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. 2024 Jan 23;13(2):68.
doi: 10.3390/biology13020068.

H3 Acetylation-Induced Basal Progenitor Generation and Neocortex Expansion Depends on the Transcription Factor Pax6

Godwin Sokpor  1   2 , Cemil Kerimoglu  3 , Pauline Antonie Ulmke  1 , Linh Pham  1 , Hoang Duy Nguyen  1 , Beate Brand-Saberi  4 , Jochen F Staiger  5 , Andre Fischer  3 , Huu Phuc Nguyen  1 , Tran Tuoc  1
Affiliations

H3 Acetylation-Induced Basal Progenitor Generation and Neocortex Expansion Depends on the Transcription Factor Pax6

Godwin Sokpor et al. Biology (Basel). .

Abstract

Enrichment of basal progenitors (BPs) in the developing neocortex is a central driver of cortical enlargement. The transcription factor Pax6 is known as an essential regulator in generation of BPs. H3 lysine 9 acetylation (H3K9ac) has emerged as a crucial epigenetic mechanism that activates the gene expression program required for BP pool amplification. In this current work, we applied immunohistochemistry, RNA sequencing, chromatin immunoprecipitation and sequencing, and the yeast two-hybrid assay to reveal that the BP-genic effect of H3 acetylation is dependent on Pax6 functionality in the developing mouse cortex. In the presence of Pax6, increased H3 acetylation caused BP pool expansion, leading to enhanced neurogenesis, which evoked expansion and quasi-convolution of the mouse neocortex. Interestingly, H3 acetylation activation exacerbates the BP depletion and corticogenesis reduction effect of Pax6 ablation in cortex-specific Pax6 mutants. Furthermore, we found that H3K9 acetyltransferase KAT2A/GCN5 interacts with Pax6 and potentiates Pax6-dependent transcriptional activity. This explains a genome-wide lack of H3K9ac, especially in the promoter regions of BP-genic genes, in the Pax6 mutant cortex. Together, these findings reveal a mechanistic coupling of H3 acetylation and Pax6 in orchestrating BP production and cortical expansion through the promotion of a BP gene expression program during cortical development.

Keywords: H3 acetylation; Pax6; basal progenitors; cortical development; epigenetic regulation; gyrification; neurogenesis.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1
H3 acetylation-induced genesis of bIPCs in the developing cortex depends on Pax6 presence. (A) Experimental scheme in which control and mutant embryos were treated with the HDAC inhibitor. (B) IHC micrographs showing Tbr2 (bIPs marker) and AP2ɣϫ (marker for APs and BPs) staining in mouse cortical sections from wild-type, BAF155cKO, and BAF155_Pax6_dcKO embryos with or without TSA treatment. Counter staining with DAPI is shown. Modified from Figure 3B in [28]. (C–E) Bar graphs showing changes in the numbers of apical and basal progenitor cells in the indicated cortical wall regions following treatment of embryos with the HDAC inhibitor TSA (C), VPA (D), or SAHA (E). Values are presented as means ± SEMs (* p < 0.05, ** p < 0.01, *** p < 0.005, NS, not significant; n = 6). Abbreviations: TSA, trichostatin A; SAHA, suberoylanilide hydroxamic acid; VPA, valproic acid; VZ, ventricular zone; SVZ, subventricular zone; IZ, intermediate zone. Scale bars = 50 µm.
Figure 2
Figure 2
HDAC inhibition induces generation of bRGCs in a Pax6-dependent manner. (A) Bar graphs showing the qPCR analysis used to confirm the upregulated expression of human-enriched bRGC markers in the TSA-treated BAF155cKO cortex and downregulation in the BAF155_Pax6_dcKO cortex, as compared with the indicated controls. (B–D) Micrographs showing immunostaining of selected human-enriched bRG markers (TNC and PTPRZ1) and Sox2 in the TSA-treated BAF155cKO and BAF155_Pax6_dcKO cortices compared with the indicated controls. Arrows point to cells co-expressing TNC and Sox2 or PTPRZ1 and Sox2. White rectangle in (B) indicates sampled cortical area imaged at high power in (C,D). Rectangular inserts in (C,D) are zoomed in images of the arrowed immunosignals. (E) Bar graphs showing quantitative analysis of cells immunostained for the bRG markers PTPRZ1 and TNC and their co-expression of Sox2 in the TSA-treated cortices compared with the untreated/vehicle-treated cortex. Values are presented as means ± SEMs (* p < 0.05, ** p < 0.01, *** p < 0.005; NS, not significant; n = 6). Abbreviations: l/d/mCx, lateral/dorsal/medial cortex; VZ, ventricular zone; IZ, intermediate zone. Scale bars = 100 μm.
Figure 3
Figure 3
Elevated H3 acetylation promotes cortical expansion and folding with Pax6 expression involvement. (A) IHC micrographs showing the developing mouse cortex stained with the indicated antibodies to reveal profiles of cortical layers and cell populations as an indication of cortical gyrification following treatment of wild-type, BAF155cKO, and BAF155_Pax6_dcKO brains with TSA. Arrows point at putative sulci. Counter staining with DAPI is shown. Modified from Figure 7C in [28]. (B) Image showing tabulations of the number of embryos with a folded cortex and the extent of folding following treatment of the wild-type, BAF155cKO, and BAF155_Pax6_dcKO brains with TSA. (C) Image showing the quantification of the Satb2-positive cortical/pallial area in the wild-type, BAF155cKO, and BAF155_Pax6_dcKO cortexes treated with TSA, as compared with the indicated controls. (D) Bar graph showing quantification of all neurons (NeuN-expressing cells), lower-layer neurons (Ctip2-expressing cells), and upper-layer neurons (Satb2-expressing cells) in the wild-type, BAF155cKO, and BAF155_Pax6_dcKO cortexes treated with TSA, as compared with the indicated controls. Values are presented as means ± SEMs (* p < 0.05, ** p < 0.01, *** p < 0.005; n = 6). Abbreviations: Veh, vehicle; TSA, trichostatin A. Scale bars = 100 μm.
Figure 4
Figure 4
The H3K9ac level and neurogenic transcriptional program are disturbed in the absence of Pax6 during corticogenesis. (A–D) Analyzed ChIP-seq data showing H3K9ac distribution along pan-gene bodies in the E15.5 Pax6cKO cortex (A), significant changes (fold change (FC) > 1.2, paired Student’s t-test p < 0.01) in the association of H3K9ac with neurogenic genes in the E15.5 Pax6cKO cortex compared with wild-type (B,C), and the distribution of H3K9ac along IPC gene bodies in the E15.5 Pax6cKO cortex (D). (E) Distribution of H3K9ac along the IPC-enriched gene bodies of Ngn1, Ngn2, Tbr2/Eomes, Cux1, and Cux2 in the Pax6cKO cortex (gray) versus control (red). Input (bottom row in black) and distributions after immunoprecipitation (top two rows) are indicated. Abbreviations: TSS, transcription start site; TES, transcription end site; IPCs, intermediated progenitor cells. Number of embryonic cortices used per group (n) = 3.
Figure 5
Figure 5
Pax6 expression is required for H3 acetylation-activated expression of Pax6 target genes in IPCs. (A) Schematic showing the experimental design used to sort TBR2-expressing cells in the Pax6cKO and wild-type cortexes for expression and promoter H3K9ac level comparison for selected bIP genes. (B) Micrographs showing cortical cells before and after isolation of TBR2-expressing cells. Counter staining with DAPI is shown. (C) Graphs showing the efficiency of TBR2+ cell sorting after the FACS experiment. (D,E) Bar graphs showing the promoter region level of H3K9ac (D) and the transcript levels (E) of the IPC-enriched genes, Ngn1, Tbr2, and Cux1, in the Pax6cKO cortex compared with the control. (F) Image showing the yeast two-hybrid assay, in which five different controls (a–d) were used in the performed two-hybrid yeast screen, as recommended by the manufacturer. Four colonies representing independent clones strongly positive for Pax6 binding revealed potential interactions between Gcn5/Kat2a and Pax6. (G) Luciferase assay demonstrating that Pax6 and H3 acetylation regulate the transcriptional program of Pax6 target genes. The promoter fragment DNA containing Pax6 binding site(s) of indicated Pax6 target genes were cloned into luciferase plasmids (Luc). Compared to control (Luc + CMV-EV), co-transfection of Pax6 (Luc + CMV-Pax6) was sufficient to activate luciferase activity. Presence of the H3 acetylation-augmenting conditions, TSA treatment, or transfection with CMV-Kat2A were important for the promoter activity of the indicated IPC genes (i.e., under pNgn2/P3-Luc, pTbr2/P3-Luc, and pCux1/P3-Luc conditions), leading to enhanced luciferase activity. Scale bar = 50 µm. Values are presented as means ± SEMs (* p < 0.05, ** p < 0.01, *** p < 0.005; NS, not significant; n = 6). Abbreviations: TSA, trichostatin A.
Figure 6
Figure 6
Schema summarizing the importance of Pax6 and H3 acetylation in the activation of IPC genes and attendant phenotypic effect in the developing cortex. In the wild-type cortex, Pax6 interacts with the H3 acetylation catalytic enzyme Kat2A to promote increased installation of H3K9ac at the promoter region of genes essential for IPC biogenesis. Thus, enhancing H3 acetylation accelerates the production of IPC in the developing cortex. This leads to expansion of the basal progenitor pool, which translates into an increase in neurogenesis, especially of upper-layer neurons, leading to cortical gyrification induction. However, in the absence of Pax6, H3K9ac levels drop, partly because of a lack of Kat2a interaction. This results in downregulation of IPC genes and a resultant reduction in upper-layer neuron production consequent to decreased IPC generation in the Pax6 mutant cortex.
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