Figure 1.
Differentiation of NTera-2 cells toward an astrocytic lineage.
NTera-2 cells were induced to cellular differentiation by infection with lentiviruses engineered to express pFUGW-Oct-4-RNAi at a multiplicity of infection of 25. A. Construction map of pFUGW-Oct-4-RNAi. B. Immunofluorescence (red) and Western blots of Oct4 expression in undifferentiated NTera-2 cells and cells 21 days after differentiation. β-Actin was used as an internal control in the Western blots. Note the substantial reduction in Oct-4 expression after differentiation. C. Phase-contrast micrographs for the morphological analysis (upper panel) and immunofluorescence staining for nestin expression (lower panel, red) of NTera-2 cells 3 and 21 days after differentiation, respectively. Magnification 400×. D. Immunofluorescence (red) and Western blots for GFAP expression in undifferentiated NTera-2 cells and NTera-2 cells 21 days after differentiation. E. Immunofluorescence staining and Western blot analyses of glutamine synthetase (GS) expression in NTera-2 cells 21 days after differentiation. The red staining for GS was compared with Hoechst dye (blue) for nuclei. β-actin was used as an internal control in the Western blots.
Figure 2.
Translocation of olig2, activation of STAT3 signaling and STAT3-p300 complex formation, and loss of Sin3A after differentiation.
A. Immunofluorescence staining for olig2 (red, upper panel), Hoechst dye (blue, middle panel), and merged images (bottom panel) are shown for undifferentiated control NTera-2 cells and NTera-2 cells 21 days after differentiation. Magnification 400×. B. STAT3 activation was examined with a specific antibody against phosphorylated STAT3, and total levels of STAT3 in lysates from undifferentiated NTera-2 cells and NTera-2 cells 21 days after differentiation (left panel) were determined. C. Lysates from cells similarly transduced were subjected to immunoprecipitation with an antibody against STAT3 and then were also probed with an anti-p300-specific antibody (right panel).
Figure 3.
Phosphoproteomic signature in cellular differentiation revealed by a label-free quantitation strategy.
A. Experiment workflow for quantitation of astrocyte phosphoproteomics. B. Functional annotation chart of the differentially phosphorylated proteins analyzed by DAVID [47], [48]; the highly represented categories are shown. Ontology terms are shown on the y axis; p-values for the significance of enrichment are graphed along the x axis. C. Analysis of the regulation of genes through label-free quantitation in NTera-2 cells 3, 7, 14 and 21 days during cell differentiation compared to undifferentiated NTera-2 cells. Data are expressed as log2 of fold change. D. Western blots showing levels of Sin3A expression in undifferentiated NTera-2 cells and NTera-2 cells 14 and 21 days after differentiation. β-actin was used as an internal control in the Western blots.
Figure 4.
Sin3A and MeCP2 co-occupy the promoter of GFAP before differentiation.
A. Schematic illustration of the structure of the proximal promoter and coding regions of GFAP. Black boxes indicate exons numbered with roman numbers. Lines connecting the exons are introns. Hatched boxes denote the 5′-untranslated region (UTR) and 3′-UTR. The start and stop codons are also indicated. qPCR primer pairs amplifying the STAT3-binding site and exon 1 regions are indicated as alphabetic letters. Region 3′ to the CpG island, h, served as a control primer pair. B. ChIP-qPCR analyses showing quantitative occupancies of Sin3A (left panel) and MeCP2 (right panel) in indicated regions of the GFAP gene in undifferentiated NTera-2 cells and at 21 days after differentiation. Multiples of enrichment are the relative abundances of the indicated regions over the control region, h. Error bars represent the means of triplicate values, and the standard deviation of one ChIP-qPCR experiment representative of two is shown. C. The methylation status of CpG sites within the STAT3 recognition sequence and GFAP gene exon 1 regions in undifferentiated NTera-2 cells and NTera-2 cells 21 days after differentiation was analyzed by bisulfite sequencing. Closed and open circles respectively indicate methylated and unmethylated CpG sites. D. Assays were performed on genomic DNA isolated from control, healthy individual blood samples. NT-2, undifferentiated NT-2 cells; astrocyte, astrocyte-like cells differentiated from NT-2 cells. The relative methylation levels of the indicated CpG sites (presented as a black bar) for the −5,000 bp upstream promoter were determined by a T-reverse cleavage reaction and MALDI-TOF/MS assay (Sequenom EpiTYPER platform).
Figure 5.
STAT3 occupies both the promoter and exon 1 of GFAP, recruitment of CBP and p300 to exon 1, and changes in histone acetylation levels at the STAT3-binding site after differentiation.
ChIP analyses were performed using anti-STAT3-P (A), RNA polymerase II (B), and H3K4me3 (C) antibodies and qPCR primer pairs (Fig. 4A) to detect the indicated regions of the GFAP gene in undifferentiated NTera-2 cells and cells 21 days after differentiation. D. Chromatin samples from undifferentiated NTera-2 cells and cells 21 days after differentiation were immunoprecipitated with anti-CBP (left panel) and anti-p300 (right panel) antibodies, and enrichment was quantitated by qPCR. E. A ChIP assay was performed as described in panel D but with active histone modifications, and anti-H3K9Ac (left panel) and anti-H3K14Ac (right panel) antibodies. Multiples of enrichment are the relative abundances of the indicated DNA fragments over the control fragment, h. Error bars represent the means of triplicate values, and the standard deviation of one ChIP-qPCR experiment representative of two is shown.
Figure 6.
Model for GFAP gene activation for induced human astrocytic differentiation.
In undifferentiated NTera-2 cells, the presence of the MeCP2 and Sin3A corepressor complex and possibly associated HDAC activity at the GFAP promoter maintain the deacetylated status of chromatin and repress GFAP transcription. Upon cellular differentiation, MeCP2 and Sin3A became dissociated from the GFAP promoter, and the subsequent recruitment of the phosphorylated STAT3 caused a conformational change in the region surrounding the GFAP transcription starting site. This, in turn, facilitated histone H3 acetylation of the promoter resulting from the recruitment of CBP/p300 to exon 1. The combination of chromatin remodeling and promoter conformational changes enabled the recruitment of RNA pol II.