Eukaryotic cells package their DNA into chromatin (Van Holde, 1988; Wolffe, 1995)--a nucleoprotein complex which facilitates gene regulation, DNA replication and other aspects of general cellular organisation such as chromatid cohesion (Bernard et al., 2001).

Heterochromatin can be distinguished from euchromatin based on its ability be stained with the DNA binding dye DAPI, which stains heterochromatin on metaphase chromosomes and interphase chromatin more strongly than euchromatin (Bickmore and Craig, 1997) . In human cells, heterochromatin (often referred to as C-band positive) is found at centromeres (Choo, 1997) and the long arm of the Y chromosome. In both mouse and human cells

centromeric heterochromatin is associated with satellite repeats. In humans the predominant repeat is a diverged 171bp a-satellite whilst in mouse (Mus musculus) two satellites are found at centromeres; the 120bp minor satellite is centromeric and the major satellite (240bp) is pericentromeric (Figure 1).
<-- Figure 1. Mouse Satellite DNA-containing chromatin

Figure 2. Heterochromatin has a compact -->
regular helical structure

Click figures to enlarge

Factors affecting the structure of heterochromatin

Using a biophysical approach we have recently demonstrated that satellite-containing heterochromatin has a more compact chromatin structure than bulk chromatin (Figure 2; Gilbert and Allan, 2001) . The factors responsible for this altered heterochromatin structure are currently poorly characterised but are probably a combination of factors including the positioning of nucleosomes on the DNA sequence in conjunction with specific chromatin associated proteins. It has been known for many years that the satellite sequences found within heterochromatic regions are able to position nucleosomes in an ordered array which might facilitate the folding of a stable higher-order chromatin fibre and allow it to form further higher-order structures. A small number of putatively structural heterochromatin proteins have been identified including Heterochromatin Protein 1 (HP1; Figure 3; Eissenberg and Elgin, 2000 ). Recent evidence suggests that HP1 binds to the chromatin fibre via a methylated residue at lysine 9 on the histone H3 tail (MetK9-H3; Reviewed in Zhang and Reinberg, 2001 ). It is probable that MetK9-H3 is able to recruit HP1 but it is also known that HP1 associates with the linker histone H1 (Nielsen et al., 2001) and this interaction might play a key role in stabilising the structure of the chromatin fibre at heterochromatic locations. Two antibodies have been generated which detect MetK9-H3. The first antibody raised against a linear methylated-lysine 9 H3 peptide (Upstate Biotech; Peters et al., 2001 ) has a dispersed staining pattern throughout the cell (Figure 4) showing there are a number of sites other than at heterochromatin which are marked by MetK9-H3.

<-- Figure 3. HP1a is localised to heterochromatin

Figure 4. Met-H3 K9 is distributed throughout the nucleus -->

Click figures to enlarge

Figure 5. Met-H3 K9 is present at heterochromatin
Click figure to enlarge

In contrast, an antibody raised against a branched methylated peptide (Peters et al., 2001 ) detects these sites but also strongly stains heterochromatic regions (Figure 5). This data indicates there might be differences in the level of MetK9-H3 at heterochromatin compared to sites within bulk chromatin or that this antibody is able to recognise an altered chromatin fibre conformation. Although MetK9-H3 might act as a primary signal for HP1 localisation co-immuno staining using antibodies against MetK9-H3 and HP1a in mouse cells clearly shows there are a number of HP1a sites which do not stain with MetK9-H3 suggesting there might be other signals responsible for directing HP1 binding (Figure 6).
Figure 6. In bulk chromatin Met-H3 K9 and HP1a are not co-localised -->

Click figure to enlarge

Heterochromatin vs. transcriptionally silent chromatin

The term heterochromatin is often used incorrectly to describe transcriptionally silent chromatin. Repressed genes do have similar protein factors associated with them as heterochromatin but in many cases only a short region of the chromatin fibre is 'closed'. Therefore, in terms of the cell nucleus these regions do not constitute domains of heterochromatin particularly when it is possible to re-open these sequences and express the genes in the loci. In contrast true heterochromatin covers a large region of chromatin which is able to form a repressive domain within the nucleus. The silencing of other genes can be facilitated by translocating them into these silenced domains (often in the centre of chromosome territories) whilst transcriptionally active genes and genes which can be reactivated are found outwith these regions of silencing (often on the surface of chromosome territories).

We are currently working on a component of the HuCHRAC chromatin remodelling complex (ACF) which has been localised to heterochromatin (Poot et al., 2000 ). One idea is that this complex facilitates the regular positioning of nucleosomes within heterochromatin thereby allowing the stable formation of a chromatin structure. Alternatively, heterochromatin might act as a sink for protein complexes which are required for repressing gene transcription such as histone deacetylases and nucleosome remodelling machines.

In our lab, one of our topics of interest is to explore heterochromatin to determine its conformation and the factors responsible for generating its structure and to understand how these might be involved in determining centromere identity (Choo, 2001; Sullivan et al., 2001).

  • Euchromatin and Heterochromatin Information--From the Euchromatin Network

  • EM Atlas: Heterochromatin --University of Mainz, Germany

  • HP1 (Suvar 205) Information-- From the Interactive Fly

  • Elgin Lab-- Heterochromatin Abstracts

  • Thomas Jenuwein Lab

    REFERENCES

    Bernard,P., Maure,J.F., Partridge,J.F., Genier,S., Javerzat,J.P., and Allshire,R.C. (2001) Requirement of heterochromatin for cohesion at centromeres. Science 294, 2539-2542

    Bickmore,W.A. and Craig,J.M. (1997). Chromosome Bands: Patterns in the Genome. (Heidelberg: Springer).

    Choo,K.H. (2001) Domain organization at the centromere and neocentromere. Dev. Cell 1, 165-177

    Choo,K.H.A. (1997). The Centromere. (Oxford: Oxford University Press).

    Eissenberg,J.C. and Elgin,S.C. (2000) The HP1 protein family: getting a grip on chromatin. Curr. Opin. Genet. Dev. 10, 204-210

    Gilbert,N. and Allan,J. (2001) Distinctive higher-order chromatin structure at mammalian centromeres. Proc. Natl. Acad. Sci. U. S. A 98, 11949-11954

    Nielsen,A.L., Oulad-Abdelghani,M., Ortiz,J.A., Remboutsika,E., Chambon,P., and Losson,R. (2001) Heterochromatin formation in mammalian cells: interaction between histones and HP1 proteins. Mol. Cell 7, 729-739

    Peters,A.H., O'Carroll,D., Scherthan,H., Mechtler,K., Sauer,S., Schofer,C., Weipoltshammer,K., Pagani,M., Lachner,M., Kohlmaier,A., Opravil,S., Doyle,M., Sibilia,M., and Jenuwein,T. (2001) Loss of the Suv39h histone methyltransferases impairs mammalian heterochromatin and genome stability. Cell 107, 323-337

    Poot,R.A., Dellaire,G., Hulsmann,B.B., Grimaldi,M.A., Corona,D.F., Becker,P.B., Bickmore,W.A., and Varga-Weisz,P.D. (2000) HuCHRAC, a human ISWI chromatin remodelling complex contains hACF1 and two novel histone-fold proteins. EMBO J. 19, 3377-3387

    Sullivan,B.A., Blower,M.D., and Karpen,G.H. (2001) Determining centromere identity: cyclical stories and forking paths. Nat. Rev. Genet. 2, 584-596.

    Van Holde,K.E. (1988). Chromatin. (New York: Springer Verlag).

    Wolffe,A.P. (1995). Chromatin structure and function. (London: Academic Press).

    Zhang,Y. and Reinberg,D. (2001) Transcription regulation by histone methylation: interplay between different covalent modifications of the core histone tails. Genes Dev. 15, 2343-2360.