BAF45D-binding to HOX genes was differentially targeted in H9-derived spinal cord neural stem cells

Chromatin accessibility has been used to define how cells adopt region-specific neural fates. BAF45D is one of the subunits of a specialised chromatin remodelling BAF complex. It has been reported that BAF45D is expressed in spinal cord neural stem cells (NSCs) and regulates their fate specification. Within the developing vertebrate spinal cord, HOX genes exhibit spatially restricted expression patterns. However, the chromatin accessibility of BAF45D binding HOX genes in spinal cord NSCs is unclear. In the present study, we found that in H9-derived spinal cord NSCs, BAF45D targets TBX6, a gene that regulates spinal cord neural mesodermal progenitors. Furthermore, BAF45D binding to the NES gene is much more enriched in H9-derived spinal cord NSCs chromatin compared to ESCs chromatin. In addition, BAF45D binding to anterior and trunk/central HOX genes, but not to lumbosacral HOX genes, was much more enriched in NSCs chromatin compared to ESCs chromatin. These results may shed new light on the role of BAF45D in regulating region-specific spinal cord NSCs by targeting HOX genes.


Over expression of GFP-tagged BAF45D in P19 cells and RA induction
P19 cell was cultured and transfected with plasmids as previously described (Liu, Zhang et al. 2015).Briefly, the cells were transfected with plasmids expressing GFP and GFP-tagged BAF45D by using Lipofectamine 2000 (Invitrogen, Carlsbad, CA).
Then the transfected P19 cells were exposured by RA and subjected to the following IB assay using the indicated antibodies.

Immunoblotting (IB) assays
The lysates of cells were subjected to IB assay according to a previous protocol (Chen 2022) .Proteins were separated by SDS-PAGE and transferred onto polyvinylidene difluoride membranes (Millipore, Temecula, CA, USA).Then, the proteins were detected using the indicated antibodies.

Gene ontology (GO) analysis
The GO assay were performed using topGO software, a package for Bioconductor (version 2.13) ( https://bioconductor.org/packages/2.13/bioc/html/topGO.html).The GO raw data include the data of both undifferentiated H9 cells and H9-derived spinal cord NSCs, as well as the data of the GO enrichment analysis of differential genes between the two cell types.The descriptions with the "neural tube" were selected first.Then the corrected P values associated with the descriptions were calculated.The top five "Log10pValues" of the selections were plotted in the bar graphs.

Function analysis of BAF45D bound regions
To address the function of BAF45D bound regions, we used publicly available Chip-seq data sets (http://cistrome.org/db/#/) of both H9 cells and H9-derived neural progenitor cells published in a previous paper (Ziller, Edri et al. 2015).The data sets were analysed using the UCSC genome browser linked to the website.The levels of H3K27ME3 and H3K27AC were plotted across the DPF2 locus.

Figure S4. Gene Ontology analysis of BAF45D binding events for some significantly enriched biological processes associated with neural tube development.
A, For the selection of GO terms,the descriptions with "neural tube" were selected first.Then "-Log10pValues" were calculated based on the corrected P-values associated with the descriptions.The top five "-Log10pValues" were plotted in the bar graphs.B-D, Functional annotation based on Gene Ontology (GO) categorization in the descriptions in the panel A. The horizontal bar represents the enrichment of the genes associated with biological processes identified in H9 cells vs. NSCs (B), and the enrichment of the same genes in H9 cells (C) and the NSCs (D), respectively.
Figure S1.BAF45D colocalised with HOXC9 in H9-derived spinal cord NSCs.A-H, IF assays were performed for the expression of HOXC9 (green)、 NESTIN (red)、PAX6 and BAF45D in H9 cells I-L, IF assay was performed for the expression of HOXC9 (green) and NESTIN (red) in the H9-derived spinal cord NSCs.M-T, IF assay was performed for the expression of HOXC9 (green) and BAF45D (red) in the H9-derived spinal cord NSCs.Q, R, S and T are the insets in M, N, O and P, respectively.Arrows indicate colocalisation of HOXC9 and BAF45D.Arrowheads indicate expression of BAF45D only.Nuclei were counterstained with DAPI (blue).Scale bar=25μm (A-D,I-P).Scale bar=50μm (E-H).U, The statistical analysis for the cells positive for HOXC9 and BAF45D. .

Figure S2 .
Figure S2.Homer known motif enrichment results of H9 cells and H9derived spinal cord NSCs.A and B, The top five motifs of BAF45D bound genes identified in the H9derived spinal cord NSCs.C and D, The top five motifs of BAF45D bound genes identified in the H9 cells.

Figure S3 .
Figure S3.Homer de novo motif results of H9 cells and H9-derived spinal cord NSCs.A and B, The top five de novo motifs for BAF45D identified in the H9-derived spinal cord NSCs.C and D, The top five de novo motifs for BAF45D identified in the H9 cells.

Figure S5 .
Figure S5.BAF45D binding to POU5F1 and HOXC13 genes was less enriched in NSCs chromatin compared to ESCs chromatin.A, Chromatin accessibility of BAF45D-bound POU5F1, a stem cell marker, was decreased in NSCs compared to H9 cells.B, Chromatin accessibility of BAF45D-bound HOXC13, a posterior spinal cord marker, was decreased in NSCs compared to H9 cells.

Figure S6 .
Figure S6.ChIP-seq enrichment profiles of H3K27ME3 and H3K27AC histone marks in across genomic regions centered at BAF45D peak summits.A, ChIP-seq enrichment profiles of H3K27ME3 histone marks in across genomic regions centered at BAF45D peak summits.B, ChIP-seq enrichment profiles of H3K27AC histone marks in across genomic regions centered at BAF45D peak summits.

Figure S7 .
Figure S7.BAF45D increased HOXC9 expression in RA treated P19 cells and H9-derived neural stem cells expressed HOXB1 but few or no HOXB13.A, Forced expression of BAF45D in P19 cells and the effect of BAF45D on HOXC9 expression in the transfected P19 cells treated by RA.B, Statistical analysis indicated that the expression of HOXC9 was significantly increased by GFP-BAF45D as compared to the GFP.C-H, Fluorescence (C and F) and phase(D and G) micrographs of the P19 cells with expression of GFP(C and E) and GFP-BAF45D (F-H).I-L, H9-derived neural stem cells expressed robust HOXB1(green, I-J) but few or no HOXB13 (red, K-L).Arrows indicate the robust expression of HOXB1.Scale bar=10μm.M，The statistical analysis for the cells positive for HOXB1 and HOXB13.