barrier DNA Sequences block the Spread of Reader–writer Complexes and thereby Separate Neighboring Chromatin Domains
The above mechanism for spreading chromatin structures raises a potential problem. Inasmuch as each chromosome contains one continuous, very long DNA molecule, what prevents a cacophony of confusing cross-talk between adjacent chromatin domains of different structure and function? Early studies of position effect variegation had suggested an answer: certain DNA sequences mark the boundaries of chromatin domains and separate one such domain from another (see Figure 4–31). Several such barrier sequences have now been identified and characterized through the use of genetic engineering techniques that allow specific DNA segments to be deleted from, or inserted in, chromosomes.
For example, in cells that are destined to give rise to red blood cells, a sequence called HS4 normally separates the active chromatin domain that contains the human β-globin locus from an adjacent region of silenced, condensed chromatin. If this sequence is deleted, the β-globin locus is invaded by condensed chromatin. This chromatin silences the genes it covers, and it spreads to a different extent in different cells, causing position effect variegation similar to that observed in Drosophila. As described in Chapter 7, the consequences are dire: the globin genes are poorly expressed, and individuals who carry such a deletion have a severe form of anemia.
In genetic engineering experiments, the HS4 sequence is often added to both ends of a gene that is to be inserted into a mammalian genome, in order to protect that gene from the silencing caused by spreading heterochromatin. Analysis of this barrier sequence reveals that it contains a cluster of binding sites for histone acetylase enzymes. Since the acetylation of a lysine side chain is incompatible with the methylation of the same side chain, and specific lysine methylations are required to spread heterochromatin, histone acetylases are logical candidates for the formation of DNA barriers to spreading (Figure 4–41). However, several other types of chromatin modifications are known that can also protect genes from silencing.
Figure 4–41 Some mechanisms of barrier action. These models are derived from experimental analyses of barrier action, and a combination of several of them may function at any one site.
The tethering of a region of chromatin to a large fixed site, such as the nuclear pore complex illustrated here, can form a barrier that stops the spread of heterochromatin.
The tight binding of barrier proteins to a group of nucleosomes can make this chromatin resistant to heterochromatin spreading. (C) by recruiting a group of highly active histone-modifying enzymes, barriers can erase the histone marks that are required for heterochromatin to spread. For example, a potent acetylation of lysine 9 on histone H3 will compete with lysine 9 methylation, thereby preventing the binding of the HP1 protein needed to form a major form of heterochromatin. (based on
A.g. west and P. Fraser, Hum. Mol. Genet. 14:R101–R111, 2005. with permission from Oxford University Press.)
The Chromatin in Centromeres Reveals How Histone variants Can Create Special Structures