Summary The majority of fast synaptic inhibition in the brain is mediated by benzodiazepine-sensitive, α1 subunit-containing GABAARs; however, our knowledge of the mechanisms neurons use to regulate their synaptic accumulation is rudimentary. Using immunoprecipitation we demonstrate that GABAARs and gephyrin are intimately associated at inhibitory synapses in cultured rat hippocampal neurons. In vitro we reveal that the E-domain of gephyrin directly binds to the α1 subunit with an affinity of ~20 μM, mediated by residues 360-375 within the intracellular domain of this receptor subunit. Mutating residues 360-375 decreases both the accumulation of α1-containing GABAARs at gephyrin-positive inhibitory synapses in hippocampal neurons and the amplitude of miniature inhibitory postsynaptic currents (mIPSCs). We also demonstrate that the affinity of gephyrin for the α1 subunit is modulated by Thr375, a putative phosphorylation site. Mutation of Thr375 to a phospho-mimetic, negatively charged amino acid decreases both the affinity of the α1 subunit for gephyrin, receptor accumulation at synapses and the amplitude of mIPSCs. Finally, single particle tracking reveals that gephyrin reduces the diffusion of α1 subunit-containing GABAARs specifically at inhibitory synapses, thereby increasing their confinement at these structures. Our results suggest that the direct binding of gephyrin to residues 360-375 of the α1 subunit and its modulation are likely to be important determinants for the stabilization of GABAARs at synaptic sites, thereby modulating the strength of synaptic inhibition.

Background: Gephyrin clusters inhibitory GABA A and glycine receptors at postsynaptic sites. Results: GABA A and glycine receptor binding to gephyrin is a mutually exclusive process relying on distantly related sequence motifs. Conclusion: Clustering of GABA A and glycine receptors is mediated by a shared binding site on gephyrin. Significance: Gephyrin-dependent synaptic clustering of chloride-permeable ligand channels at synaptic sites relies on an evolutionarily conserved mechanism.

GABAA receptors are clustered at synaptic sites to achieve a high density of postsynaptic receptors opposite the input axonal terminals. This allows for an efficient propagation of GABA mediated signals, which mostly result in neuronal inhibition. A key organizer for inhibitory synaptic receptors is the 93 kDa protein gephyrin that forms oligomeric superstructures beneath the synaptic area. Gephyrin has long been known to be directly associated with glycine receptor β subunits that mediate synaptic inhibition in the spinal cord. Recently, synaptic GABAA receptors have also been shown to directly interact with gephyrin and interaction sites have been identified and mapped within the intracellular loops of the GABAA receptor α1, α2, and α3 subunits. Gephyrin-binding to GABAA receptors seems to be at least one order of magnitude weaker than to glycine receptors (GlyRs) and most probably is regulated by phosphorylation. Gephyrin not only has a structural function at synaptic sites, but also plays a crucial role in synaptic dynamics and is a platform for multiple protein-protein interactions, bringing receptors, cytoskeletal proteins and downstream signaling proteins into close spatial proximity.

The transcription factor ∆Np63 is a master regulator of epithelial cell identity and essential for the survival of squamous cell carcinoma (SCC) of lung, head and neck, oesophagus, cervix and skin. Here, we report that the deubiquitylase USP28 stabilizes ∆Np63 and maintains elevated ∆NP63 levels in SCC by counteracting its proteasome‐mediated degradation. Impaired USP28 activity, either genetically or pharmacologically, abrogates the transcriptional identity and suppresses growth and survival of human SCC cells. CRISPR/Cas9‐engineered in vivo mouse models establish that endogenous USP28 is strictly required for both induction and maintenance of lung SCC. Our data strongly suggest that targeting ∆Np63 abundance via inhibition of USP28 is a promising strategy for the treatment of SCC tumours.

SummaryMYC proteins bind globally to active promoters and promote transcriptional elongation by RNA polymerase II (Pol II). To identify effector proteins that mediate this function, we performed mass spectrometry on N-MYC complexes in neuroblastoma cells. The analysis shows that N-MYC forms complexes with TFIIIC, TOP2A, and RAD21, a subunit of cohesin. N-MYC and TFIIIC bind to overlapping sites in thousands of Pol II promoters and intergenic regions. TFIIIC promotes association of RAD21 with N-MYC target sites and is required for N-MYC-dependent promoter escape and pause release of Pol II. Aurora-A competes with binding of TFIIIC and RAD21 to N-MYC in vitro and antagonizes association of TOP2A, TFIIIC, and RAD21 with N-MYC during S phase, blocking N-MYC-dependent release of Pol II from the promoter. Inhibition of Aurora-A in S phase restores RAD21 and TFIIIC binding to chromatin and partially restores N-MYC-dependent transcriptional elongation. We propose that complex formation with Aurora-A controls N-MYC function during the cell cycle.

Background: The molecular basis of GABA A receptor ␣3 subtype-specific synaptic localization is unknown. Results: GABA A R ␣3 interacts with the gephyrin E domain via defined intracellular motifs that partially overlap with glycine receptor binding determinants. Conclusion: GABA A R subtypes containing ␣3 are clustered at postsynaptic specializations via direct interactions with gephyrin. Significance: Distinct binding properties of GABA A R and GlyRs to gephyrin may govern mixed glycinergic/GABAergic transmission.

Highlights d MYC enhances productive transcription by defining the protein composition of Pol II d MYC directly binds SPT5 and hands it over to Pol II in a CDK7dependent manner d Transfer of SPT5 increases speed and processivity of Pol II d MYC's effects on Pol II function shape its tumor-specific gene expression profile

Fast inhibitory synaptic transmission is mediated by γ-aminobutyric acid type A receptors (GABAARs) that are enriched at functionally diverse synapses via mechanisms that remain unclear. Using isothermal titration calorimetry and complementary methods we demonstrate an exclusive low micromolar binding of collybistin to the α2-subunit of GABAARs. To explore the biological relevance of collybistin-α2-subunit selectivity, we generate mice with a mutation in the α2-subunit-collybistin binding region (Gabra2-1). The mutation results in loss of a distinct subset of inhibitory synapses and decreased amplitude of inhibitory synaptic currents. Gabra2–1 mice have a striking phenotype characterized by increased susceptibility to seizures and early mortality. Surviving Gabra2-1 mice show anxiety and elevations in electroencephalogram δ power, which are ameliorated by treatment with the α2/α3-selective positive modulator, AZD7325. Taken together, our results demonstrate an α2-subunit selective binding of collybistin, which plays a key role in patterned brain activity, particularly during development.