Locus Control Regions(LCRs)Michael Bulger, University of Rochester, Rochester, New York, USA

Mark Groudine, University of Rochester, Rochester, New York, USA

Locus control regions (LCRs) are defined as deoxyribo-

nucleic acid sequence elements that confer high-level,

tissue-specific expression to stably integrated transgenes

in a position-independent manner. As such, LCRs mediate

gene activation and render a high proportion of genomic

integration sites permissive for such expression, and are

distinguished from transcriptional enhancers, which like

LCRs are capable of mediating gene activation over large

genomic distances. The mechanistic basis for the func-

tional distinction between LCRs and enhancers is unclear.

LCRs appear to subsume the separate functions of both

transcriptional enhancers and chromatin insulators, but

may be qualitatively different from either of these classes

of regulatory elements. At their native loci, however, LCRs

mayonly berequiredfora subsetof theactivities that they

display in transgenic contexts.

Definition and Characteristics

Locus control regions (LCRs) comprise a class of cis-actingdeoxyribonucleic acid (DNA) regulatory element that isdefined by activity in transgenic assays. Many tissue-spe-cific genes are subject to position effects upon stable inte-gration in the genome; thus, gene promoters by themselvesmay drive expression in only a small proportion of mouselines (Figure 1), corresponding to a limited number of per-missive integration sites. An element is defined as anLCR ifit confers high-level gene expression onto a linked gene in aposition-independent manner – that is, if it protects atransgene from position effects and activates transcriptionabove basal levels. AnLCR is therefore able to rendermostgenomic integration sites permissive for gene expression, inaddition to activating gene transcription. This activity isnot absolute; expression levels from the same LCR-con-taining transgene can still vary at different integration sites,but this variability is much less severe, and nonexpressinglines are almost never observed. To date, regulatory

elements satisfying these criteria have only been charac-terised in vertebrates.The first LCR was identified within the mammalian b-

globin locus by the presence of a groupof erythroid-specificnuclease hypersensitive sites (HSs) distributed over a 20 kbregion of DNA located upstream of the gene cluster; theseHSs formed regardless of which b-like globin gene wasactive at any developmental stage (Forrester et al., 1986,1987). These sites were subsequently shown to conferposition-independent expression by gain-of-function inhuman b-globin transgenes in mice (Grosveld et al., 1987).A number of elements with similar activities in transgeniccontexts have since been derived from other loci, and soLCRsappear tobe a general feature of tissue-specific genes.Although the b-globin LCR is a complex set of sequencesspread over a large region, other LCRs such as theimmunoglobulin intronic enhancer Em are relatively small(1 kb), discrete sequences. There are no known generalrules for predicting LCR function from DNA sequence.

Enhancers Compared with LCRs

The ability of LCRs to confer high-level, tissue-specificexpression on linked genes is similar to the activity ofenhancers. Enhancers are cis-regulatory elements thatactivate transcription independent of their orientation orposition with respect to linked promoters. These elementsare defined in specific functional assays such as transienttransfection of cells in culture, which do not involve stableintegration into the genome. (Though the term ‘enhancer’is often used to refer to any DNA sequence that activatestranscription of a linkedpromoter, here it refers only to thislimited functional definition.) Thus, though mostenhancers will activate transcription of transgenes or evenincrease the proportion of mouse lines that express atransgene, they donot exhibit the comprehensive, position-independent behaviour that characterises LCRs (Figure 1).See also: Enhancers; PromotersLCRs in turn appear to subsume the function of

enhancers. The b-globin LCR, for example, contains atleast one region (HS2) that acts as an enhancer in transientassays, and other LCRs are known to possess enhanceractivity as well. This leads to the question: Does an LCRconsist of the same activity as an enhancer, but muchstronger, or does an LCR comprise a different class ofactivity? Evidence exists to support both possibilities. HS2

Advanced article

Article Contents

. Definition and Characteristics

. Enhancers Compared with LCRs

. Mechanisms of LCR Function

. LCRs at their Native Loci

Online posting date: 13th June 2013

eLS subject area: Molecular Biology

How to cite:Bulger, Michael; and Groudine, Mark (June 2013) Locus ControlRegions (LCRs). In: eLS. John Wiley & Sons, Ltd: Chichester.

DOI: 10.1002/9780470015902.a0005034.pub2

eLS & 2013, John Wiley & Sons, Ltd. www.els.net 1

of the b-globinLCR, for example, can also confer position-independent expression, but only when HS2-containingtransgenes are integrated with copy numbers of more than3–4 (Ellis et al., 1993). One interpretation of this behaviouris that a sufficient number of HS2 enhancer elements canadd up to an LCR, but this possibility has never beeninvestigated systematically. In addition, deletions per-formed within the endogenous murine b-globin locus haverevealed a purely additive effect of the individual HSs thatcomprise the LCRwith respect to b-globin gene expression(Bender et al., 2012).

On the other hand, the b-globin LCR also includes atleast two sequences – HSs 3 and 4 – that exhibit minimal orno activity in transient assays, but stimulate transcriptionof linked genes when stably integrated (Hardison et al.,1997). Many examples exist of regulatory regions derivedfrom other loci that behave similarly; the activity of theseelements suggests that transient and stable assays of geneexpression can reveal different processes of gene activation,and that LCRs can function in ways that are distinct fromenhancers.Additional indications of a fundamental difference

between LCR and enhancer activity are provided bystudies of the immunoglobulin enhancer Em (Forresteret al., 1994) and the thymic enhancer of the adenosinedeaminase gene (Aronow et al., 1995). In both cases, coresequences possess classic enhancer activity in transientassays, but do not function as LCRs in transgenic assays.LCR activity is obtained when additional flanking elem-ents, which possess no measurable activity on their own,are included with the core enhancers.Analyses of genome-wide mapping of covalent histone

modifications have suggested that distal transcriptionalenhancers can be identified by a characteristic ‘signature’consisting of monomethylated histone H3 lysine 4, amongothermarks (Bulger andGroudine, 2011).As yet, however,

histone ‘signatures’ of this sort have not been extensivelyinvestigated for potential relationships to different aspectsof enhancer function. Thus, correlations have not yet beenmade between specific histone modification patterns andLCR function.

Mechanisms of LCR Function

LCRs exhibit both the function of classical enhancers and adistinct activity that is only revealed upon stable inte-gration in the genome. A fundamental question of generegulation therefore concerns the mechanisms by whicheither function is accomplished. This question can bereduced to two problems: (1) how do enhancers mediatelong-distance gene activation and (2) howdoLCRs protecttransgenes from position effects?Most models for enhancer function assume that gene

activation requires the physical juxtaposition of proteinsbound to both enhancer and promoter, with looping out ofthe intervening DNA. There are several variants of this‘looping’ model, differing mostly in how the necessaryjuxtaposition is brought into being. Evidence for directinteractions between enhancers (and LCRs) and their tar-get promoters has been provided by the ‘chromosomeconformation capture’ (3C) assay and its genome-widederivatives, in which interactions between two genomicregions are identified by crosslinking, restriction endonu-clease digestion, intermolecular ligation and PCR analysisof the resulting products (Miele and Dekker, 2008). Usingthis assay, LCRs and enhancers are commonly found to bemore proximal to active gene promoters than othersequences that are located closer to the promoter on thelinear genome (Miele and Dekker, 2008; de Laat et al.,2008). This correlation is generally assumed to reflect adirect interaction that results in gene activation.

Enhancer

(a) (b) (c)

TransgeneTransgene LCRTransgene

Figure 1 Expression in mice of transgenes containing different regulatory elements. A hypothetical transgene is integrated at random genomic sites in mice

linked to only its promoter (a), the promoter and a classical enhancer (b), or the promoter and a locus control region (LCR; (c)). The sorts of expression

patterns that might be expected among different mice, each containing the transgene in a different integration site, in each case are shown: the darker the

shading the higher the level of expression; no shading indicates no expression.

Locus Control Regions (LCRs)

eLS & 2013, John Wiley & Sons, Ltd. www.els.net2

In a more general sense, LCR function has also beencorrelated with nuclear localisation. In the b-globinlocus, the LCR is required for the extrusion of the locusfrom the specific region of the nucleus occupied by thechromosome on which it resides, which is termed thechromosomal territory (CT) (Ragoczy et al., 2003).Ectopic integration of the b-globin LCR into a gene-dense region of the mouse genome also resulted in morefrequent extrusion of this region away from its CT(Noordermeer et al., 2008). In addition, LCR-mediatedb-globin gene activation has been correlated withmigration of the locus away from the nuclear periphery,a region associated with heterochromatin and genesilencing, and towards the interior of the nucleus, whereactive genes are generally located (Ragoczy et al., 2006).Thus, LCRs may accomplish at least some of theirobserved effects by influencing the nuclear subcompart-ments to which linked genes are directed. Notably,however, enhancers have also been associated withchanges in nuclear localisation, and so it is not clear thatsuch changes comprise a basis for the distinctive activityof LCRs compared with enhancers in transgenic con-texts. See also: Chromatin in the Cell Nucleus: Higher-order Organisation; Chromatin Structure and Domains;Chromosomes and ChromatinProtection from position effects is commonly thought to

involve a dominant ability of an LCR to mediate the for-mation of a permissive or ‘open’ chromatin structure. Thisview originates in part from studies of position effects inDrosophila, where mutations that affect the formation ofheterochromatin also modulate the severity of positioneffects. Similarly, mutations in mammalian homologues ofheterochromatin-associated factors also modify transgeneposition effects, for example, in human b-globin transgenesharbouring a mutated and thus partially functional LCR(McMorrow et al., 2000). Thus, an intact LCR eitherinhibits the formation of repressive chromatin entirely orblocks the spread of repressive chromatin to the transgene.A block to repressive chromatin is a characteristic ofregulatory regions termed insulators or boundary elem-ents; genes flanked by insulators are not as subject toposition effects. LCRs, as complex regulatory elements,could subsume the function of insulators as well asenhancers.The integration-dependent activity of HS3 of the b-glo-

bin LCR, or of the flanking regions of the Em or adenosinedeaminase thymic enhancers, does not seem to be related toinsulator activity, however. Studies of the Em enhancersuggest a more direct effect on chromatin structure: byitself, the Em core enhancer is accessible to T7 or T3 ribo-nucleic acid polymerase only locally. Inclusion of theflanking sequences, required for LCR activity, results inaccessibility and in histone acetylation farther from theenhancer as well (Fernandez et al., 2001). Thus, LCRactivity is correlated with the propagation of a moreaccessible or ‘open’ chromatin structure, which providesyet another potential mechanism in addition to ‘looping’that might be applied to such elements. See also:

Heterochromatin and Euchromatin; The Role of Insu-lators in Genome Organization and Gene Expression

LCRs at their Native Loci

LCRs are defined by their ability to overcome transgeneposition effects. Endogenous gene loci, however, have noneed to ensure gene expression within more than onechromosomal environment, so it is not obvious how LCRactivity in transgenic assays relates to the function of theseelements within the loci from which they are derived. Theeffects of deletion of the immunoglobulin enhancer Emfrom its native locus conform in some respects to themodelpresented by transgenic studies (Ronai et al., 1999). Geneexpression from the mutant immunoglobulin locus isvariegated – that is, transcription is only observed in asmall proportion of cells, whereas in the majority of cellsexpression is completely silenced. Such heterocellularexpression is one hallmark of transgene position effects.Onthe other hand, deletion of all but the core enhancer regionof Em resulted only in a partial decrease in gene expressionlevels in all cells, whereas transgenic studies would havepredicted a heterocellular effect in this case also.Deletion of the mouse b-globin LCR similarly leads to a

severe reduction in gene expression levels (Bender et al.,2000). These levels are consistent among all cells, however,the nuclease-sensitive chromatin structure and histoneacetylation pattern throughout the remainder of the locusare maintained (Schubeler et al., 2001). Thus, targeteddisruption of the b-globin LCR fails to reveal a dominantrole for this element in the formation of a permissivechromatin structure within the locus, whereas studies ofb-globin transgenes predicted such a function. See also:Tissue-specific Locus Control: Structure and FunctionSuch discrepancies could arise from a number of causes,

most prominent being the possible existence of additionaland perhaps redundant activating sequences at theendogenous loci. Nevertheless, though LCR function intransgenic assays defines a regulatory function distinctfrom that of classical enhancers, it is not necessarily pre-dictive of the role of such elements at their native loci.

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Further Reading

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establishing barriers to set heterochromatin boundaries. Epi-

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integration hubs in development: lessons from genomics.

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region function and heterochromatin-induced position effect

variegation. Science 271: 1123–1125.

Jenuwein T, Forrester WC, Fernandez-Herrero LA et al. (1997)

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ment regions. Nature 385: 269–272.

Li Q, Harju S and Peterson KR (1999) Locus control regions:

coming of age at a decade plus. Trends in Genetics 15: 403–408.

Locus Control Regions (LCRs)

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