PAPER Tissue-specific regulation and function of Grb10 during COLLOQUIUM growth and neuronal commitment Robert N. Plasschaert and Marisa S. Bartolomei1 Department of Cell & Developmental Biology, Perelman School of Medicine at the University of Pennsylvania, Philadelphia, PA 19104 Edited by Eric B. Keverne, University of Cambridge, Cambridge, United Kingdom, and accepted by the Editorial Board September 23, 2014 (received for review July 29, 2014) Growth-factor receptor bound protein 10 (Grb10) is a signal factor, which is recruited in a DNA methylation-sensitive manner adapter protein encoded by an imprinted gene that has roles in and has been implicated in the regulation of imprinted expression growth control, cellular proliferation, and insulin signaling. Addi- at other loci (Fig. 1A). tionally, Grb10 is critical for the normal behavior of the adult Thus, Grb10 is an imprinted gene with multiple functions and mouse. These functions are paralleled by Grb10’s unique tissue- a complex tissue-specific imprinted expression pattern. Here, we specific imprinted expression; the paternal copy of Grb10 is review work that has illuminated the functional roles of Grb10 in expressed in a subset of neurons whereas the maternal copy is embryonic development, cellular growth, and behavior. We also expressed in most other adult tissues in the mouse. The mecha- review the various regulatory mechanisms that have been implicated nism that underlies this switch between maternal and paternal in the control of Grb10 tissue-specific expression and imprinting. expression is still unclear, as is the role for paternally expressed Additionally, we present data addressing the regulation of Grb10 Grb10 in neurons. Here, we review recent work and present com- expression during neuronal development in vitro. We show that in plementary data that contribute to the understanding of Grb10 vitro differentiation of mouse embryonic stem cells (mESCs) into gene regulation and function, with specific emphasis on growth motor neurons results in the switch of Grb10 promoter use. Spe- and neuronal development. Additionally, we show that in vitro cifically, the maternally expressed major promoter is repressed differentiation of mouse embryonic stem cells into alpha motor whereas the paternally expressed neuron-specific promoter is acti- neurons recapitulates the switch from maternal to paternal ex- vated, concordant with neuronal maturation. We postulate that this BIOLOGY pression observed during neuronal development in vivo. We switch in expression is required for the appropriate function of DEVELOPMENTAL postulate that this switch in allele-specific expression is related Grb10 in the nervous system. to the functional role of Grb10 in motor neurons and other neuronal tissues. Grb10 Binding Partners Define Its Role in Development In mouse and human, Grb10 has five major protein domains: the genomic imprinting | epigenetics | Grb10 | motor neurons | N-terminal proline-rich region (PR), a centrally located pleck- neurodevelopment strin homology domain (PH), a Ras-associating domain (RA), a C-terminal Src homology 2 domain (SH2), and the family- rb10 is an adapter protein that is a member of the Grb7/ specific BPS domain, so named because it is between the PH and GGr10/Grb14 protein family. These proteins interact with SH2 domains (4). Each domain is associated with protein binding numerous receptor tyrosine kinases, impacting a variety of sig- partners that define the functions of Grb10 in development and naling pathways (1). Specifically, Grb10 has been implicated in growth (Fig. 2 and Table S1). the regulation of cell proliferation, apoptosis, and metabolism, The SH2 domain enables Grb10 to interact with phosphory- among other functions (2–4). The first characterized Grb10 lated tyrosine residues of other proteins and acts as the re- knockout mouse exhibited overgrowth in numerous organs and cruitment point for a variety of signaling molecules. Many of tissues, suggesting that Grb10 acts as a potent growth suppressor these proteins are receptors associated with growth, including in vivo (5). Other Grb10 knockout mouse models have addi- the insulin receptor (IR) and the growth hormone receptor tionally shown that Grb10 plays an important role in social (GHR) (9, 10). The SH2 domain is critical for the role of Grb10 dominance behavior in mice (6). in the mitogen-activated protein kinase (MAPK) pathway, hav- Interestingly, these phenotypes are dependent on the parental ing direct interactions with numerous critical components in- origin of the Grb10 mutation. The overgrowth phenotype is cluding MEK1 and RAF1 (11). The SH2 domain also interacts exhibited only when the mutation is transmitted through the ma- with many receptor proteins implicated in cell proliferation, ternal lineage whereas the behavior phenotype is revealed upon differentiation, and control of the cell cycle, including RET, paternal transmission of the mutation. These surprising allele- KIT, MET, PDGFR, EGFR, FLT3, and VEGFR-2 (9, 12–16). specific phenotypes can be explained by the tissue-specific genomic Additionally, the SH2 domain has been implicated in Grb10 imprinting of Grb10. Genomic imprinting refers to an infrequent regulatory phenomenon in which a gene is expressed in a parent- – of-origin specific manner. Genomic imprinting is dependent on This paper results from the Arthur M. Sackler Colloquium of the National Academy of cis-regulatory elements called imprinting control regions (ICRs) Sciences, “Epigenetic Changes in the Developing Brain: Effects on Behavior,” held March that are epigenetically modified on a single parental allele to 28–29, 2014, at the National Academy of Sciences in Washington, DC. The complete pro- induce allele-specific expression (7). Grb10 is expressed from gram and video recordings of most presentations are available on the NAS website at www.nasonline.org/Epigenetic_changes. the maternal chromosome in most tissues in the mouse but is Author contributions: R.N.P. and M.S.B. designed research; R.N.P. performed research; R.N.P. expressed from the paternal allele in a subset of neurons. Maternal contributed new reagents/analytic tools; R.N.P. and M.S.B. analyzed data; and R.N.P. and M.S.B. expression occurs from the Grb10 major promoter whereas Grb10 wrote the paper. paternal expression comes from three downstream alternative The authors declare no conflict of interest. promoters (Fig. 1). The region surrounding one of these alternative This article is a PNAS Direct Submission. E.B.K. is a guest editor invited by the Editorial promoters has been identified as an ICR, which exhibits DNA Board. methylation only on the maternal allele in all examined tissues (8). 1To whom correspondence should be addressed. Email: [email protected]. On the paternal allele, the unmethylated Grb10 ICR is bound by This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. CCCTC-binding factor (CTCF), a multifunctional transcription 1073/pnas.1411254111/-/DCSupplemental. www.pnas.org/cgi/doi/10.1073/pnas.1411254111 PNAS | June 2, 2015 | vol. 112 | no. 22 | 6841–6847 Downloaded by guest on September 26, 2021 A Grb10 (chr11:11930499-12037420; mm10) 1A (Major) A.S. ICR 4R CTCF 1B2 (Neuronal) 1B1 (Neuronal) 1C (Neuronal/Embryonic) B Whole Whole E14 Spinal C Kidney Brain Brain Liver mESC Cord (Cast x B6) (Cast x B6) Cac8I - + - + 1A-4R 311bp 1C-4R 199bp 1B1-4R 1B2-4R 112bp Fig. 1. Grb10 is expressed maternally from most adult tissues and paternally in neuronal tissues. (A) A schematic of the Grb10 locus is shown, with the allelic- specific expression of alternative promoters indicated (maternal and paternal alleles, arrow above the line and below the line, respectively). The imprinting control region (ICR) is designated by a yellow box. Maternal allele-specific DNA methylation, evident in all characterized tissues, is indicated by black lollipops, above the line. The reverse primer location for expression analysis is shown by its name, 4R. The location of the amplicon used for allele-specific (A.S.) analysis is similarly indicated. (B) Expression of transcripts from alternative promoters of Grb10 is shown in adult brain, adult liver, mESCs, and adult spinal cord. (C) Allele-specific expression as measured via SNP-dependent Cac8I restriction enzyme digests is shown in brain and liver from Mus Castaneus/EiJ (Cast) × C57BL/6J (B6) hybrid adult mice. Expression of the maternal allele is indicated by the 199-bp and 112-bp bands after restriction digest whereas the 311-bp band indicates paternal expression. homodimerization, which is hypothesized to enable Grb10 to a variety of metabolic consequences affecting the storage and integrate signals from multiple pathways through the formation synthesis of glucose and fatty acids, in addition to promoting of larger protein complexes (17). cellular growth through regulating proteins important for the cell The BPS domain, specific to the Grb7/10/14 protein family, cycle and other mitogenic factors. Additionally, both pathways has also been shown to be important for Grb10’s role in insulin can activate mTORC1, a protein complex that integrates growth signaling through its binding to IR and additionally binds IGF1R signals and acts positively upon cell proliferation (24). (14). The BPS domain interacts with the 14-3-3 protein, another Grb10 potently inhibits cellular growth by acting at multiple protein scaffold with numerous protein partners, and BCL2L11, levels in these pathways, among others. Grb10 inhibits insulin