Complexes of Transcription as Pharmacological Targets

Head : Monique Erard

 

Our main objective has been the structural study of the dysregulation of genetic expression by transcription complexes involved in cellular or viral proliferation. By combining molecular modelling with biochemistry and molecular biology methods, we have identified structural determinants of critical proteinnucleic acid interactions which are potential therapeutic targets.

Keyplayers in the genesis of neuroendocrine tumours and melanoma

N-Oct-3, the human equivalent of the mouse Brn‐2 (‘Brain‐2’) protein, is a neuronal transcription factor widely expressed in the developing central nervous system, and necessary to maintain neural cell differentiation. Any imbalance in N‐Oct‐3 expression has critical consequences. Whilst the deletion of the Brn-2 gene results in the loss of specific neuronal lineages in the endocrine hypothalamus, N-Oct-3 overexpression in melanocytes, cells derived from the neural crests, leads to tumorigenesis via the dysregulation of a number of genes. This key role for N‐Oct‐3 is the consequence of the structural plasticity of its DNA‐binding domain (DBD), which comprises two highly conserved sub‐domains, termed ‘POUs’ and ‘POUh’, connected by a flexible linker. All the POU domains bind specifically to the prototypic octamer ATGCAAAT; POUs interacting with the tetramer ATGC in a similar manner to the phage repressors, and POUh with the tretramer AAAT in a similar manner to homeodomains. Despite the robustness of the fit between the POU domain and the octamer sequence, most POU domains can also recognize various other AT‐rich sequences due to the flexibility of the linker joining the two sub‐domains. Another important functional implication of the structural plasticity of the POU domain is the possibility of different patterns of homo‐ and heterodimerization. Based on the crystallographic structures of various Pit‐1 or Oct‐1 POU/DNA complexes, two major types of POU homodimerization with palindromic or pseudo‐palindromic DNA targets have been described; the “PORE-” (Palindromic Oct-1 Responsive Element) and “MORE- ”(More palindromic Oct‐1 Responsive Element) induced modes.

Transcriptional dysregulation and human immunodeficiency virus, HIV1

Expression of the HIV RNA genome first requires retro‐transcription and then integration into the host cell genome, thereby leading to transcriptional dysregulation. The viral transactivator Tat binds to TAR, an RNA structure present at the 5’ termini of all viral messengers, which serves as an adapter to contact the components of the transcriptional machinery. In cotransfection assays, the TAR RNA‐binding protein, TRBP, was shown to act synergistically with Tat to stimulate HIV‐1 promoter expression. Since TRBP can also bind RRE, the objective of our study has been to investigate how to target the interactions between HIV TAR and RRE RNAs and their respective viral partners, Tat and Rev, as well as their common cellular ligand, TRBP (Blaud, Manival et al., 2009). This project has been performed in collaboration with David Barker (LIPM, INRA, Toulouse) and Anne Gatignol (McGill AIDS Centre, Montreal).

We had previously delineated the minimal 15‐residue KR‐helix motif ‘TR13’ in the C‐terminus of the second TRBP double‐stranded RNA‐binding domain which is necessary and sufficient to bind to the high affinity upper‐stem/loop site of TAR. We now demonstrate that TRBP dsRBD2 and TR13 exert opposite effects on TAR structure, leading to backbone opening or closing respectively (studied by a combination of circular dichroism and flexible docking). A Lys‐to‐Arg mutation in TR13 (the TR33 peptide) induces an even more closed structure, incapable of binding either Tat or TRBP. TR33 also elicits a closed RRE structure. We have thus identified a key peptide with the potential to inhibit both transcriptional transactivation and RNA splicing.

Macromolecular flexibility and docking

The results described above emphasize the necessity to employ a structural reading of nucleic acid regulatory sequences and to integrate information about protein flexibility when predicting functional structures. In collaboration with the team directed by Thierry Siméon (LAAS, Toulouse) within the framework of an ITAV (Institut des Technologies Avancées en sciences du Vivant) contract, we have focused on approaches which address the critical issue of the indirect readout of promoter DNA sequences, and on emerging concepts and methods which explore protein flexibility and allostery. By combining molecular dynamics with the use of modified nucleotides, we have recently explained why the octamer sequence can be read in two different ways by POU proteins (Blaud et al., 2009). Although the coupling of molecular mechanics with SAXS experiments has turned out to be a very powerful tool to address the question of protein plasticity (Alazard et al., 2007; Roblin et al., 2008, in collaboration with Lionel’s Mourey’s team), the main limitation is the time‐consuming “torsion driving”. Robotics‐based methods now offer efficient alternatives to explore conformational space (Cortes et al., 2009).

Predictive methods have an important role to play in the functional identification and structural characterization of transcriptional networks. Since transcription factor families are generally specified by highly conserved consensus DNA‐binding domains as well as common strategies of interaction with target DNA, homology modeling is a particularly relevant approach. Equally, the prepositioning of a DBD within its DNA binding site can often be inferred by homology, a step that most docking programs cannot yet address ab initio. Such an approach based on molecular mimicry has been applied to the biologically significant [IL- 33]-nucleosome complex (Roussel et al., 2008).