4. Silent cassettes at HML and HMR are repressed

17.4 Silent cassettes at HML and HMR are repressed




Figure 17.6 Silent cassettes have the same sequences as the corresponding active cassettes, except for the absence of the extreme flanking sequences in HMRa. Only the Y region changes between a and a types.

The transcription map in Figure 17.6 reveals an intriguing feature. Transcription of either MATa or MATα initiates within the Y region. Only the MAT locus is expressed; yet the same Y region is present in the corresponding nontranscribed cassette (HML or HMR). This implies that regulation of expression is not accomplished by direct recognition of some site overlapping with the promoter. A site outside the cassettes must distinguish HML and HMR from MAT.


Deletion analysis shows that sites on either side of both HML and HMR are needed to repress their expression. They are called silencers. The sites on the left of each cassette are called the E silencers, and the sites on the right side are called the I silencers. These control sites can function at a distance (up to 2.5 kb away from a promoter) and in either orientation. They behave like negative enhancers (enhancers are elements that activate transcription, and are discussed in 20 Initiation of transcription; for review see Laurenson and Rine, 1992).


Can we find the basis for the control of cassette activity by identifying genes that are responsible for keeping the cassettes silent? We would expect the products of these genes to act on the silencers. A convenient assay for mutation in such genes is provided by the fact that, when a mutation allows the usually silent cassettes at HML and HMR to be expressed, both a and α functions are produced, so the cells behave like MATa/MATα diploids.


Mutations in several loci abolish silencing and lead to expression of HML and HMR. The first to be discovered were the four SIR loci (silent information regulators). All four wild-type SIR loci are needed to maintain HML and HMR in the repressed state; mutation in any one of these loci to give a sir V allele has two effects. Both HML and HMR can be transcribed. And both the silent cassettes become targets for replacement by switching. So the same regulatory event is involved in repressing a silent cassette and in preventing it from being a recipient for replacement by another cassette.


Other loci required for silencing include RAP1 (which is also required to maintain telomeric heterochromatin in its inert state) and the genes coding for histone H4. Deletions of the N-terminus of histone H4 or individual point mutations activate the silent cassettes. The effects of these mutations can be overcome either by introducing new mutations in SIR3 or by over-expressing SIR1, which suggests that there is a specific interaction between H4 and the SIR proteins.


The general model suggested by these results is that the SIR proteins act on chromatin structure to prevent expression of the genes. Because mutations in the SIR proteins have the same effects on genes that have been inactivated by the proximity of telomeric heterochromatin, it seems likely that SIR proteins are involved generally in interacting with histones to form heterochromatic (inert) structures. We discuss this in more detail in 19 Nucleosomes.


There is an interesting connection between repression at the silencers and DNA replication. Each silencer contains an ARS sequence (an origin of replication). The ARS is bound by the ORC (the origin recognition complex) that is involved in initiating replication. Mutations in ORC genes prevent silencing, indicating that ORC protein binding at the silencer required for silencing.


There are two separate types of connection between silencing and the replication apparatus. If a Sir1 protein is localized at the silencer (by linkage to another protein that is bound there), the binding of ORC is no longer necessary. This means that the role of ORC is solely to bring in Sir1; it is not required to initiate replication. The role of ORC could therefore be to provide an initiating center from which the silencing effect can spread. This is different from its role in replication.


However, passage through S phase is necessary for silencing to be established. This does not require initiation to occur at the ARS in the silencer. The effect could depend on the passage of a replication fork through the silencer, perhaps in order to allow the chromatin structure to be changed. (This would contrast with the ability to remodel chromatin at promoters without replication; see 19 Nucleosomes.)



Reviews
Laurenson, P. and Rine, J. (1992). Silencers, silencing, and heritable transcriptional states. Microbiol. Rev. 56, 543-560.



Genes VII
Genes VII
ISBN: B000R0CSVM
EAN: N/A
Year: 2005
Pages: 382

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