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

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 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 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 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 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 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 norvegicus: NP_001009178 [6] Mus musculus: NP_038661 [7] Drosophila melanogaster: NP_523558 [8] Caenorhabditis elegans: NP_492304 [9] Danio rerio: NP_571187 [10] Gallus gallus: NP_001103648

Fig. 2. (continued).

POU family genes in the chicken genome, and 15 POU family species (Fig. 3A). Detailed comparison revealed that the dif- genes in the genomes of human, mouse and rat, respectively. ferences among mammalian Oct4 proteins were mostly mu- All these genes encoded proteins with the POU domain, tations resulting in single amino acid changes, whereas both and majority of them also contained the homeobox domain the N-terminal and C-terminal sequences of the zebrafish and (Fig. 2). Both of the POU and homeobox domains have DNA chicken Oct4 proteins differed a lot from the mammalian ones. binding abilities. The human POU domain class 1 protein, We next studied the relationships among different POU POU1F1, also contained the Lsr2 domain, which may be family proteins in human. Consistent with their grouping responsible for the association with nucleoid (Gordon et al., criteria, proteins of the same class were grouped together, with 2010). the class 2 and class 3 POU family proteins had higher As the human POU family genes have been grouped into 6 sequence similarity than other classes (Fig. 3B). For proteins classes in the previous studies (Cook and Sturm, 2008), we within each class, POU2F1 and POU2F2 had higher sequence tried to classify genes in other species according to their homology than POU2F3; POU3F2, POU3F3 and POU3F4 similarity to the human POU family genes. Except for C. were more similar than POU3F1; POU4F2 and POU4F3 were elegans and D. melanogaster genomes which only had one more similar than POU4F1 (Fig. 3B). POU class 1 gene and one POU class 2 gene, respectively, all the six classes of POU family genes could be found in the Expression analysis of human POU family genes studied mammalian genomes (Table 1). In addition, the number of genes in each POU class was similar across species, To compare the expression patterns of POU family genes, with all studied mammalian genomes having one class 1 gene we resorted to the Gene Expression Atlas database, which and comparable numbers of genes in other classes (Table 1). combines the data from a subset of tissue-related gene The lack of some POU family genes in the chimpanzee expression experiments from the ArrayExpress archive and genome may be due to the incompleteness of its genome reports the expression profiles of genes according to their sequence. detection frequency in those experiments. The summarized expression profiles of all human POU family genes are showed Phylogenetic analysis of POU family proteins in Fig. 4. Brain and other neuronal organs, liver, lung, mam- mary gland and kidney were the tissues with over two thirds of We used the sequences of Oct4 proteins as an example to POU family genes expressed. Consistent with the previous study its evolutionary pattern in the 10 species. The Oct4 report (Herman et al., 2012), the POU1F1 protein was most protein was present in 8 species and was highly conserved frequently detected in the pituitary gland. In addition, higher during evolution (Fig. S1). Phylogenetic analysis revealed that expression of POU1F1 was also detected in trigeminal gan- the sequence similarity of Oct4 proteins was in the same glion, superior cervical ganglion, as well as lung, liver and pattern as the evolutionary relationship of their corresponding kidney. The POU2F2 class genes, especially POU2F2, were Table 1 Classification of POU family genes in 10 species

POU1 POU2 POU3 POU4 POU5 POU6

H. sapiens ENSG00000064835 ENSG00000143190 ENSG00000185668 ENSG00000152192 ENSG00000204531 ENSG00000184271 (POU1F1) (POU2F1) (POU3F1) (POU4F1) (POU5F1) (POU6F1) ENSG00000028277 ENSG00000184486 ENSG00000151615 ENSG00000248483 ENSG00000106536 (POU2F2) (POU3F2) (POU4F2) (POU5F2) (POU6F2) ENSG00000137709 ENSG00000198914 ENSG00000091010 (POU2F3) (POU3F3) (POU4F3) ENSG00000196767 (POU3F4) P. troglodytes ENSPTRG00000015123 ENSPTRG00000004264 ENSPTRG00000030679 ENSPTRG00000017374 ENSPTRG00000017953 ENSPTRG00000004950 (POU1F1) (POU2F1) (POU3F1) (POU4F1) (POU5F1) (POU6F1) 399 (2013) 40 Genomics and Genetics of Journal / al. et Zhang X. ENSPTRG00000029496 ENSPTRG00000016492 ENSPTRG00000019105 (POU2F2) (POU4F2) (POU6F2) ENSPTRG00000001633 (POU2F3) S. scrofa ENSSSCG00000011996 ENSSSCG00000006310 ENSSSCG00000003646 ENSSSCG00000025334 ENSSSCG00000001393 ENSSSCG00000000220 (POU1F1) (POU2F1) (POU3F1) (POU4F1) (POU5F1) (POU6F1) ENSSSCG00000028237 ENSSSCG00000004346 ENSSSCG00000026015 ENSSSCG00000024471 (POU2F2) (POU3F2) (POU4F2) (POU6F2) ENSSSCG00000004040 ENSSSCG00000022096 ENSSSCG00000014412 (POU2F3) (POU3F3) (POU4F3) ENSSSCG00000012454 (POU3F4) B. taurus ENSBTAG00000009128 ENSBTAG00000024534 ENSBTAG00000012061 ENSBTAG00000005648 ENSBTAG00000021111 ENSBTAG00000016362 (POU1F1) (POU2F1) (POU3F1) (POU4F1) (POU5F1) (POU6F1) ENSBTAG00000008556 ENSBTAG00000024773 ENSBTAG00000018817 ENSBTAG00000013648 (POU2F2) (POU3F2) (POU4F2) (POU6F2) ENSBTAG00000021340 ENSBTAG00000015712 (POU2F3) (POU3F3) M. musculus ENSMUSG00000004842 NSMUSG00000026565 ENSMUSG00000090125 ENSMUSG00000048349 ENSMUSG00000024406 ENSMUSG00000009739 (POU1F1) (POU2F1) (POU3F1) (POU4F1) (POU5F1) (POU6F1) e

ENSMUSG00000008496 ENSMUSG00000095139 ENSMUSG00000031688 ENSMUSG00000093668 ENSMUSG00000009734 412 (POU2F2) (POU3F2) (POU4F2) (POU5F2) (POU6F2) ENSMUSG00000032015 ENSMUSG00000045515 ENSMUSG00000024497 (POU2F3) (POU3F3) (POU4F3) ENSMUSG00000056854 (POU3F4) R. norvegicus ENSRNOG00000000715 ENSRNOG00000003581 ENSRNOG00000002784 ENSRNOG00000009669 ENSRNOG00000046487 ENSRNOG00000004595 (POU1F1) (POU2F1) (POU3F1) (POU4F1) (POU5F1) (POU6F1) ENSRNOG00000020381 ENSRNOG00000006908 ENSRNOG00000012167 ENSRNOG00000013844 ENSRNOG00000013237 (POU2F2) (POU3F2) (POU4F2) (POU5F2) (POU6F2) ENSRNOG00000009118 ENSRNOG00000038909 ENSRNOG00000018842 (POU2F3) (POU3F3) (POU4F3) M72711 (POU3F4)

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ubiquitously expressed in most tissues. Both of the POU3 and POU4 family genes were enriched in brain related tissues, in concert with their neural-specific functions. We also found that POU6F1 tended to be co-expressed with the POU3 class genes, also with enrichment in neuronal tissues. As embryos were not included in these samples, the POU5 class genes were barely detected. But liver seemed to have a relatively POU6 (POU6F1) high expression of Oct4, probably due to the fast regenerative ability of liver. As Oct4 is the key regulatory factor of ES cells, we examined the expression of POU family genes in human ES cells using the public microarray data. As expected, the Oct4 gene had very high expression in human ES cells (Fig. 5). Also, POU2F1 had relatively high expression in human ES cells, indicating that it may also have some similar functions POU5 ENSDARG00000044774 (POU5F1) DQ867024 (POU5F1) ENSGALG00000012303 as Oct4 (Fig. 5).

Functions and interaction networks of POU family proteins

To examine the functions and relationships of POU family proteins, we used the Pathway Studio database to construct the interaction network of Oct4 family proteins. The network was built by combining reported proteins that had known regula- POU4 ENSDARG00000070220 (POU4F1) (POU4F1) ENSGALG00000014195 (POU4F2) tory or binding relationships with the Oct4 family proteins. The analysis results showed that POU1F1, POU2F1 and POU2F2 were all involved in the regulation of DNA replica- tion, chromatin remodeling, apoptosis and cell differentiation processes. These three protein classes had some common binding and regulatory proteins. Besides, both POU2F1 and POU2F2 can bind to their co-activator, POU2AF1 (Fig. 6A), which was considered to enhance the function of POU2F1 and POU2F2 (Gstaiger et al., 1996). POU3 ENSDARG00000095896 (POU3F1) ENSDARG00000076262 (POU3F2) ENSDARG00000009823 (POU3F3) Gga.32081 (POU3F1) ENSGALG00000010008 The POU3F1 and POU3F2 proteins were not only co- expressed in brain related tissues, but also had crosstalk in their functions through the common partner, involucrin (IVL) (Fig. 6B). IVL is a protein involved in the formation of cell envelope, and the binding of POU family proteins on the promoter of IVL gene suppressed its expression (Welter et al., 1996; Shi et al., 2010). POU4F1, POU4F2 and POU4F3 were found to co-interact POU2 ENSDARG00000052387 (POU2F1) ENSDARG00000036816 (POU2F2) ENSGALG00000015446 (POU2F1) ENSGALG00000006809 (POU2F2) ENSGALG00000006673 (POU2F3) FBgn0004394 (POU2) with the internexin neuronal intermediate filament protein (INA) (Fig. 6C), which is a major component of the intermediate filament network in small interneurons and cerebellar granule cells. Both POU4F1 and POU4F3 can activate the expression of INA gene, whereas POU4F2 represses it (Fig. 6C). The other interacting proteins of POU4F1 included TP53, SNAP25, BCL2, etc. It has been shown that the interaction between BCL2 and POU4F1 requires the N-terminal transactivation domain of POU4F1; therefore it cannot be activated by other POU4 family POU1 ENSDARG00000058924 (POU1F1) ENSGALG00000015495 (POU1F1) K02B12.1 (POU1) genes (Smith et al., 1998).

) POU5F1 (Oct4) is involved in a complicated regulation network in stem cells and interacts with lots of other proteins (Fig. 6D). Oct4 is co-localized with Sox2 and interacts with continued Klf4 (Li et al., 2007). It was reported that the interaction of endogenous Klf4 with Oct4/Sox2 is required for the reprog- Table 1 ( D. rerio G. gallus D. melanogaster C. elegans IDs from the Ensembl database are used. ramming process (Wei et al., 2009). Another key protein of the X. Zhang et al. / Journal of Genetics and Genomics 40 (2013) 399e412 405

A 99 NP_777005 (B. taurus) 91 NP_001106531 (S. scrofa) NP_002692 (H. sapiens) 100 100 NP_00123897 (P. troglodytes) NP_001009178 (R. norvegicus) 100 NP_038661 (M. musculus) NP_492304 (C. elegans) 100 NP_523558 (D. melanogaster) NP_571187 (D. rerio) 87 NP_001103648 (G. gallus)

NP_001193955 (POU2F2 isoform 3) B 100 NP_00268 (POU2F2 isoform 2) NP_001193954 (POU2F2 isoform 1) 94 NP_001234923 (POU2F2 isoform 4) NP_001185712 (POU2F1 isoform 2) 100 NP_002688 (POU2F1 isoform 1) 100 NP_001185715 (POU2F1 isoform 3) NP_055167 (POU2F3 isoform 1) 54 100 NP_001231611 (POU2F3 isoform 2) NP_002690 (POU3F1 ) NP_005595 (POU3F2) 88 100 NP_006227 (POU3F2) 78 NP_000298 (POU3F4) NP_694948 (POU5F2) NP_976034 (POU5F1 isoform 2) 98 100 NP_002692 (POU5F1 isoform 1) 80 NP_001153014 (POU5F1 B) NP_000297 (POU1F1 isoform alpha) 100 NP_001116229 (POU1F1 isoform beta)

60 NP_004566 (POU4F2) 100 NP_002691 (POU4F3) NP_006228 (POU4F1 ) NP_002693 (POU6F1 ) 100 NP_001159490 (POU6F2 isoform 2) 100 NP_009183 (POU6F2 isoform 1)

Fig. 3. Phylogenetic tree of the Oct4 family proteins. A: phylogenetic tree of the Oct4 protein in the 10 studied species. B: phylogenetic tree of the human POU family proteins. network is SALL4. Silencing of SALL4 can cause significant predicted binding sites of all known human miRNAs on the 30 down-regulation of both Oct4 and Sox2 (Yang et al., 2012). UTR sequences of human POU family genes (Table 2). Except Correlated with its core role in stem cells, POU5F1 was found for POU3F3, POU4F3 and POU6F2, other human POU family to be involved in the regulation of many cell processes, genes all contained putative miRNA binding sites. Different including cell fate, embryonal development and chromatin isoforms of the same POU family genes usually contain the remodeling. binding sites of the same miRNA. Both POU2F2 and POU4F2 were the putative targets of hsa-miR-29 family Regulation of POU family genes by miRNAs miRNAs, but the other POU family genes had no common pairing miRNAs. Most identified miRNAs listed in Table 2 miRNAs are post-transcriptional regulators that bind to had similar tissue-specific expression patterns as their corre- target mRNAs via sequence complementarity, usually result- sponding genes. For example, the pairing miRNA of POU1F1, ing in translational repression or mRNA degradation of target hsa-miR-4251, was reported to be expressed in neuronal tis- genes (Kusenda et al., 2006; Bartel, 2009). To investigate the sues, in concert with the expression profile of POU1F1 (Goff relationship between miRNAs and POU family genes, we et al., 2009). To the contrary, miRNAs regulating the POU 406 X. Zhang et al. / Journal of Genetics and Genomics 40 (2013) 399e412

POU1POU2 POU2 POU2 POU3 POU3 POU3POU3 POU4POU4 POU4 POU5 POU5POU6POU6 Organ name F1 F1 F2 F3 F1 F2 F3 F4 F1 F2 F3 F1 F2 F1 F1 Dorsal root ganglion 1 4 1 1 4 1 3 1 1 4 3 34 3 4 5 3 4 3 2 1 3 111 3 4 3 11 Trigeminal ganglion 1 4 1 3 3 4 1 1 4 1 4 Skin 1 1 113 2 1 1 1 1 12 3 1 3 5 5 2 5 5 5 5 2 2 5 Ovary 2 12 1 11 1 4 3 1 1 1 1 6 665 6 6 2 6 5 Salivary gland 1 1 1 1 4 4 4 1 2 1 1 1 1 Testes 2 4 1 1 1 1 5 Uterus 3 1 1 1 1 4 1 3 4 1 112 1 2 1 1 1 Skeletal muscle tissue 1 4 1 117 1 Corpus uteri 1 1 1 1 1 1 Appendix 1 1 1 1 1 2 1 4 4 1 1 2 3 1 Brain 5 6 6 3 3 4 4 445 Cerebellum 2 2 11 32 1 1 11 11 1 1 1 1 3 1 1 4 4 4 4 4 5 3 4 4 4 10 4 4 1 1 1 Medulla 1 1 1 1 1 1 1 1 Midbrain 1 1 1 1 1 1 1 1 Superior cervical ganglion 1 1 1 1 1 1 Uterus corpus 1 4 1 1 1 1 1 Ventral tegmental area 1 1 1 1 1 1 2 2 2 1 Prefrontal cortex 1 2 2 2 1 Colon 11 1 1 2 1 1 4 5 5 4 4 2 1 1 1 2 Thyroid gland 3 2 3 3 Prostate gland 1 1 1 1 Lung 1 3 4 13 3 1 2221 1 11 4 11 10 10 12 11 10 12 10 11 10 9 7 9 Hepatocellular carcinoma 1 1 1 1 11 Thymus 1 1 1 4 3 3 Mammary gland 1 2 1 1 1 1 1 1 1 1 4 3 3 4 4 3 4 1 2 3 3 1 2 1 1 1 2 3 Peripheral blood 6 5 6 6 6 6 6 1 1 1 1 1 1 Thyroid 2 1 2 3 1 2 1 1 1 1 Frontal cortex, superior motor cortex, Brodmann’s Area 9 1 1 1 1 1 Frontal cortex, primary motor cortex, Brodmann’s Area 4 1 1 1 1 1 Kidney 1 2 4 1 1 2 4 1 1 1 4 5 1 1 6 6 877 6 5 5 10 7 7 1 1 White blood cell 1 1 1 1 1 1 1 1 1 221 24 1 1 2 Blood 8 8 7 6 8 8 7 3 1 2 1 N 1 1 1 1 1 1 Pituitary gland 3 3 3 2 3 3 4 1 3 Liver 1123 42 2 2 3 1 2 2 1 3 2911 5 9 11 11 11 10 9 11 9 10 8 23 4 8 Cerebral cortex 2 2 1 1 2 1 1 2 2 2 5 1 1 Spinal cord 1 1 1 2 1 1 4 1 2 4 4 4 4 Heart 2 3 3 2 3 1 2 1 4 1 1 1 8 3 Spleen 1 3 111311 3 331 2 2 7

Fig. 4. Expression patterns of POU family genes in normal human tissues. Numbers in red/blue/white background indicate the number of published studies in which a gene is with increased/decreased/unchanged expression pattern compared to the overall mean expression level of the gene in all experiments. Blank boxes indicate that no expression of a gene was detected in the corresponding tissue. class 2 genes were expressed in many tissues, consistent with DISCUSSION the ubiquitous expression pattern of the POU class 2 genes (Sempere et al., 2004). We also identified a putative binding The Oct4 protein is a key transcription factor in establish- site of hsa-miR-335 in the Oct4 gene. It has been proven that ing and maintaining the pluripotency of ES cells and iPS cells. miR-335 can regulate Oct4 expression in mouse ES cells Among the various combinations of factors that are capable of (Schoeftner et al., 2013). Our results indicated that similar producing iPS cells, Oct4 seems to be the only factor that regulatory relationship is also conserved in human. cannot be replaced so far (Lau et al., 2009; Sterneckert et al., X. Zhang et al. / Journal of Genetics and Genomics 40 (2013) 399e412 407

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POU3F1POU3F2POU3F3POU3F4POU4F1POU4F2POU4F3 POU5F2POU6F1 POU1F1 beta POU1F1 alpha POU2F1POU2F2 isoform1POU2F3 isoform1 isoform1 POU5F1POU5F1 isoform1 isoform2 POU6F2 isoform1

Fig. 5. Expression patterns of the POU family genes in H9 human embryonic stem cells according to the microarray dataset GSE21244 of the GEO database. The Y-axis represents the normalized gene expression data obtained by the RMA method.

2012). As Oct4 belongs to the multiple member POU gene constructed their interaction networks and investigated their family, it is worth knowing what is the relationship of Oct4 relationships with miRNAs. All of them will shed light on the with other POU family genes and how they are conserved future functional studies of these genes. through evolution. However, there is no report to systemati- cally address these questions so far. MATERIALS AND METHODS In this work, we have performed systematic analysis of POU family genes from both the evolutionary and functional Identification of POU family proteins in 10 species aspects. The results demonstrated that the expansion of the POU family genes occurred in vertebrates, with an additional Ten animal species from lower to higher complexity and increment in mammals. Such expansion indicated that the with complete genome sequences were included in this study, functions of POU family genes should be related to the gained namely Caenorhabditis elegans, Drosophila melanogaster, organism complexity in vertebrates. It is unclear yet whether Danio rerio, Gallus gallus, Bos taurus, Sus scrofa, Rattus the single POU family gene in C. elegans and D. melanogaster norvegicus, Pan troglodytes, Mus musculus and Homo sapiens. genomes can carry out the functions of other POU family To search for POU family proteins, the annotated protein se- genes in higher species, or the functions of those expanded quences of the above mentioned genomes were downloaded POU family genes are not needed in C. elegans and D. from the NCBI database. The POU family proteins in each melanogaster. species were first identified by their annotations (Oct4 or POU Previous studies on the POU family genes were mainly family class). These identified POU family proteins were used focused on single gene or genes of the same POU class, as template sequences to search against the annotated proteins without whole family or cross species comparison. In this of each species for homologous sequences by the BLASTP work, we performed the genome-wide identification of program. Sequences with an e-value <10 20 and >40% different POU class genes in the studied species, and also alignment coverage on the template sequences as well as with compared the expression of different classes of POU family identifiable POU domain were selected as POU family pro- genes in human. These results provide a thorough picture teins and used for further analysis. about the POU family genes and their functional differences. miRNAs have been proven to be important regulators of eukaryotic genes, especially for transcription factors (Naeem Domain identification and sequence conservation et al., 2011). Here, we provided a relatively complete rela- analysis tionship between miRNAs and POU family genes. The results showed that different isoforms of the same mRNA share The functional domains of all proteins were analyzed using common miRNA target sites, indicating that the requirement the sequence search function of the Pfam website (Punta et al., of miRNA regulation is essential for the functions of POU 2012). Domains with an e-value <0.01 were reported. The family genes. diagrams of domain composition and distribution of each As a class of key transcription factors, the POU family protein were produced by Perl programming language. The genes have many essential functions (Assa-Munt et al., 1993; conservation status of proteins was analyzed using the Andersen and Rosenfeld, 2001), yet relationships among these BLASTP program by comparing to the proteins of all species genes have been less studied. Here, we provided a thorough included in this study. Phylogenetic tree was constructed by evolutionary and functional analysis of POU family genes, MEGA 5 (Tamura et al., 2011) using the Neighbor-Joining identified POU family genes from lower to higher animal method with bootstrap value of 1000, with 50% as the cut- species, analyzed their sequence and expression relationships, off value. SST A GHRL GH1 GHR GHRH HR CSH2 PROP CREB LHX3 NR1l2 1 BP Cell EP300 GATA2 proliferation NFKBIA SYT1 PITX2

R TSHB NR1l3 PITX1 ET

Adenophysis NCOR Embryonal ETS1 development development 1

DR D2 FOS JUN CTNNB Phosphate POU1 1 import F1 IL8 PARP1 THRB NCOR 2 Cell Chromatin DNA Apoptosis differentiation HBG1 remodeling REN replication CREB 1 LPL SP1

POU2 VCAM1 F2 PRL POU2 GNRH1 AR F1 GADD4 5A PBX1 GNR HR NR3C1 CCND1

SNAP C4 ADR NOS2 IL3 B2

GTF2B IVL POU2 CDX2 AF1 DNA NGF CC TH recombination SPI1 R5 TBP IL5 CD86 IL2 CD CXC HLA- VWF 36 R5 CALCA DRA CSN2

KRT14 B SST MBP

GNRH1 EGR2

POU3 F1

Cell Myelination differentiation IVL

POU3 FLG F2 MITF

BRAF NES

TNC CRH

LOR

Fig. 6. Interactions between Oct4 family members. Different line colors represent the types of evidence for the association. A: interaction network of POU1F1, POU2F1 and POU2F2. B: relationship between the interaction networks of POU class 3 proteins (POU3F1 and POU3F2). C: network of POU class 4 proteins (POU4F1, POU4F2, POU4F3). D: network of proteins directly interacting with POU5F1 (Oct4). X. Zhang et al. / Journal of Genetics and Genomics 40 (2013) 399e412 409

NEFH C BCL2L1 TAC1

BCL2

SNAP25

Cell differentiation

POU4F BAX 1

Hair cell POU4F differentiation 3

TP53

INA Cell proliferation

P21

POU4F 2 HSPB1

ISL1

Cell fate SMARC A2 ATOH7 WT1

EPAS NOBO MTOR D ESRR 1 X B PARP1 SALL4 NR6A 1 SOX2 ROCK1 ROCK2 SUMO NR2F 1 1 NR5A 1 NANO G WWP2 RARA SMAR CA4

TP53 CDX2 POU5 Mesoderm F1 Stem cell development ELP2 differentiation MYBL CTNNB 2 1 Cell FOXA NR5A differentiation 2 2

UTF1 FOXA Chromatin 1 remodeling Cell ZFP42 proliferation STK40 Embryonal CGB CGB5 development ZSCA Apoptosis FGF4 MYOD 1 EED N10 SPP1 Cell fate

Fig. 6. (continued). 410 X. Zhang et al. / Journal of Genetics and Genomics 40 (2013) 399e412

Table 2 Putative regulatory relationships of miRNAs and the POU family genes

Gene name Gene ID miRNA

POU1F1 ENSG00000064835 hsa-miR-4251 POU2F1 ENSG00000143190 hsa-miR-23c, hsa-miR-23b, hsa-miR-23a hsa-miR-23c, hsa-miR-23a, hsa-miR-23b POU2F2 ENSG00000028277 hsa-miR-29a, hsa-miR-29c, hsa-miR-29b POU2F3 ENSG00000137709 hsa-miR-27a, hsa-miR-27b POU3F1 ENSG00000185668 hsa-miR-144 POU3F2 ENSG00000184486 hsa-miR-19a, hsa-miR-19b POU3F3 ENSG00000198914 NA POU3F4 ENSG00000196767 hsa-miR-4691-5p, hsa-miR-1825 hsa-miR-937, hsa-miR-4693-3p, hsa-miR-186 POU4F1 ENSG00000152192 hsa-miR-133a, hsa-miR-133b POU4F2 ENSG00000151615 hsa-miR-29a, hsa-miR-29c, hsa-miR-29b POU4F3 ENSG00000091010 NA POU5F1 ENSG00000204531 hsa-miR-3606, hsa-miR-335, hsa-miR-3658, hsa-miR-3657 POU5F2 ENSG00000248483 hsa-miR-4749-3p, hsa-miR-4668-5p POU6F1 ENSG00000184271 hsa-miR-372, hsa-miR-520e, hsa-miR-520b, hsa-miR-520c-3p, hsa-miR-520d-3p, hsa-miR-520a-3p, hsa-miR-373, hsa-miR-302e, hsa-miR-302c, hsa-miR-302d, hsa-miR-302a, hsa-miR-302b POU6F2 ENSG00000106536 NA

NA: no known miRNA was identified.

Expression analysis target sites that were conserved across at least 6 mammals were reported in this work. The expression profiles of POU family genes in human ES cells were obtained from the microarray dataset GSE21244 ACKNOWLEDGEMENTS (Mayshar et al., 2010) of the NCBI GEO database (Wilhite and Barrett, 2012), which included gene expression data of The work was supported by the grants from the Ministry of undifferentiated human ES cells and iPS cells. Raw expression Science and Technology of China (No. 2011CBA01101 to data from the Affymetrix microarray experiments are pre- X.-J.W.) and from the Chinese Academy of Sciences (Nos. processed in R using RMA, by the oligo package of Bio- XDA01020105, KSCX2-EW-R-01-03 and 2010-Biols-CAS- conductor (http://bioconductor.org/). The normalized expres- 0303 to X.-J.W.). sion values of the studied genes were extracted and plotted. The expression profiles of POU family genes in human tis- sues were collected from the Gene Expression Atlas (http://www. SUPPLEMENTARY DATA ebi.ac.uk/gxa/), which is a curated database of publicly available gene expression data collected in the ArrayExpress archive. Fig S1. Multiple alignment of Oct4 protein sequences in 8 species. Supplementary data related to this article can be found at Construction of protein interaction networks http://dx.doi.org/10.1016/j.jgg.2013.04.011.

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