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New research findings show that embryonic stem cells unable to fully compact
the DNA inside them cannot complete their primary task: differentiation into
specific cell types that give rise to the various types of tissues and
structures in the body.
Researchers from the Georgia Institute of Technology and Emory University
found that chromatin compaction is required for proper embryonic stem cell
differentiation to occur. Chromatin, which is composed of histone proteins and
DNA, packages DNA into a smaller volume so that it fits inside a cell.
A study published in PLoS Genetics found that embryonic stem cells
lacking several histone H1 subtypes and exhibiting reduced chromatin compaction
suffered from impaired differentiation under multiple scenarios and
demonstrated inefficiency in silencing genes that must be suppressed to induce
differentiation.
“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,” said Yuhong Fan, an assistant professor
in the Georgia Tech School of Biology.
Fan and Todd McDevitt, an associate professor in the Wallace H. Coulter
Department of Biomedical Engineering at Georgia Tech and Emory University,
led the study with assistance from Georgia Tech graduate students Yunzhe Zhang
and Kaixiang Cao, research technician Marissa Cooke, and postdoctoral fellow
Shiraj Panjwani.
The work was supported by the National Institutes of Health’s National
Institute of General Medical Sciences (NIGMS), the National Science Foundation,
a Georgia Cancer Coalition Distinguished Scholar Award, and a Johnson &
Johnson/Georgia Tech Healthcare Innovation Award.
To investigate the impact of linker histones and chromatin folding on stem
cell differentiation, the researchers used embryonic stem cells that lacked
three subtypes of linker histone H1—H1c, H1d, and H1e—which is the structural
protein that facilitates the folding of chromatin into a higher-order
structure. They found that the expression levels of these H1 subtypes increased
during embryonic stem cell differentiation, and embryonic stem cells lacking
these H1s resisted spontaneous differentiation for a prolonged time, showed
impairment during embryoid body differentiation and were unsuccessful in
forming a high-quality network of neural cells.
“This study has uncovered a new, regulatory function for histone H1, a
protein known mostly for its role as a structural component of chromosomes,”
said Anthony Carter, who oversees epigenetics grants at NIGMS. “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.”
During spontaneous differentiation, the majority of the H1 triple-knockout
embryonic stem cells studied by the researchers retained a tightly packed
colony structure typical of undifferentiated cells and expressed high levels of
Oct4 for a prolonged time. Oct4 is a pluripotency gene that maintains an
embryonic stem cell’s ability to self-renew and must be suppressed to induce
differentiation.
“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,” explained Fan. “While a
significant reduction in H1 levels does not interfere with embryonic stem cell
self-renewal, it appears to impair differentiation.”
The researchers also used a rotary suspension culture method developed by
McDevitt to produce with high efficiency homogonous 3D clumps of embryonic stem
cells called embryoid bodies. Embryoid bodies typically contain cell types from
all three germ layers—the ectoderm, mesoderm, and endoderm—that give rise to
the various types of tissues and structures in the body. However, the majority
of the H1 triple-knockout embryoid bodies formed in rotary suspension culture
lacked differentiated structures and displayed gene expression signatures
characteristic of undifferentiated stem cells.
“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.” noted McDevitt.
The embryoid bodies also lacked the epigentic changes at the pluripotency
genes necessary for differentiation, 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,” explained
Fan. “The epigenetic regulation of Oct4 expression by H1 was also evident in
mouse embryos.”
In another experiment, the researchers provided an environment that would
encourage embryonic stem cells to differentiate into neural cells. However, the
H1 triple-knockout cells were defective in forming neuronal and glial cells and
a neural network, which is essential for nervous system development. Only 10%
of the H1 triple-knockout embryoid bodies formed neurites and they produced on
average eight neurites each. In contrast, half of the normal embryoid bodies
produced, on average, 18 neurites.
In future work, the researchers plan to investigate whether controlling H1
histone levels can be used to influence the reprogramming of adult cells to
obtain induced pluripotent stem cells, which are capable of differentiating
into tissues in a way similar to embryonic stem cells.
