Definition of Chromatin
In eukaryotes, on the contrary of prokaryotes, the DNA is packaged in the form of a nucleoprotein complex called "chromatin", which carries the hereditary message. It is located in a nucleus and is organised in several separate entities, the chromosomes.
The Concept of Heterochromatin
In 1928, based on histological observations, Emil HEITZ defined heterochromatin (HC) as being the chromosomal segments which appear extremely condensed and dark in colour in the interphase nucleus. In fact, chromatin consists of a tangle of fibres, the diameter of which not only vary during the cell cycle, but also depend on the region of the chromosome observed.
The active euchromatin consists of a fibre with a diameter corresponding to that of a nucleosome, a double strand DNA segment, wound around homodimers of the histones H2A, H2B, H3, and H4 . In inactive euchromatin, this fibre can wind itself into a solenoid thanks to histones H1. It is further organised through interactions with non-histone proteins (topoisomerase II, scaffold protein 2, lamins...). As regards the heterochromatin, as defined above, its constituent fibre is more condensed and often appears to be composed of aggregates. It involves numerous additional proteins, including the HP1 proteins (Heterochromatin Protein 1).
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: Properties which allow to differentiate constitutive from facultative heterochromatin
II.1 Constitutive heterochromatin
II.2 Facultative heterochromatin
Despite the differences described above, constitutive HC and facultative HC have very similar properties.
III.1 Heterochromatin is condensed
This 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 inaccessible to DNAse 1 and to other restriction enzymes in general.
III.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. HC late replication results, on the one hand, from its high degree of condensation, which prevents the replicating machinery from easily accessing the DNA, and, on the other hand, from its location in a peripheral nuclear domain that is poor in active elements.
III.3 Heterochromatin DNA is methylated
III.4 In heterochromatin, histones are hypo-acetylated
Histones may undergo post-translational modifications of their N-terminal ends which may affect the genetic activity of the chromatin.
III.5 Histones from heterochromatin are methylated on lysine 9
Methylation of the histone H3 lysine 9 (H3-K9) has only very recently been found to be involved in the process of heterochromatinisation of the genome, both in constitutive and facultative HC.
III.6 Heterochromatin is transcriptionally inactive
III.7 Heterochromatin does not participate in genetic recombination
III.8 Heterochromatin has a gregarious instinct
The study of various organisms has shown that constitutive HC has a genuine tendency to aggregate during interphase.
This tendency of the heterochromatin to aggregate appears to be strongly linked to the presence of satellite DNA sequences, but it may also involve other additional sequences.
Certain observations have led to the identification of various elements that have an important role in the formation of heterochromatin, be it constitutive or facultative.
IV.1 Large arrays of tandemly repeated sequences.
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. Such repeated sequences could allow the chromatin to be compacted to a greater extent, by forming characteristic structures. These structures could be recognised by specific proteins, such as the HP1 proteins, which in turn direct the formation of a higher-order chromatin.
IV.2 Methylation of DNA
Large repetitions of transgenes do not all lead to a transcriptional inactivation of the transgene. The silencing induced by tandem repeats appears to be linked to the presence of prokaryotic DNA sequences, rich in CpG, likely to be methylated. Then the base composition of the tandem repeats could therefore play an important role in the formation of heterochromatin.
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).
IV.3 Hypo-acetylation of Histones
We have seen that hypo-acetylation of histones is a characteristic of silent chromatin, whether it is heterochromatin or not. 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
IV.4 Methylation of H3-K9
Methylation of the histone H3 on lysine 9 is an epigenetic modification that has recently been shown to be involved in the process of heterochromatinisation, not only in constitutive HC but also on the inactive X. The enzyme responsible for this methylation is the histone methyltransferase SUV39H1.
Figure 2: Histone H3-K9 methylation induces DNA methylation, modification which characterizes DNA in heterochromatin or 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 Lysine 9 of Histone H3; HP1=Heterochromatin Protein 1; DNMT=DNA Methyl transferase).
IV.5 HP1 proteins
The HP1 proteins do appear 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 these proteins.
It is interesting to note that even where a transgene is repressed, not as a result of a centromeric effect but as a result of its presence in multiple copies, HP1 proteins are also found to be associated with the repressed chromatin.
HP1 proteins appear to be an essential link in the formation of heterochromatin, and could have the role of chromatin domain organisers. These proteins appear to be able to recognise particular structures that are created by the pairing and/or the association of repeated DNA sequences. In addition, thanks to the chromodomain (CD) and the chromoshadow domain (CSD), they are able to establish secondary interactions with a large number of other proteins.
IV.6 Nuclear RNAs
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.
V.1 Role of HC in the organisation of nuclear domains
V.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.
It is supposed that centromeric HC might, de facto, create a compartment by increasing the local concentration of the centromeric histone variant, CENP-A, and by promoting the incorporation of CENP-A rather than the histone H3 during replication.
V.3 Role of HC in gene repression (epigenetic regulation)
Gene expression may be controlled at two levels:
Mechanism of inactivation in cis :
Following a chromosomal rearrangement, a euchromatic region may be juxtaposed with a heterochromatic region. Where the rearrangement removes certain normal barriers that protect the euchromatin, the heterochromatic structure is able to propagate in cis to the adjacent euchromatin, thus inactivating the genes contained therein. This mechanism has been observed in position effect variegation (PEV) in Drosophila and also in the inactivation of certain transgenes in mouse.
Mechanism of inactivation
During cell differentiation, certain active genes are likely to be transposed into a heterochromatic nuclear domain, thus causing them to become inactive. Such a mechanism has been proposed as an explanation for the co-localisation in lymphocyte nuclei of the protein IKAROS and the genes of which it controls the expression, with centromeric heterochromatin.
VI.1 Diseases of the constitutive heterochromatin
These diseases are generally the result of an alteration in the process of cell differentiation.
VI.2 Diseases of the facultative heterochromatin
In conclusion, although heterochromatin is apparently amorphous and isolated at the periphery of the nucleus, it appears to have an absolutely essential role in the organisation and function of the genome.
Throughout this review we have mainly presented the characteristics linked with heterochromatin, be it constitutive or facultative. We have shown that the properties of constitutive HC are not fundamentally different from those of facultative HC. It therefore seems clear that the mechanisms involved in facultative heterochromatinisation, which are epigenetic mechanisms, are the same mechanisms that intervene in the repression of euchromatin in general.
Contributors : Marie-Geneviève Mattei, Judith Luciani *
*Judith Luciani was supported by a grant from the Fondation Electricité de France.
|Written||01-2003||Marie-Geneviève Mattei, Judith Luciani|
|This paper should be referenced as such :|
|Mattei MG, Luciani J . Heterochromatin, from Chromosome to Prote. Atlas Genet Cytogenet Oncol Haematol. January 2003 .|
|URL : http://AtlasGeneticsOncology.org/Educ/HeterochromEng.html|
The various updated versions of this paper are referenced and archived by INIST as such :
|© Atlas of Genetics and Cytogenetics in Oncology and Haematology||indexed on : Mon Dec 2 18:01:58 CET 2013|
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