LHX2 Interacts with the NuRD Complex and Regulates Cortical Neuron Subtype Determinants Fezf2 and Sox11

Abstract

In the developing cerebral cortex, sequential transcriptional programs take neuroepithelial cells from proliferating progenitors to differentiated neurons with unique molecular identities. The regulatory changes that occur in the chromatin of the progenitors are not well understood. During deep layer neurogenesis, we show that transcription factor LHX2 binds to distal regulatory elements of Fezf2 and Sox11, critical determinants of neuron subtype identity in the mouse neocortex. We demonstrate that LHX2 binds to the nucleosome remodeling and histone deacetylase histone remodeling complex subunits LSD1, HDAC2, and RBBP4, which are proximal regulators of the epigenetic state of chromatin. When LHX2 is absent, active histone marks at the Fezf2 and Sox11 loci are increased. Loss of LHX2 produces an increase, and overexpression of LHX2 causes a decrease, in layer 5 Fezf2 and CTIP2-expressing neurons. Our results provide mechanistic insight into how LHX2 acts as a necessary and sufficient regulator of genes that control cortical neuronal subtype identity.

Significance statement: The functional complexity of the cerebral cortex arises from an array of distinct neuronal subtypes with unique connectivity patterns that are produced from common progenitors. This study reveals that transcription factor LHX2 regulates the numbers of specific cortical output neuron subtypes by controlling the genes that are required to produce them. Loss or increase in LHX2 during neurogenesis is sufficient to increase or decrease, respectively, a particular subcerebrally projecting population. Mechanistically, LHX2 interacts with chromatin modifying protein complexes to edit the chromatin landscape of its targets Fezf2 and Sox11, which regulates their expression and consequently the identities of the neurons produced. Thus, LHX2 is a key component of the control network for producing neurons that will participate in cortical circuitry.

Keywords: cell fate; chromatin; epigenetics; lamination; progenitor; specification.

Figure 1.

Cortex-specific loss of Lhx2 alters the expression of neuronal subtype markers in layers 5 and 6. A, Expression of Lhx2 in control brains is seen in the ventricular zone at E12.5 and E15.5 (white asterisks) but not in postmitotic deep layer neurons at E15.5 and P7 (black asterisks). B, C, Expression of neuronal subtype markers at P7 in control and LHX2cKO brains reveals a decrease in the Tbr1-expressing population and an increase in the Fezf2, ER81, and Id2-expressing population. B, Boxed regions are shown in C. D, E, The numbers of cells expressing TBR1 or CTIP2, expressed as a percentage of all DAPI-stained cells in layer 5 (L5) + layer 6 (L6). Error bars indicate SEM. Scale bars, 500 μm. *p < 0.05. **p < 0.001.

Figure 2.

LHX2 occupies enhancer elements of cortical neuron subtype regulators Fezf2 and Sox11. A, Diagram illustrating dissection of cortical primordia. B, The LHX2 binding site sequence reported in the literature was also found in the LHX2 binding regions of Fezf2 and Sox11. C–F, ChIP-Seq data showing UCSC genome browser tracks of the LHX2 occupancy profile at the Fezf2 (C) and the Sox11 (E) loci. Each LHX2-binding region was validated by ChIP followed by qPCR analysis (D,F). G, H, Examination of Fezf2 and Sox11 at E12.5 (G), 1 d after cortex-specific loss of LHX2, reveals an increased expression of Fezf2 and Sox11 in the ventricular zone (white asterisks) and an increased accumulation of Fezf2- and Sox11-expressing postmitotic cells (arrowhead). High-magnification images are displayed alongside. H, By E13.5, the increase in the ventricular zone has attenuated (white asterisks), but the expression in the cortical plate has expanded (arrowhead). Error bars indicate SEM. Scale bar, 100 μm. *p < 0.05. **p < 0.001.

Figure 3.

LHX2 binds specific subunits of the NuRD complex. A, A silver-stained gel showing control (IgG) and LHX2 IP from cortical tissue. Boxes represent bands that were excised and from which the proteins were analyzed using mass spectrometry. B, Putative LHX2 binding partners identified in the mass spectrometry (HDAC2, LSD1, and RBBP4) were validated by Western blot (immunoblot) analysis of LHX2 IP material using antibodies against each candidate partner. C, Reverse validation of interactions was performed by performing IP for each binding partner and probing the Western blot using anti-LHX2. D, Diagram illustrating the NuRD complex and members that bind LHX2. E, ChIP-qPCR-based occupancy analysis demonstrating the binding of LSD1, HDAC2, and RBBP4 to either the TSS and/or the LHX2 binding region (LHX2 BR) on Fezf2 and/or Sox11. y-axis indicates fold enrichment over IgG at the respective loci. CHD4, Chromodomain helicase DNA binding protein 4; HDAC2, histone deacetylase 2; LSD1, lysine-specific histone demethylase1; MDB, methyl CpG binding domain protein; MTA1/2, metastasis-associated protein1/2; RBBP4, retinoblastoma binding protein 4. A, The image of the silver-stained gel has been cropped to remove the lanes corresponding “unbound” fractions of control IgG and LHX2 IP that were on the left of the marker lane. B, C, The Western blots are cropped from full-length Western blots. The original uncropped images are available upon request. Error bars indicate SEM. *p < 0.05. **p < 0.001. ***p < 0.0001.

Figure 4.

Loss of LHX2 causes an increase in active epigenetic histone marks on Fezf2 and Sox11. A, ChIP-qPCR for active histone marks H3K4me3 and H3K9ac in E12.5 control versus LHX2cKO cortical tissue. y-axis indicates fold change over control at the respective loci. B, Diagram depicting proposed model of chromatin looping bringing distant regulatory elements and TSS near each other. We propose that LHX2 binding to the regulatory elements of its target genes Fezf2 and Sox11 recruits the NuRD subunits LSD1 and HDAC2, which associate with the TSS and the LHX2 binding region, leading to erasure of active marks. In the absence of LHX2, the active marks are enriched. Error bars indicate SEM. *p < 0.05. **p < 0.001. ***p < 0.0001.

Figure 5.

LHX2 is necessary and sufficient to regulate molecular subtype identity. A, B, Electroporation of control GFP (A) or LHX2-GFP (B) at E12.5 and examination at P5 reveal GFP-expressing cells in the region of electroporation, overlapping with CTIP2-expressing layer 5 cells. Individual high-magnification confocal images of GFP, CTIP2, SATB2, and the corresponding merged images of GFP/CTIP2 and GFP/SATB2 are shown alongside the low-magnification GFP/CTIP2 image. C, Diagram illustrating in utero electroporation at E12.5, and examination of brain sections at P5. D, The percentage of electroporated (GFP-expressing) cells that also express CTIP2 or SATB2 reveals a striking decrease in CTIP2-expressing cells and an increase in SATB2-expressing cells upon electroporation of LHX2-GFP. E, A gradient of Lhx2 expression is seen in a series of sagittal sections at E12.5. Scale bars: A, B, 500 μm (low-magnification images), 50 μm (high-magnification images); E, 500 μm. Error bars indicate SEM. **p < 0.001.