Transcriptional cooperation of PBX1 and PAX6 in adult neural progenitor cells

PAX6 is a highly conserved transcription factor and key regulator of several neurogenic processes, including the continuous generation of dopaminergic/GABAergic interneurons in the adult ventricular-subventricular (V-SVZ) neurogenic system in mice. Here we report that PAX6 cooperates with the TALE-homeodomain transcription factor PBX1 in this context. Chromatin-immunoprecipitation showed that PBX1 and PAX6 co-occupy shared genomic binding sites in adult V-SVZ stem- and progenitor cell cultures and mouse embryonic stem cells, while depletion of Pbx1 revealed that association of PAX6 with these sites requires the presence of PBX1. Expression profiling together with viral overexpression or knockdown of Pax6 or Pbx1 identified novel PBX1-PAX6 co-regulated genes, including several transcription factors. Computational modeling of genome wide expression identified novel cross-regulatory networks among these very transcription factors. Taken together, the results presented here highlight the intimate link that exists between PAX6 and TALE-HD family proteins and contribute novel insights into how the orchestrated activity of transcription factors shapes adult V-SVZ neurogenesis.

To obtain passage 1 aNS, primary spheres were dissociated after five days in culture by treatment with Accutase (Sigma Aldrich) for 15 min at 37°C.
For immunohistochemical analyses, cellular differentiation was induced as follows: First-passage aNS were dissociated and cultured in EGF/FGF2-containing medium for another 48 hours. Cell numbers were then determined by dissociating and counting cells in a small aliquot of each aNS culture, and spheres were plated at a density corresponding to 7-8x10 4 cells per cm 2 in medium without EGF/FGF2 but supplemented with 20 ng/ml brain-derived neurotrophic factor (BDNF; PeproTech) on laminincoated tissue coverslips for another 24 hours. For ChIP or Affymetrix analysis, differentiation was induced by plating dissociated, single cells at a density of 1-2x10 5 cells per cm 2 in medium without EGF/FGF2 but supplemented with 20 ng/ml BDNF on laminin-coated tissue culture dishes as described in Hau et al., 2017. For subsequent Affymetrix analysis, cells were harvested after 10 hours of differentiation, for chromatin immunoprecipitation at the times indicated.
For retro-and lentiviral transduction, aNS were dissociated in Accutase (Sigma Aldrich), 5 million cells per sample were incubated in a fresh 10cm tissue cell culture dish in approximately 6ml aNS culture medium, containing EGF/FGF2 but without penicillin/streptomycin, and incubated for at least 5 hours at 37°C in the presence of the viral stocks at 4-8x10 5 CFU / ml (see below). Transduced cells were pelleted by centrifugation, washed twice in culture medium containing EGF and FGF2, and grown for additional 48 hours as free-floating spheres in the presence of growth factors prior to fixation for ChIP or differentiation.
For siRNA-mediated knockdown of Pbx1, first passage aNS were transfected with Silencer® Select siRNAs (5`-guuggaccaacgugcaau-3; 50pmol transfected per 2x10 6 cells; Thermo Fisher Scientific) or negative control siRNAs No1 (Thermo Fisher Scientific). RNA duplexes were transfected with Metafectene Pro (Biontex). When used for ChIP, aNS cells were grown for 48 hours as free-floating spheres following siRNA transfection. When used for Affymetrix gene expression arrays, cells were transduced with Pax6-expressing retroviruses as described above four hours after siRNA transfection, allowed to grow as free-floating spheres for additional 48 hours before differentiation was induced by growth factor withdrawal and plating on laminin-coated cell culture dishes. Knockdown with shRNAexpressing lentiviruses was performed with pGIPZ lentiviral vectors carrying shRNAs directed against

Production of retro-or lentiviral particles.
Retroviral transduction was carried out with pCLIG, a GFP-expressing vector virus [1]. pCLIG-Pax6 contains the coding sequence of canonical Pax6, pCLIG-Pbx1 contains the coding region of Pbx1b.

Immunohistochemical assays: Immunostaining, FACS, Western Blot
For immunofluorescence analysis of in vitro grown aNS or neurons, the cells were fixed with 4% paraformaldehyde (PFA) in phosphate buffered saline pH 7.5 (PBS) for 10 minutes at RT and washed in PBS at 4°C. aNS were allowed to attach to poly-D-lysine-coated coverslips for 30min at 37°C prior to fixation. Primary and secondary antibodies were diluted in 10% goat serum and 0.5% TritonX-100 in PBS. Primary antibodies were applied over night at 4°C. Samples were washed three times with PBS for 5-10 minutes each. Secondary antibodies were applied for one hour at RT. The samples were washed with PBS, cell nuclei were stained with 4',6-diamidino-2-phenylindole (DAPI), and samples were mounted with Aqua Poly Mount (Polyscience Inc.  and analysed for Gene Ontology enrichment using the online resource DAVID (https://david.ncifcrf.gov/) [3]. GO terms together with corresponding p-values were exported from DAVID and further summarised as enriched biological processes using the Revigo online tool (http://revigo.irb.hr/) [4].
Draw Venn Diagramm (http://bioinformatics.psb.ugent.be/webtools/Venn/) was used to create intersecting lists of DEGs for different comparisons. The array data were submitted to NCBI Gene Expression Omnibus under the accession number GSE172449.

ISMARA analysis
ISMARA was used to potentially identify key transcription factors which might be involved in driving the gene expression changes observed in the transcriptome expression data from Affymetrix GeneChip arrays [5].    hybridization for four TFs, which are known to bind to motifs whose activity was predicted by ISMARA 11 to account for gene expression differences between the Pax6OE, Pax6OE/Pbx1KD and vector ctrl samples. Coronal sections through the forebrain of adult mice are shown for Rfx3, Rfx4 and Pbx3, a sagital section for Nfia. The boxed areas indicating the V-SVZ in the coronal sections or the transition from V-SVZ to RMS in sagital section are shown at higher magnifications on the right hand side. Note the prominent expression of all four TFs in the V-SVZ germinal niche. Images are taken from the Allen Mouse Brain Atlas (https://mouse.brain-map.org/).        Extracts I-III were loaded at 5µg and 25µg protein per lane on the gel shown in Fig. S2a; extracts I and II were loaded twice at 25µg per lane on the gel shown in Fid. S2d. Red boxes mark the areas of the membranes shown in Fig. S2a and S2d, respectively.