Prediction of novel pluripotent proteins involved in reprogramming of male Germline stem cells (GSCs) into multipotent adult Germline stem cells (maGSCs) by network analysis

Highlights

• Various proteins such as Pou5f1 and Prdm14 are involved in the Germline stem cell maintenance.

• Five Identified clusters were ranked according to the score.

• Lama3, Lamc2, and Lama1 in cluster 4 involve in the P13K-Akt signaling pathway plays an important role in pluripotency.

Abstract

Germline stem cells (GSCs) are known to transmit genetic information from parents to offspring. These GSCs can undergo reprogramming to transform themselves into pluripotent stem cells, called as Multipotent adult Germline stem cells (maGSCs). The mechanism of the reprogramming of GSCs to maGSCs is elusive. To investigate novel factors that may govern the process of reprogramming, the RNA-seq data of both GSCs and maGSCs were retrieved and subjected to Tuxedo protocol using Galaxy server. Total 1558 differentially expressed genes were identified from the analysis. Protein sequence in the FASTA format of all 1558 differentially expressed genes was retrieved and submitted to Pluripred web server to predict whether the proteins were pluripotent or not. A total of 232 proteins were predicted as pluripotent, and to identify the novel proteins, these were submitted to STRING database to obtain an interaction map. The obtained interaction map was submitted to Cytoscape, and various apps such as MCODE and Centiscape were used to identify the clusters and centrality measures between the nodes of the generated network. Five clusters were identified and ranked according to their score. Novel pluripotent proteins like cadherin related cdh5, cdh10 were predicted. Phox2b, Nrp2, Dll1, Shh, Gbx2, Nodal, Lefty1, Wnt7b, Pitx2, fgf4, Pou5f1, Nanog, Tet1, trim8, alx2, Dppa2, Prdm14,Sox11, Esrrb were predicted to be involved in the stem cell development. Dppa2, Sox11, Sox2, Bmp4, Shh, and Otp were predicted to be involved in positive regulation of the stem cell proliferation. Pathway analysis further revealed that signaling pathways such as Wnt, Jak-Stat and PI3K may play important role in the pluripotency of the maGSCs. Novel proteins involved in pluripotency, which were predicted by our findings, can be experimentally researched in future.

Introduction

The Germline stem cells (GSCs) give rise to germ cells, which exhibit the peculiar property of inheriting genetic characteristics from generation to generation (Kim and Belmonte, 2011). GSCs acquire the potential to undergo unipotent differentiation into spermatozoa in adult male gonad (Kim and Belmonte, 2011; Liu et al., 2016). Experimental evidence depicts that the cellular state of GSCs is likely to be similar to that of embryonic stem cells (ESCs). Hence, it was suggested that GSCs can undergo certain spontaneous reprogramming mechanisms, under different culture conditions, to acquire pluripotency by transforming themselves into pluripotent stem cells (PSCs) termed as multipotent adult Germline stem cells (maGSCs) (Kanatsu-Shinohara et al., 2004; Ko et al., 2009). At present, PSC researchers employ the use of stem cells of embryonic origin; called ESCs, which are derived from inner cell mass of blastocyst (Kim and Belmonte, 2011; Kanatsu-Shinohara et al., 2004; Seandel et al., 2007). ESCs entail their usage with ethical issues and safety concern. GSCs offer promising substitutes of ESCs for their use as PSCs, as their use does not pose any immune rejections due to their autologous nature. Hence, the use of GSCs is also free of any ethical complications (Kim and Belmonte, 2011). Additional advantages come from GSCs having higher genome integrity than induced Pluripotent stem cells (iPSCs) (Ko et al., 2009) and having better gene expression profiles than iPSCs (Kim and Belmonte, 2011). The PSCs can also be derived from other mammalian germ cells such as primordial germ cells (PGCs) and, spermatogonial stem/progenitor cells (SSCs, SPCs) from adult mice and humans (Geijsen and Leanne Jones, 2008; Kerr et al., 2006). Experimental evidence suggests that during in vitro expansion, mouse GSCs of spermatogonial origin, having unipotent potential, undergoes spontaneous reprogramming and converts into maGSCs with a near pluripotent state (Kanatsu-Shinohara et al., 2004; Seandel et al., 2007). Although it has been established that maGSCs acquire pluripotency, the mechanism underlying the same is still unknown. But, it can be postulated that certain genetic and epigenetic changes are responsible for the same (Liu et al., 2016).

It can be suggested that GSCs possess a latent ESCs-like gene profile and it loses its commitment to developing into spermatozoa; while converting into maGSCs. During germline specification in embryo, certain specific transcription factors of ESCs are transcriptionally activated, and their expression is preserved in GSCs or SSCs, in turn, repressing the function of somatic genes in the embryo (Pesce et al., 1998; Arnold et al., 2011; Zheng et al., 2009). For example, transcription factors like Pou5f1 (also known as Oct4) along with Sox2 and Nanog forms the core transcription factors regulating the pluripotent machinery in ESCs required for stem cell renewal and controlling the expression of many differentiated genes (Boyer et al., 2005; Chew et al., 2006). It was observed that these core transcription factors regulate pluripotency of maGSCs along with other novel pluripotent factors. Another reason for pluripotency of maGSCs might be modifications of histones which are associated with transcription mechanism (Wang et al., 2007; Surani et al., 2007; Brownell et al., 1996).

In recent days, computational techniques like Bioinformatics, Machine learning methods and network-based approaches are being used to understand the relationships between the vast amounts of genomic data available. To identify the proteins involved in the maintenance of the pluripotency in maGSCs, we used computational methods to explain this elusive mechanism. In this study, RNA-Seq data of GSCs and maGSCs were retrieved and it was observed that maGSCs are distinguished from GSCs by repression of certain spermatogenesis regulators, inactivating their conversion to spermatozoa, in turn, activating certain somatic cell lineage genes establishing pluripotent nature of maGSCs. Based on these observations, we predicted certain novel transcription factors responsible for a pluripotent state of maGSCs. The up-regulated genes required for conversion of GSCs to maGSCs were identified using Galaxy server, and protein sequences of all up-regulated genes were submitted to Pluripred to predict pluripotent proteins amongst the up-regulated ones. The interconnections between different pluripotent proteins were studied in STRING. However, it was very difficult to predict the novel pluripotent factors required for the pluripotent state of maGSCs amongst the interactions between different proteins in the entire network. Hence, we further employed Cytoscape for network analysis of our proteins. Centiscape refined our analysis by including certain centralities using MCODE and Centiscape thereby, predicting certain novel transcription factors responsible for establishing the pluripotent state in maGSCs.