Impact of Transcription Factors on Fibroblast Specific Gene Silencing in Somatic Cell Hybrids

As totipotent cells differentiate to specific cell types in multicellular organisms, certain sets of genes are turned off (gene silencing/gene extinction) while others are turned on (gene activation). Cell type specific gene silencing and gene activation is the basis of cell differentiation. Somatic cell hybrids which are produced by fusing different types of somatic cells from two different tissues have been used as a valuable resource to discern the phenomenon of lineage- specific gene extinction. Identification of regulatory factors that mediate gene extinction in hybrids are key to understanding regulatory mechanisms that govern cell differentiation in a multicellular organism. Transcription factors (TFs) have been implicated as powerful activators and/or repressors of gene expression in mammalian cells. In our prior work on hepatoma-specific gene extinction we used a hepatoma X fibroblast somatic cell hybrid model to explore liver specific gene silencing and confirmed that loss of hepatoma phenotype was in large part a consequence of loss of hepatoma-specific TFs. Based on these prior findings we hypothesized that loss of fibroblast phenotype in somatic hybrids may be in part due to loss of fibroblast-specific TFs. Using a hepatoma X fibroblast somatic cell model, we subjected the parental hepatoma cells (FT02B), fibroblast cells (RA T-2) and somatic cell hybrids FR(2) to whole genome transcriptional microarray profiling and assigned a fivefold or more increase in expression of a gene as a selection criterion to identify fibroblast-enriched genes. We identified candidate fibroblast-specific genes from among the fibroblast-enriched genes by their decrease in fold expression in hybrids and validated the repression of the putative gene candidates (fibroblast-specific TFs like Prrx1, Snai2 and Shox2, signaling proteins like Bmp3, Opn, Co11a1 and Sema3a etc.) in somatic cell hybrids using q-RT-PCR. Our findings confirm that loss of these fibroblast-lineage specific TFs in hybrids likely affects fibroblast transcriptional regulatory networks (TRN\u27s) and contributes towards the loss of fibroblast phenotype in somatic cell hybrids. We also report that ectopic overexpression of our candidate TFs Prxx1 and Snai2 in somatic cell hybrid leads to induction of fibroblast-specific cell reprograming in the somatic hybrids and significant restoration of fibroblast-specific traits. Forced expression of Prrx1 in FR(2) hybrid cells and selection of clones that had at least five fold or more expression of Prrx1 than the parental hybrid demonstrated a significant change in expression of other key fibroblast associated TFs such as Shox2, c-Fos and Twist1. Individual overexpression of Snai2 in FR(2) hybrid cells led to a large fold increase of TFs c-Fos, Prrx1, Twist1 and Shox2. As a result of forced overexpression of Prrx1 and Snai2 individually in somatic cell hybrids FR(2) the cells re-acquired a spindle-shaped morphology and more significantly an enhanced migration capability, which is reminiscent of parental fibroblast cells. Thus it appears that ectopic expression of candidate fibroblast lineage specific transcription factors in somatic cell hybrids contributes to significant restoration of the fibroblast phenotype. In conclusion, in this study, we identify fibroblast-specific target genes as well as putative fibroblast lineage-specific TF regulatory genes. Our findings suggest that observed gene extinction in somatic cell hybrids may be attributed, in large measure, to the shutdown of fibroblast regulatory networks governed by master lineage-specific transcriptional regulators of the fibroblast phenotype