Stem Cell Differentiation Requires Proper Compaction of DNA

Human cells have a compartment that houses the chromosomes known as the nucleus. The nucleus in human cells contains so much DNA that if the DNA was laid out end to end, it would stretch out for about one meter. Think of that – almost every cell in your body has one meter’s length of DNA in it. In order to properly package all that DNA into the nucleus of the cell, it must be assembled into a structure called “chromatin.”

Chromatin involves winding the DNA around small spools, and the spools are coiled into a fiber and then the fiber is looped into rosettes. The spools are composed of proteins called “histones.” Histone proteins are extremely conserved from one organism to another. In fact, histone proteins from cow peas and extremely similar to histones from cows. This tells scientists that the function of histones is exactly the same from one organism to another. The tiny spools are composed of four “core histones” known as H2A, H2B, H3 and H4. Each tiny spool has two copies of each core histone protein. The DNA is then wound around each of these histone complexes 1.8 times. The core histone complex with its DNA wound about it is either called a “core particle,” or a “nucleosome.”

To form chromatin, the nucleosomes are bound together by another histone proteins called H1. H1 winds the nucleosomes together into a chromatin fiber that is about 30 nanometers in diameter. These chromatin fibers are looped into a 300 nanometer coiled chromatin fiber, and these coiled chromatin fibers are then wound into 700 nanometer condensed chromatin. A chromosome that has been observed during the height of cell division (a phase called metaphase for those who are interested) has a diameter of 1400 nanometers, and since there are two chromatids present during cell division that compose each chromosome, these measurements agree completely with other work.

All of this might seem rather dry and uninteresting, except that researchers at the Georgia Institute of Technology and Emory University have demonstrated that chromatin compaction is essential for embryonic stem cell differentiation. Embryonic stem cells (ESCs) express several different types of H1 subtypes, and ESCs that fail to express these H1 subtypes show reduced chromatin compaction and impaired differentiation. The diminished differentiation capacity of these genes seems to derived from the inability of these cells to properly silence particular genes.

Yuhong Fan, assistant professor in the Georgia Tech School of Biology said, “While researchers have observed that embryonic stem cells exhibit a relaxed, open chromatin structure and differentiated cells exhibit a compact chromatin structure, our study is the first to show that this compaction is not a mere consequence of the differentiation process but is instead a necessity for differentiation to proceed normally,”

In this study, which was led by Fan and Todd McDevitt, who is an associate professor in the Wallace H. Coulter Department of Biomedical Engineering at Georgia Tech and Emory University, a ESC strain was used that lack three H1 subtypes; H1c, H1d, and H1e. These three H1 subtypes show increased expression levels during ESC differentiation, and the ESC strains triply deficient for these three H1 subtypes were unable to differentiate, even under standard culture conditions that normally induce differentiation. They also failed to differentiate in embryoid bodies, and could not form neural cells.

Anthony Carter, who oversees grants at National Institutes of Health’s National Institute of General Medical Sciences that deal with the effects of chromatin structure on gene expression, made these comments about this work: “This study has uncovered a new, regulatory function for histone H1, a protein known mostly for its role as a structural component of chromosomes. By showing that H1 plays a part in controlling genes that direct embryonic stem cell differentiation, the study expands our understanding of H1’s function and offers valuable new insights into the cellular processes that induce stem cells to change into specific cell types.”

When the triply-deficient ESCs were subjected to differentiation protocols, they remained tightly packed together, much like the early embryo. The cells also expressed genes that are found in the inner cell mass of the early embryo, such as Oct4. In order for the cells of the early embryo to properly differentiate, genes like Oct4 must undergo programmed down-regulation.

Accord to Fan, ““H1 depletion impaired the suppression of the Oct4 and Nanog pluripotency genes, suggesting a novel mechanistic link by which H1 and chromatin compaction may mediate pluripotent stem cell differentiation by contributing to the epigenetic silencing of pluripotency genes. While a significant reduction in H1 levels does not interfere with embryonic stem cell self-renewal, it appears to impair differentiation.”

In order to make embryoid bodies, this study utilized a rotary suspension technique that was developed by McDevitt and his co-workers. Normally, scientists use the so-called “hanging drop” method in which cells are placed in a drop of medium with few growth factors (20% serum usually), and suspended upside down on a microscope slide in a sealed chamber that prevents desiccation. under these conditions, the ESCs will form roundish little balls of cells that differentiate on the inside. These are known as embryoid bodies, and McDevitt’s technique forms three-dimensional embryoid bodies at very high-efficiency.

Embryoid bodies contain cell types of all three primary embryonic germ layers (endoderm, mesoderm and ectoderm). However, when the triply-deficient ESC line was subjected the McDevit’s embryoid body-making protocol, that lacked differentiated cell types and largely resisted differentiation.

As noted by McDevitt, “H1 triple-knockout embryoid bodies displayed a reduced level of activation of many developmental genes and markers in rotary culture, suggesting that differentiation to all three germ layers was affected.”

Fan and MCDevitt’s groups tried to add back H1 subtypes to the triply-deficient ESC strain. According to Fan, “When we added one of the deleted H1 subtypes to the embryoid bodies, Oct4 was suppressed normally and embryoid body differentiation continued. The epigenetic regulation of Oct4 expression by H1 was also evident in mouse embryos.”

This work also examined the ability of the triply-deficient ESC strain to differentiate into neural cells. However, the H1 triple-knockout ESCs could form neither neuronal nor glial cells, and unable to contribute to the formation of a neural network. Only 10% of the H1 triple-knockout embryoid bodies formed neurites and they produced on average eight neurites each in comparison to normal embryoid bodies, which produced, on average, 18 neurites.

In he future, Fan and McDevitt would like to investigate if controlling H1 histone levels can be used to influence the reprogramming of adult cells to form induced pluripotent stem cells, which have the capacity to differentiate into tissues in a way similar to embryonic stem cells.