERα may bind to, there are also non-coding RNAs and particularly lncRNAs [ 17 ], whose implications in BC progression and induction were previously demonstrated. Recently, the ERα-lncRNA interaction was further investigated from a biochemical point of view, identifying the amino acid region of ERα as most likely involved in the direct association with RNA molecules [ 144 ]. This adds another level of complexity to the already complicated regulation of estrogen signaling by ERα.…”

Section: Discussionmentioning

confidence: 99%

Functional Relationships between Long Non-Coding RNAs and Estrogen Receptor Alpha: A New Frontier in Hormone-Responsive Breast Cancer Management

Melone

Salvati

Brusco

et al. 2023

IJMS

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In the complex and articulated machinery of the human genome, less than 2% of the transcriptome encodes for proteins, while at least 75% is actively transcribed into non-coding RNAs (ncRNAs). Among the non-coding transcripts, those ≥200 nucleotides long (lncRNAs) are receiving growing attention for their involvement in human diseases, particularly cancer. Genomic studies have revealed the multiplicity of processes, including neoplastic transformation and tumor progression, in which lncRNAs are involved by regulating gene expression at epigenetic, transcriptional, and post-transcriptional levels by mechanism(s) that still need to be clarified. In breast cancer, several lncRNAs were identified and demonstrated to have either oncogenic or tumor-suppressive roles. The functional understanding of the mechanisms of lncRNA action in this disease could represent a potential for translational applications, as these molecules may serve as novel biomarkers of clinical use and potential therapeutic targets. This review highlights the relationship between lncRNAs and the principal hallmark of the luminal breast cancer phenotype, estrogen receptor α (ERα), providing an overview of new potential ways to inhibit estrogenic signaling via this nuclear receptor toward escaping resistance to endocrine therapy.

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“…vector. Protein expression and purification methods were described previously (Steiner et al 2022). Starting with a single transformed colony of BL21(DE3)pLysS E. coli, expression cultures were grown at 37°C (with 50 μg/mL kanamycin and 50 μg/mL chloramphenicol) using 2x YT rich media to an OD600 of 0.8-1.0 and cold shocked on ice for 20 min.…”

Section: Protein Expression and Purificationmentioning

confidence: 99%

Transcription factors ERα and Sox2 have differing multiphasic DNA and RNA binding mechanisms

Hemphill,

Steiner,

Kominsky

et al. 2024

Preprint

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Many transcription factors (TFs) have been shown to bind RNA, leading to open questions regarding the mechanism(s) of this RNA binding and its role in regulating TF activities. Here we use biophysical assays to interrogate the kon, koff, and Kd for DNA and RNA binding of two model human transcription factors, ERα and Sox2. Unexpectedly, we found that both proteins exhibited multiphasic nucleic acid binding kinetics. We propose that Sox2 RNA and DNA multiphasic binding kinetics could be explained by a conventional model for sequential Sox2 monomer association and dissociation. In contrast, ERα nucleic acid binding exhibited biphasic dissociation paired with novel triphasic association behavior, where two apparent binding transitions are separated by a 10-20 min "lag" phase depending on protein concentration. We considered several conventional models for the observed kinetic behavior, none of which adequately explained all the ERα nucleic acid binding data. Instead, simulations with a model incorporating sequential ERα monomer association, ERα nucleic acid complex isomerization, and product "feedback" on isomerization rate recapitulated the general kinetic trends for both ERα DNA and RNA binding. Collectively, our findings reveal that Sox2 and ERα bind RNA and DNA with previously unappreciated multiphasic binding kinetics, and that their reaction mechanisms differ with ERα binding nucleic acids via a novel reaction mechanism.

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An Extended DNA Binding Domain of the Estrogen Receptor Alpha Directly Interacts with RNAs in Vitro

Cited by 16 publications

References 35 publications

PRC2 direct transfer from G-quadruplex RNA to dsDNA: Implications for RNA-binding chromatin modifiers

PRC2 direct transfer from G-quadruplex RNA to dsDNA: Implications for RNA-binding chromatin modifiers

Functional Relationships between Long Non-Coding RNAs and Estrogen Receptor Alpha: A New Frontier in Hormone-Responsive Breast Cancer Management

Transcription factors ERα and Sox2 have differing multiphasic DNA and RNA binding mechanisms

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