Predicting involvement of polycomb repressive complex 2 in direct conversion of mouse fibroblasts into induced neural stem cells

Yaqubi et al. Stem Cell Research & Therapy (2015) 6:42 DOI 10.1186/s13287-015-0045-x RESEARCH Open Access Predicting involvement of polycomb repressive complex 2 in direct conversion of mouse fibroblasts into induced neural stem cells Moein Yaqubi1†, Abdulshakour Mohammadnia2† and Hossein Fallahi3,4* Abstract Introduction: Mouse fibroblasts could be directly converted into induced neural stem cells (iNSCs), by introducing a set of known transcription factors (TFs). This process, known as direct reprogramming, is an alternative source of NSCs production for cell therapy applications, hence, more common sources for such cells including embryonic stem cell (ESCs) and induced pluripotent stem cell (iPSCs) are also in use. Despite their importance, the exact role of different TFs involved in the conversion of fibroblasts into iNSCs and the interactions between these factors has not been studied. Methods: Here, we have used available microarray data to construct a gene regulatory network to understand the dynamic of regulatory interactions during this conversion. We have implemented other types of data such as information regarding TFs binding sites and valid protein-protein interactions to improve the network reliability. The network contained 1857 differentially expressed (DE) genes, linked by11054 interactions. The most important TFs identified based on topology analysis of the network. Furthermore, in selecting such TFs, we have also considered their role in the regulation of nervous system development. Results: Based on these analyses, we found that Ezh2, Jarid2, Mtf2, Nanog, Pou5f1, Sall4, Smarca4, Sox2, Suz12, and Tcf3 are the main regulators of direct conversion of mouse fibroblasts into iNSCs. Because, members of the polycomb repressive complex 2 (PRC2) were present in the most effective TFs’ list, we have concluded that this complex is one of the major factors in this conversion. Additionally, gene expression profiling of iNSCs, obtained from a different data sets, showed that Sox2 and Ezh2 are two main regulators of the direct reprogramming process. Conclusions: Our results provide an insight into molecular events that occur during direct reprogramming of fibroblasts into iNSCs. This information could be useful in simplifying the production of iNSCs, by reducing the number of required factors, for use in regenerative medicine. Introduction Embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs) are the two main sources of induced neural stem cells (iNSCs) generation. Recently direct reprogramming has been introduced as an alternative approach to produce NSCs for use in regenerative medicine. In this approach, over-expression of a set of pre* Correspondence: † Equal contributors 3 Department of Biology, School of Science, Razi University, Kermanshah, Iran 4 Medical Biology Research Center, Kermanshah University of Medical Sciences, Kermanshah, Iran Full list of author information is available at the end of the article defined transcription factors (TFs) reprograms fully differentiated fibroblasts into neural stem cells (NSCs) and neurons. Production of iPSCs from somatic cells and their subsequent differentiation into NSCs is a rather slow process (one or two months for each step). Direct reprogramming, on the other hand, is a much faster approach to generate NSCs. This process involves only one step, while generating NSCs via iPSCs requires first reprogramming and then differentiation. In addition, cells produced via ESCs and iPSCs show different degrees of genetic instability and harbor cancer development risks [1]. © 2015 Yaqubi et al.; licensee BioMed Central. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly credited. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Yaqubi et al. Stem Cell Research & Therapy (2015) 6:42 Recently, several groups have produced NSCs and neurons from terminally differentiated cells, including fibroblasts [2-9], sertoli cells [10], and astrocytes [11]. Interestingly, neurons were also produced from fibroblasts [12-19] by introducing certain types of TFs or a combination of TFs and miRNA [18,19]. Two different approaches have been successfully used for direct reprogramming. One uses four well-known TFs: OCT4, KLF4, SOX2, and MYC similar to Yamanaka’s method, in production of iPSCs. In this procedure, additional factors are required for generation of NSCs and neurons, which should be applied before iPSCs clonal formation [20]. In the second approach, direct conversion of fibroblasts into NSCs was achieved by introducing a list of alternative TFs. Systematic elimination of TFs resulted in identification of a minimum set of TFs that are essential for successful direct conversion in this method [20]. Thus, previous findings highlight the role of TFs in differentiation, fate specification, and direct reprogramming. Fortunately, computational methods are able to predict the most important TFs involved in such cellular processes [21,22]. To this aim, a gene regulatory network has been constructed for mouse ESCs using expression and TFs binding data [23]. However, the dynamics of the gene regulatory network during direct conversion of fibroblasts into iNSCs has not been studied. Here, we have constructed a gene regulatory network for conversion of mouse fibroblasts into iNSCs and investigated the role of differentially expressed TFs in this process. We have used publicly available data obtained from wet-lab experiments, including microarray expression profiles, information regarding TFs binding sites and valid protein-protein interactions to construct our gene regulatory network. Statistical analysis of this network unveiled a central role for several TFs. Finally, we have extracted and introduced the most important TFs that are involved in regulating the conversion of fibroblasts into iNSCs. Methods Microarray availability and analysis Microarray data for direct conversion of adult mouse fibroblasts into iNSCs was obtained from the Gene Expression Omnibus (GEO) using GSE31598 accession number [2]. Raw data were normalized using the robust multi-array averaging (RMA) algorithm in the FlexArray [24]. Differentially expressed (DE) genes were detected by comparing the gene expression profiles of mouse fibroblasts and iNSCs, using a fold change algorithm implemented in the FlexArray software [25]. A very restrictive fold change = 3 was set as the threshold for detection of DE genes, in order to eliminate background noises. Annotation of the probe sets was achieved using annotation file number HGU133_Plus_2.na33.annot, which was obtained from [26]. Page 2 of 13 The origina (...truncated)