Chromatin Remodeling and Gene Expression
Control of DNA transcription in eukaryotes was thought to involve the assembly of many proteins at the promoter into a pre-initiation complex (PIC). Once assembled, RNA polymerase could bind and transcription would be initiated. But wait a minute! Isn't DNA packaged in the nucleus into chromatin in which 147 BP of DNA is wound around a core of 4 pairs of positively charged histone proteins - including H2A, 2B, 3, and 4 - to form a nucleosome, seen under a microscope as beads on a string?
- Jmol: nucleosome
Isn't this chromatin further wound into fibers which result in the classic picture of sister chromatids ready to separate at cell division? How could the transcription factors and RNA polymerase recognize target sites on DNA given this degree of "folding" and condensation of the DNA?
- Animation: Packing of DNA from the Walter + Eliza Hall Institute of Medical Research
Clearly the complex compacted state of DNA and its interaction with the histone proteins must be "remodeled" to allow interactions of the transcription factors and RNA polymerase (which is about the same size as a nucleosome). The regulation of this chromatin remodeling clearly affects gene transcription, and is another example of epigenetic changes that can affect phenotype. The state of chromatin structure is regulated by enzymes that affect histone structure and function by chemically modifying the histone proteins (through acetylation, methylation, and phosphorylation). Likewise, the DNA at the promoter region is changed by enzymes that remodel the DNA through an ATP dependent series of modifications. For example when histones are modified by histone acetyltransferase (HAT's), other modeling factors (SWI/SNF) are recruited to the chromatin. Chromatin remodeling would also be affected by that cell cycle stage of the cell. For example, chromatin condensed in sister chromatids ready for cells division would have different remodeling requirements for gene transcription than might chromatin in the form of bead on a string. Likewise remodeling efforts would also be gene-specific.
The figure below shows how remodeling is coupled to formation of the pre-initiation complex for three genes:
yeast HO gene: Swi5p activator binding results in the interaction of the SWI/SNF ATP-dependent remodeling enzyme, which leads to the binding of histone acetyltransferase (HAT). These facilitate formation of the pre-initiation complex.
human interferon-bgene: gene sequences known as activators, 5' to the promoter, bind HATs. When histones are acetylated, SWI/SNF interacts to remodel the chromatin and facilitate PIC formation.
human a-1 antitrypsin gene: the PIC is preformed and recruits HAT and SWI/SNF, which leads to gene transcription.
Alternations in chromatin remodeling could lead to changes in gene expression, in some cases causing cancer. SNF5 is a component of the SWI/SNF complex and in its normal form acts to suppress tumors (i.e. its gene is a tumor suppressor gene). Mutations in SNF5 are associated with rare and aggressive childhood tumors. Stuart Orkin has developed a technique to alter the gene in some mouse cells to produce an inverted gene which produces no functional SNF5. Cells with this mutation become tumor cells almost immediately.
DNA winds around the histone core to form the nucleosome. However, histone tails not associated with DNA binding protrude from the nucleosome, and the function of these tails is just being unraveled. The amino acids in these tails are clearly sites for posttranslational modifications, including methylation, acetylation, and phosphorylation. When modified, these tails would provide additional binding sites for protein which could regulate transcription and chromatin modeling, thus modifying the "genetic code". Understanding the "histone code" and how it affects gene transcription becomes important. For example, the methylation of Lys 9 on histone 3 leads to binding of heterochromatin-associated protein, leading to inhibition of gene transcription (an example of epigenetic silencing). Acetylation of the tails generally leads to activation of gene transcription at that site. Acetylation of Lys residues converts them to amides and removes the positive charge of the amine. This would lead to decreased electrostatic interactions between the DNA and histones proteins, making the DNA more available for interaction with transcription factors and RNA polymerase.
Epigenetic changes (through methylation of DNA or acetylation, methylation, and phosphorylation of histone proteins) causing chromatin remodeling may change phenotype (characteristics of the individual) as evidenced by the fact that identical twins can eventually diverge in ways that effect their propensities to disease. Differences in diet and lifestyle, which can alter disease propensity, might exert their effects through epigenetic changes in gene expression. The Human Epigenome Consortium is developing a catalog of methylation pattern differences in the human genome which might be correlated with disease risk.
The nucleosome core is about the same size as RNA polymerase. How can RNA polymerase bind to its promoter site if it is wrapped around a nucleosome? One obvious answer is that nucleosomes are not evenly distributed on chromosomal DNA, and perhaps not even found at promoter sites on the DNA. Rando et al. have studied the distribution of nucleosomes along the yeast genome. They cleaved internucleosomal DNA with nucleases leaving behind the nuclease protected-DNA. They separated the bound DNA from the nucleosome proteins, and labeled it with fluorescein. Next, total yeast DNA was isolated, fragmented, and labeled with rhodamine. They added both fluorescently labeled fragments to microarrays situated with overlapping 50 bp yeast chromosome 3 fragments. Equal red and green fluorescence at a given site on the array would arise if the DNA fragments labeled with fluorescein were protected by the nucleosome protein core particle. Low green to red fluorescence would arise if the fluorescein-labeled DNA was not protected by the nucleosome core.
From a thermodynamic viewpoint, binding affinities for the nucleosome protein core should be the same anywhere along the chromosomal DNA. This would lead to the prediction that nucleosomes would bind randomly along the DNA at all locations, leading to a constant ratio of green to red fluorescence across the array. That is, there would not be district signals from the array, but rather a smeared-out signal when the DNA was extracted from many yeast cells. The actual data showed sharp flourescein/rhodamine signals and was consistent with fact that 70% of the nucleosomes were positioned at the same position in the DNA in different cells. Promoter sites for active genes were generally not occupied by nucleosomes. It was unclear if these sites are always free of nucleosomes or whether protein transcription factors and RNA polymerase cause the nucleosomal core proteins to slide away from the promoter sites.
Recent work suggests that positions of nucleosomes along the DNA is encoded in part by the DNA sequence itself, adding yet another "genetic code" that controls gene expressions. DNA must bend around the nucleosome core. Certain dsDNA sequences are more bendable than others, and they would be expected to have a greater chance of being involved in nucleosome complexes and less accessible for transcription. Segal et al. isolated nucleosome bound DNA sequences and developed a computation model to predict which sequence of DNA would be bendable and hence be able to easily form nucleosome complexes. In other words, they calculated which DNA sequences would have high affinity for nucleosomes. They concluded that 50% of the positioning of nucleosomes can be accounted for by certain DNA sequences having higher affinity of the histone octamer. They found low nucleosome occupancy at important regulatory sites such as transcription initiation sites. Regions of the chromosome coding for tRNA and rRNA, which are highly expressed, were found to have low nucleosome occupancy.
Acetylation of histones is obviously an important method in the control of gene transcription. A recent study by Choudhary et al. investigated the effect and prevalence of lysine acetylation in a range of other cellular pathways. The study discovered over 3600 acetylation sites on 1750 different proteins comprising the acetylome using high resolution mass spectrometery. These regulate a wide variety of dissimilar cellular functions and showed acetylation as a prevalent form of post translational modification falling in terms of its frequency between the phosphoproteome and the spectrum of ubiquitinated proteins. It is a highly conserved process occurring in many different cellular lines from prokaryotes to human beings, and being as prevalent as phosphoproteins found in the evolutionary tree. The authors found that compared to phosphorylation sites in proteins, which are found in more disordered regions, acetylation sites are found in more structured regions with significant secondary structure.
Acetylation eliminates the positive charge on lysine side chains in a reversible process. It has already been established that acetylation of lysine side chains was a key component of DNA damage repair as it modifies histone protein tails found in the DNA. However, recently it has been shown that acetylation’s effects extend to regulation of other cellular functions. Most commonly acetylation plays a role in nearly all nuclear functions, but it also plays a surprisingly big role in cytoplasmic functions. One of the new cellular functions investigated was the involvement of acetylation in regulating macromolecular complexes within the cell pertaining to functions such as signal transduction, DNA damage repair, and the cell cycle. One example protein included in the study is the 14-3-3 protein which binds specifically to phosphoserine or phosphothreonine in phosphorylated peptides. Four different lysines in the protein were mutated to glutamine in an attempt to determine the effect of acetylation on the protein’s binding. Acetylation was found to regulate binding as the enzyme’s activity was severely harmed by mutation. This has been seen in other cellular processes where acetylation has lead to regulation of enzymatic activity. In addition, the study discovered that there is important interaction between phosphorylation and acetylation. This interaction or “cross talk” between acetylation and other post translational modification methods in regulating cellular activity has been observed in the protein p53 as well which plays an important role in repairing damaged DNA. Acetylation data can be found at Phosida.