Locus control regions are defined as gene regulatory sequences that are able to direct high-level and tissue-specific expression of a transgene, independent of its site of integration the host genome. These regions influence the mechanisms of gene activation and silencing through both trans-acting and cis-acting elements that are not completely understood, but seem to involve both long-distance gene regulation and major chromatin remodeling events.
The locus control region was first identified in studies of severe cases of beta-globin deficiencies in human known as gamma, delta, or beta-thalassemia. These diseases involve natural deletions far upstream of the human beta-globin locus and result in transcription silencing of the gene, even though its enhancer, promoter and protein-coding region are normal. Transcription of each gene in the beta-globin locus is restricted to specific developmental stages and to erythroid cells, and both gene proximal and gene distal elements have been implicated in this tissue-specific and developmental regulation. The tissue specific expression of the beta-globin locus correlate with a chromatin opening activity and the manifestation of a generalized DNAase I sensitivity along several sites upstream of this locus, which all are lost in the thalassemias. The region containing these hypersensitive sites thus identified was designated locus control region (LCR) and functionally defined by its ability to overcome repressive effects of condensed heterochromatin on the expression of the beta-globin gene. The importance of the LCR was demonstrated by experiments, which showed that the linkage of large DNA fragments containing the hypersensitive sites to recombinant human transgenes led to high-level and tissue-specific expression of these genes in the mice. This was in contrast to the long observed phenomenon that a eukaryotic recombinant gene that would be expressed at high level in bacteria will show an expression profile in a eukaryotic cell or in the transgenic mice that is dependant on the site of its integration in the host chromosome. The generalized DNAase I hypersensitivity within locus control regions was which is thought to generate extensive chromatin remodeling was experimentally distinguished from the localized hypersensitivity that occur during promoter and enhancer activation at transcribed regions. In fact enhancers are only capable of gene activation at a subset of gene loci and are unable to overcome the repressive effect of heterochromatin at the site of chromosomal integration of linked recombinant transgenes.
The molecular composition and structure of the higher order euchromatin and heterochromatin structures that seem to be the target of the LCR action are largely unknown. The various models that have been invoked to explain the effects of heterochromatin on gene expression are based mainly on genetic data obtained from studies of the fruit fly Drosophila. In this model organism, a specific form of silencing was shown to be imposed by heterochromatin on genes placed adjacent to it. Such silencing which occurs in a random or stochastic way and affects genes that are supposed to be expressed in specific cell lineages is known in Drosophila as position effect variegation (PEV). Similar mechanisms involving silencing through heterochromatin were also described in yeast, where they seem to be especially implicated in regulating genes associated with mating and probably aging processes. Based on studies in transgenic mice that involved the breakdown of the LCR, cases of PEV were also demonstrated in mammals.
Distinct sets of chromatin-associated proteins are involved in the establishment of a higher level of condensation. They are thought to be responsible for the prevention of nonspecific gene activation by rigorously limiting the accessibility of the packaged DNA to transcription factors. The process of gene activation through LCR is assumed to be mediated by complex interactions with these proteins with subsequent decondensation or unfolding of the chromatin and the establishment of discrete domains, which are characterized by the creation of nuclease hypersensitive sites. Thus, LCR are hypothesized to act in a similar fashion but at a higher level of complexity than the classical enhancers.
The basic principle of gene activation by proximal promoters, which was derived from studies in bacteria, is the recruitment of DNA binding transcription factors. Enhancers mediated activation extends this model to recruit, through DNA looping, complexes of transcription factors to the proximal promoter. The LCR is hypothesized to act in a similar fashion but at even higher levels of complexity by involving chromatin remodeling and interactions with still poorly characterized cis- and trans-acting elements to promote gene expression. Both LCR and enhancers seem to involve similar specific molecular mechanisms in gene activation like recruitment of histone modifying enzymes. Both are also proposed to use DNA looping to act at long distances from their target genes. But, very few trans-acting factors have been identified so far that specifically interact with the LCR, and the DNA looping model is difficult to test experimentally. It is likely that besides chromatin remodeling, the LCR are also involved in positioning and targeting of specific DNA segments to specialized compartments in the nucleus. Experimental tools that will allow testing of these possibilities are under development. Techniques such as chromatin immunoprecipitation (CHIP) and the yeast one system are proving valuable information in identifying transacting factors that interact with LCR. The studies of positioning and dynamics of different factors involved in chromatin remodeling in the different compartments of the living nucleus will be facilitated by novel microscopy and imaging techniques like the fluorescence recovery after photobleaching (FRAP) and the fluorescence loss after photobleaching (FLIP).
By studying LCR structure and function, researchers are now gaining new knowledge about the role of chromatin dynamics and nuclear organization in transcription and gene regulation. Furthermore, the growing evidence implicating position effect variegation in human diseases involving chromosomal translocation and the promises of the use of LCR as gene regulatory elements in gene therapy vectors are encouraging more investigations about the in vivo function of these regulatory elements. Most studies have so far examined the function of the LCR at the site of integration in transgenic mice. This meant that the LCR effect was tested at chromatin sites that are different from their endogenous sites. Recent experiments that targeted the LCR regions by deleting all the endogenous hypersensitive sites of the beta-globin locus suggest that the reported effects on chromatin remodeling may not be the major role of the LCR but revealed what seem to be novel properties of these intriguing sequences. LCR are thus, shown to be directly involved in increasing the elongation efficiency of transcription initiated at the beta-globin promoter. The LCR might, therefore, be responsible for the conversion of expression from a basal to a highly activated step with a subsequent effect on chromatin remodeling that needs to be further analyzed.
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