Histones Might Hold the Key to the Generation of Totipotent Stem Cells


Reprogramming adult cells into pluripotent stem cells remains a major challenge to stem cell research. The process remains relatively inefficient and slow and a great deal of effort has been expended to improve the speed, efficiency and safety of the reprogramming procedure.

Researchers from RIKEN in Japan have reported one piece of the reprogramming puzzle that can increase the efficiency of reprogramming. Shunsuke Ishii and his colleagues from RIKEN Tsukuba Institute in Ibaraki, Japan have identified two variant histone proteins that dramatically enhance the efficiency of induced pluripotent stem cell (iPS cell) derivation. These proteins might be the key to generating iPS cells.

Terminally-differentiated adult cells can be reprogrammed into a stem-like pluripotent state either by artificially inducing the expression of four factors called the Yamanaka factors, or as recently shown by shocking them with sublethal stress, such as low pH or pressure. However, attempts to create totipotent stem cells capable of giving rise to a fully formed organism, from differentiated cells, have failed.  However, a paper recently published in the journal Nature has shown that STAP or stimulus-triggered acquisition of pluripotency cells from mouse cells have the capacity to form placenta in culture and therefore, are totipotent.

The study by Shunsuke Ishii and his RIKEN colleagues, which was published in the journal Cell Stem Cell, attempted to identify molecules in mammalian oocytes (eggs) that induce the complete reprograming of the genome and lead to the generation of totipotent embryonic stem cells. This is exactly what happens during normal fertilization, and during cloning by means of the technique known as Somatic-Cell Nuclear Transfer (SCNT). SCNT has been used successfully to clone various species of mammals, but the technique has serious limitations and its use on human cells has been controversial for ethical reasons.

Ishii’s research group focused on two histone variants named TH2A and TH2B, which are known to be specific to the testes where they bind tightly to DNA and influence gene expression.

Histones are proteins that bind to DNA non-specifically and act as little spool around which the DNA winds.  These little wound spools of DNA then assemble into spirals that form thread-like structures.  These threads are then looped around a protein scaffold to form the basic structure of a chromosome.  This compacted form of DNA is called “chromatin,” and the DNA is compacted some 10,000 to 100,000 times.  Histones are the main arbiters of chromatin formation.  In the figure below, you can see that the “beads on a string” consist of histones with DNA wrapped around them.

DNA_to_Chromatin_Formation

There are five “standard” histone proteins: H1, H2A, H2B, H3, and H4.  H2A, H2B, H3 and H4 form the beads and the H1 histone brings the beads together to for the 30nm solenoid.  Variant histones are different histones that assemble into beads that do not wrap the DNA quite as tightly or wrap it differently than the standard histones.  Two variant histones in particular, TH2A and TH2B, tend to allow DNA wrapped into chromatin to form and more loosely packed structure that allows the expression of particular genes.

When members of Ishii’s laboratory added these two variant histone proteins, TH2A/TH2B, to the Yamanaka cocktail (Oct4, c-Myc, Sox2, and Klf4) to reprogram mouse fibroblasts, they increased the efficiency of iPSC cell generation about twenty-fold and the speed of the process two- to threefold. In fact, TH2A and TH2B function as substitutes for two of the Yamanaka factors (Sox2 and c-Myc).

Ishii and other made knockout mice that lacked the genes that encoded TH2A and TH2B. This work demonstrated that TH2A and TH2B function as a pair, and are highly expressed in oocytes and fertilized eggs. Furthermore, these two proteins are needed for the development of the embryo after fertilization, although their levels decrease as the embryo grows.

Graphical Abstract1 [更新済み]

In early embryos, TH2A and TH2B bind to DNA and induce an open chromatin structure in the paternal genome (the genome of sperm cells), which contributes to its activation after fertilization.

These results indicate that TH2A/TH2B might induce reprogramming by regulating a different set of genes than the Yamanaka factors, and that these genes are involved in the generation of totipotent cells in oocyte-based reprogramming as seen in SCNT.

“We believe that TH2A and TH2B in combination enhance reprogramming because they introduce a process that normally operates in the zygote during fertilization and SCNT, and lead to a form of reprogramming that bears more similarity to oocyte-based reprogramming and SCNT” explains Dr. Ishii.

Polycomb Proteins Pave the Way for Proper Stem Cell Differentiation


Embryonic stem cells have the ability to differentiate into one of the more than 200 cell types. Differentiation requires a strictly regulated program of gene expression that turns certain genes on at specific times and shuts other genes off. Loss of this regulatory circuit prevents stem cells from properly differentiating into adult cell types, and an inability to differentiate has also been linked to the onset of cancer.

Researchers at the BRIC, University of Copenhagen have identified a crucial role of the molecule Fbx110 in embryonic stem cell differentiation. Kristian Helin from the BRIC said, “Our new results show that this molecule is required for he function of one of the most important molecular switches that constantly regulated the activity of our genes. If Fbx110 is not present in embryonic stem cells, the cells cannot differentiate properly and this can lead to developmental defects.”

What is the function of Fbx110? Fbx110 recruits members of the “Polycomb” gene family to DNA. Polycomb proteins, in particular PRC1 & 2, are known to modulate the structure of chromatin, even though they do not bind DNA. Fbx110,, however binds DNA, but it also binds PRC1 . Therefore, Fbx110 seems to serve as an adapter that recruits Polycomb proteins to DNA.

Polycomb proteins bound to nucleosomes
Polycomb proteins bound to nucleosomes

Postdoctoral fellow Xudong Wu, who led the experimental part of this investigation, said, “Our results show that Fbx110 is essential for recruiting PRC1 to genes that are to be silenced in embryonic stem cells. Fbx110 binds directly to DNA and to PRC1, and this way it serves to bring PRC1 to specific genes. When PRC1 is bound to DNA it can modify the DNA associated proteins, which lead to silencing of the gene to which it binds.”

Timing of gene activity is crucial during development and must be maintained throughout the lifespan of any cell. Particular genes are active at a certain times and inactive at other times, and PRC1 seems to be part of the reason for this coordination of gene activity. PRC1 is dynamically recruited to and dissociates from genes according to the needs of the organism.

When cancer arises, this tight regulation of gene activity is often lost and the cells are locked in an inchoate state. This loss of terminal differentiation causes increases cell proliferation and the accumulation of other mutations that allow the cancer cells to undergo continuous self-renewal through endless cell divisions. Such an ability is denied to mature cells because of their tightly controlled programs of gene expression.

Wu added, “Given the emerging relationship between cancer and stem cells, our findings may implicate that an aberrant activity of Fbx110 can disturb PRC function and promote a lack of differentiation in our cells. This makes it worth studying whether blocking the function of Fbx110 could be a strategy for tumor therapy.”

In collaboration with a biotech company called EpiTherapeutics, the BRIC researchers want to develop Fbx110 inhibitors as potential novel therapies for cancer.