The Unconserved Groucho Central Region Is Essential for Viability and Modulates Target Gene Specificity

Groucho (Gro) is a Drosophila corepressor required by numerous DNA-binding repressors, many of which are distributed in gradients and provide positional information during development. Gro contains well-conserved domains at its N- and C-termini, and a poorly conserved central region that includes the GP, CcN, and SP domains. All lethal point mutations in gro map to the conserved regions, leading to speculation that the unconserved central domains are dispensable. However, our sequence analysis suggests that the central domains are disordered leading us to suspect that the lack of lethal mutations in this region reflects a lack of order rather than an absence of essential functions. In support of this conclusion, genomic rescue experiments with Gro deletion variants demonstrate that the GP and CcN domains are required for viability. Misexpression assays using these same deletion variants show that the SP domain prevents unrestrained and promiscuous repression by Gro, while the GP and CcN domains are indispensable for repression. Deletion of the GP domain leads to loss of nuclear import, while deletion of the CcN domain leads to complete loss of repression. Changes in Gro activity levels reset the threshold concentrations at which graded repressors silence target gene expression. We conclude that co-regulators such as Gro are not simply permissive components of the repression machinery, but cooperate with graded DNA-binding factors in setting borders of gene expression. We suspect that disorder in the Gro central domains may provide the flexibility that allows this region to mediate multiple interactions required for repression

Crosstalk between the EGFR and other signalling pathways at the level of the global transcriptional corepressor Groucho/TLE

In this minireview, we briefly revisit the Drosophila Notch and epidermal growth factor receptor pathways, and relate to the relationship between them. We then mainly focus on the involvement of Groucho (Gro)/TLE, a global developmental corepressor, in these pathways. In particular, we discuss Gro/TLE's role at the junction between these two signal transduction cascades

Establishment of Motor Neuron-V3 Interneuron Progenitor Domain Boundary in Ventral Spinal Cord Requires Groucho-Mediated Transcriptional Corepression

Background: Dorsoventral patterning of the developing spinal cord is important for the correct generation of spinal neuronal types. This process relies in part on cross-repressive interactions between specific transcription factors whose expression is regulated by Sonic hedgehog. Groucho/transducin-like Enhancer of split (TLE) proteins are transcriptional corepressors suggested to be recruited by at least certain Sonic hedgehog-controlled transcription factors to mediate the formation of spatially distinct progenitor domains within the ventral spinal cord. The aim of this study was to characterize the involvement of TLE in mechanisms regulating the establishment of the boundary between the most ventral spinal cord progenitor domains, termed pMN and p3. Because the pMN domain gives rise to somatic motor neurons while the p3 domain generates V3 interneurons, we also examined the involvement of TLE in the acquisition of these neuronal fates. Methodology and Principal Findings: A combination of in vivo loss- and gain-of-function studies in the developing chick spinal cord was performed to characterize the role of TLE in ventral progenitor domain formation. It is shown here that TLE overexpression causes increased numbers of p3 progenitors and promotes the V3 interneuron fate while suppressing the motor neuron fate. Conversely, dominant-inhibition of TLE increases the numbers of pMN progenitors and postmitotic motor neurons. Conclusion: Based on these results, we propose that TLE is important to promote the formation of the p3 domain an

PET quantification of brain O-GlcNAcase with [F-18]LSN3316612 in healthy human volunteers

BACKGROUND: Previous studies found that [18F]LSN3316612 was a promising positron emission tomography (PET) radioligand for imaging O-GlcNAcase in nonhuman primates and human volunteers. This study sought to further evaluate the suitability of [18F]LSN3316612 for human clinical research. METHODS: Kinetic evaluation of [18F]LSN3316612 was conducted in a combined set of baseline brain scans from 17 healthy human volunteers and test-retest imaging was conducted in 10 of these volunteers; another 6 volunteers had whole-body scans to measure radiation exposure to body organs. Total distribution volume (VT) estimates were compared for the one- and two-tissue compartment models with the arterial input function. Test-retest variability and reliability were evaluated via mean difference and intraclass correlation coefficient (ICC). The time stability of VT was assessed down to a 30-min scan time. An alternative quantification method for [18F]LSN3316612 binding without blood was also investigated to assess the possibility of eliminating arterial sampling. RESULTS: Brain uptake was generally high and could be quantified as VT with excellent identifiability using the two-tissue compartment model. [18F]LSN3316612 exhibited good absolute test-retest variability (12.5%), but the arithmetic test-retest variability was far from 0 (11.3%), reflecting a near-uniform increase of VT on the retest scan in nine of 10 volunteers. VT values were stable after 110 min in all brain regions, suggesting that no radiometabolites accumulated in the brain. Measurements obtained using only brain activity (i.e., area under the curve (AUC) from 150-180 min) correlated strongly with regional VT values during test-retest conditions (R2 = 0.84), exhibiting similar reliability to VT (ICC = 0.68 vs. 0.64). Estimated radiation exposure for [18F]LSN3316612 PET was 20.5 ± 2.1 μSv/MBq, comparable to other 18F-labeled radioligands for brain imaging. CONCLUSIONS: [18F]LSN3316612 is an excellent PET radioligand for imaging O-GlcNAcase in the human brain. Alternative quantification without blood is possible, at least for within-subject repeat studies. However, the unexplained increase of VT under retest conditions requires further investigation

EAR motif-mediated transcriptional repression in plants: An underlying mechanism for epigenetic regulation of gene expression

Ethylene-responsive element binding factor-associated amphiphilic repression (EAR) motif-mediated transcriptional repression is emerging as one of the principal mechanisms of plant gene regulation. The EAR motif, defined by the consensus sequence patterns of either LxLxL or DLN xxP, is the most predominant form of transcriptional repression motif so far identified in plants. Additionally, this active repression motif is highly conserved in transcriptional regulators known to function as negative regulators in a broad range of developmental and physiological processes across evolutionarily diverse plant species. Recent discoveries of co-repressors interacting with EAR motifs, such as TO PLESS (TPL) and AtSA P18, have begun to unravel the mechanisms of EAR motif-mediated repression. The demonstration of genetic interaction between mutants of TPL and AtHDA 19, co-complex formation between TPL-related 1 (TPR1) and AtHDA 19, as well as direct physical interaction between AtSA P18 and AtHDA 19 support a model where EAR repressors, via recruitment of chromatin remodeling factors, facilitate epigenetic regulation of gene expression. Here, we discuss the biological significance of EAR -mediated gene regulation in the broader context of plant biology and present literature evidence in support of a model for EAR motif-mediated repression via the recruitment and action of chromatin modifiers. Additionally, we discuss the possible influences of phosphorylation and ubiquitination on the function and turnover of EAR repressors

Prolyl isomerase Pin1 and protein kinase HIPK2 cooperate to promote cortical neurogenesis by suppressing groucho/TLE:Hes1-mediated inhibition of neuronal differentiation

The Groucho/transducin-like Enhancer of split 1 (Gro/TLE1):Hes1 transcriptional repression complex acts in cerebral cortical neural progenitor cells to inhibit neuronal differentiation. The molecular mechanisms that regulate the anti-neurogenic function of the Gro/TLE1:Hes1 complex during cortical neurogenesis remain to be defined. Here we show that prolyl isomerase Pin1 (peptidyl-prolyl cis-trans isomerase NIMA-interacting 1) and homeodomain-interacting protein kinase 2 (HIPK2) are expressed in cortical neural progenitor cells and form a complex that interacts with the Gro/TLE1:Hes1 complex. This association depends on the enzymatic activities of both HIPK2 and Pin1, as well as on the association of Gro/TLE1 with Hes1, but is independent of the previously described Hes1-activated phosphorylation of Gro/TLE1. Interaction with the Pin1:HIPK2 complex results in Gro/TLE1 hyperphosphorylation and weakens both the transcriptional repression activity and the anti-neurogenic function of the Gro/TLE1:Hes1 complex. These results provide evidence that HIPK2 and Pin1 work together to promote cortical neurogenesis, at least in part, by suppressing Gro/TLE1:Hes1-mediated inhibition of neuronal differentiation

Groucho related gene 5 (GRG5) is involved in embryonic and neural stem cell state decisions

Groucho related gene 5 (GRG5) is a multifunctional protein that has been implicated in late embryonic and postnatal mouse development. Here, we describe a previously unknown role of GRG5 in early developmental stages by analyzing its function in stem cell fate decisions. By both loss and gain of function approaches we demonstrate that ablation of GRG5 deregulates the Embryonic Stem Cell (ESC) pluripotent state whereas its overexpression leads to enhanced self-renewal and acquisition of cancer cell-like properties. The malignant characteristics of teratomas generated by ESCs that overexpress GRG5 reveal its pro-oncogenic potential. Furthermore, transcriptomic analysis and cell differentiation approaches underline GRG5 as a multifaceted signaling regulator that represses mesendodermal-related genes. When ESCs exit pluripotency, GRG5 promotes neuroectodermal specification via Wnt and BMP signaling suppression. Moreover, GRG5 promotes the neuronal reprogramming of fibroblasts and maintains the self-renewal of Neural Stem Cells (NSCs) by sustaining the activity of Notch/Hes and Stat3 signaling pathways. In summary, our results demonstrate that GRG5 has pleiotropic roles in stem cell biology functioning as a stemness factor and a neural fate specifier