Fetal Timeline      Maternal Timeline     News     News Archive    Sep 16, 2015 

Researchers have developed a method that enables the regulation
of a single gene's behavior without changing the genome itself.
Image Credit: Professor Otonkoski, University of Helsinki

A new method to turn genes off and on

Researchers at the University of Helsinki, Finland, have developed a new method to enable activation of genes in a cell. All without changing the genome.

Young researchers Diego Balboa and Jere Weltner, developed the techniques while working on their doctoral dissertations in Professor Timo Otonkoski's lab at the Meilahti medical campus of the University of Helsinki. Their research is published in the journal Stem Cell Reports, a leading publication in the field of stem cell research.

The hottest topic in stem cell research is the regulation of differentiation in cells. This process is based on how genes in a cell are activated or deactivated, researchers want ways to turn genes on and off - at specific times.

"We can produce undifferentiated stem cells from specialised cells - also known as induced pluripotent stem cells (iPSc). We can also regulate their differentiation by providing them with the right kinds of growth environments. However, we cannot control the differentiation process completely. The process may be going smoothly, but at the very end a single gene won't activate on time, and the cell remains immature."

Timo Otonkoski PhD, Professor, Research Programs Unit, Molecular Neurology, Biomedicum Stem Cell Center, University of Helsinki, and Children’s Hospital, University of Helsinki and Helsinki University Central Hospital, Helsinki, Finland

A system now being used called clustered regularly interspaced short palindromic repeats, or CRISPR, allows genes to be edited by cutting DNA at certain points. This method can remove a faulty gene from a cell or introduce a transplanted gene into a cell, and the gene will activate at a desired time.

Otonkoski's researchers' new method enables regulation of a single gene without cutting the DNA. The method employs CRISPR technology, but gene regulation itself is controlled chemically. The target gene is made receptive to new chemicals by placing bits of RNA into the cell so the RNA will bind to the activator protein in a gene's regulatory area. When chemicals that regulate the activator protein are put into a cell, the gene will turn on - or activate.

"In our research, we used two common antibiotics, doxycycline and trimethoprim, which enabled us to regulate the expression of many genes precisely and effectively. The method worked on all cells we tested, including stem cells - and we used human cells in our development.The basic idea has now been developed, and the method has been demonstrated to be viable, and I believe that it can become a very important research tool. In my laboratory we use the method to regulate the differentiation of stem cells, but it has many potential applications in other research fields - for example, in cancer biology."

Timo Otonkoski PhD

Professor Otonkoski emphasises that the method is currently used in experimental models - it is far too early to discuss therapeutic applications.

Abstract
Highlights
•dCas9VP192 targeted to proximal promoters activates transcription in different loci
•Transgenic OCT4 can be replaced by dCas9VP192
•Destabilized dCas9VP192 enables temporal control of gene expression by TMP
•TMP and DOX can be combined to control multiple genes in differentiation

Summary
CRISPR/Cas9 protein fused to transactivation domains can be used to control gene expression in human cells. In this study, we demonstrate that a dCas9 fusion with repeats of VP16 activator domains can efficiently activate human genes involved in pluripotency in various cell types. This activator in combination with guide RNAs targeted to the OCT4 promoter can be used to completely replace transgenic OCT4 in human cell reprogramming. Furthermore, we generated a chemically controllable dCas9 activator version by fusion with the dihydrofolate reductase (DHFR) destabilization domain. Finally, we show that the destabilized dCas9 activator can be used to control human pluripotent stem cell differentiation into endodermal lineages.

This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).

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