Neurons of the peripheral nervous system have long been known to require survival factors to prevent their death during development. But why they selectively become dependent on secretory molecules has remained a mystery, as is the observation that in the central nervous system, most neurons do not show this dependency. Using engineered embryonic stem cells, we show here that the neurotrophin receptors TrkA and TrkC (tropomyosin receptor kinase A and C, also known as Ntrk1 and Ntrk3, respectively) instruct developing neurons to die, both in vitro and in vivo. By contrast, TrkB (also known as Ntrk2), a closely related receptor primarily expressed in the central nervous system, does not. These results indicate that TrkA and TrkC behave as dependence receptors, explaining why developing sympathetic and sensory neurons become trophic-factor-dependent for survival. We suggest that the expansion of the Trk gene family that accompanied the segregation of the peripheral from the central nervous system generated a novel mechanism of cell number control.

ATP-dependent chromatin remodeling activities function to manipulate chromatin structure during gene regulation. One of the ways in which they do this is by altering the positions of nucleosomes along DNA. Here we provide support for the ability of these complexes to move nucleosomes into positions in which DNA is unraveled from one edge. This is expected to result in the loss of histone-DNA contacts that are important for retention of one H2A/H2B dimer within the nucleosome. Consistent with this we find that several chromatin remodeling complexes are capable of catalyzing the exchange of H2A/H2B dimers between chromatin fragments in an ATP-dependent reaction. This provides eukaryotes with additional means by which they may manipulate chromatin structure.

Kapinos et al. show that nuclear pore complex permeability and cargo release functionalities are concomitantly regulated by karyopherin occupancy and turnover in a systematic continuum. This highlights increasingly important roles for the soluble nucleocytoplasmic transport machinery that depart from established views of the nuclear pore complex selectivity mechanism.

Previous studies have identified sin mutations that alleviate the requirement for the yeast SWI/SNF chromatin remodelling complex, which include point changes in the yeast genes encoding core histones. Here we characterise the biochemical properties of nucleosomes bearing these mutations. We find that sin mutant nucleosomes have a high inherent thermal mobility. As the SWI/SNF complex can alter nucleosome positioning, the higher mobility of sin mutant nucleosomes provides a means by which sin mutations may substitute for SWI/SNF function. The location of sin mutations also provides a new opportunity for insights into the mechanism for nucleosome mobilisation. We find that both mutations altering histone DNA contacts at the nucleosome dyad and mutations in the dimertetramer interface influence nucleosome mobility. Furthermore, incorporation of H2A.Z into nucleosomes, which also alters dimer-tetramer interactions, affects nucleosome mobility. Thus, variation of histone sequence or subtype provides a means by which eukaryotes may regulate access to chromatin through alterations to nucleosome mobility. The EMBO Journal (2004) IntroductionNucleosomes are the universal molecular packaging state of DNA in nuclei. They are responsible for compacting eukaryotic genomes to allow them to fit into the limited volume of the cell nucleus. The nucleosome core particle and an additional variable length of unbound linker DNA together comprise the fundamental repeating unit of chromatin (Kornberg, 1974). The nucleosome core particle consists of a core of eight polypeptides, two copies each of the four highly conserved histone proteins H2A, H2B, H3 and H4, around which 147 bp of DNA are wrapped in 1.7 superhelical turns (Luger et al, 1997). H3 and H4 associate to form histone fold dimers as do histones H2A and H2B. Each histone fold dimer associates to form an octameric spiral with dyad symmetry.The H2AÀH2B histone fold dimer units are less stably associated within the octamer than the two H3ÀH4 histone fold dimers (Eickbush and Moudrianakis, 1978). A number of extra protein elements decorate the regular spiral of histone fold dimers, including unstructured 'tails' that extend outside the DNA superhelix, the additional H3 aN helix that organises the most exterior turn of bound DNA, a structured H2A C-terminal extension that passes over the top face of the histone octamer, and an H2B aC helix lying above the histone dimer (Luger and Richmond, 1998).A consequence of the organisation of DNA into nucleosomes is that all genetic processes must contend with a chromatin substrate. In the case of gene regulation, wrapping into nucleosomes makes DNA sequences differentially accessible to transcription factors (Owen-Hughes and Workman, 1994). Accessibility of a particular site depends on the absolute 'position' of the nucleosome (Polach and Widom, 1995), and many cases have been recognised in which nucleosome positioning affects genomic accessibility (Simpson, 1990;Lomvardas and Thanos, 2002;Miller and Widom, 2003). In this way, nucleoso...

Nuclear pore complexes (NPCs) discriminate nonspecific macromolecules from importin and exportin receptors, collectively termed “karyopherins” (Kaps), that mediate nucleocytoplasmic transport. This selective barrier function is attributed to the behavior of intrinsically disordered phenylalanine-glycine nucleoporins (FG Nups) that guard the NPC channel. However, NPCs in vivo are typically enriched with different Kaps, and how they impact the NPC barrier remains unknown. Here, we show that two major Kaps, importinβ1/karyopherinβ1 (Kapβ1) and exportin 1/chromosomal maintenance 1 (CRM1), are required to fortify NPC barrier function in vivo. Their enrichment at the NPC is sustained by promiscuous binding interactions with the FG Nups, which enable CRM1 to compensate for the loss of Kapβ1 as a means to maintain NPC barrier function. However, such a compensatory mechanism is constrained by the cellular abundances and different binding kinetics for each respective Kap, as evidenced for importin-5. Consequently, we find that NPC malfunction and nucleocytoplasmic leakage result from poor Kap enrichment.

Ran is a small GTPase whose nucleotide-bound forms cycle through nuclear pore complexes (NPCs) to direct nucleocytoplasmic transport (NCT). Generally, Ran guanosine triphosphate (RanGTP) binds cargocarrying karyopherin receptors (Kaps) in the nucleus and releases them into the cytoplasm following hydrolysis to Ran guanosine diphosphate (RanGDP). This generates a remarkably steep Ran gradient across the nuclear envelope that sustains compartment-specific cargo delivery and accumulation. However, because NPCs are permeable to small molecules of comparable size, it is unclear how an uncontrolled mixing of RanGTP and RanGDP is prevented. Here, we find that an NPCenriched pool of karyopherin subunit beta 1 (KPNB1, hereafter referred to as Kapβ1) selectively mediates Ran diffusion across the pore but not passive molecules of similar size (e.g. GFP). This is due to RanGTP having a stronger binding interaction with Kapβ1 than RanGDP. For this reason, the RanGDP importer, nuclear transport factor 2, facilitates the return of RanGDP into the nucleus following GTP hydrolysis. Accordingly, the enrichment of Kapβ1 at NPCs may function as a retention mechanism that preserves the sharp transition of RanGTP and RanGDP in the nucleus and cytoplasm, respectively.

The Polycomb Repressive Complex 2 (PRC2) plays important roles in the epigenetic regulation of cellular development and differentiation through H3K27me3-dependent transcriptional repression. Aberrant PRC2 activity has been associated with cancer and neurodevelopmental disorders, particularly with respect to the malfunction of sits catalytic subunit EZH2. Here, we investigated the role of the EZH2-mediated H3K27me3 apposition in neuronal differentiation. We made use of a transgenic mouse model harboring Ezh2 conditional KO alleles to derive embryonic stem cells and differentiate them into glutamatergic neurons. Time course transcriptomics and epigenomic analyses of H3K27me3 in absence of EZH2 revealed a significant dysregulation of molecular networks affecting the glutamatergic differentiation trajectory that resulted in: (i) the deregulation of transcriptional circuitries related to neuronal differentiation and synaptic plasticity, in particular LTD, as a direct effect of EZH2 loss and (ii) the appearance of a GABAergic gene expression signature during glutamatergic neuron differentiation. These results expand the knowledge about the molecular pathways targeted by Polycomb during glutamatergic neuron differentiation.

Fetal akinesia deformation sequence (FADS) represents the severest form of congenital myasthenic syndrome (CMS), a diverse group of inherited disorders characterised by impaired neuromuscular transmission. Most CMS originate from defects in the muscle nicotinic acetylcholine receptor, but the underlying molecular pathogenesis is only poorly understood. Here we show that RNAi-mediated silencing of FADS-related proteins rapsyn and NUP88 in foetal fibroblasts alters organisation of the actin cytoskeleton. We show that fibroblasts from two independent FADS individuals have enhanced and shorter actin stress fibre bundles, alongside with an increased number and size of focal adhesions, with an otherwise normal overall connectivity and integrity of the actin-myosin cytoskeleton network. By proximity ligation assays and bimolecular fluorescence complementation, we show that rapsyn and NUP88 localise nearby adhesion plaques and that they interact with the focal adhesion protein paxillin. Based on these findings we propose that a respective deficiency in rapsyn and NUP88 in FADS alters the regulation of actin dynamics at focal adhesions, and thereby may also plausibly dictate myofibril organisation and contraction in skeletal muscle of FADS individuals.