Development of a Multi-Omics Approach to Study Differential Gene Regulation- a Potential Workflow for the Development and Validation of Synthetic Genetic Circuits

The characteristic functionality of a biological system is governed by its proteome profile. This in turn is determined by the transcriptome profile which is governed by the nucleus. Transcriptionally active regions of the chromatin mainly defines the genetic circuit of a cell type and determines its function as well as identity. Any change in genetic circuit of a cell type (principally done in any synthetic biology study) would alter its entire transcriptome and proteome profile. Development and validation of such altered genetic circuits demands use of multi-omics approach to ensure appropriate tracking of the entire transcriptome and proteome. To this end, we developed a computationally integrated transcriptomics and Next-Generation Proteomics (SWATH-MS) approach for the holistic visualization of the molecular components of a particular cell type.

We used infant (5-days old) and pubertal (12-days old) rat Sertoli cell (Sc) as a model to establish such a workflow. Infant Sc is immature and incapable of supporting germ cell differentiation whereas, pubertal Sc is mature and can support robust spermatogenesis. Sc at two different developmental stages are functionally distinct with respect to each other and they have distinct genetic circuits.

Infant and pubertal Sc were cultured in-vitro and their RNA was used for DNA microarray. In pubertal Sc, 663 genes were upregulated and 735 genes were downregulated. The cellular abundance of these transcripts are governed by their respective promoters. Promoters (+/- 2kb from Transcriptional Start Site) of all these differentially expressed genes were retrieved and interrogated for the abundance of various Transcription Factor Binding Sites (TFBS), as compared topromoter sets which are not expressed differentially, using vertebrate non-redundant Positional Weight Matrixes (PWM) from TRANSFAC database  employing MATCH™ utility. The discrepancy between the frequency of individual TFBS on up and down-regulated gene-set promoters (observed occurrence) and that of control promoter sets (expected by chance) was evaluated independently by determining the statistical variable chi-square (χ2). Considering a significance level of p<0.05, we identified 61 TFBS on up-regulated and 56 TFBS on down-regulated gene-set promoters. Out of these 19 and 14 TFBS were found exclusive on up-regulated and down-regulated gene-set promoters respectively. Interactome analysis using string database suggested 75 genes to be associated with the TFBS in case of downregulated-gene-set and 87 genes in case of upregulated-gene-set. Nuclear and cytoplasmic fractionation of both the cell types were used for mass spectrometry for Data Dependent Acquisition (DDA) and SWATH-MS analysis. DDA data obtained from each sample was merged and processed to generate a comprehensive rat SWATH-assay library. This assay library was interrogated using the SWATH-MS data for comprehensive proteome relative quantification. This helped in cataloguing the list of proteins which were enriched in nucleus (in infant and pubertal Sc) and how many of them were associated with the enriched TFBS obtained in each case.   This study revealed a holistic picture of differential gene regulation by the TFBS in coordination with the nuclear proteome of infant and pubertal Sc. The workflow can be applied for the development and validation of gene regulation in synthetic genetic circuits.