FOXG1 targets BMP repressors and cell cycle inhibitors in human neural progenitor cells

Abstract

FOXG1 is a critical transcription factor in human brain where loss-of-function mutations cause a severe neurodevelopmental disorder, while increased FOXG1 expression is frequently observed in glioblastoma. FOXG1 is an inhibitor of cell patterning and an activator of cell proliferation in chordate model organisms but different mechanisms have been proposed as to how this occurs. To identify genomic targets of FOXG1 in human neural progenitor cells (NPCs), we engineered a cleavable reporter construct in endogenous FOXG1 and performed chromatin immunoprecipitation (ChIP) sequencing. We also performed deep RNA sequencing of NPCs from two females with loss-of-function mutations in FOXG1 and their healthy biological mothers. Integrative analyses of RNA and ChIP sequencing data showed that cell cycle regulation and Bone Morphogenic Protein (BMP) repression gene ontology categories were over-represented as FOXG1 targets. Using engineered brain cell lines, we show that FOXG1 specifically activates SMAD7 and represses CDKN1B. Activation of SMAD7 which inhibits BMP signaling may be one way that FOXG1 patterns the forebrain, while repression of cell cycle regulators such as CDKN1B may be one way that FOXG1 expands the NPC pool to ensure proper brain size. Our data reveal novel mechanisms on how FOXG1 may control forebrain patterning and cell proliferation in human brain development.

Figure 1

Genes regulating BMP signaling, development and cilium assembly are differentially expressed in neural progenitor cells (NPCs) with pathogenic FOXG1 mutations. (A) Illustrative diagram of FOXG1 Syndrome cases, their genetic mutations and matched controls. (B) Representative immunofluorescence images of NPCs stained for standard forebrain neural progenitor markers and the pluripotency marker, OCT4. The scale bar represents 50 μm. (C) Western blot detecting endogenous FOXG1 in human NPCs demonstrating loss of FOXG1 in Case A and B NPCs compared to sex-matched family controls. (D) Volcano plot of differentially expressed genes that are up-regulated and down-regulated according to the nominal P-value. (E). Representative immunofluorescence of BAMBI with DAPI stain in forebrain NPCs. The scale bar represents 25 μm. (F) Quantification of BAMBI-positive NPCs in cases versus matched controls (n = 2 controls and 2 cases; 6 images from 6 independent replicates quantified per line for a total of 24 experiments). Error bars denote SEM. Significance stars are based on student's t-test (^**P < 0.005). (G) Representative immunofluorescence of SMAD6 with DAPI stain in forebrain NPCs. The scale bar represents 25 μm. (H) Quantification of SMAD6-positive NPCs in all cases versus matched controls (n = 2 controls and 2 cases; 6 images from 6 independent replicates quantified per line for a total of 24 experiments).

Figure 2

Immunoprecipitation (IP) and ChIPseq of endogenous FOXG1-tdTomato in human neural progenitor cells (NPCs) identifies peaks at forkhead binding motifs across genomic features. (A) Detection of transfected FOXG1-FLAG vector in HEK293 cells which do not endogenously express FOXG1. IP for FLAG and western blot for FOXG1 confirms antibody detection of FOXG1 at about ~ 62 kDa (1 = pcDNA3.1 empty vector; 2 = pcDNA3.1 FOXG1-FLAG). (B) IP for FOXG1 and western blot for FLAG confirms antibody detection of FOXG1 and ability to pull down FOXG1 using ab196868 antibody (1 = pcDNA3.1 empty vector; 2 = pcDNA3.1 FOXG1 FLAG). (C) Schematic diagram of FOXG1-reporter construct integrated into the FOXG1 locus in a control NPC line with CRISPR HDR. A tdTomato (tandem dimer Tomato) reporter was integrated into the 3′ end of FOXG1 for visualization of FOXG1 in live cells. The components of the construct are as follows: RA = right arm; LA = left arm; NLS = nuclear localization signal; T2A and F2A = two types of 2A signaling to enable translational gapping between two proteins; Stop = stop codon; Puro = puromycin resistance gene. (D) Representative images of FOXG1-tdTomato visualized in FOXG1 reporter NPCs. tdTomato expression is visualized using RFP fluorescent channel settings and under phase-contrast microscopy conditions (10× magnification) in FOXG1 reporter NPCs. (E) Pie plot visualization of genomic features annotated to called ChIP peaks. (F) Bar plot quantifying the number of peaks with one or more FOXG1 DNA-binding motifs. About 28% of peaks had two or more FBD motifs.

Figure 3

Integrated analysis of RNAseq and ChIPseq reveal enrichment for regulators of the cell cycle. (A) Flow chart demonstrating the number of genes differentially expressed in the RNAseq and genes annotated to peaks in the ChIPseq analyses that overlap. (B) Top enriched Gene Ontology terms ranked according to fold enrichment. (C) Visualization of ChIPseq peaks with FOXG1 DNA-binding (FDB) motifs highlighted for genes also identified in the RNAseq analysis with significant roles in cell cycle and cell proliferation (CDKN1B, CDKN1C, CCNDBP1, MASTL, CDC25A and CCND3). Y-axis represents normalized read depth, where the height of peaks reflects read depth above normalized baseline.

Figure 4

Loss of FOXG1 is associated with decreased expression of BMP repressor genes and increased expression of neurogenic and axon guidance genes. (A). Venn diagram highlighting the number of genes identified from RNAseq at q < 0.05 that are down-regulated and ChIPseq with peaks at FOXG1 DNA-binding (FDB) motifs. (B) Visualization of ChIPseq peaks with FDB motifs highlighted for genes identified in the integrated analysis with significant roles in SMAD and BMP signaling pathways (SMAD6, SMAD7, BAMBI and GDF7). (C) Venn diagram highlighting the number of genes identified from RNAseq at q < 0.05 that are up-regulated and ChIPseq with peaks at FOXG1 DNA-binding (FDB) motifs. (D) Visualization of ChIPseq peaks with FOXG1 DNA-binding (FDB) motifs highlighted for genes identified in the integrated analysis with significant roles in SMAD and BMP signaling pathways (NRXN3, NRN1, SEMA3D and SLIT1).

Figure 5

Molecular tuning of SMAD7 and CDKN1B levels in human neural progenitor cells (NPCs) by FOXG1. (A) Western blot experiments from two cases and two controls showing reduction of SMAD7 and increase of CDKN1B in NPCs from FOXG1 syndrome cases. (B) Transient transfection of FOXG1 in NPCs from one case and one control increases SMAD7 and decreases CDKN1B in both FOXG1 mutant and wild-type cells. (C) Example of cells that endogenously over-express FOXG1-FLAG-RFP from the CLYBL safe harbor locus. (D) FOXG1 Over-Expression (OE) in Case B with doxycycline application at the NPC stage increases SMAD7 and decreases CDKN1B.