Lack Of Cul4b In Mouse Nervous System Leads To Increased Abundance Of GFAP-positive Cells

CUL4B is a member of cullin family, which are components of Cullin-RING ligases (CRLs) complexes, the largest known class of ubiquitin ligases. The CRL complexes formed by CUL4B (CRL4B) recognizes and targets various substrates for proteosomal degradation, thereby regulating a broad variety of physiologically and developmentally controlled processes. Recently, we showed that CRL4B complex can also catalyze H2AK119 monoubiquitination and, by coordinating with PRC2 complex, promote H3K27 trimethylation, leading to the transcriptional repression of targeted genes. Mutations in human CUL4B gene led to syndromic X-linked mental retardation (XLMR) associated with short stature, abnormal gait, impaired speech and other abnormalities. Unlike in humans, constitutive knockout of Cul4b in mice led to embryonic lethality. However, the mice that lack Cul4b only in epiblast but not in extraembryonic tissues (Cul4bSox2-Cre mice) were viable and showed impaired spatial learning, which potentially mimics mental retardation.In this thesis, we show that CUL4B represses the expression of GFAP in neural progenitor cells (NPCs) during brain development. Lack of Cul4b in NPCs in cultures led to increased generation of astrocytes, marked by GFAP and S100β. The GFAP+ cells were also found to be more abundant in the brains of nervous system specific Cul4b knockout mice in vivo. Moreover, we demonstrated that the increased generation of GFAP+cells from Cul4b-null NPCs was mediated by an upregulation of prostaglandin D2 synthase PTGDS. We showed that the increased GFAP+cells can be attenuated by pharmacological inhibition of the PTGDS enzymatic activity or by shRNA-mediated knockdown of Ptgds. Importantly, exogenously added PTGDS could promote the generation of GFAP+cells from wild type NPCs. We further observed that Ptgds is targeted and repressed by the CUL4B/PRC2 complex. Together, our results demonstrate CUL4B as a negative regulator of GFAP+cell differentiation during neural development.PART ONE Lack of CUL4B in mouse nervous system leads to increased abundance of GFAP-positive cellsTo determine whether CUL4B regulates NPCs differentiation, we generated nervous system specific Cul4b knockout mice (Cul4bNestin-Cre mice) by crossing Cul4b floxed mice with Nestin-Cre transgenic mice, and isolated NPCs from El 5.5 Cul4bNes-Cre and littermate control mice forebrains for in vitro studies. The proliferation and apoptosis assays revealed no significant difference between Cul4b-null and wild-type NPCs, suggesting that CUL4B is probably dispensable for the expansion of NPCs. NPCs were induced to undergo differentiation by growth factor deprivation for 4-8 days. Cul4b-null NPCs resulted in increased generation of GFAP-positive cells compared with control NPCs. Similar results were obtained with immunostaining of S100β. On the other hand, Cul4b-null NPCs showed reduced neuronal differentiation, as shown by TUJ1-positive cells, when compared with control NPCs.We then examined the roles of Cul4b in GFAP-positive cells in vivo. Consistent with the in vitro result, the numbers of GFAP+cells were significantly increased in multiple brain regions of Cul4bNestin-Cre mice, including cortex, hippocampus, thalamus, hypothalamus and striatum, when compared to those in littermate controls.PART TWO The regulation of GFAP-positive cells differentiation by CUL4B is mediated by PTGDSThe JAK-STAT signaling pathway is known to regulate GFAP gene expression and neuron-to-astrocyte cell fate switch of neural progenitor cells. Therefore, we examined whether the function of CUL4B in NPC differentiation was also through JAK-STAT pathway. Indeed, phosphorylation of Stat3 at Y705, the active form of Stat3, during the in vitro differentiation of NPCs was more pronounced for Cul4b null than for wild-type cells. p-STAT3 was also enhanced in the cortex and hippocampus of Cul4bNes-Cre mice compared to littermate controls. Moreover, pharmacological inhibition of p-STAT3 could attenuate the increased astrocytic differentiation of Cul4b-null NPCs. These results suggested that increased astrocytic differentiation of Cul4b-null NPCs was dependent on activation of JAK-STAT pathway.We recently demonstrated that CUL4B-Ring E3 ligase complex (CRL4B) can catalyze H2AK119 monoubiquitination and, in coordination with PRC2, repress the transcription of various target genes. H3K27me3 level was significantly decreased in cortex of Cul4bNes-Cre mice compared to that of littermate controls. We next carried out a microarray transcriptome analysis to identify the genes that are deregulated in the brains of Cul4bNes-Cre mice, and found that expression of Ptgds was greatly elevated in Cul4bNes-Cre brains compared to that in littermate controls. Expression change of Ptgds was verified by quantitative real-time RT-PCR, Western blotting, and immunostainingThe greatly elevated expression of Ptgds in Cul4b-null brains and NPCs promoted us to investigate whether PTGDS mediates the increased percentages of GFAP+cells. Treatment with AT56, an inhibitor of PTGDS activity, efficiently attenuated the increased percentages of GFAP+cells. Moreover, knockdown of Ptgds expression in Cul4b-null NPCs by lentiviral particles containing shRNA could also attenuate the increased percentages of GFAP+cells. Strikingly, addition of exogenous PTGDS protein to the cultures of wild type NPCs greatly increased the percentages of GFAP+cells. Together, these data suggested that the increased percentages of GFAP+ cells in the absence of CUL4B was mediated by PTGDS.We next performed chromatin immunoprecipitation (ChIP) assay to examine whether Ptgds gene is repressed by CRL4B complex. As expected, ChIP assay showed that CUL4B could directly bind to the promoter of Ptgds. CUL4B, EZH2 as well as H3K27me3 were all enriched at the promoter of Ptgds in wild-type brain cells. Importantly, loss of CUL4B nearly abolished the binding of EZH2 to Ptgds, indicating that the binding of PRC2 complex to Ptgds in the brain depends on CRL4B complex. ChIP assay using NPCs also confirmed decreased H3K27me3, as well as increased H3K4me3, in the promoter of Ptgds in Cul4b-null NPCs compared to those in control NPCs. Taken together, these results demonstrated that CRL4B/PRC2 complexes can repress Ptgds by promoting H2AK119 monoubiquitination and H3K27 trimethylation.