A Next-Generation Multi-Omics Approach to Dissect Cellular and Molecular Reprogramming

Cellular functions are known to be governed by its proteome profile. The proteome profile is further regulated by the transcriptome profile which is eventually defined by the gene expression programme of the cell type. Very often cells undergo transitions leading to gain of new or altered function. In order to gain an insight into such cellular transition, single omics approach is relatively less efficient in deciphering cellular reprogramming at molecular level. To this end, we developed a multi-omics approach to understand cellular transitions at molecular level with higher resolution. In order to develop and establish multi-omics approach, we chose rat Sertoli cell (Sc) as a model system. Sc is an important cell type of testis and is indispensable for spermatogenesis. During infancy (5 days old), the Sc are immature and cannot support spermatogenesis. However, from puberty (~12 days post birth) it becomes functionally mature and can support spermatogenesis. To understand this functional transition from immature to matured state, we implemented various high throughput technologies- transcriptomics, genomics and proteomics. We started with DNA microarray analysis of infant (immature) and pubertal (mature) Sc and found that 663 genes were up regulated 735 genes were down regulated in pubertal Sc as compared to that of infant Sc. The promoters of these differentially expressed genes (along with the control promoters selected randomly from the genome) were interrogated against the TRANFAC database for Transcription factor binding site (TFBS) predictions. Our TFBS analysis revealed that 61 and 56 TFBS were enriched over the promoters of the up regulated genes and down regulated genes respectively. The TFBS analysis were followed by proteomics (SWATH-MS) analysis of the nuclear and cytoplasmic fractions of the infant and pubertal Sc. Our TFBS and proteomics analysis suggested YY1 and Retinoic acid receptor-related orphan receptor (ROR) alpha as crucial factors for the maintenance of the functional status of the Sc. Binding sites of these TFBS were abundant over the promoters of the genes upregulated in puberty. These proteins were also found to be nuclear localized in pubertal Sc as shown by our SWATH-MS analysis and immunostaining studies. We generated transgenic rats knocking down these genes specifically in Sc from puberty onwards which led to compromised spermatogenesis. Gene Ontology and KEGG pathway analysis of these TFBS and the differentially expressed genes followed by network analysis using cytoscape is being undertaken which may enable us to look into the differentially acting signaling pathways acting on these two (infant and pubertal Sc) cellular states. The role of those differentially acting signaling pathways can be further validated by in-vitro and/or in-vivo studies.