SUMO control of nervous system development.

In the last decades, the post-translational modification system by covalent attachment of the SUMO polypeptide to proteins has emerged as an essential mechanism controlling virtually all the physiological processes in the eukaryotic cell. This includes vertebrate development. In the nervous system, SUMO plays crucial roles in synapse establishment and it has also been linked to a variety of neurodegenerative diseases. However, to date, the involvement of the modification of specific targets in key aspects of nervous system development, like patterning and differentiation, has remained largely elusive. A number of recent works confirm the participation of target-specific SUMO modification in critical aspects of nervous system development. Here, we review pioneering and new findings demonstrating the essential role SUMO plays in neurogenesis and other facets of neurodevelopment, which will help to precisely understand the variety of mechanisms SUMO utilizes to control most fundamental processes in the cell.

BETting on a Transcriptional Deficit as the Main Cause for Cornelia de Lange Syndrome.

Cornelia de Lange Syndrome (CdLS) is a human developmental syndrome with complex multisystem phenotypic features. It has been traditionally considered a cohesinopathy together with other phenotypically related diseases because of their association with mutations in subunits of the cohesin complex. Despite some overlap, the clinical manifestations of cohesinopathies vary considerably and, although their precise molecular mechanisms are not well defined yet, the potential pathomechanisms underlying these diverse developmental defects have been theoretically linked to alterations of the cohesin complex function. The cohesin complex plays a critical role in sister chromatid cohesion, but this function is not affected in CdLS. In the last decades, a non-cohesion-related function of this complex on transcriptional regulation has been well established and CdLS pathoetiology has been recently associated to gene expression deregulation. Up to 70% of CdLS cases are linked to mutations in the cohesin-loading factor NIPBL, which has been shown to play a prominent function on chromatin architecture and transcriptional regulation. Therefore, it has been suggested that CdLS can be considered a transcriptomopathy. Actually, CdLS-like phenotypes have been associated to mutations in chromatin-associated proteins, as KMT2A, AFF4, EP300, TAF6, SETD5, SMARCB1, MAU2, ZMYND11, MED13L, PHIP, ARID1B, NAA10, BRD4 or ANKRD11, most of which have no known direct association with cohesin. In the case of BRD4, a critical highly investigated transcriptional coregulator, an interaction with NIPBL has been recently revealed, providing evidence on their cooperation in transcriptional regulation of developmentally important genes. This new finding reinforces the notion of an altered gene expression program during development as the major etiological basis for CdLS. In this review, we intend to integrate the recent available evidence on the molecular mechanisms underlying the clinical manifestations of CdLS, highlighting data that favors a transcription-centered framework, which support the idea that CdLS could be conceptualized as a transcriptomopathy.

The Sumo proteome of proliferating and neuronal-differentiating cells reveals Utf1 among key Sumo targets involved in neurogenesis.

Post-translational modification by covalent attachment of the Small ubiquitin-like modifier (Sumo) polypeptide regulates a multitude of processes in vertebrates. Despite demonstrated roles of Sumo in the development and function of the nervous system, the identification of key factors displaying a sumoylation-dependent activity during neurogenesis remains elusive. Through a SILAC (stable isotope labeling by/with amino acids in cell culture)-based proteomic approach, we have identified the Sumo proteome of the model cell line P19 under proliferation and neuronal differentiation conditions. More than 300 proteins were identified as putative Sumo targets differentially associated with one or the other condition. A group of proteins of interest were validated and investigated in functional studies. Among these, Utf1 was revealed as a new Sumo target. Gain-of-function experiments demonstrated marked differences between the effects on neurogenesis of overexpressing wild-type and sumoylation mutant versions of the selected proteins. While sumoylation of Prox1, Sall4a, Trim24, and Utf1 was associated with a positive effect on neurogenesis in P19 cells, sumoylation of Kctd15 was associated with a negative effect. Prox1, Sall4a, and Kctd15 were further analyzed in the vertebrate neural tube of living embryos, with similar results. Finally, a detailed analysis of Utf1 showed the sumoylation dependence of Utf1 function in controlling the expression of bivalent genes. Interestingly, this effect seems to rely on two mechanisms: sumoylation modulates binding of Utf1 to the chromatin and mediates recruitment of the messenger RNA-decapping enzyme Dcp1a through a conserved SIM (Sumo-interacting motif). Altogether, our results indicate that the combined sumoylation status of key proteins determines the proper progress of neurogenesis.

The Cornelia de Lange Syndrome-associated factor NIPBL interacts with BRD4 ET domain for transcription control of a common set of genes.

Mutations in NIPBL are the major cause of Cornelia de Lange Syndrome (CdLS). NIPBL is the cohesin-loading factor and has recently been associated with the BET (bromodomains and extra-terminal (ET) domain) proteins BRD2 and BRD4. Related to this, a CdLS-like phenotype has been described associated to BRD4 mutations. Here, we show direct interaction of NIPBL with different BET members in yeast, and selective interaction with BRD4 in cells, being the ET domain involved in the interaction. To understand the relationship between NIPBL and BET proteins, we have performed RNA-Seq expression analysis following depletion of the different proteins. Results indicate that genes regulated by NIPBL largely overlap with those regulated by BRD4 but not with those regulated by BRD2. ChIP-Seq analysis indicates preferential NIPBL occupancy at promoters, and knockdown experiments show mutual stabilization of NIPBL and BRD4 on co-regulated promoters. Moreover, human fibroblasts from CdLS probands with mutations in NIPBL show reduced BRD4 at co-occupied promoters. Functional analysis in vivo, using mutants of Drosophila melanogaster, confirmed the genetic interaction between Nipped-B and fs(1)h, the orthologs of human NIPBL and BRD4, respectively. Thus, we provide evidence for NIPBL and BRD4 cooperation in transcriptional regulation, which should contribute to explain the recently observed CdLS-like phenotype associated with BRD4 mutations.

Pleiotrophin antagonizes Brd2 during neuronal differentiation.

Bromodomain-containing protein 2 (Brd2) is a BET family chromatin adaptor required for expression of cell-cycle-associated genes and therefore involved in cell cycle progression. Brd2 is expressed in proliferating neuronal progenitors, displays cell-cycle-stimulating activity and, when overexpressed, impairs neuronal differentiation. Paradoxically, Brd2 is also detected in differentiating neurons. To shed light on the role of Brd2 in the transition from cell proliferation to differentiation, we had previously looked for proteins that interacted with Brd2 upon induction of neuronal differentiation. Surprisingly, we identified the growth factor pleiotrophin (Ptn). Here, we show that Ptn antagonized the cell-cycle-stimulating activity associated with Brd2, thus enhancing induced neuronal differentiation. Moreover, Ptn knockdown reduced neuronal differentiation. We analyzed Ptn-mediated antagonism of Brd2 in a cell differentiation model and in two embryonic processes associated with the neural tube: spinal cord neurogenesis and neural crest migration. Finally, we investigated the mechanisms of Ptn-mediated antagonism and determined that Ptn destabilizes the association of Brd2 with chromatin. Thus, Ptn-mediated Brd2 antagonism emerges as a modulation system accounting for the balance between cell proliferation and differentiation in the vertebrate nervous system.

Lack of association of morphologic and functional retinal changes with motor and non-motor symptoms severity in Parkinson's disease.

Visual symptoms are common among the nonmotor symptoms in Parkinson's disease. The aims of this study were to assess the diagnostic accuracy and relationship of retinal morphologic and functional changes with motor and non-motor symptoms disturbances in Parkinson's disease. Thirty patients with Parkinson's disease, with a median Hoehn-Yahr stage of 2 (1-4), were compared to 30 age- and gender-matched controls. Retinal thinning and function were measured using optical coherence tomography (OCT), visual evoked potentials (VEP), and pattern electroretinography. Motor impairment and motor laterality were measured using the Short Parkinson's Evaluation Scale/Scales for Outcomes in Parkinson's disease, and non-motor symptoms severity using the nonmotor symptoms questionnaire. Only pattern electroretinography, P50 and N95 amplitudes, were lower in patients with Parkinson's disease, compared to controls (p = 0.01, respectively). Age, disease duration, levodopa dose, motor, and non-motor impairment were not significantly associated with retinal thinning and functional changes. The patients vs. controls area under the curve of OCT, VEP, and pattern electroretinography receiver-operating-characteristic curves were<0.50. In conclusion, morphologic and functional retina changes are not significantly correlated with motor and non-motor symptoms impairment severity, and do not discriminate between Parkinson's disease and controls.

Association of bromodomain BET proteins with chromatin requires dimerization through the conserved motif B.

BET (bromodomain and extra terminal domain) family proteins are unique among bromodomain-containing proteins in that they not only associate with acetylated chromatin in interphase, but also remain attached to chromosomes during mitosis. Although the two tandem bromodomains are essential to display this behaviour, they do not suffice. In this work we report that a small conserved domain, motif B, is also required. A deletion mutant of this domain dissociates from mitotic chromosomes. However, inhibition of histone deacetylases alleviates dissociation. We also show that motif-B-dependent association with chromosomes is not restricted to mitosis. Interestingly, our results indicate that motif B constitutes a surface for homo- and hetero-dimerization between BET proteins. Finally, linked to the prominent role BET proteins play in cell proliferation, we report that ectopic expression of the family member Brd2 interferes with neuronal differentiation in P19 cells and in the vertebrate neural tube, probably because of preservation of adequate levels of cyclin A2 and cyclin D1. By contrast, a deletion mutant of motif B fails to perform in this way, highlighting the relevance of this domain for Brd2 function.

Control of neuronal differentiation by sumoylation of BRAF35, a subunit of the LSD1-CoREST histone demethylase complex.

The LSD1-CoREST histone demethylase complex is required to repress neuronal genes in nonneuronal tissues. Here we show that sumoylation of Braf35, one of the subunits of the complex, is required to maintain full repression of neuron-specific genes and for occupancy of the LSD1-CoREST complex at its gene targets. Interestingly, expression of Braf35 was sufficient to prevent neuronal differentiation induced by bHLH neurogenic transcription factors in P19 cells and in neuronal progenitors of the chicken embryo neural tube. Sumoylation of Braf35 is required for this antineurogenic activity. We also show that iBraf, a paralogue of Braf35, forms heterodimers with Braf35. Braf35-iBraf heterodimerization impairs Braf35 interaction with the LSD1-CoREST complex and inhibits Braf35 sumoylation. Consistent with these results, iBraf prevents the antineurogenic activity of Braf35 in vivo. Our data uncover a mechanism of regulation of the LSD1-CoREST complex and provide a molecular explanation for the antagonism between Braf35 and iBraf in neuronal differentiation.

The transcription factor Krox20 is an E3 ligase that sumoylates its Nab coregulators.

Covalent attachment of small ubiquitin-like modifier (SUMO) to proteins regulates many processes in the eukaryotic cell. This reaction is similar to ubiquitination and usually requires an E3 ligase for substrate modification. However, only a few SUMO ligases have been described so far, which frequently facilitate sumoylation by bringing together the SUMO-conjugating enzyme Ubc9 and the target protein. Ubc9 is an interaction partner of the transcription factor Krox20, a key regulator of hindbrain development. Here, we show that Krox20 functions as a SUMO ligase for its coregulators--the Nab proteins--and that Nab sumoylation negatively modulates Krox20 transcriptional activity in vivo.