New tools aid in understanding how nucleosome works
Nucleosomes are the basic packaging units of DNA or genetic material, each made of eight core proteins called histones and about 147 base pairs of DNA. Nucleosomes can be considered the protector or barrier to unwinding the DNA to allow it to the transcription into the genetic code used by the cell’s protein making machinery.
The dynamics of how the nucleosome protects the DNA or is removed to allow transcription are being unraveled by the laboratory of Dr. Wei Li, associate professor in the NCI-designated Dan L. Duncan Cancer Center at Baylor College of Medicine. Li credits Dr. Kaifu Chen, a postdoctoral associate in Li’s laboratory, with spearheading much of the research involved in a series of three recently published studies in Genome Research and Cell, respectively.
Changes function of genome
In a recent article in Genome Research, Li, members of his laboratory and colleagues from the University of Texas MD Anderson Cancer Center describe a new tool DANPOS that allows researchers to understand how nucleosomes orient themselves in regard to the DNA. Regulatory proteins can alter the position of a nucleosome or deplete it all together to affect the way that genes are expressed.
"Any changes in the nucleosome changes the function of the genome," said Li. "Nucleosomes are the fundamental building block of the genome."
Nucleosomes affected by these regulatory proteins are called dynamic nucleosomes but their exact position with regard to the DNA is fuzzy because their exact position can differ among samples. DANPOS overcomes this difficulty by evaluating position in regard to single nucleotides, making its resolution high and then decreasing experimental variation between samples through mathematical means, making it possible to identify the position of the nucleosomes within the DNA.
This method can be used to look at the characteristics of dynamic nucleosomes including shifts in position, changes in fuzziness, and changes in occupancy (presence of the nucleosome at given positions).
Others who took part in this research include Yuanxin Xi, Xuewen Pan and Xiangwei He, all of BCM; and Zhaoyu Li, Klaus Kaestner, Jessica Tyler and Sharon Dent of MD Anderson.
In another Genome Research paper, Li and his colleagues, including those from MD Anderson and the University of Vermont, used special techniques, including DANPOS, that allowed them to recover a promoter nucleosome in an area that they previously considered a "nucleosome-free region," he said.
"One goal of our software is to understand the fundamental molecular biology of nucleosome heterogeneity," he said. Under normal conditions, these promoter nucleosomes are stabilized by a co-repressor molecule such as Cyc8 or Tup1 to block gene transcription. However, when the cell is stressed, it loses the co-repressors (in this case, the Cyc8-Tup1 complex, which can regulate gene expression by positioning nucleosomes over the promoters of some target genes to prevent their transcription).
Li and his colleagues found that Cyc8 and Tup1 bind to crucial promoters that are enriched in genes that are not repressed when the two co-repressors are lost.
"When stress comes in, the promoter nucleosome is gone and the nucleosome-free region can recruit an activator to turn on gene expression," he said.
Others who took part in this research include Marenda A. Wilson, Calley Hirsch, Shoudan Liang, Yue Lu and Sharon Y.R. Dent, all of MD Anderson Cancer Center and Anjanette Watson of the University of Vermont.
DANPOS plays role
In a third report that appears in the journal Cell, Li, his BCM laboratory and colleagues from the University of Pennsylvania identified two proteins that played an important role in genome-wide nucleosome dynamics that involved nucleosome depletion and gene activation during the period in which embryonic stem cells differentiate into cells that eventually make up the different tissues in an organism. Again, DANPOS plays an important role in data analysis.
H2A.Z is one of the histone variant proteins binding to the nucleosome or linker DNA between nucleosomes, effectively condensing the size of chromosomes (one of the units of DNA) and Foxa2 is a protein that helps the DNA unwrap from the histone so that it can be transcribed into a form from which proteins are usually made. This unwrapping process effectively "depletes the nucleosome," exposing the DNA to the genetic promoters and enzyme that can allow it to do its job to start the process that results in production of proteins, the workhorses of the cell.
Li and his colleagues show that nucleosome depletion occurs when the embryonic stem cell is differentiating into a more adult form of stem cell called an endoderm/hepatic progenitor cell. These adult stem cells need the genes "protected" by the nucleosome to be exposed and expressed to function.
Critical step in differentiation
During this depletion, H2A.Z releases the DNA and Foxa2 bind it at the site of the genes that need to be expressed in the differentiated cells. Both proteins (H2A.Z and Foxa2) also interact directly with proteins known to be involved in nucleosome remodeling, strengthening evidence for their role in the process.
A high level of Foxa2 increases the rate of nucleosome depletion. If Foxa2 is lacking, the cell does not differentiate, further affirming the finding that it is a critical step in differentiation.
In other words, the differentiation of embryonic stem cells into endoderm/hepatic precursor cells takes three steps:
- The embryonic stem cell receives a signal that it should differentiate, sparking expression of Foxa2.
- Foxa2 finds the DNA where the histone H2A.Z is bound and there is a nucleosome, and
- Foxa2 and H2A.Z interact with other proteins involved in nucleosome depletion in areas where genes required for differentiation are expressed.
Others who took part in this research include Zhaoyu Li, Paul Gadue, Yang Jiao, Geetu Tuteja, Jonathan Schug and Klaus H. Kaestner, all of the University of Pennsylvania School of Medicine in Philadelphia.
Funding for this work comes from the U.S. National Institute of Diabetes, Digestive and Kidney Diseases (P01-DK049210), NSREC, the Juvenile Diabetes Research Foundation, the Cancer Prevention and Research Institute of Texas, the U.S. Department of Defense, 973 project 2010CB944900 of China, and the National Institutes of Health (HG004840, R01GM51189), the U.S. National Institute of Child Health and Human Development (2 T32 HD07325), the University of Texas MD Anderson Cancer Center Senior Research Trust, the National Center for Research Resources (2P20RR016462 to the Vermont Genetics Network).