Human Septin 3 on Chromosome 22q13.2 Is Upregulated by Neuronal Differentiation Axel Methner, 1 Frank Leypoldt, Patrick Joost, and Jan Lewerenz Department of Neurology and Zentrum fu ¨ r Molekulare Neurobiologie, University Hospital Hamburg, Falkenried 94, D-20251 Hamburg, Germany Received March 13, 2001 An expression sequence tag identified in a screen for genes upregulated by retinoic acid induced neuronal differentiation of the human teratocarcinoma cell line Ntera2/D1 was found in close genomic proximity to a region of high sequence homology to the septin sub- family of GTPase genes. We could show that the tag corresponds to the 3 untranslated region of this novel gene named septin 3 and cloned three isoforms A (2191 bp), B (4378 bp), and C (1896 bp) from human Ntera2/D1 cDNA. We present the genomic localization and organization on chromosome 22q13.2, a chromo- somal hot spot for translocations implicated in leuke- mia. Interestingly, MSF the closest paralog of septin 3 is a fusion partner in a therapy-related acute myeloid leukemia. Quantitative PCR confirmed the upregula- tion of the putative septin by neuronal differentiation and northern blotting showed only one band corre- sponding to sep3B with a neurospecific expression pattern in adult human tissues. © 2001 Academic Press Key Words: Ntera2cl.D/1 (NT2) cells; neuronal differ- entiation; septin; leukemia. Neurons and glia derive from pluripotent precursor cells and differentiate in response to environmental and intrinsic factors. This process can be studied in vitro by retinoic acid (RA) induced differentiation of the human teratocarcinoma cell line NTera-2/D1 (NT2). NT2 cells remain as undifferentiated mitotic precursor cells, when maintained under defined tissue culture conditions. Upon exposure to RA and the use of differ- ential adhesion matrices and mitotic inhibitors, the cells develop the morphological and cytoskeletal char- acteristics of postmitotic central nervous system (CNS) neurons (1). This seems to model the in vivo situation, as high concentrations of RA have been detected in the embryonic CNS and the developing spinal cord (2– 4). RA binding proteins and its receptors are present in the developing nervous system suggesting a role in neurogenesis (5– 8). A screen for genes regulated by RA-induced differ- entiation of NT2 cells generated I-U#19, an expressed sequence tag (EST) with no homology to known genes (9). The progress of the human genome project as- signed this EST to human chromosome 22q13.2 in close genomic proximity to a region of high sequence homology to the septin subfamily of GTPase genes. This putative gene shares strong similarity to the mouse neuronal-specific septin 3 (10) and rat brain- specific G-septin (11). The septins are a family of 40 –50 kDa GTPases that were first identified in Saccharomy- ces cerevesiae as proteins required for the completion of the cell cycle (12). These proteins share several char- acteristics, e.g., a P-loop nucleotide-binding consensus sequence for GTP binding near the N terminus (13), and most are predicted to have a coiled-coil domain at the C terminus. Mutations of yeast septins lead to the disruption of a characteristic set of filaments associ- ated with the plasma membrane and result in cell cycle arrest and defective cytokinesis (14). Yeast septins form hetero-oligomeric filaments that contribute to bud site selection and neck stability (reviewed in (15)). Sep- tin proteins from Drosophila also assemble into hetero- oligomeric filaments (16) and are required for cytoki- nesis (17). The mammalian septin NEDD5 localizes near the contractile ring during cytokinesis in HeLa cells, and microinjection of anti-NEDD5 antibodies into dividing cells results in binucleated cells, suggest- ing that NEDD5 plays an essential role during cytoki- nesis (18). New roles for neuronal septins are emerging in synaptic transmission neurons (19). Other septin genes known in mammalian tissues are mouse DIFF6 (20), mouse PNUTL2/H5 (21), human CDC10 (22), hu- man CDCrel-1/PNUTL1 (23, 24), human SEP2-like (25), human SEPTIN 6 (26), human MSF/mouse SINT1 (27, 28). In this study, we used the sequence information from I-U#19 to clone a novel human septin with high homol- ogy to mouse neuronal-specific septin 3 and rat 1 To whom correspondence should be addressed. Fax: +49 40 4 28 03 51 01. E-mail: methner@uke.uni-hamburg.de. Biochemical and Biophysical Research Communications 283, 48 –56 (2001) doi:10.1006/bbrc.2001.4741, available online at http://www.idealibrary.com on 48 0006-291X/01 $35.00 Copyright © 2001 by Academic Press All rights of reproduction in any form reserved.