New research findings show that embryonic stem cells unable to fully compact the DNA inside them cannot complete theirprimary task: differentiation into specific cell types that giverise to the various types of tissues and structures in the body. Researchers from the Georgia Institute of Technology and EmoryUniversity found that chromatin compaction is required for properembryonic stem cell differentiation to occur. Chromatin, which iscomposed of histone proteins and DNA, packages DNA into a smallervolume so that it fits inside a cell. A study published in the journal PLoS Genetics found that embryonic stem cells lacking several histone H1subtypes and exhibiting reduced chromatin compaction suffered fromimpaired differentiation under multiple scenarios and demonstratedinefficiency in silencing genes that must be suppressed to inducedifferentiation.
"While researchers have observed that embryonic stem cells exhibita relaxed, open chromatin structure and differentiated cellsexhibit a compact chromatin structure, our study is the first toshow that this compaction is not a mere consequence of thedifferentiation process but is instead a necessity fordifferentiation to proceed normally," said Yuhong Fan, an assistantprofessor 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 andEmory University, led the study with assistance from Georgia Techgraduate students Yunzhe Zhang and Kaixiang Cao, researchtechnician Marissa Cooke, and postdoctoral fellow Shiraj Panjwani. The work was supported by the National Institutes of Health'sNational Institute of General Medical Sciences (NIGMS), theNational Science Foundation, a Georgia Cancer CoalitionDistinguished Scholar Award, and a Johnson & Johnson/Georgia TechHealthcare Innovation Award. To investigate the impact of linker histones and chromatin foldingon stem cell differentiation, the researchers used embryonic stemcells that lacked three subtypes of linker histone H1 -- H1c, H1dand H1e -- which is the structural protein that facilitates thefolding of chromatin into a higher-order structure. They found thatthe expression levels of these H1 subtypes increased duringembryonic stem cell differentiation, and embryonic stem cellslacking these H1s resisted spontaneous differentiation for aprolonged time, showed impairment during embryoid bodydifferentiation and were unsuccessful in forming a high-qualitynetwork of neural cells.
"This study has uncovered a new, regulatory function for histoneH1, a protein known mostly for its role as a structural componentof chromosomes," said Anthony Carter, who oversees epigeneticsgrants at NIGMS. "By showing that H1 plays a part in controllinggenes that direct embryonic stem cell differentiation, the studyexpands our understanding of H1's function and offers valuable newinsights into the cellular processes that induce stem cells tochange into specific cell types." During spontaneous differentiation, the majority of the H1triple-knockout embryonic stem cells studied by the researchersretained a tightly packed colony structure typical ofundifferentiated cells and expressed high levels of Oct4 for aprolonged time. Oct4 is a pluripotency gene that maintains anembryonic stem cell's ability to self-renew and must be suppressedto induce differentiation. "H1 depletion impaired the suppression of the Oct4 and Nanogpluripotency genes, suggesting a novel mechanistic link by which H1and chromatin compaction may mediate pluripotent stem celldifferentiation by contributing to the epigenetic silencing ofpluripotency genes," explained Fan. LED Tube Light Fixture
"While a significant reductionin H1 levels does not interfere with embryonic stem cellself-renewal, it appears to impair differentiation." The researchers also used a rotary suspension culture methoddeveloped by McDevitt to produce with high efficiency homogonous 3Dclumps of embryonic stem cells called embryoid bodies. Embryoidbodies typically contain cell types from all three germ layers -the ectoderm, mesoderm and endoderm - that give rise to the varioustypes of tissues and structures in the body. However, the majorityof the H1 triple-knockout embryoid bodies formed in rotarysuspension culture lacked differentiated structures and displayedgene expression signatures characteristic of undifferentiated stemcells. "H1 triple-knockout embryoid bodies displayed a reduced level ofactivation of many developmental genes and markers in rotaryculture, suggesting that differentiation to all three germ layerswas affected." noted McDevitt. The embryoid bodies also lacked the epigentic changes at thepluripotency genes necessary for differentiation, according to Fan. China LED Lighting Panels
"When we added one of the deleted H1 subtypes to the embryoidbodies, Oct4 was suppressed normally and embryoid bodydifferentiation continued," explained Fan. "The epigeneticregulation of Oct4 expression by H1 was also evident in mouseembryos." In another experiment, the researchers provided an environment thatwould encourage embryonic stem cells to differentiate into neuralcells. However, the H1 triple-knockout cells were defective informing neuronal and glial cells and a neural network, which isessential for nervous system development. Only 10 percent of the H1triple-knockout embryoid bodies formed neurites and they producedon average eight neurites each. In contrast, half of the normalembryoid bodies produced, on average, 18 neurites. LED Fluorescent Tubes Manufacturer
In future work, the researchers plan to investigate whethercontrolling H1 histone levels can be used to influence thereprogramming of adult cells to obtain induced pluripotent stemcells, which are capable of differentiating into tissues in a waysimilar to embryonic stem cells. Additional References Citations.
"While researchers have observed that embryonic stem cells exhibita relaxed, open chromatin structure and differentiated cellsexhibit a compact chromatin structure, our study is the first toshow that this compaction is not a mere consequence of thedifferentiation process but is instead a necessity fordifferentiation to proceed normally," said Yuhong Fan, an assistantprofessor 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 andEmory University, led the study with assistance from Georgia Techgraduate students Yunzhe Zhang and Kaixiang Cao, researchtechnician Marissa Cooke, and postdoctoral fellow Shiraj Panjwani. The work was supported by the National Institutes of Health'sNational Institute of General Medical Sciences (NIGMS), theNational Science Foundation, a Georgia Cancer CoalitionDistinguished Scholar Award, and a Johnson & Johnson/Georgia TechHealthcare Innovation Award. To investigate the impact of linker histones and chromatin foldingon stem cell differentiation, the researchers used embryonic stemcells that lacked three subtypes of linker histone H1 -- H1c, H1dand H1e -- which is the structural protein that facilitates thefolding of chromatin into a higher-order structure. They found thatthe expression levels of these H1 subtypes increased duringembryonic stem cell differentiation, and embryonic stem cellslacking these H1s resisted spontaneous differentiation for aprolonged time, showed impairment during embryoid bodydifferentiation and were unsuccessful in forming a high-qualitynetwork of neural cells.
"This study has uncovered a new, regulatory function for histoneH1, a protein known mostly for its role as a structural componentof chromosomes," said Anthony Carter, who oversees epigeneticsgrants at NIGMS. "By showing that H1 plays a part in controllinggenes that direct embryonic stem cell differentiation, the studyexpands our understanding of H1's function and offers valuable newinsights into the cellular processes that induce stem cells tochange into specific cell types." During spontaneous differentiation, the majority of the H1triple-knockout embryonic stem cells studied by the researchersretained a tightly packed colony structure typical ofundifferentiated cells and expressed high levels of Oct4 for aprolonged time. Oct4 is a pluripotency gene that maintains anembryonic stem cell's ability to self-renew and must be suppressedto induce differentiation. "H1 depletion impaired the suppression of the Oct4 and Nanogpluripotency genes, suggesting a novel mechanistic link by which H1and chromatin compaction may mediate pluripotent stem celldifferentiation by contributing to the epigenetic silencing ofpluripotency genes," explained Fan. LED Tube Light Fixture
"While a significant reductionin H1 levels does not interfere with embryonic stem cellself-renewal, it appears to impair differentiation." The researchers also used a rotary suspension culture methoddeveloped by McDevitt to produce with high efficiency homogonous 3Dclumps of embryonic stem cells called embryoid bodies. Embryoidbodies typically contain cell types from all three germ layers -the ectoderm, mesoderm and endoderm - that give rise to the varioustypes of tissues and structures in the body. However, the majorityof the H1 triple-knockout embryoid bodies formed in rotarysuspension culture lacked differentiated structures and displayedgene expression signatures characteristic of undifferentiated stemcells. "H1 triple-knockout embryoid bodies displayed a reduced level ofactivation of many developmental genes and markers in rotaryculture, suggesting that differentiation to all three germ layerswas affected." noted McDevitt. The embryoid bodies also lacked the epigentic changes at thepluripotency genes necessary for differentiation, according to Fan. China LED Lighting Panels
"When we added one of the deleted H1 subtypes to the embryoidbodies, Oct4 was suppressed normally and embryoid bodydifferentiation continued," explained Fan. "The epigeneticregulation of Oct4 expression by H1 was also evident in mouseembryos." In another experiment, the researchers provided an environment thatwould encourage embryonic stem cells to differentiate into neuralcells. However, the H1 triple-knockout cells were defective informing neuronal and glial cells and a neural network, which isessential for nervous system development. Only 10 percent of the H1triple-knockout embryoid bodies formed neurites and they producedon average eight neurites each. In contrast, half of the normalembryoid bodies produced, on average, 18 neurites. LED Fluorescent Tubes Manufacturer
In future work, the researchers plan to investigate whethercontrolling H1 histone levels can be used to influence thereprogramming of adult cells to obtain induced pluripotent stemcells, which are capable of differentiating into tissues in a waysimilar to embryonic stem cells. Additional References Citations.