Biology subjects

Leen, E.

Publications and source records attributed to Leen, E..

3 recordsLinked to original sources

bioRxiv2024

Mechanism of Interaction Between the Transactivation Domain of N-MYC and the DNA-Binding Surface of TFIIIC5

N-myc is a member of the myc family of transcription factors, which are powerful drivers of cellular growth and consequently, important oncoproteins. N-myc interacts with many factors and complexes to affect transcription. One such complex is the RNA Polymerase III assembly factor, TFIIIC, a six-member complex that is essential for the transcription of small, structured RNA. TFIIIC and N-myc mutually restrict each others chromatin association, and their complex contributes to quality control in mRNA transcription. We previously demonstrated that the largely intrinsically disordered transactivation domain of N-myc interacts directly with a sub-complex of TFIIIC, {tau}A. Structural studies by others show that DNA binding of {tau}A is largely mediated by TFIIIC3, which suggests that TFIIIC5 is at most a secondary binding site for DNA. Here we identified the DNA binding domain of TFIIIC5 as a key binding site for N-myc. We used an integrated approach combining NMR, HDX mass spectrometry, pull-downs and biophysical assays to elucidate the molecular basis of the interaction. Two sequences in the transactivation domain of N-myc bind to the DNA binding interface of TFIIIC5. AlphaFold modelling predicts a high-confidence binding mode for the higher affinity N-myc motif that overlaps with the predicted intramolecular binding site of the C-terminal acidic plug of TFIIIC5, removal of which enhances the binding of N-myc. The same two motifs in N-myc also interact with Aurora-A kinase, which competes with N-myc for TFIIIC binding during S-phase. This model elucidates how the N-myc:TFIIIC5 interaction competes with other interactions, providing a basis for their mutual censoring function and regulation. GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=125 SRC="FIGDIR/small/619198v1_ufig1.gif" ALT="Figure 1"> View larger version (33K): org.highwire.dtl.DTLVardef@1e4a76aorg.highwire.dtl.DTLVardef@ab349corg.highwire.dtl.DTLVardef@af85dorg.highwire.dtl.DTLVardef@37dd2f_HPS_FORMAT_FIGEXP M_FIG C_FIG

molecular biology↗

bioRxiv2024

Exploring the dynamics and interactions of the N-myc transactivation domain through solution NMR

The myc family of proteins (c-, N- and L-myc) are transcription factors (TFs) responsible for maintaining the proliferative program in cells. They consist of a C-terminal domain that mediates heterodimerisation with Max and DNA binding, and an N-terminal disordered region culminating in the transactivation domain (TAD). The TAD participates in many protein-protein interactions, notably with kinases that promote stability (Aurora-A) or degradation (ERK1, GSK3) via the ubiquitin-proteasome system. Structural characterization of the TAD of N-myc, is very limited, with the exception of a crystal structure of Aurora-A bound to a helical region of N-myc. We probed the structure, dynamics and interactions of N-myc TAD using nuclear magnetic resonance (NMR) spectroscopy following its complete backbone assignment enabled by a truncation approach. Chemical shift analysis revealed that N-myc has two regions with clear helical propensity: one region within Trp77-Glu86 and the second between Ala122-Glu132. These regions also have more restricted ps-ns motions than the rest of the TAD, and, along with another known interaction site (myc box I), have comparatively high transverse (R2) 15N relaxation rates, indicative of slower timescale dynamics and/or chemical exchange. Collectively these features suggest differential propensities for structure and interaction, either internal or with binding partners, across the TAD. Solution studies on the interaction between N-myc and Aurora-A revealed a previously uncharacterised binding site. The specificity and kinetics of sequential phosphorylation of N-myc by ERK1 and GSK3 were characterised using NMR and showed no significant structural changes through the rest of the TAD. When doubly phosphorylated on residues Ser62 and Thr58, N-myc formed a robust interaction with the Fbxw7-Skp1 complex. Our study provides foundational insights into N-myc TAD dynamics and a backbone assignment that will underpin future work on the structure, dynamics, interactions and regulatory post-translational modifications of this key oncoprotein.

biophysics↗

bioRxiv2023

Association with TFIIIC limits MYCN localization in hubs of active promoters and chromatin accumulation of non-phosphorylated RNA Polymerase II

MYC family oncoproteins regulate the expression of a large number of genes and broadly stimulate elongation by RNA polymerase II. While the factors that control the chromatin association of MYC proteins are well understood, much less is known about how interacting proteins mediate MYCs effects on transcription. Here we show that TFIIIC, an architectural protein complex that controls the three-dimensional chromatin organization at its target sites, binds directly to the amino-terminal transcriptional regulatory domain of MYCN. Surprisingly, TFIIIC has no discernible role in MYCN-dependent gene expression and transcription elongation. Instead, MYCN and TFIIIC preferentially bind to promoters with paused RNAPII and globally limit the accumulation of non-phosphorylated RNAPII at promoters. Consistent with its ubiquitous role in transcription, MYCN broadly participates in hubs of active promoters. Depletion of TFIIIC further increases MYCN localization to these hubs. This increase correlates with a failure of the nuclear exosome and BRCA1, both of which are involved in nascent RNA degradation, to localize to active promoters. Our data suggest that MYCN and TFIIIC exert an censoring function in early transcription that limits promoter accumulation of inactive RNAPII and facilitates promoter-proximal degradation of nascent RNA.

molecular biology↗