Chromatin remodelling factor Mll1 is essential for neurogenesis from postnatal neural stem cells

Abstract

Epigenetic mechanisms that maintain neurogenesis throughout adult life remain poorly understood. Trithorax group (trxG) and Polycomb group (PcG) gene products are part of an evolutionarily conserved chromatin remodelling system that activate or silence gene expression, respectively. Although PcG member Bmi1 has been shown to be required for postnatal neural stem cell self-renewal, the role of trxG genes remains unknown. Here we show that the trxG member Mll1 (mixed-lineage leukaemia 1) is required for neurogenesis in the mouse postnatal brain. Mll1-deficient subventricular zone neural stem cells survive, proliferate and efficiently differentiate into glial lineages; however, neuronal differentiation is severely impaired. In Mll1-deficient cells, early proneural Mash1 (also known as Ascl1) and gliogenic Olig2 expression are preserved, but Dlx2, a key downstream regulator of subventricular zone neurogenesis, is not expressed. Overexpression of Dlx2 can rescue neurogenesis in Mll1-deficient cells. Chromatin immunoprecipitation demonstrates that Dlx2 is a direct target of MLL in subventricular zone cells. In differentiating wild-type subventricular zone cells, Mash1, Olig2 and Dlx2 loci have high levels of histone 3 trimethylated at lysine 4 (H3K4me3), consistent with their transcription. In contrast, in Mll1-deficient subventricular zone cells, chromatin at Dlx2 is bivalently marked by both H3K4me3 and histone 3 trimethylated at lysine 27 (H3K27me3), and the Dlx2 gene fails to properly activate. These data support a model in which Mll1 is required to resolve key silenced bivalent loci in postnatal neural precursors to the actively transcribed state for the induction of neurogenesis, but not for gliogenesis.

Figure 1. Mll1 is required for normal SVZ-olfactory bulb neurogenesis

a, Schematics of SVZ-olfactory-bulb neurogenesis. Left, coronal section of the olfactory bulb (OB) indicating the region where newly born neuroblasts (red dots) initially arrive from the SVZ. GCL, granule cell layer. Middle, sagittal section showing paths of neuroblast migration from the SVZ to the olfactory bulb. Right, coronal section indicating the germinal SVZ (red dots); the blue box indicates regions shown in c. b, Haematoxylin and eosin (H&E)-stained coronal sections through the P25 olfactory bulb of control (left) and hGFAP-Cre;Mll1^F/F (right) mice. The black box indicates the olfactory bulb core comprised of recently born neuroblasts. c, DCX (red) and BrdU (green) immunohistochemistry of the SVZ is shown. The size of the SVZ is indicated by a yellow doubleheaded arrow. LV, lateral ventricle; St, striatum. d, Quantification of BrdU⁺ DCX⁺ SVZ cells. hpf, high power field. Error bars, s.e.m.; three mice per group; *P = 0.025. Scale bars, 200 μm (b) and 20 μm (c).

Figure 2. Mll1-deletion impairs postnatal SVZ-olfactory-bulb neurogenesis but not gliogenesis

a, Immunohistochemistry for the astrocyte marker GFAP (green) in P25 coronal brain sections of control (left) and hGFAP-Cre;Mll1^F/F (right) mice. Nuclei are counterstained with 4,6-diamidino-2-phenylindole (DAPI; blue). b, Immunohistochemistry for the S100⁺ (green) ependymal cells. c, Immunohistochemistry for markers of oligodendrocytes, OLIG2 (green) and MBP (red). CC, corpus callosum. d, SVZ cultures after 4 days of differentiation from control (left) and Mll1^Δ/Δ (right) mice immunostained for the neuronal marker Tuj1 (red). e, O4⁺ (green) oligodendrocytes in the same fields of view as those in d. f, Quantification of cell differentiation. Error bars, s.e.m. of triplicate cultures; *P = 0.016. Scale bars, 20 μm.

Figure 3. Mll1-dependent DLX2 expression is required for postnatal SVZ neurogenesis

a, Immunocytochemistry for Tuj1⁺ (red) neuroblasts in FACS-isolated GFP⁺ ZEG;control SVZ cells after differentiation. b, ZEG;Mll1^Δ/Δ cultures stained for Tuj1. c, Quantification of Tuj1⁺ neuronal differentiation and activated caspase 3⁺ cells after 4 days of differentiation. **P = 0.002. d, e, Quantification of glial differentiation. The number of O4⁺oligodendrocytes (d) and GFAP⁺astrocytes (e) was counted after 4–5 days of differentiation. f, Quantification of MASH1⁺ and DLX2⁺ cells after 2 days of differentiation. *P = 0.02. g, h, Immunohistochemistry for DLX2 (green) and DCX (red) in SVZ coronal brain sections of control (g) and hGFAP-Cre;Mll1^F/F mice (h). i–k, Enforced Dlx2 expression can rescue neurogenesis in Mll1^Δ/Δ SVZ cultures. Immunocytochemistry for Tuj1 (red) and GFP (green) after transfection of both pCAG-Dlx2 and pCAG-GFP plasmids (i) and pCAG-GFP plasmid alone (j). k, Quantification of neuronal lineage rescue by Dlx2 transfection. **P = 0.004; error bars, s.e.m.; 3–6 replicates per group. Scale bars, 10 μm (g, h) and 20 μm (a, b, i, j).

Figure 4. Dlx2 is trimethylated at both H3K4 and H3K27 in Mll1^Δ/Δ cells

At the top is a schematic indicating the location of the primer sets used for qChIP. a, MLL1 qChIP of the Dlx2 locus. b, qRT–PCR analysis of Dlx2, Mash1 and Olig2 in wild-type (grey bars) and Mll1^Δ/Δ (black bars) cells during early differentiation. c, qChIP analysis of H3K4me3 levels at Dlx2, Mash1 and Olig2 loci. d, qChIP for H3K27me3 levels. e, Model of Mll1 function in the specification of the neuronal lineage from NSCs. NSCs have bivalent chromatin domains at key neurogenic genes (for example, Dlx2). In this state, precursors can form astrocytes and oligodendrocytes (blue arrows). In order for neurogenesis to proceed (red arrow), MLL1 is required for the resolution of specific bivalent loci, possibly by recruiting H3K27-specific demethylases (K27DM). Error bars, s.d.; n = 3.