Available online at www.sciencedirect.com JGG Journal of Genetics and Genomics 40 (2013) 399e412 ORIGINAL RESEARCH Evolutionary and Functional Analysis of the Key Pluripotency Factor Oct4 and Its Family Proteins Xinmin Zhang a,1, Yuzhen Ma e,1, Xiuying Liu b,d, Qi Zhou c,*, Xiu-Jie Wang b,* a Computer & Information Engineering College, Inner Mongolia Normal University, Inner Mongolia, Hohhot 010022, China b State Key Laboratory of Plant Genomics, Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, Beijing 100101, China c State Key Laboratory of Reproductive Biology, Institute of Zoology, Chinese Academy of Sciences, Beijing 100101, China d University of Chinese Academy of Sciences, Beijing 100101, China e Centre of Reproductive Medicine, Inner Mongolia Hospital, Inner Mongolia, Hohhot 010017, China Received 28 March 2013; revised 14 April 2013; accepted 15 April 2013 Available online 13 June 2013 ABSTRACT Oct4 is one of the key pluripotent factors essential for embryonic stem cells and induced pluripotent stem (iPS) cells. Oct4 belongs to the POU domain family, which contains multiples genes with various important functions. Although the function of Oct4 has been extensively studied, detailed comparison of Oct4 with other POU family genes and their evolutionary analysis are still lacking. Here, we systematically identified POU family genes from lower to higher animal species. We observed an expansion of POU family genes in vertebrates, with an additional increment in mammalian genomes. We analyzed the phylogenetic relationship, tissue specific expression profiles and regulatory networks of POU family genes of the human genome, and predicted the putative binding microRNAs of human POU family genes. These results provide the first comprehensive evolutionary and comparative analysis of POU family genes, which will help to better understand the relationships among POU family genes and shed light on their future functional studies. KEYWORDS: Oct4; Induced pluripotent stem cell; POU family; MicroRNA; Evolutionary analysis INTRODUCTION implantation (Pesce and Scholer, 2001). Oct4 shares a sub- stantial fraction of target genes with the other key pluripotent Oct4 is a key transcription factor regulating the pluripotency regulatory factors, namely Sox2 and Nanog. Genes co- of embryonic stem cells. By cooperating with other proteins, regulated by these three factors and with functions in pluripo- Oct4 plays essential roles in the establishment and maintenance tency maintenance are actively transcribed in ES cells, whereas of pluripotency in embryonic stem (ES) cells (Babaie et al., those regulating differentiation and development are tran- 2007). Endogenous Oct4 is initially activated as a maternal scriptionally repressed (Loh et al., 2006). Oct4 is the central factor in oocytes and remains active in embryos throughout the factor for the generation of induced pluripotent stem (iPS) cells pre-implantation period (Looijenga et al., 2003). The expres- (Zhao et al., 2009; Wang et al., 2012a, 2012b). Overexpression sion of Oct4 decreases during the trophoblast differentiation of Oct4 with other key transcription factors, typically Sox2, process and remains only in primordial germ cells after Klf4 and c-Myc, can convert differentiated somatic cells back to the embryonic stem cell-like stage (Yamanaka and Blau, 2010). Among these reprogramming factors, Oct4 seems to be indis- þ þ * Corresponding authors. Tel: 86 10 6480 6590, fax: 86 10 6480 6595 pensable for high efficient iPS cell production (Shi and Jin, (X.-J. Wang); Tel: þ86 10 6480 7299, fax: þ86 10 6480 7858 (Q. Zhou). E-mail addresses: [email protected] (Q. Zhou); [email protected] 2010). (X.-J. Wang). Oct4 belongs to the Octamer transcription factor family 0 0 1 These authors contributed equally to this work. which binds to the consensus 5 -ATTTGCAT-3 motif, and 1673-8527/$ - see front matter Copyright Ó 2013, Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, and Genetics Society of China. Published by Elsevier Limited and Science Press. All rights reserved. http://dx.doi.org/10.1016/j.jgg.2013.04.011 400 X. Zhang et al. / Journal of Genetics and Genomics 40 (2013) 399e412 hence it is named as “Oct”. The Oct4 protein contains the interferon-induced transcriptional responses (Hofmann et al., homeodomain and POU domains (Hammachi et al., 2012). 2010). The other POU domain class 3 factors, namely The homeodomain is a protein structural domain binding DNA POU3F2, POU3F3 and POU3F4, are all brain-specific homeo- or RNA, and is thus commonly found in transcription factors box factors with neuronal development related functions (Urrutia, 1997). The name of POU domain is derived from the (Andersen and Rosenfeld, 2001). Similar to the POU3 class names of pituitary-specific domain, Octamer transcription proteins, the POU domain class 4 factors also mainly function in factor domain and neural Unc-86 transcription factor domain regulating the development of sensory nervous system (Erkman from Caenorhabditis elegans. The human genome contains et al., 1996; McEvilly et al., 1996). Besides Oct4, the POU many POU family proteins. They were grouped into 6 classes domain class 5 also contains two other members. POU5F1B is a according to their sequence similarities. Oct4 belongs to the processed pseudogene of Oct4 and may also play roles in the class 5 of POU domain transcription factors, and therefore is regulation of cell pluripotency and cancer development also named as POU5F1. (Crowther-Swanepoel et al., 2010; Kastler et al., 2010). Another The various members of the POU family have a wide variety member of the POU domain class 5 genes is POU5F2, whose of functions, mainly related to the regulation of the neuroen- function is unconfirmed yet. The POU domain class 6 proteins docrine system (Assa-Munt et al., 1993) and the development of are less studied, both members mainly function in brain and may an organism (Andersen and Rosenfeld, 2001). The member of also be implicated in the development of certain cancers (Perotti the POU domain class 1, POU1F1 (Pit1), is expressed in pitui- et al., 2004; Zhang et al., 2011). tary and serves as the key regulator for human growth hormone Although the importance of Oct4 in regulating embryonic expression (Hunsaker et al., 2012). POU2F1, also known as stem cell pluripotency and inducing somatic cell reprogram- Oct1, is ubiquitously expressed in both embryonic and adult ming has been well recognized, systematic analysis on the mouse tissues, and has multiple functions in growth regulation POU family proteins through the evolutionary point of view is and disease development (Sreenivasan and Viljoen, 2013). still lacking. Here, we performed genome-wide identification POU2F2 (Oct2) is a key factor for B-cells, functioning by of POU family members in 10 animal species. With the pri- directly activating the anti-apoptotic gene bcl-2 (Heckman et al., mary focus of the POU family proteins in human, we sys- 2006). POU3F1 (Oct6) is essential for terminal myelination of tematically studied their domain structure, evolutionary Schwann cells (Ryu et al., 2007) and can be involved in conservation, expression profile, interacting network and Fig. 1. Phylogenetic tree of the 10 studied species and the numbers of POU family genes in each species. Numbers in red represent the identified POU family genes. The names of studied species are shown in italic type, and the classification information of species is shown in bold. X. Zhang et al. / Journal of Genetics and Genomics 40 (2013) 399e412 401 relationship with microRNAs (miRNAs). These findings will genome sequences in this work, including Caenorhabditis be helpful for a thorough functional understanding of the POU elegans, Drosophila melanogaster, zebrafish, chicken, cow, family members. pig, mouse, rat, chimpanzee and human (Fig. 1). By searching for proteins with identifiable POU domain and sequence ho- RESULTS mology to the Oct4 protein, genes encoding POU family proteins were identified in these species. In nematode Identification of POU family genes in 10 species C. elegans and the arthropod D. melanogaster, only one POU family gene was found in each of their genomes. However, a To systematically identify POU family genes in animals, big expansion of the POU family genes occurred in verte- we selected 10 commonly studied species with complete brates, with 8 POU family genes in the zebrafish genome, 9 A NP_000297 (POU1F1 isoform alpha) NP_001116229 (POU1F1 isoform beta) NP_002688 (POU2F1 isoform1) NP_001185712 (POU2F1 isoform2) NP_001185715 (POU2F1 isoform3) NP_001193954 (POU2F2 isoform1) NP_002689 (POU2F2 isoform2) NP_001193955 (POU2F2 isoform3) NP_001234923 (POU2F2 isoform4) NP_055167 (POU2F3 isoform1) NP_001231611 (POU2F3 isoform2) NP_002690 (POU3F1) NP_005595 (POU3F2) NP_006227 (POU3F3) NP_000298 (POU3F4) NP_006228 Pou (POF4F1) NP_004566 Homeobox (POU4F2) NP_002691 (POU4F3) Lsr2 NP_002692 (POU5F1 isoform1) DUF1183 NP_976034 (POU5F1 isoform2) NP_001153014 DUF521 (POU5F1B) NP_694948 (POU5F2) NP_002693 (POU6F1) NP_009183 (POU6F2 isoform1) NP_001159490 (POU6F2 isoform2) Fig. 2. Domain analysis of POU family proteins. A: domain composition and distribution of human POU family proteins. B: domain composition and distribution of Oct4 protein in 10 studied species. 402 X. Zhang et al. / Journal of Genetics and Genomics 40 (2013) 399e412 B [1] Homo sapiens: Pou NP_002692 [2] Pan troglodytes: Homeobox XP_001135162 [3] Sus scrofa: DUF521 NP_001106531 [4] Bos taurus: NP_777005 [5] Rattus