Chromatin is the stuff chromosomes are made of. Microscopists used the term long before the discovery was made that DNA is the genetic material. Today, the word "chromatin" is mostly used by molecular geneticists to describe DNA associated with any of 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 or to duplicate the chromosomal DNA (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 the ~2 nm DNA string (Kornberg, 1974; Olins and Olins, 1974) about once every 200 bp. Each bead is a nucleosome core particle (install the plug-in to view this link) that includes ~146 bp of DNA wrapped almost twice around a core histone octamer (2 molecules each of Histones H2A, H2B, H3 and H4). 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 fibers ~30 nm in diameter (Finch and Klug, 1976; Thoma et al., 1979). Viewed under the electron microscope, these 30-nm fibers 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.

Levels of chromosome organization beyond the 30-nm chromatin filaments are less well understood, but stunning electron micrographs made by Laemmli and his colleagues in the late 1970s (Marsden and Laemmli, 1979; Paulson and Laemmli, 1977) provide the basis for current models. Their images of HeLa cell metaphase chromosomes stripped of histones show DNA spooling out in 30 to 90-kb loops from a proteinaceous "scaffold" that still retains the X 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 viewed at high magnification

Chromatin modifications and gene regulation

Core histones can be reversibly modified by acetylation, methylation, phosphorylation, ubiquitination or ADP-ribosylation (Strahl and Allis, 2000) and these modifications have consequences for gene activation, gene repression and chromosome replication (Ng and Bird, 2000; Grunstein, 1997; Struhl et al., 1998). Lysines at the amino-terminal ends of the core histones are the predominant sites of known regulatory modifications. Active genes are preferentially associated with highly acetylated histones whereas inactive genes are associated with hypoacetylated histones. Histone acetylation and deacetylation are thought to exert their regulatory effects on gene expression by altering the accessibility of nucleosomal DNA to DNA-binding transcription activators, other chromatin modifying enzymes or multi-subunit chromatin remodeling complexes capable of displacing nucleosomes (Guschin et al., 2000) (Fuks et al., 2000)

An example of the interconnections among histone modifications is the finding that deacetylation of lysine K9, located nine amino acids from the amino terminus of histone H3, is a prerequisite for methylation of this same lysine. Methylation of K9, in turn, recruits the binding of repressor proteins, such as HP1 (Heterochromatin Protein 1) that help establish highly compacted and transcriptionally inactive regions of chromatin known as heterochromatin (Rice and Allis, 2001). The inter-connectedness of histone modifications that collectively influence a web of regulatory events has led to the hypothesis for a "histone code" controlling chromatin dynamics (Jenuwein and Allis, 2001; Strahl and Allis, 2000; Turner, 2000). Such a code would allow post-translational modifications of various amino acids within the core histones to carry informational content and instructions that help specify which genes are to be activated or repressed during development.

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 (Richards, 1997). High levels of methylation is typically correlated with gene silencing, and is particularly evident in the silencing of transposable elements and multi-copy transgenes (Kass et al., 1997; Martienssen and Colot, 2001). A variety of DNA methyltransferases exist to modify the DNA in a variety of 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. Other DNA methyltransferases can add methyl groups to DNA strands that have no pre-existing methylation. Methylation of DNA may silence genes by preventing the binding of transcription factors. However, it is likely that cytosine methylation exerts most 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 and several are found in close association with one or more histone deacetylases. These findings suggest models whereby cytosine methylation brings about local histone deacetylation, which could facilitate methylation of one or more deacetylated lysines on the histones and subsequent recruitment of repressor proteins that prevent transcription factors from gaining access to affected genes (Ng and Bird, 1999).

Epigenetic phenomenon and chromatin modifications

The laboratories collaborating to carry out the NSF-funded functional genomics project described at this website share a common fascination with epigenetic phenomena. 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" of "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). Our consortium is specifically interested in epigenetic phenomena related to gene silencing and genetic transformation in plants. Chromatin modifications including cytosine methylation and histone modifications are known to be involved in many such epigenetic phenomena (Habu et al., 2001). This has inspired us to team up to create a community resource of transgenic Arabidopsis and maize plants representing insertional and dominant knock-outs of major classes of chromatin modifying proteins. A variety of epigenetic assays can be conducted using these plants.

Some major protein families targeted in our project include:

DNA methyltransferases (METs,CMTs,DRMs)
    These are the enzymes that methylate DNA in various patterns.

Methylcytosine Binding Domain Proteins (MBDs)
    These proteins are thought to bind to methylated DNA to mediate other chromatin modifying events.

Histone acetyltransferases (HACs)
    These enzymes add acetyl groups to histones.

Histone deacetylases (HDAs)
    These enzymes remove acetyl groups from histones.

Chromatin remodeling activities (CHR, CHB, CHC etc)
    These large multi-protein complexes use energy derived from the hydrolysis of ATP to alter the positioning of nucleosomes on DNA.

SET Domain containing proteins (SDGs)
    SET domains are common within proteins that methylate histones.

Chromodomain containing proteins
    Chromodomains are found in histone-binding repressor proteins such as Heterochromatin Protein 1.

Bromodomain containing proteins
    Bromodomains are found in proteins that bind acetylated lysines.

High Mobility Group (HMG) Proteins
    HMG proteins are abundant non-histone chromosomal proteins that bind and bend DNA and sereve "architectural" roles.

Please explore this website for detailed information concerning plant chromatin protein genes and vectors designed to disrupt their functions. The website is a work-in-progress and new data are being added on a regular basis. We hope this will be a useful resource for the scientific community.

References

Aalfs, J. D., and Kingston, R. E. (2000). What does 'chromatin remodeling' mean?, Trends Biochem Sci 25, 548-55.

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

Fuks, F., Burgers, W. A., Brehm, A., Hughes-Davies, L., and Kouzarides, T. (2000). DNA methyltransferase Dnmt1 associates with histone deacetylase activity, Nat Genet 24, 88-91.

Grant, P. A., and Berger, S. L. (1999). Histone acetyltransferase complexes, Semin Cell Dev Biol 10, 169-77.

Guschin, D., Wade, P. A., Kikyo, N., and Wolffe, A. P. (2000). ATP-Dependent histone octamer mobilization and histone deacetylation mediated by the Mi-2 chromatin remodeling complex, Biochemistry 39, 5238-45.

Habu, Y., Kakutani, T., and Paszkowski, J. (2001). Epigenetic developmental mechanisms in plants: molecules and targets of plant epigenetic regulation, Curr Opin Genet Dev 11, 215-20.

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

Kass, S. U., Pruss, D., and Wolffe, A. P. (1997). How does DNA methylation repress transcription?, Trends Genet 13, 444-9.

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

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

Martienssen, R. A., and Colot, V. (2001). DNA methylation and epigenetic inheritance in plants and filamentous fungi, Science 293, 1070-4.

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 Biophys Chem 15, 457-475.

Ng, H. H., and Bird, A. (1999). DNA methylation and chromatin modification, Curr Opin Genet Dev 9, 158-63.

Ng, H. H., and Bird, A. (2000). Histone deacetylases: silencers for hire, Trends Biochem Sci 25, 121-6.

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.

Rice, J. C., and Allis, C. D. (2001). Histone methylation versus histone acetylation: new insights into epigenetic regulation, Curr Opin Cell Biol 13, 263-73.

Richards, E. J. (1997). DNA methylation and plant development, Trends Genet 13, 319-23.

Strahl, B. D., and Allis, C. D. (2000). The language of covalent histone modifications, Nature 403, 41-5. Struhl, K. (1998). Histone acetylation and transcriptional regulatory mechanisms, Genes Dev 12, 599-606.

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.