Downloaded from genesdev.cshlp.org on October 7, 2021 - Published by Cold Spring Harbor Laboratory Press Phf21b imprints the spatiotemporal epigenetic switch essential for neural stem cell differentiation Amitava Basu,1 Iván Mestres,2 Sanjeeb Kumar Sahu,3 Neha Tiwari,4 Bimola Khongwir,1 Jan Baumgart,5 Aditi Singh,6 Federico Calegari,2 and Vijay K. Tiwari6 1Institute of Molecular Biology, 55128 Mainz, Germany; 2Center for Regenerative Therapies Dresden (CRTD), School of Medicine, Technische Universität Dresden, 01307 Dresden, Germany; 3Salk Institute for Biological Studies, La Jolla, California 92037, USA; 4Institute of Physiological Chemistry, University Medical Center Johannes Gutenberg-University Mainz, 55128 Mainz, Germany; 5Translational Animal Research Center (TARC), University Medical Centre, Johannes Gutenberg-University, 55131 Mainz, Germany; 6Wellcome-Wolfson Institute for Experimental Medicine, School of Medicine, Dentistry, and Biomedical Science, Queens University Belfast, Belfast BT9 7BL, United Kingdom Cerebral cortical development in mammals involves a highly complex and organized set of events including the transition of neural stem and progenitor cells (NSCs) from proliferative to differentiative divisions to generate neurons. Despite progress, the spatiotemporal regulation of this proliferation-differentiation switch during neuro- genesis and the upstream epigenetic triggers remain poorly known. Here we report a cortex-specific PHD finger protein, Phf21b, which is highly expressed in the neurogenic phase of cortical development and gets induced as NSCs begin to differentiate. Depletion of Phf21b in vivo inhibited neuronal differentiation as cortical progenitors lacking Phf21b were retained in the proliferative zones and underwent faster cell cycles. Mechanistically, Phf21b targets the regulatory regions of cell cycle promoting genes by virtue of its high affinity for monomethylated H3K4. Subse- quently, Phf21b recruits the lysine-specific demethylase Lsd1 and histone deacetylase Hdac2, resulting in the simultaneous removal of monomethylation from H3K4 and acetylation from H3K27, respectively. Intriguingly, mutations in the Phf21b locus associate with depression and mental retardation in humans. Taken together, these findings establish how a precisely timed spatiotemporal expression of Phf21b creates an epigenetic program that triggers neural stem cell differentiation during cortical development. [Keywords: cortical development; epigenetics; genomics; gene regulation; neurogenesis] Supplemental material is available for this article. Received October 18, 2019; revised version accepted July 21, 2020. The development of the mammalian cerebral cortex is a Orford and Scadden 2008; Hardwick et al. 2015). Misregu- highly coordinated process that relies on a complex inter- lation of molecules involved in these cortical development play between a variety of regulatory factors including epi- pathways is known to be associated with cortical malfor- genetic regulators and transcriptional factors that control mations caused by abnormal proliferation, migration de- gene expression (Guillemot et al. 2006; Itoh et al. 2013; Flo- fects or altered connectivity (Guerrini and Parrini 2010; rio and Huttner 2014; Imayoshi and Kageyama 2014; Shi- Wollnik 2010; Bozzi et al. 2012; Yang et al. 2012; Gilmore bata et al. 2015; Thakurela et al. 2015; Pataskar et al. and Walsh 2013; Homem et al. 2015). The regulation of cell 2016; Urbán et al. 2016; Kishi and Gotoh 2018; Tsuboi cycle, specifically the G1 phase, was also shown to play a et al. 2018; Zhang et al. 2019). During embryonic develop- crucial role in controlling area-specific rates of neuron pro- ment of the mammalian brain, neural stem and progenitor duction and the generation of cytoarchitectonic maps cells (NSC) progressively switch from proliferative to dif- (Dehay and Kennedy 2007). For example, lengthening G1 ferentiative divisions to generate neurons and glia that of neural stem and progenitor cells triggered premature populate the cortical layers. The molecular control of the neurogenesis (Calegari and Huttner 2003), while shorten- switch from proliferation to differentiation is of vital im- ing G1 inhibited neurogenesis and promoted the portance for proper neurogenesis during cortical develop- ment (Dehay and Kennedy 2007; Herrup and Yang 2007; © 2020 Basu et al. This article is distributed exclusively by Cold Spring Harbor Laboratory Press for the first six months after the full-issue publi- cation date (see http://genesdev.cshlp.org/site/misc/terms.xhtml). After Corresponding author: [email protected] six months, it is available under a Creative Commons License (Attribu- Article published online ahead of print. Article and publication date are tion-NonCommercial 4.0 International), as described at http://creative- online at http://www.genesdev.org/cgi/doi/10.1101/gad.333906.119. commons.org/licenses/by-nc/4.0/. 1190 GENES & DEVELOPMENT 34:1190–1209 Published by Cold Spring Harbor Laboratory Press; ISSN 0890-9369/20; www.genesdev.org Downloaded from genesdev.cshlp.org on October 7, 2021 - Published by Cold Spring Harbor Laboratory Press Epigenetic regulation of cortical development expansion of the progenitor pool (Lange et al. 2009; Pilaz a master regulator of cortical development by controlling et al. 2009). Several factors are well characterized to con- the epigenetic program underlying the spatiotemporal trol the progression through G1, including retinoblastoma switch from proliferation to differentiation during neuro- (Rb), which becomes hyperphosphorylated and allows E2F genesis. Corroborating our findings of a critical role of transcription factors to induce several downstream tar- Phf21b during cortical development, a deletion encom- gets, notably cyclins, required for cell cycle progression passing PHF21B locus was shown to be associated with (Poppy Roworth et al. 2015). However, despite an in- the neurodevelopmental disorder Phelan-McDermid syn- creased understanding of the downstream gene regulatory drome (Sarasua et al. 2014) and a rare single nucleotide programs controlling cell cycle progression (Lee 1997; Shi- variation near PHF21B gene was linked with an increased bata et al. 2015), the upstream epigenetic triggers that risk of major depressive disorder (Wong et al. 2017). initiate the Rb–E2F cascade and essential for NSC differen- tiation and brain development remain elusive. The PHD proteins comprise an important set of epige- Results netic readers that are known to play diverse roles including Phf21b is induced during neurogenesis and exhibits a in cell cycle regulation in different contexts (Baker et al. distinct spatiotemporal expression pattern 2008; Gatchalian et al. 2016). Along these lines, genetic ab- errations that target PHD fingers of certain genes (e.g., In search of novel PHD-containing proteins relevant for RAG2, ING, NSD1, and ATRX) have been associated embryonic neurogenesis, we compiled a list of 75 candi- with a wide range of human pathologies including immu- dates using Interpro database (Hunter et al. 2009) and an- nological disorders, cancers, and neurological diseases, po- alyzed the expression of the corresponding genes using tentially arising from a misinterpretation of epigenetic transcriptome (RNA-seq) data sets of several tissues dur- marks (Baker et al. 2008). Depending on the stoichiometry, ing development including the cortical areas ventricular the PHD proteins recognize specific histone modifications zone (VZ), subventricular zone (SVZ), and cortical plate and regulate gene expression (Sanchez and Zhou 2011). For (CP) as well as the specific cell populations of proliferative example, PHD fingers from BPTF and ING2 recognize versus neurogenic progenitors and neurons (Fietz et al. H3K4me3 mark (Jones et al. 2000; Lan et al. 2007), while 2012; Aprea et al. 2013). This revealed that 27 out of 75 the PHD fingers in BHC80 and DNMT3L bind to unmod- PHD-containing genes are expressed at higher levels in ified histone H3 tails. Interestingly, a PHD protein Phf6 the cortex compared with other tissues (Fig. 1A; Supple- also regulates neuronal migration and its mutations are as- mental Fig. S1a). Out of the 27 shortlisted genes, 10 sociated with intellectual disability (Zhang et al. 2013). were not expressed or very lowly expressed in VZ and 13 Another PHD protein Phf21a (Bhc80) was shown to recog- were not differentially expressed. Of the remaining four nize H3K4me0 (Lan et al. 2007) and repress nonneuronal genes, we shortlisted Phf21b for further investigation giv- genes in cooperation with REST (Monaghan et al. 2017). en its genetic association with a neurodevelopmental dis- However, the expression pattern of Phf21a is not brain-spe- order (Phelan-McDermid syndrome) as well as depression cific and Phf21a knockout mice died due to suckling dys- in humans. Surprisingly, no study has investigated the function without any noticeable brain defects (Iwase function of Phf21b in cortical development, or its role as et al. 2006). an epigenetic regulator in any other context. Here we used a comprehensive bioinformatics analysis Phf21b contains a single PHD and a nuclear localization in combination with global gene expression profiling of signal (NLS) predicted at its N terminal end (Fig. 1B). In several tissues derived from the three germ layers, which situ hybridization data (Visel 2004) allowed us to validate revealed certain PHD finger proteins that are specifically the expression of Phf21b selectively within the cortex and expressed