Dual role of ZIC2 during neural induction: from pioneer transcription factor to enhancer activator [Multiome]

ZIC2, a member of the Zinc Finger of the Cerebellum family of transcription factors (TFs), plays crucial roles during neural development. In humans, defects in ZIC2 cause holoprosencephaly, a congenital brain malformation characterized by the defective cleavage of cerebral hemispheres due to problems in midline patterning. However, the gene regulatory network (GRN) controlled by ZIC2 and the regulatory mechanisms it employs during neural development remain largely unexplored. Here, we combined a mouse embryonic stem cell (mESC) in vitro differentiation model towards anterior neural progenitors (AntNPCs) with genome editing approaches, bulk and single cell (i.e. Multiome scATAC + scRNAseq) genomic methods to elucidate the precise GRN controlled by ZIC2 and the underlying mechanisms. We found that ZIC2 shows widespread binding throughout the genome already in mESC as well as upon pluripotency exit and neural induction. Despite its extensive binding in mESC, ZIC2 function is dispensable in pluripotent cells due to compensation by ZIC3. In contrast, ZIC2 plays a major regulatory function during neural induction, directly controlling the expression of master regulators implicated in the patterning and morphogenesis of specific brain regions, such as the midbrain (e.g., En1, Lmx1b, Pax2, Wnt1) and the roof plate (e.g., Lmx1a, Wnt3a). Mechanistically, ZIC2 plays a dual role in neural differentiation: (i) during pluripotency exit, ZIC2 acts as a pioneer TF, binding de novo to distal enhancers and promoting their chromatin accessibility; (ii) during neural induction, ZIC2 is essential for the activation of a subset of the previously primed enhancers, which in turn control the expression of major neural patterning regulators and signaling pathways (i.e. WNT) that prevent the premature differentiation of neural progenitors. Overall, our work shows that, by sequentially acting as a promiscuous pioneer and selective activator of enhancer elements, ZIC2 canalizes pluripotent cells towards neural progenitors with rostro-dorsal identities. Multiome 10X experiment (snRNA-seq and snATAC-seq) was performed using WT and Zic2-/- (Z2KO) day 3 (D3) progenitor cells and day 6 (D6) AntNPCs, obtained from a 6 days in-vitro differentiation from mESCs (D0) towards AntNPCs (D6). To do so, the initial nuclei isolation started by washing cells twice with PBS before detaching them from the tissue culture plate with Stempro™ Accutase™ Cell Dissociation Reagent (Thermo Scientific, A1110501). The Accutase incubation times were adjusted to the cell type (6 min for D3 progenitors and 10 min for D6 AntNPCs). Next, Accutase was inactivated with BSA 0.04% in PBS. Cell clumps were disrupted by gently pipetting up and down the cell suspension in the collection tubes. Cell suspension was then centrifuged for 5 min at 300 rcf. The supernatant was aspirated without disrupting the cell pellet and cells were washed twice in BSA 0.04% in PBS. Then, cells were diluted 1/10 in BSA 0.04% in PBS and counted. Next, 106 cells were transferred to a DNA LoBind® 1.5 ml microtube (Eppendorf, 0030108051). Cells were centrifuged again at 300 rcf for 5 min at 4ºC. Next, the supernatant was carefully aspirated and cells were lysed by adding 175 µl of pre-chilled Lysis Buffer (10X Genomics) to the pellet while smoothly pipetting up and down 10 times.After incubation for 4 min on ice, suspension was centrifuged at 500 rcf for 5 min at 4º C. Then, supernatant was completely removed without disrupting the nuclei pellet. Nuclei suspension was cleaned up by adding Wash Buffer (10X Genomics) followed by centrifugation (500 rcf, 5 min at 4ºC). Assuming ~50% nuclei loss during cell lysis, cells were resuspended in chilled Diluted Nuclei Buffer (10X Genomics) to achieve the desired final nuclei concentration. The rest of the steps of the scATAC-seq and scRNA-seq coupled protocol were performed exactly as indicated in the Chromium Next GEM Single Cell Multiome ATAC + Gene Expression User Guide CG000338 Rev F (https://cdn.10xgenomics.com/image/upload/v1666737555/support-documents/CG00033_ChromiumNextGEM_Multiome_ATAC_GEX_User_Guide_RevF.pdf).