Cofactor-Activated Phosphorylation Is Required for Inhibition of Cortical Neuron Differentiation by Groucho/TLE1

BACKGROUND:Transcriptional co-repressors of the Groucho/transducin-like Enhancer of split (Gro/TLE) family regulate the expression of a variety of genes and are involved in numerous developmental processes in both invertebrate and vertebrate species. More specifically, Gro/TLE1 participates in mechanisms that inhibit/delay the differentiation of cerebral cortex neural progenitor cells into neurons during mammalian forebrain development. The anti-neurogenic function of Gro/TLE1 depends on the formation of protein complexes with specific DNA-binding transcription factors that engage Gro/TLE1 through WRP(W/Y) sequences. Interaction with those transcription partners results in Gro/TLE1 recruitment to selected DNA sites and causes increased Gro/TLE1 phosphorylation. The physiological significance of the latter event, termed "cofactor-activated phosphorylation," had not been determined. Therefore, this study aimed at clarifying the role of cofactor-activated phosphorylation in the anti-neurogenic function of Gro/TLE1. METHODS AND PRINCIPAL FINDINGS:A combination of site-directed mutagenesis, mass spectrometry, biochemistry, primary cell culture, and immunocytochemical assays was utilized to characterize point mutations of Ser-286, a residue that is phosphorylated in vivo and is located within the serine/proline-rich (SP) domain of Gro/TLE1. Mutation of Ser-286 to alanine or glutamic acid does not perturb the interaction of Gro/TLE1 with DNA-binding partners, including the basic helix-loop-helix transcription factor Hes1, a prototypical anti-neurogenic WRP(W/Y) motif protein. Ser-286 mutations do not prevent the recruitment of Gro/TLE1 to DNA, but they impair cofactor-activated phosphorylation and weaken the interaction of Gro/TLE1 with chromatin. These effects are correlated with an impairment of the anti-neurogenic activity of Gro/TLE1. Similar results were obtained when mutations of Ser-289 and Ser-298, which are also located within the SP domain of Gro/TLE1, were analyzed. CONCLUSION:Based on the positive correlation between Gro/TLE1 cofactor-activated phosphorylation and ability to inhibit cortical neuron differentiation, we propose that hyperphosphorylation induced by cofactor binding plays a positive role in the regulation of Gro/TLE1 anti-neurogenic activity

The Level of the Transcription Factor Pax6 Is Essential for Controlling the Balance between Neural Stem Cell Self-Renewal and Neurogenesis

Neural stem cell self-renewal, neurogenesis, and cell fate determination are processes that control the generation of specific classes of neurons at the correct place and time. The transcription factor Pax6 is essential for neural stem cell proliferation, multipotency, and neurogenesis in many regions of the central nervous system, including the cerebral cortex. We used Pax6 as an entry point to define the cellular networks controlling neural stem cell self-renewal and neurogenesis in stem cells of the developing mouse cerebral cortex. We identified the genomic binding locations of Pax6 in neocortical stem cells during normal development and ascertained the functional significance of genes that we found to be regulated by Pax6, finding that Pax6 positively and directly regulates cohorts of genes that promote neural stem cell self-renewal, basal progenitor cell genesis, and neurogenesis. Notably, we defined a core network regulating neocortical stem cell decision-making in which Pax6 interacts with three other regulators of neurogenesis, Neurog2, Ascl1, and Hes1. Analyses of the biological function of Pax6 in neural stem cells through phenotypic analyses of Pax6 gain- and loss-of-function mutant cortices demonstrated that the Pax6-regulated networks operating in neural stem cells are highly dosage sensitive. Increasing Pax6 levels drives the system towards neurogenesis and basal progenitor cell genesis by increasing expression of a cohort of basal progenitor cell determinants, including the key transcription factor Eomes/Tbr2, and thus towards neurogenesis at the expense of self-renewal. Removing Pax6 reduces cortical stem cell self-renewal by decreasing expression of key cell cycle regulators, resulting in excess early neurogenesis. We find that the relative levels of Pax6, Hes1, and Neurog2 are key determinants of a dynamic network that controls whether neural stem cells self-renew, generate cortical neurons, or generate basal progenitor cells, a mechanism that has marked parallels with the transcriptional control of embryonic stem cell self-renewal

Regulation of cerebral cortical neurogenesis by the Pax6 transcription factor

Understanding brain development remains a major challenge at the heart of understanding what makes us human. The neocortex, in evolutionary terms the newest part of the cerebral cortex, is the seat of higher cognitive functions. Its normal development requires the production, positioning and appropriate interconnection of very large numbers of both excitatory and inhibitory neurons. Pax6 is one of a relatively small group of transcription factors that exert high-level control of cortical development, and whose mutation or deletion from developing embryos causes major brain defects and a wide range of neurodevelopmental disorders. Pax6 is very highly conserved between primate and non-primate species, is expressed in a gradient throughout the developing cortex and is essential for normal corticogenesis. Our understanding of Pax6’s functions and the cellular processes that it regulates during mammalian cortical development has significantly advanced in the last decade, owing to the combined application of genetic and biochemical analyses. Here we review the functional importance of Pax6 in regulating cortical progenitor proliferation, neurogenesis, and formation of cortical layers and highlight important differences between rodents and primates. We also review the pathological effects of PAX6 mutations in human neurodevelopmental disorders. Finally, we discuss some aspects of Pax6’s molecular actions including its own complex transcriptional regulation, the distinct molecular functions of its splice variants and some of Pax6’s known direct targets which mediate its actions during cortical development

The neural progenitor to neuron transition : role and regulation of GrouchoTLE proteins

Groucho/transducin-like Enhancer of split (Gro/TLE) family proteins are corepressors found as part of multiple transcriptional complexes that play significant roles during many developmental processes, including neurogenesis. This thesis sought to characterize the molecular mechanisms underlying the biological activity of Gro/TLE1. More specifically, the aim was to clarify the contribution of different transcriptional cofactors, as well as phosphorylation events induced by cofactor binding, to Gro/TLE1 ability to inhibit neuronal differentiation from proliferating neural progenitor cells.By characterizing specific point mutations within the C-terminal domain of Gro/TLE1, we were able to selectively impair binding of Gro/TLE1 to different classes of DNA-binding proteins and then assess the effect of those mutations on Gro/TLE1 anti-neurogenic function. These studies showed that the inhibition of cerebral cortex (cortical) neuron differentiation by Gro/TLE1 requires interaction with transcription factors that use short tetrapeptide sequences, WRP(W/Y), to recruit Gro/TLE1. In contrast, interactions with proteins that either interact with the C-terminal domain of Gro/TLE1 using a different type of binding sequence, termed engrailed homology 1 (Eh1) motif, or bind to the N-terminal part of the protein, are not required for Gro/TLE1 anti-neurogenic function.Using a similar strategy based on mutation analysis, we characterized point mutations that block the hyperphosphorylation of Gro/TLE1 induced by transcription cofactor binding ("cofactor-activated phosphorylation") without impairing cofactor binding and transcriptional corepression ability. These mutations map at phosphorylatable serine residues, Ser-286, Ser-289, and Ser298. Mutation of those residues to alanine blocks/reduces both cofactor-activated phosphorylation and anti-neurogenic activity of Gro/TLE1, demonstrating that cofactor-activated phosphorylation is required for that function. Tandem mass spectroscopy analysis showed further that Ser-286 is phosphorylated. Taken together, these findings characterize the role of cofactor-activated phosphorylation and identify residues important for this mechanism.Our studies also showed that homeodomain-interacting protein kinase 2 (HIPK2) mediates phosphorylation of Gro/TLE1 when the latter is complexed with transcriptional partners of the WRP(W/Y) motif family. However, HIPK2 is not involved in Gro/TLE1 cofactor-activated phosphorylation. Rather, HIPK2--mediated phosphorylation is antagonistic to the latter and decreases the ability of Gro/TLE1 to interact and repress transcription with WRP(W/Y) motif proteins.Taken together, these results improve significantly our understanding of the mechanisms underlying the anti-neurogenic function of Gro/TLE1. This information provides new insight into the regulation of mammalian neuronal development and, possibly, other developmental processes controlled by Gro/TLE proteins

COCO/DAND5 inhibits developmental and pathological ocular angiogenesis

Abstract Neovascularization contributes to multiple visual disorders including age‐related macular degeneration (AMD) and retinopathy of prematurity. Current therapies for treating ocular angiogenesis are centered on the inhibition of vascular endothelial growth factor (VEGF). While clinically effective, some AMD patients are refractory or develop resistance to anti‐VEGF therapies and concerns of increased risks of developing geographic atrophy following long‐term treatment have been raised. Identification of alternative pathways to inhibit pathological angiogenesis is thus important. We have identified a novel inhibitor of angiogenesis, COCO, a member of the Cerberus‐related DAN protein family. We demonstrate that COCO inhibits sprouting, migration and cellular proliferation of cultured endothelial cells. Intravitreal injections of COCO inhibited retinal vascularization during development and in models of retinopathy of prematurity. COCO equally abrogated angiogenesis in models of choroidal neovascularization. Mechanistically, COCO inhibited TGFβ and BMP pathways and altered energy metabolism and redox balance of endothelial cells. Together, these data show that COCO is an inhibitor of retinal and choroidal angiogenesis, possibly representing a therapeutic option for the treatment of neovascular ocular diseases

FoxG1 and TLE2 act cooperatively to regulate ventral telencephalon formation

FoxG1 is a conserved transcriptional repressor that plays a key role in the specification, proliferation and differentiation of the telencephalon, and is expressed from the earliest stages of telencephalic development through to the adult. How the interaction with co-factors might influence the multiplicity and diversity of FoxG1 function is not known. Here, we show that interaction of FoxG1 with TLE2, a Xenopus tropicalis co-repressor of the Groucho/TLE family, is crucial for regulating the early activity of FoxG1. We show that TLE2 is co-expressed with FoxG1 in the ventral telencephalon from the early neural plate stage and functionally cooperates with FoxG1 in an ectopic neurogenesis assay. FoxG1 has two potential TLE binding sites: an N-terminal eh1 motif and a C-terminal YWPMSPF motif. Although direct binding seems to be mediated by the N-terminal motif, both motifs appear important for functional synergism. In the neurogenesis assay, mutation of either motif abolishes functional cooperation of TLE2 with FoxG1, whereas in the forebrain deletion of both motifs renders FoxG1 unable to induce the ventral telencephalic marker Nkx2.1. Knocking down either FoxG1 or TLE2 disrupts the development of the ventral telencephalon, supporting the idea that endogenous TLE2 and FoxG1 work together to specify the ventral telencephalon

Inhibition of Cortical Neuron Differentiation by Groucho/TLE1 Requires Interaction with WRPW, but Not Eh1, Repressor Peptides*

In both invertebrates and vertebrates, transcriptional co-repressors of the Groucho/transducin-like Enhancer of split (Gro/TLE) family regulate a number of developmental mechanisms, including neuronal differentiation. The pleiotropic activity of Gro/TLE depends on context-specific interactions with a variety of DNA-binding proteins. Most of those factors engage Gro/TLE through two different types of short peptide motifs, the WRP(W/Y) tetrapeptide and the Engrailed homology 1 (Eh1) sequence (FXIXXIL). The aim of this study was to elucidate the contribution of WRP(W/Y) and Eh1 motifs to mammalian Gro/TLE anti-neurogenic activity. Here we describe point mutations within the C-terminal WD40 repeat domain of Gro/TLE1 that do not perturb protein folding but disrupt the ability of Gro/TLE1 to inhibit the differentiation of cerebral cortex neural progenitor cells into neurons. One of those mutations, L743F, selectively blocks binding to Hes1, an anti-neurogenic basic helix-loop-helix protein that harbors a WRPW motif. In contrast, the L743F mutation does not disrupt binding to Engrailed1 and FoxG1, which both contain Eh1 motifs, nor to Tcf3, which binds to the Gro/TLE N terminus. These results demonstrate that the recruitment of transcription factors harboring WRP(W/Y) tetrapeptides is essential to the anti-neurogenic function of Gro/TLE1

Myeloid‐resident neuropilin‐1 promotes choroidal neovascularization while mitigating inflammation

Abstract Age‐related macular degeneration (AMD) in its various forms is a leading cause of blindness in industrialized countries. Here, we provide evidence that ligands for neuropilin‐1 (NRP1), such as Semaphorin 3A and VEGF‐A, are elevated in the vitreous of patients with AMD at times of active choroidal neovascularization (CNV). We further demonstrate that NRP1‐expressing myeloid cells promote and maintain CNV. Expression of NRP1 on cells of myeloid lineage is critical for mitigating production of inflammatory factors such as IL6 and IL1β. Therapeutically trapping ligands of NRP1 with an NRP1‐derived trap reduces CNV. Collectively, our findings identify a role for NRP1‐expressing myeloid cells in promoting pathological angiogenesis during CNV and introduce a therapeutic approach to counter neovascular AMD