Marie-Geneviève Mattei and Judith Luciani
INSERM U 491, Faculté de Médecine, Bd Jean Moulin, 13385 Marseille, France
(Paper co-edited with E.C.A. Newsletter)
I THE CONCEPT OF HETEROCHROMATIN
Definition of Chromatin
In prokaryotes such as Escherichia coli there is no detectable heterochromatin, for the simple reason that there is no chromatin. The hereditary message is carried by a circular molecule of naked DNA, and there is no separate nuclear compartment.
In eukaryotes, however, the DNA is packaged in the form of a nucleoprotein complex called "chromatin". The hereditary message is, therefore, carried by the chromatin. It is located in a nucleus and is organised in several separate entities, the chromosomes.
II TWO TYPES OF HETEROCHROMATIN
There are two types of heterochromatin, constitutive HC and facultative HC, which differ slightly, depending on the DNA that they contain. The richness in satellite DNA determines the permanent or reversible nature of the heterochromatin, its polymorphism and its staining properties (Table I).
Table I: Properties which allow to differentiate constitutive from facultative heterochromatin.
1) Richness in satellite DNA
4) C-band Staining :
III PROPERTIES OF HETEROCHROMATIN
Despite the differences described above, constitutive HC and facultative HC have very similar properties.
1) Heterochromatin is condensedThis is in fact what defines heterochromatin, and it is applicable to both constitutive HC and facultative HC. This high condensation renders it strongly chromophilic, and the intensity of HC staining is directly proportional to its degree of condensation.
2) Heterochromatin DNA is late replicating
The incorporation of various nucleotide analogues shows that the DNA from
both constitutive and facultative HC, is late replicating.
The DNA of the inactive X is very late replicating, as it replicates at the end of the late S phase. The incorporation of 5-Bromodeoxyuridine four hours before harvesting allows this to be clearly seen.
As regards the heterochromatic centromere regions, their replication precedes that of the inactive X and takes place at the beginning of the late S phase. The centromeric DNA must, therefore, replicate sufficiently early to allow the formation, with the centromeric proteins, of a nuclear-protein complex that will be functional during mitosis.
3) Heterochromatin DNA is methylated
The DNA of constitutive HC is highly methylated, with
the methylation occurring exclusively on the cytosines. An anti-5-methyl cytosine
antibody therefore strongly labels all the regions of constitutive
HC, thus showing both its richness in cytosines and their methylation.
As regards facultative HC, the methylation of the DNA is more discrete, and cannot be detected using an antibody directed against methylated cytosines. Nevertheless, restriction enzymes that are sensitive to methylation can be used to reveal strong methylation of the CpG islands, which are specifically located in the control regions of the genes.
Histones may undergo post-translational modifications of their N-terminal ends which may affect the genetic activity of the chromatin.
4) In heterochromatin, histones are hypo-acetylated:
5) Histones from heterochromatin are methylated on lysine 9:This modification of the N-terminal tail of histones has only very recently been discovered and found to be involved in the process of heterochromatinisation of the genome. It characterises both constitutive HC and facultative HC.
6) Heterochromatin is transcriptionally inactive:
7) Heterochromatin does not participate in genetic recombination:It is generally accepted that constitutive HC does not participate in genetic recombination. The reason for this is that there is no preliminary pairing of the homologous heterochromatic regions, even though some aggregation of these regions is often observed. In any case, the polymorphism that characterises the heterochromatic regions would render such pairing difficult, if not impossible. Moreover, this is the reason why the pericentric inversion of the secondary constriction of chromosome 9 has no effect in terms of chromosome mechanics and can be considered as a normal variant.
8) Heterochromatin has a gregarious instinct:
The study of various organisms has shown that constitutive HC has a genuine tendency to aggregate during interphase.
Thus, in the interphase nuclei of the salivar glands of Drosophila larvae, the centromeres of polytene chromosomes, which are rich in heterochromatin, aggregate to form the chromocentres.
In the mouse, the number of heterochromatic blocks that can be observed in interphase nuclei is always lower than the number of heterochromatic regions visualised on the metaphase chromosomes. The relationship between the size and the number of HC blocks in interphase suggests a coalescence of the heterochromatic regions.
In the human, the short arms of the acrocentric chromosomes, which are mainly formed from heterochromatin, carry the nucleolar organiser regions. They are frequently associated in the interphase nucleus, and this does not appear to result solely from their common function. Indeed, numerous other chromosomes are involved in this association, and the participation of each of them is all the more marked because it carries a large HC block, as is the case for chromosomes 1, 9 or 16.
This tendency of the heterochromatin to aggregate appears to be strongly linked to the presence of satellite DNA sequences, but this is a property which may not be exclusive and may involve other additional sequences.
IV FACTEURS INVOLVED in HETEROCHROMATINISATION
There are probably various diverse ways of organising a genomic region into heterochromatin. Certain observations have, however, led to the identification of various elements that have an important role in the formation of heterochromatin, be it constitutive or facultative.
1) Large arrays of tandemly repeated sequences.
The fact that in the human genome, as well as in the genome of other organisms,
the localisation of the satellite DNA visualised by FISH corresponds exactly
to that of the constitutive heterochromatin stained by DAPI, highlights
the potential role of satellite DNA in the formation of this type of heterochromatin.
Indeed, this type of DNA sequence has the distinctive feature of bending and
folding upon itself, and this may be an important factor in determining the
extremely compact structure of the constitutive DNA.
However, this does not only concern satellite DNA. In plants, Drosophila, and also in the mouse, certain multicopy transgenes are barely expressed, or are not expressed at all, even when they are not subject to centromere repression.
These different observations suggest that the tandem repetition of a DNA sequence in a large number of copies is sufficient on its own to direct the formation of heterochromatin. The presence of repetitive DNA, such as satellite DNA, appears to simply allow the chromatin to be compacted to a greater extent, as is the case for constitutive heterochromatin. The mechanism would appear to be as follows: the large arrays of tandemly repeated DNA sequences appear to be able to pair, thus forming characteristic structures. These structures would then appear to be recognised by specific proteins, such as the HP1 proteins, which in turn direct the formation of a higher-order chromatin.
2) Methylation of DNA
Although the presence of tandemly repeated sequences is important, it is
not the only factor, as large repetitions of transgenes do not all lead to
a transcriptional inactivation of the transgene. Most often, the silencing
induced by the tandem repeats appears to be linked to the presence of prokaryotic
DNA sequences that are rich in CpG and, therefore, likely to be methylated
(for example, the lacZ gene). The base composition of the tandem
repeat and, in particular, its ability to be methylated could therefore play
an important role in the formation of heterochromatin.
Interestingly, it has recently been shown that there is a direct relationship between the methylation of DNA and the de-acetylation of histones, both of which characterise heterochromatic structures. The methyl binding protein MeCP2, which normally binds to DNA containing methylated cytosines, has thus been shown to be able to recruit histone de-acetylases (Figure 1). Methylation of the DNA could therefore induce a de-acetylation of histones and thus promote heterochromatisation.
Figure 1: DNA methylation induces Histone de-acetylation, modification which characterizes histones in both heterochromatin and repressed euchromatin.
MeCP2 specifically binds to methylated DNA, and recruits an HDAC which de-acetylates histones (Ac= Acetyl; Me= Methyl; MeCP2= Methyl-CpG binding Protein 2; HDAC= Histone De-Acetylase).
However, the methylation of DNA is not indispensable for the formation of heterochromatin. It could be an element involved in stabilisation, as has been shown for the facultative HC of the inactive X. Indeed, in marsupials, the inactive X is not methylated and is much less stable than in eutherian mammals.
3) Regular organisation of nucleosomesWe have seen that sequences of prokaryotic DNA inserted into eukaryotes have the ability to be methylated and can induce the formation of heterochromatin. Heterochromatisation may not, however, depend solely on the methylation of the CpGs contained in the DNA.
4) Hypo-acetylation of Histones
We have seen that hypo-acetylation of histones is a characteristic of silent chromatin, whether it is heterochromatin or not. In vitro, the modification of the acetylation of the histones has a direct effect on the stability and compaction of the nucleosomes. Thus, blocking the de-acetylation of the histones by adding trichostatine A induces hyper-acetylation of the histones, which causes a more open chromatin structure.
The mechanism involved is simple: in the N-terminal tail of the histones, the basic amino-acids such as lysine are positively-charged at cellular pH and therefore interact with the negative charges of the DNA phosphates.
5) Methylation of H3-K9
Methylation of the histone H3 on lysine 9 is an epigenetic modification of histones that has recently been shown to be involved in the process of heterochromatinisation. This has been demonstrated not only in constitutive HC but also on the inactive X. The enzyme responsible for the methylation of H3-K9 in constitutive HC is the histone methyltransferase SUV39H1.
Figure 2: Histone H3-K9 methylation induces DNA methylation, modification which characterizes DNA in heterochromatin and repressive euchromatin.
SUVAR39H is a methyltransferase which specifically methylates the Lysine 9 of histone H3. Such a methylation creates a binding site for the Heterochromatin Protein HP1 which recruits a DNA methyl transferase, capable to methylate the CpG in DNA (Me= Methyl; Methyl H3-K9= Methyl on Lysin 9 of Histone H3; HP1=Heterochromatin Protein 1; DNMT=DNA Methyl transferase).
6) HP1 proteins
Many different heterochromatin proteins are to be found in mammals, and at the present time little is known about them. The HP1 proteins do appear, however, to have a particular role in the organisation of heterochromatin. Studies of the variegation by position effect (PEV effect) in Drosophila and studies of transgenes in Drosophila and mouse have allowed a better understanding of the role of HP1 proteins.
7) Nuclear RNAs
It is already well established that certain nuclear RNAs are able to contribute to the formation of facultative HC. The transcripts of the XIST (X inactive Specific Transcripts) gene are nuclear RNAs that have an essential role in the facultative inactivation of one X chromosome, which occurs in the somatic cells in female mammals. This XIST RNA is necessary for the initiation of X inactivation, but not its maintenance. Other nuclear RNAs, such as H19, have been shown to be involved in the regulation of genes that are subject to genomic imprinting.
Some recent studies in mouse have suggested that nuclear transcripts may also be involved in the formation of constitutive HC. In mouse, as in most other species, the centromeric HC is particularly stable. It is characterised by the presence of a high concentration of methylated H3-K9 histone and heterochromatic HP1 proteins, which co-localise in the nuclei with regions strongly stained with DAPI. However, incubation of permeabilised mouse cells with RNAse A causes rapid de-localisation of the HP1 and methylated H3-K9 signals, in relation to the heterochromatin foci. These data suggest that a nuclear RNA may be an essential structural component of constitutive HC. The RNA may either facilitate compaction of the centromeric HC or may serve as an additional binding site for the proteins that associate with the chromatin. Surprisingly, the treatment with RNAse A does not alter the methylated H3-K9 signals at the level of the Barr body. In fact, on the inactive X chromosome, the methylation of H3-K9 does not appear to lead to the binding of HP1 proteins. This suggests that the facultative heterochromatinisation of the inactive X may require a different mechanism to come into play from that of constitutive HC.
V FUNCTIONS OF HETEROCHROMATIN
The precise role of heterochromatin in the human genome long remained a mystery, as its frequent polymorphisms did not appear to have any functional or phenotypic effect.
1) Role of HC in the organisation of nuclear domains
2) Role of HC in the centromeric function
In most eukaryotes, the centromeres are loaded with a considerable mass of heterochromatin. It has been suggested that centromeric HC is necessary for the cohesion of sister chromatids and that it allows the normal disjunction of mitotic chromosomes.
3) Does centromeric HC act as a transporter?Many proteins have been shown to be associated with centromeric HC, in particular on metaphase chromosomes. It can be assumed that certain proteins that must be present and functional at the very beginning of the G1 phase will not be able to be synthesised due to a lack of time. Following this hypothesis, binding to the centromere would be an ideal means for such proteins to traverse cell division unhindered, in order to be available at the beginning of G1.
4) Role of HC in gene repression (epigenetic regulation)
VI HETEROCHROMATIN DISEASES
1) Diseases of the constitutive heterochromatin
These diseases are generally the result of an alteration in the process of cell differentiation.
2) Diseases of the facultative heterochromatin
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|Written||2003-01||Marie-Genevièvee Mattei, Judith Luciani|
|U 491, Faculté de Médecine, Bd Jean Moulin, 13385 Marseille, France|
|This paper should be referenced as such :|
|Mattei, MG ; Luciani, J|
|Heterochromatin, from chromosome to protein|
|Atlas Genet Cytogenet Oncol Haematol. 2003;7(2):135-143.|
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|On line version : http://AtlasGeneticsOncology.org/Deep/HeterochromatineDEEPID20044.htm|
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