Chromatin for Beginners

What is Chromatin?

Chromatin is the stuff chromosomes are made of. Microscopists used the term long before the discovery that DNA is the genetic material. Today, the word "chromatin" is mostly used by molecular geneticists to describe DNA associated with the numerous proteins that help organize, activate or repress DNA.

Chromatin and chromosome structure

Stuffing the long strands of chromosomal DNA into a eukaryotic nucleus requires that the DNA be compacted in length approximately 10,000 to 50,000 -fold. Incredibly, cells achieve this tight packing of the DNA while still maintaining the chromosomes in a form that allows regulatory proteins to gain access to the DNA to turn on (or off) specific genes, to repair DNA damage or to duplicate the chromosomes (replication). This engineering feat is accomplished by a variety of chromatin proteins, the most abundant of which are the histones. Histones associate with DNA to accomplish the first step in chromatin assembly, forming protein-DNA structures known as nucleosomes. When viewed under the electron microscope, nucleosomes are beads of ~10 nm in diameter that are distributed along a ~2 nm string of DNA approximately once every 200 basepairs (bp) (Kornberg, 1974; Olins and Olins, 1974). Each bead on the string is a nucleosome core particle that includes ~146 bp of DNA wrapped not-quite-twice around an octamer of core histones composed of 2 molecules each of Histones H2A, H2B, H3 and H4 (Luger et al. 1997). Nucleosome formation compacts the DNA approximately six-fold in its linear dimension. Histone H1 or a related "linker" histone binds to the 40-70 bp of linker DNA that separates adjacent core particles and helps compact the beads-on-a-string into higher order structures. For metaphase chromosomes, fibers of ~30 nm in diameter are apparent (Finch and Klug, 1976; Thoma et al., 1979) and appear to be helical structures with approximately six nucleosomes per turn, an arrangement in which the DNA has been compacted ~40-fold in its linear dimension. However, it is not clear that 30 nm fibers are ever formed in interphase nuclei, with modern high resolution imaging techniques failing to detect interphase chromatin fibers thicker than 10 nm even within the most highly compacted chromatin (Fussner et al. 2010).

Levels of chromosome organization beyond the 10-nm beads-on-a-string filaments are poorly understood in interphase nuclei, as discussed above, but the stunning electron micrographs HeLa cell metaphase chromosomes made by Laemmli and his colleagues in the late 1970s have provided a basis for current models of metaphase chromatin organization (Marsden and Laemmli, 1979; Paulson and Laemmli, 1977). Their images of chromosomes stripped of histones show DNA spooling out in 30 to 90-kb loops from a proteinaceous "scaffold" that still retains the shape of the paired sister chromatids (Paulson and Laemmli, 1977). The loops appear to emanate from, and return to, the same point, suggesting that the DNA is tethered to the scaffold at the base of the loops. Methods that do not remove the histones from the DNA reveal loops of chromatin made up of 180 to 300 nucleosomes coiled in 30-nm fibers (Marsden and Laemmli, 1979). Organized in this way, each loop would account for ~700-fold packing of the DNA relative to the long axis of the chromosome. In cross section, the loops appear to radiate from the scaffold as if tracing the outline of the petals on a daisy flower. Adjacent loop attachment sites are thought to be arranged in a helical spiral along the long axis of the metaphase scaffold (Marsden and Laemmli, 1979). Organizing 15 to 18 such loops per turn along the chromatid would account for ~1.2 million bp of DNA (Nelson et al., 1986). This arrangement predicts the stacking of loops into a cylinder of chromatin ~800 to 1000 nm in thickness, which is in good agreement with the diameter of the metaphase chromosome (Marsden and Laemmli, 1979; Nelson et al., 1986). This model also accounts for the dimensions of metaphase chromosomes, which are ~10, 000-fold shorter and 400 to 500-fold thicker than the double stranded DNA helices contained within them. Twisting the cylinder into a superhelix would further compress it in the linear dimension and account for the corkscrew appearance of metaphase chromosomes when viewed at high magnification.

Chromatin modifications and gene regulation

Core histones can be reversibly modified by post-translational modifications that include acetylation, methylation, phosphorylation, ubiquitination or ADP-ribosylation and specific modifications are clearly interconnected with processers such as gene activation, gene repression, DNA repair or chromosome replication (Wang et al. 2004; Berger 2007; Liu et al. 2010). For instance, lysines at the amino-terminal ends of the core histones are hotspots of reversible modifications that include acetylation, deacetylation, methylation and demethylation, and the modifications occur in patterns that correlate with gene activity states. Active genes are preferentially associated with histones whose amino terminal lysines tend to be highly acetylated. By contrast, inactive genes tend to be associated with histones that are hypoacetylated. Histone modifications can influence gene expression by altering the accessibility of nucleosomal DNA to regulatory proteins, such as activators or repressors, while serving as direct binding sites for other regulatory proteins. For instance, proteins bearing chromodomains, chromo barrel domains, PHD domains or MBT domains bind histones H3 or H4 that are methylated on specific lysines; proteins with Tudor domains bind histones dimethylated on arginines; proteins with bromodomains interact with histones with acetylated lysines; and 14-3-3 proteins bind histones with specific phosphorylated amino acids (Taverna et al. 2007). The identification of more than 120 histone modifications with potential regulatory significance has led to a "histone code" hypothesis by which different combinations of modifications serve as signals, or part of a  "language" that influences the activity state of the chromatin (Jenuwein and Allis, 2001; Strahl and Allis, 2000; Turner, 2000) (Berger 2007; Lee et al. 2010). Understanding the degree to which histone modifications cause, or reflect, gene expression states is an area of active investigation.

In addition to modifying the histones that wrap the DNA into nucleosomes and higher-order structures, the DNA itself can be modified, most notably by the addition of methyl groups to cytosines (Klose and Bird 2006; Suzuki and Bird 2008; Law and Jacobsen 2010). A high level of promoter methylation is typically correlated with gene silencing, and is particularly evident in the silencing of transposable elements and multi-copy transgenes. A variety of DNA methyltransferases exist to modify the DNA in different patterns. Some DNA methyltransferases act primarily in conjunction with replication to perpetuate methylation patterns from "mother" strands to newly synthesized "daughter" strands of a chromosome. This is known as maintenance methylation. Other DNA methyltransferases can add methyl groups to DNA strands that have no pre-existing methylation, a process known as de novo methylation. In plants, de novo DNA methylation is guided by a class of 24 nt short interfering RNAs (siRNAs) that associate with specific Argonaute family proteins (Zaratiegui et al. 2007; Matzke et al. 2009; Law and Jacobsen 2010). 

Methylation of DNA may silence genes by preventing the binding of transcription factors. However, it is more likely that cytosine methylation exerts its negative effects on gene regulation via the involvement of other proteins that bind specifically to DNA when it is methylated. Indeed, a number of methylcytosine binding proteins have been identified, often in association with other chromatin modifiers. Like histone modifications, DNA methylation is reversible. In plants, several DNA glycosylases are involved in cytosine demethylation, nicking the DNA to initiate a repair process in which methylated cytosines are replaced by unmethylated cytosines (Zhu 2009).

Epigenetic phenomena, chromatin modifications and epigenomics

Broadly defined, epigenetic phenomena are heritable (or propagated), alternative states of gene expression, molecular function, or organization specified by the same genetic instructions (the primary DNA sequence). Examples include unpredictable "on" or "off" expression patterns of wild-type (non-mutant) genes, alternative states of protein folding that can be propagated from one molecule to the next (e.g. prions of neurodegenerative disorders), or alternative, self-perpetuating developmental patterns (e.g. cilia orientation in Paramecium).  Chromatin modifications are key aspects of epigenetic phenomena that involve transcriptional gene silencing, including cytosine methylation, repressive histone modifications, or both (Feng et al. 2010)(Bonasio et al. 2010). The field of epigenomics involves genome-wide analyses of chromatin modification states and endeavors to understand the underlying causes of these modifications (Schones and Zhao 2008)(Fingerman et al. 2011).

Some important chromatin modifying activities:

DNA methyltransferases. These are the enzymes that methylate DNA in various patterns.

Methylcytosine binding proteins. These proteins bind to methylated DNA to mediate other chromatin modifying events.

DNA demethylases. These are glycosylases that nick the DNA, leading to repair processes that replace methylated cytosines with unmethylated cytosines.

Histone acetyltransferases. These enzymes add acetyl groups to histones.

Histone deacetylases. These enzymes remove acetyl groups from histones.

Histone methyltransferases. These enzymes, which contain SET domains, can add as many as three methyl groups to lysines or up to two methyl groups to arginines.

Histone demethylases. These enzymes remove methyl groups from histones.

Chromatin remodeling activities. These large multi-protein complexes use energy derived from the hydrolysis of ATP to alter the positioning of nucleosomes on DNA, or displace histones from DNA.

A huge body of literature is devoted to exploring the role of chromatin modifications in epigenetic regulation. I hope that this brief primer will serve as a useful starting point for delving into the literature.

Prof. Craig S. Pikaard

Department of Biology and Department of Molecular and Cellular Biochemistry

Indiana University,

Bloomington, Indiana 47405





Berger, S.L. 2007. The complex language of chromatin regulation during transcription. Nature 447: 407-412.

Bonasio, R., Tu, S., and Reinberg, D. 2010. Molecular signals of epigenetic states. Science 330: 612-616.

Feng, S., Jacobsen, S.E., and Reik, W. 2010. Epigenetic reprogramming in plant and animal development. Science 330: 622-627.

Finch, J. T., and Klug, A. (1976). Solenoid model for superstructure in chromatin, Proc Natl Acad Sci USA 73, 1897-1901.

Fingerman, I.M., McDaniel, L., Zhang, X., Ratzat, W., Hassan, T., Jiang, Z., Cohen, R.F., and Schuler, G.D. 2011. NCBI Epigenomics: a new public resource for exploring epigenomic data sets. Nucleic Acids Res 39: D908-912.

Fussner, E., Ching, R.W., and Bazett-Jones, D.P. 2010. Living without 30nm chromatin fibers. Trends Biochem Sci.

Jenuwein, T., and Allis, C. D. (2001). Translating the histone code, Science 293, 1074-80.

Klose, R.J. and Bird, A.P. 2006. Genomic DNA methylation: the mark and its mediators. Trends Biochem Sci 31: 89-97.

Kornberg, R. (1974). Chromatin structure: a repeating unit of histones and DNA, Science 184, 868-871.

Law, J.A. and Jacobsen, S.E. 2010. Establishing, maintaining and modifying DNA methylation patterns in plants and animals. Nat Rev Genet 11: 204-220.

Lee, J.S., Smith, E., and Shilatifard, A. 2010. The language of histone crosstalk. Cell 142: 682-685.

Liu, C., Lu, F., Cui, X., and Cao, X. 2010. Histone methylation in higher plants. Annu Rev Plant Biol 61: 395-420.

Marsden, M. P., and Laemmli, U. K. (1979). Metaphase chromosome structure: evidence for a radial loop model, Cell 17, 849-858.

Matzke, M., Kanno, T., Daxinger, L., Huettel, B., and Matzke, A.J. 2009. RNA-mediated chromatin-based silencing in plants. Curr Opin Cell Biol 21: 367-376.

Nelson, W. G., Pienta, K. J., Barrack, E. R., and Coffey, D. S. (1986). The role of the nuclear matrix in the organization and function of DNA, Ann Rev Biophys Chem 15, 457-475.

Olins, A. L., and Olins, D. E. (1974). Spheroid chromatin units (u -bodies), Science 183, 330-332.

Paulson, J. R., and Laemmli, U. K. (1977). The structure of histone-depleted metaphase chromosomes, Cell 12, 817-828.

Schones, D.E. and Zhao, K. 2008. Genome-wide approaches to studying chromatin modifications. Nat Rev Genet 9: 179-191.

Strahl, B. D., and Allis, C. D. (2000). The language of covalent histone modifications, Nature 403, 41-5.

Suzuki, M.M. and Bird, A. 2008. DNA methylation landscapes: provocative insights from epigenomics. Nat Rev Genet 9: 465-476.

Taverna, S.D., Li, H., Ruthenburg, A.J., Allis, C.D., and Patel, D.J. 2007. How chromatin-binding modules interpret histone modifications: lessons from professional pocket pickers. Nat Struct Mol Biol 14: 1025-1040.

Thoma, F., Koller, T., and Klug, A. (1979). Involvement of H1 in the organization of the nucleosome and of salt-dependent superstructures of chromatin, J Cell Biol 83, 403-427.

Turner, B. M. (2000). Histone acetylation and an epigenetic code, Bioessays 22, 836-45.

Wang, Y., Wysocka, J., Perlin, J.R., Leonelli, L., Allis, C.D., and Coonrod, S.A. 2004. Linking covalent histone modifications to epigenetics: the rigidity and plasticity of the marks. Cold Spring Harb Symp Quant Biol 69: 161-169.

Zaratiegui, M., Irvine, D.V., and Martienssen, R.A. 2007. Noncoding RNAs and gene silencing. Cell 128: 763-776.

Zhu, J.K. 2009. Active DNA demethylation mediated by DNA glycosylases. Annu Rev Genet 43: 143-166.

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