Journal of Molecular Biology
Research ArticleHigh Conformational Flexibility of the E2F1/DP1/DNA Complex
Graphical abstract
Introduction
RNA production is regulated by a plethora of sequence-specific Transcription Factors -TFs- that recognize short DNA sequences in enhancers and promoters of genes, forming modules dictated by the arrangement of the DNA sites. Binding of TFs to regulatory regions leads to the recruitment of cofactors endowed with chromatin remodeling and histone modification activities, which make chromatin accessible for RNA Polymerase processivity.1 TFs typically require a minimum of a sequence-specific DNA-binding domain (DBD) and a Trans-Activation domain (TAD).
E2Fs constitute a family of eight evolutionarily conserved TFs, whose founding member -E2F1- was originally identified as a master regulator of cell-cycle progression.2, 3 In particular, E2F1 is essential for the G1/S transition and S-phase progression4; E2F4, as part of the DREAM complex, is also involved in repression of G2/M genes in G1/S, and cell-cycle exit during terminal differentiation,5 but it is an activator in mES cells.6 In addition to the regulation of cell-cycle genes, E2F1 has a global role in the regulation of apoptosis, the DNA-damage response, metabolism, and differentiation.7 E2F1 is usually catalogued as an activator, but depending on the cellular context, it can be also a repressor in differentiation.8 E2F1, among others, is targeted by the Retinoblastoma -Rb- and by other “pocket” proteins, as part of repressive complexes that include other corepressors: genetic mutations in Rb, as found in tumors, eliminate or weaken interaction, leading to uncontrolled E2F1 activation, cell cycle progression and cell growth.9 Somatic mutations in the E2F1 gene are relatively rare in cancer, but, as many other family members, the gene expression is often upregulated in transformed cells.10
E2Fs bind DNA as heterodimers with the structurally related DP1/2/3 subunits. In addition to DBDs, E2Fs have a coiled-coil domain (CC) involved in dimerization with the partner DPs, a marked-box (MB) domain responsible for the interaction with pocket proteins, and a C-terminal TAD. A similar structural scheme is shared by DP proteins, except that they lack a TAD. The DBD and CC domains are typically connected by a short linker.3 From the structural viewpoint, two parts have been characterized by crystallography: the minimal DBDs of E2F4/DP2, and of E2F8 in complex with “canonical” DNA sites11, 12; the CC-MB domains of E2F1/DP1 and of E2F5/DP1, including in complex with Rb pocket proteins peptides.13, 14 Such structures refer to DBDs separated from CC-MB: a coherent view of the relative topology of the linked domains is not available to date (Figure 1). For this reason, we set out to produce and characterize, both biochemically and structurally, the ensembles of E2F1 and DP1 DBD, CC, and MB domains. In so doing, we provide evidence for a high level of flexibility of the heterodimer, including a newly identified region stabilizing DNA contacts.
Section snippets
The E2F1/DP1/DNA complex displays a spectrum of flexible conformations
The structural hallmarks of E2F and DP proteins consist of a DBD connected through a short linker to the CC-MB domains. As the available 3D structures from distinct E2F/DP family members only refer to the separated domains, to have a global view of the DNA-bound full-length heterodimers, we produced recombinant proteins including the full conserved regions of E2F1 and DP1 (DBD-CC-MB, hereafter E2F1DCM/DP1DCM) (Figure 1(A)). Singularly, DP1DCM was insoluble and E2F1DCM soluble, possibly binding
Discussion
Sequence-specific TFs are proteins with minimally two essential parts: a DNA-binding domain (DBD) and a transcriptional activation domain (TAD). Most of them feature additional parts, which regulate their activities. In general, DBDs are well structured and indeed many of them have been detailed in complex with DNA by 3D studies over the last 30 years; other parts, including TADs, often characterized by a large content of disordered regions,26 remain less understood but are generally thought to
Protein co-expression and purification
E2F1 was co-produced with DP1, both containing the DBD, the linker region, the coiled-coil (CC) heterodimerization domain, and the marked-box (MB) domain (DBD-CC-MB; E2F1DCM/DP1DCM) (Figure 1(A)). Human E2F1 cDNA (a kind gift of L. Vandel)40 encoding residues Ser121-Glu301 (E2F1DCM) was amplified by PCR and subcloned into the pmcnYC vector.41 The cDNA sequence encoding residues Arg105-Thr353 (DP1DCM), or residues Arg111-Thr353 (ΔN-DP1DCM), of human DP1 was amplified by PCR from pCMV-Neo-Bam-Dp1
Research data
The input files used to generate the structural models, as well as the bundle of structures shown in Figure 3, are deposited in PLUMED-NEST63 as plumID:20.029. The SAXS data and the single model shown in Figure 2 are deposited in the SASBDB64 as SASDKH2.
Accession numbers
RefSeq protein IDs: NP_009042 (transcription factor Dp-1 [Homo sapiens]); NP_005216 (transcription factor E2F1 [Homo sapiens]). PDB ID: 2AZE_C (Rb C-terminal domain); PDB ID: 1CF7; PDB ID: 2AZE
CRediT authorship contribution statement
Dana Saad: Investigation, Visualization, Formal analysis. Cristina Paissoni: Investigation, Visualization, Formal analysis. Antonio Chaves-Sanjuan: Investigation, Visualization. Marco Nardini: Conceptualization, Supervision, Resources. Roberto Mantovani: Conceptualization, Supervision, Resources, Funding acquisition. Nerina Gnesutta: Conceptualization, Supervision, Resources, Project administration. Carlo Camilloni: Conceptualization, Supervision, Resources, Project administration.
Acknowledgements
We thank Diamond staff from the B21 beamline and iNEXT (PID: 5912) for financial support. We thank Laurence Vandel, Université Clermont Auvergne (Clermont-Ferrand I, France) for kind gift of plasmids. We acknowledge PRACE for awarding us access to Piz Daint at CSCS, Switzerland.
Funding
This work was supported by Fondazione AIRC per la Ricerca sul Cancro [grant number IG-19050 to R.M.].
Conflict of Interest
Authors declare no conflicts of interest.
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