Briefly, hESCs were dissociated into single cells using Accutase, and plated at high density on Matrigel (Corning). neurons showed a significant reduction in cell numbers starting at day 40 of differentiation (Figure 1H). Interestingly, we observed that the cell loss in SATB1KO cultures stabilized by day 40 and was maintained at that level over the 60 days of differentiation. We speculate that the loss of live SATB1KO DA neurons is at least in part a consequence of detachment from the culture dish due to fundamental reorganization of their gross morphology including the cytoskeleton. Consistent with this, we found that surviving SATB1KO DA neurons showed significantly decreased neurite outgrowth and complexity at day 60, following normal development at earlier days of differentiation (Figure 1I). ML 228 SATB1 Acts Predominantly as ML 228 a Gene Repressor in DA Neurons. To understand the functional role of SATB1 in DA neurons, we performed concurrent RNA-Seq and ChIP-Seq experiments (Figure 2A). We used ChIP-Seq to compare the genome-wide binding profiles of SATB1 in early and mature DA neurons (Figure 2B). We found that SATB1-binding had the highest intensity in mature DA neurons. We confirmed this finding by analysis of the expression profile changes caused by SATB1KO in DA neurons. Open in a separate window Figure 2. SATB1 Plays Discrete Regulatory Roles in early and mature DA Neurons.(A) Outline of the experimental approach comparing expression, DNA-binding, and regulator profile of SATB1 in DA neurons. (B) Genome-wide heatmaps of SATB1-ChIP-Seq experiments comparing binding patterns in early and mature DA neurons (ChIP-Seq experiments performed in 4 independent experiments). RNA-Seq expression profile comparing WT vs. SATB1KO of early DA neurons (C) (n=4) and mature DA neurons (D) (n=3). Red dots indicate significantly changed genes (FDR 0.05, 2-fold expression change). BETA plots of combined computational analysis of SATB1-ChIP-Seq and RNA-Seq data of ML 228 early DA neurons (E) and mature DA neurons (F). Black line: static background, red line: repressive function, blue line: activating function. See also Figure S2. Comparison of WT and SATB1KO DA neurons at an early timepoint (day 30) revealed few changes in gene expression (Figure 2C). At this timepoint, the cells were phenotypically comparable to WT. At day 50 of differentiation, when surviving SATB1KO neurons showed a phenotype, much greater gene expression changes were observed (Figure 2D). The KO of SATB1 has a more dramatic effect in mature DA neurons than in early DA neurons. Next, we Mouse monoclonal to CD154(FITC) used the binding and expression target analysis (BETA) software (Wang et al., 2013) to incorporate the ChIP-Seq and RNA-Seq data. This analysis showed that SATB1 has no significant effects as a gene regulator in early DA neurons (Figure 2E). In mature DA neurons SATB1 acts as a gene repressor (p = 0.000236) (Figure 2F). Interestingly, network analysis of enriched gene ontologies (GO) in DA neurons revealed that the loss of SATB1 activates connected transcriptional programs that underlie cytoskeleton remodeling as seen in (Figure 1I, S2). Surprisingly, in these postmitotic cells, ontologies related to the negative regulation of cell proliferation were enriched (Figure S2). Loss of SATB1 in Dopamine Neurons Results in a Senescence Phenotype Amongst the GO pathways enriched in SATBKO versus WT DA neurons, we found the cellular senescence pathway. The DA neuron enrichment was further confirmed by GSEA of the mature SATB1KO DA neuron transcriptome (Figure 3A). Given this, we sought to investigate if SATB1KO DA neurons present the classical features of cellular senescence. First, we observed a dramatic increase in acidic lysosomal senescence associated beta-Galactosidase (SA-Gal) activity, the hallmark senescence biomarker (Figure 3B). Another key feature of senescent cells is the activation of the SASP. To determine if SATB1KO DA neurons present this phenotype, we evaluated the expression of the described key SASP factors (Coppe et al., 2008). We found an upregulation of the majority of the SASP factors at 50 days of differentiation in the SATB1KO DA neurons versus WT neurons (Figure 3C). We confirmed SASP activation by western blotting. In the conditioned media of SATB1KO neurons, we found IGFBP7, which was absent in the media of WT neurons (Figure 3D). In fact,.