Published in : Lewin B, "Genes VII", pp. 674-675, New York: Oxford University Press Inc, 2000:
http://www.oup.com/genesvii

Some genes respond to additional regulatory elements as well as to promoters and enhancers. The existence of these elements was identified by the inability of a region of DNA including a gene and all known regulatory elements to be properly expressed when introduced into an animal as a transgene. The best characterized example is provided by the mouse beta-globin cluster. Recall from Figure 4.1 that the alpha-globin and beta-globin genes in mammals each exist as clusters of related genes, expressed at different times during embryonic and adult development.

These genes are provided with a large number of regulatory elements, which have been analyzed in detail. In the case of the adult human beta-globin gene, regulatory sequences are located both 5' and 3' to the gene and include both positive and negative elements in the promoter region, and additional positive elements within and downstream of the gene.

But a human beta-globin gene containing all of these control regions is never expressed in a transgenic mouse within an order of magnitude of wild-type levels. Some further regulatory sequence is required. Regions that provide the additional regulatory function are are identified by DNAase I hypersensitive sites that are found at the ends of the cluster.

The map of Figure 21.22 shows that the 20 kb upstream of the epsilon-gene contains a group of 5 sites; and there is a single site 30 kb downstream of the beta-gene. Transfecting various constructs into mouse erythroleukemia cells shows that sequences between the individual sites in the 5' region can be removed without much effect, but that removal of any of the sites reduces the overall level of expression.

The 5' regulatory sites are the primary regulators, and the cluster of hypersensitive sites is called the LCR (locus control region). We do not know whether the 3' site has any function. The LCR is absolutely required for expression of each of the globin genes in the cluster. Each gene is then further regulated by its own specific controls. Some of these controls are autonomous: expression of the epsilon- and gamma-genes appears intrinsic to those loci in conjunction with the LCR. Other controls appear to rely upon position in the cluster, which provides a suggestion that gene order in a cluster is important for regulation.

The entire region containing the globin genes, and extending well beyond them, constitutes a chromosomal domain. It shows increased sensitivity to digestion by DNAase I. Deletion of the 5' LCR restores normal resistance to DNAase over the whole region. It is possible that the LCR is required to "open up" the whole domain and make it available for transcription.

Does this model apply to other gene clusters ? The alpha-globin locus has a similar organization of genes that are expressed at different times, with a group of hypersensitive sites at one end of the cluster, and increased sensitivity to DNAase I throughout the region. Only a small number of other cases are known in which an LCR controls a group of genes.

Does a domain have a discrete boundary that marks its end: or do effects emanating from discrete points within it decline gradually at the ends of the region ?

Elements that prevent the passage of activating or inactivating effects are called insulators, and have been identified in two circumstances. When an insulator is placed between an enhancer and a promoter, it prevents the enhancer from activating the promoter. This may explain how the action of an enhancer is limited to a particular promoter. When an insulator is placed between an active gene and heterochromatin, it protects the gene against the inactivating effect that spreads from the heterochromatin. Those insulators that have been characterized so far possess both these properties, suggesting that they affect the general organization of chromatin.

The leftmost hypersensitive site of the chick beta-globin LCR (HS4) is an insulator that marks the 5' end of the functional domain. HS4 lies within a 250 bp region that has the properties of an (unmethylated) CpG island (although it is not associated with a promoter). We do not know yet what identifies the other end of the domain.

Additional References:

1. "Ultrastructural Continuity between Active and Repressed Chromatin".

2. "Nuclear Polyanions as De-Repressors of Synthesis of Ribonucleic Acid".

3. "A Model of Specific De-Repression within Interphase Chromatin".

4. "Single-Cell Analysis of DNase I-Sensitive Sites During Neoplastic and Normal Cell Differentiation within Human Bone Marrow".

5. "Nuclear RNA Species Activate DNA Transcription within Chromatin".

6. "Oncogenes as Molecular Targets within Active Chromatin".

For Further Information or Feedback:
E-mail: frenster@euchromatin.net
John H. Frenster, M.D.
Physicians' Educational Series
247 Stockbridge Avenue
Atherton, CA 94027-5446
Voice: 650/367-6483
Fax: 650/364-1773