Disturbance of Transcription Factor Dynamics in Mammalian Cells: Knock-In, Knock-Down, Knock-Out or Anchor-Away

Abstract

Transcription factors (TFs) are proteins that bind DNA and thereby can influence the activity of genes. TFs help determination of cell identity (liver cell vs blood cell). They can do this to regulate the activity of groups of genes that can together suppress one identity and promote another identity. If TFs become active in the wrong cell through genome mutations/translocations, this can lead to disease like cancer. An important goal in cell biological research is to identify target genes of TFs to obtain an overview of what genes need to be active for cells to obtain a specific identity.

A way to determine the target genes of a specific TF is by knocking down the mRNA of the gene by RNA interference (RNAi). After the RNA and later on the TF itself are gone from the target cells, gene expression analysis is performed to see what genes are differentially expressed after knock down. Although RNAi has been a successful technique, it has a major drawback: knocking down the RNA is fast, but it takes the cellular machinery in general at least 48 hours before the TF is cleared. During this 48 hour the cell is in a state where the TF is slowly disappearing from the cell and in this time the level of expression of target genes is adjusted. However, among the direct target genes are also other TFs. Because these other TFs too change expression levels, their target genes are also differentially expressed. These last group of genes are indirect or secundary effects of the knock down, but cannot be discerned from the direct target genes.

To overcome this restriction, Anchor-Away for mammalian cells is developed. In this work I show Anchor-Away can disrupt target nuclear targets (LDB1 mostly in this work) within four hours by sequestering the targets in the cytoplasm. Since the target TF is not anymore in the nucleus, it cannot bind to DNA anymore and therefore cannot perform its normal (nuclear) function. Anchor-Away is at least 10 times faster than RNAi and can therefore be used to obtain gene expression analysis results after TF disruption that contain significantly fewer secundary effects than is possible with RNAi.