MicroRNA and stem cells II

MicroRNA and stem cells are both pretty hot topics these days. But curiously, there haven't been a whole lot of reports that involve the two together. My last discussion of both, back in March, is here.

However, the combination of microRNA and stem cells just was back in the news, as briefly noted here.

As you recall, microRNA refers to small single-stranded RNA molecules, generally about 21 to 23 nucleotides in length. Each different microRNA (miRNA for short) is transcribed from a DNA gene like any other gene, but the resulting RNA isn't translated into a protein. Instead, the typical miRNA functions by downregulating the expression of another gene that codes for a protein.

An embryonic stem cell (ESC) has the property of pluripotency, which means that it is capable of giving rise to essentially any type of cell in the body of a multicellular organism. Whenever an ESC divides, the resulting daughter cells may also by ESCs (hence pluripotent) or they may be more specialized cells that will eventually give rise to some type of adult body cell.

In any particular ESC, the determination of remaining pluripotent or instead heading down the path to a more specialized cell type depends on what set of genes are expressed. Since miRNAs downregulate gene expression, they can keep an ESC in its pluripotent state, if they are active and suppress a gene that would make an ESC more specialized. But then if such a miRNA is blocked from being expressed, the ESC can start to become more specialized.

On the other hand, as we shall see, some miRNAs may block transcription factors that are needed to maintain a pluripotent state. Such an miRNA needs to be silent in an ESC, so some other protein needs to suppress its expression. (The miRNA called miR-21, discussed later, is an example.)

So the name of the game in studying ESCs, as far as miRNA is concerned, is to figure out what causes a miRNA gene to be expressed or not. Like other genes in an ESC, which genes are expressed is strongly controlled by a few master transcription factors.

There are four such transcription factors which seem to be especially important in ESCs: Oct4, Sox2, Nanog, and Tcf3. As discussed here, the first three of these factors have been found capable of playing a role in turning an ordinary adult cell into a pluripotent stem cell (called an "induced pluripotent stem cell").

Currently there are 336 mature mouse miRNAs known, and 441 mature human miRNAs. It is simple (given the known, complete sequences of mouse and human genomes) to locate the genes for each miRNA. However, in order to determine when a transcription factor regulates the miRNA gene, the promoter for the gene (a separate portion of DNA) must also be located. In order for a gene to be expressed, the right transcription factors have to bind to the gene's promoter.

Finding promoters is a lot harder, but there are techniques that involve searching for methylation of histone proteins that make up the nucleosomes around which cellular DNA is wrapped.

It was known, before the recent research we're discussing, that there were 14,230 sites in the genome where all four of the named master transcription factors could bind simultaneously. Most of those sites were not promoters of some miRNA, but it was straightforward to identify those that were. Of those miRNAs that appeared to be regulated by Oct4, Sox2, Nanog, and Tcf3, it was found that most are in fact preferentially expressed in ESCs. This set of miRNAs would seem to be good candidates for maintaining ESC pluripotency by downregulating other genes.

On the other hand, some of the miRNAs mediated by the transcription factors are silent in ESCs. Subsequent research found that another type of proteins (polycomb proteins) also bind to the miRNA promoters. These proteins were already known to block transcription by binding to gene promoters. But it turns out that some of these silenced miRNAs become active once the ESC loses its pluripotency and begins to differentiate.

The next step will be to figure out what each of these miRNAs regulated by Oct4, Sox2, Nanog, and Tcf3 actually does – either in the ESC or a differentiated cell. That should be very interesting, as the press release suggests:

Putting microRNAs on the stem cell map (8/7/08)

“We now have a list of what microRNAs are important in embryonic stem cells,” says Alex Marson, co-lead author on the paper and an MD/PhD student in the Young lab. “This gives us clues of which microRNAs you might want to target to direct an embryonic stem cell into another type of cell. For example, you might be able to harness a microRNA to help drive an embryonic stem cell to become a neuron, aiding with neurodegenerative disease or spinal cord injury.”

Moreover, the results give scientists a better platform for analyzing microRNA gene expression in cancer and other diseases. “We and others are finding that the overall gene circuitry for embryonic stem cells and cancer cells is very similar,” notes Marson. “Now that we have connected the circuitry to microRNAs, we can begin to compare microRNAs that are regulated in embryonic stem cells to those in cancer cells.”

Here's a somewhat more detailed description of the research: Stem Cell microRNA, Transcription Factor Interplay Uncovered (8/8/08)

Other research on miRNA and ESCs that has appeared since the previous discussion (here) gives a small taste of what may be learned about the miRNAs silenced in ESCs:

Protein Protects Embryonic Stem Cells' Versatility And Self-renewal (3/23/08)

A protein known as REST blocks the expression of a microRNA that prevents embryonic stem cells from reproducing themselves and causes them to differentiate into specific cell types, scientists at The University of Texas M. D. Anderson Cancer Center report in the journal Nature.

Researchers show RE1-silencing transcription factor (REST) plays a dual role in embryonic stem cells, said senior author Sadhan Majumder, Ph.D., professor in M. D. Anderson's Department of Cancer Genetics. "It maintains self-renewal, or the cell's ability to make more and more cells of its own type, and it maintains pluripotency, meaning that the cells have the potential to become any type of cell in the body."

The details are particularly interesting:

In studies using mouse embryonic stem cells, the researchers found that REST disarms a specific microRNA called microRNA-21 or miR-21. MicroRNAs are tiny pieces of RNA that control gene expression by binding to the gene's messenger RNA.

The team found that MiR-21 suppresses embryonic stem cell self-renewal and is associated with a corresponding loss of expression of critical self-renewal regulators, such as Oct4, Nanog, Sox2 and c-Myc. REST counters this by suppressing miR-21 to preserve the cells' self-renewal and pluripotency.

The researchers discovered the roles of REST and miR-21 in a series of experiments using cultured mouse embryonic stem cells in either a self-renewal state or a differentiating state. They found that REST expression was significantly higher in the self-renewal state. Withdrawing REST reduced the stem cells' ability to reproduce themselves and started differentiation -- even when the cells were grown under conditions conducive to self-renewal. Adding REST to differentiating cells maintained their self-renewal.

These experiments also revealed that REST is bound to the gene chromatin of a set of microRNAs with the potential to target self-renewal genes. REST controls transcription of 11 microRNAs.

Is anything special known about miR-21? Yes, in fact – it is known to play a role in cancers of the colon, liver, and thyroid. (See here.)

Tags: microRNA, stem cells, embryonic stem cells