Unlocking Tn3-family transposase activity in vitro unveils an asymetric pathway for transposome assembly.

The Tn3 family is a widespread group of replicative transposons that are notorious for their contribution to the dissemination of antibiotic resistance and the emergence of multiresistant pathogens worldwide. The TnpA transposase of these elements catalyzes DNA breakage and rejoining reactions required for transposition. It also is responsible for target immunity, a phenomenon that prevents multiple insertions of the transposon into the same genomic region. However, the molecular mechanisms whereby TnpA acts in both processes remain unknown. Here, we have developed sensitive biochemical assays for the TnpA transposase of the Tn3-family transposon Tn4430 and used these assays to characterize previously isolated TnpA mutants that are selectively affected in immunity. Compared with wild-type TnpA, these mutants exhibit deregulated activities. They spontaneously assemble a unique asymmetric synaptic complex in which one TnpA molecule simultaneously binds two transposon ends. In this complex, TnpA is in an activated state competent for DNA cleavage and strand transfer. Wild-type TnpA can form this complex only on precleaved ends mimicking the initial step of transposition. The data suggest that transposition is controlled at an early stage of transpososome assembly, before DNA cleavage, and that mutations affecting immunity have unlocked TnpA by stabilizing the protein in a monomeric activated synaptic configuration. We propose an asymmetric pathway for coupling active transpososome assembly with proper target recruitment and discuss this model with respect to possible immunity mechanisms.

HOXA1 binds RBCK1/HOIL-1 and TRAF2 and modulates the TNF/NF-κB pathway in a transcription-independent manner.

HOX proteins define a family of key transcription factors regulating animal embryogenesis. HOX genes have also been linked to oncogenesis and HOXA1 has been described to be active in several cancers, including breast cancer. Through a proteome-wide interaction screening, we previously identified the TNFR-associated proteins RBCK1/HOIL-1 and TRAF2 as HOXA1 interactors suggesting that HOXA1 is functionally linked to the TNF/NF-κB signaling pathway. Here, we reveal a strong positive correlation between expression of HOXA1 and of members of the TNF/NF-κB pathway in breast tumor datasets. Functionally, we demonstrate that HOXA1 can activate NF-κB and operates upstream of the NF-κB inhibitor IκB. Consistently, we next demonstrate that the HOXA1-mediated activation of NF-κB is non-transcriptional and that RBCK1 and TRAF2 influences on NF-κB are epistatic to HOXA1. We also identify an 11 Histidine repeat and the homeodomain of HOXA1 to be required both for RBCK1 and TRAF2 interaction and NF-κB stimulation. Finally, we highlight that activation of NF-κB is crucial for HOXA1 oncogenic activity.

KPC2 relocalizes HOXA2 to the cytoplasm and decreases its transcriptional activity.

Regulation of transcription factor activity relies on molecular interactions or enzymatic modifications which influence their interaction with DNA cis-regulatory sequences, their transcriptional activation or repression, and stability or intracellular distribution of these proteins. Regarding the well-conserved Hox protein family, a restricted number of activity regulators have been highlighted thus far. In the framework of a proteome-wide screening aiming at identifying proteins interacting with Hoxa2, KPC2, an adapter protein constitutive of the KPC ubiquitin-ligase complex, was identified. In this work, KPC2 was confirmed as being a genuine interactor of Hoxa2 by co-precipitation and bimolecular fluorescence complementation assays. At functional level, KPC2 diminishes the transcriptional activity and induces the nuclear exit of Hoxa2. Gene expression analyses revealed that Kpc2 is active in restricted areas of the developing mouse embryo which overlap with the Hoxa2 expression domain. Together, our data support that KPC2 regulates Hoxa2 by promoting its relocation to the cytoplasm.

Post-translational modifications of HOX proteins, an underestimated issue.

Post-translational modifications (PTMs) are important determinants which contribute to modulating the turn-over, intracellular localisation, molecular interactions and activity of most eukaryotic proteins. Such modifications and their consequences have been extensively examined for some proteins or classes of proteins. This is not the case for the HOX transcription factors which are crucial regulators of animal development. In this review, we provide a survey of the literature and data repositories pertaining to HOX-associated PTMs. This highlights that HOX proteins are also likely widely post-translationally modified, and defines HOX PTMs as an under-valued facet of their biology.

PRDM14, a putative histone methyl-transferase, interacts with and decreases the stability and activity of the HOXA1 transcription factor.

Understanding how the activity of transcription factors like HOX proteins is regulated remains a widely open question. In a recent screen for proteins interacting with HOXA1, we identified a PRDM protein family member, PRDM14, which is known to be transiently co-expressed with HOXA1 in epiblast cells before their specification towards somatic versus germ cell fate. Here, we confirm PRDM14 is an interactor of HOXA1 and we identify the homeodomain of HOXA1 as well as the PR domain and Zinc fingers of PRDM14 to be required for the interaction. An 11-His repeat of HOXA1 previously highlighted to contribute to HOXA1-mediated protein-protein interactions is also involved. At a functional level, we provide evidence that HOXA1 displays an unexpectedly long half-life and demonstrate that PRDM14 can reduce the stability and affect the transcriptional activity of HOXA1.

The O-GlcNAc transferase OGT interacts with and post-translationally modifies the transcription factor HOXA1.

HOXA1 belongs to the HOX family of transcription factors which are key regulators of animal development. Little is known about the molecular pathways controlling HOXA1. Recent data from our group revealed distinct partner proteins interacting with HOXA1. Among them, OGT is an O-linked N-acetylglucosamine (O-GlcNAc) transferase modifying a variety of proteins involved in different cellular processes including transcription. Here, we confirm OGT as a HOXA1 interactor, we characterise which domains of HOXA1 and OGT are required for the interaction, and we provide evidence that OGT post-translationally modifies HOXA1. Mass spectrometry experiments indeed reveal that HOXA1 can be phosphorylated on the AGGTVGSPQYIHHSY peptide and that upon OGT expression, the phosphate adduct is replaced by an O-GlcNAc group.