Eur. J. Biochem. 269, 1589–1599 (2002) Ó FEBS 2002

REVIEW ARTICLE The human b-globin locus control region A center of attraction

Padraic P. Levings and Jo¨ rg Bungert Department of Biochemistry and Molecular Biology, Gene Therapy Center, Center for Mammalian , College of Medicine, University of Florida, Gainesville, FL, USA

The human b-globin gene locus is the subject of intense and capable of recruiting, with great efficiency, chromatin- study, and over the past two decades a wealth of information modifying, , and transcription complexes. These has accumulated on how tissue-specific and stage-specific complexes are used to establish accessible chromatin expression of its genes is achieved. The data are extensive and domains, allowing basal factors to be loaded on to specific it would be difficult, if not impossible, to formulate a com- globin gene promoters in a developmental stage-specific prehensive model integrating every aspect of what is cur- manner. We conceptually divide this process into four steps: rently known. In this review, we introduce the fundamental (a) generation of a highly accessible LCR holocomplex; characteristics of globin locus regulation as well as questions (b) recruitment of transcription and chromatin-modifying on which much of the current research is predicated. We then complexes to the LCR; (c) establishment of chromatin outline a hypothesis that encompasses more recent results, domains permissive for transcription; (d) transfer of tran- focusing on the modification of higher-order chromatin scription complexes to globin gene promoters. structure and recruitment of transcription complexes to the Keywords: chromatin domains; globin genes; intergenic globin locus. The essence of this hypothesis is that the locus transcription; locus control region; transcription. control region (LCR) is a genetic entity highly accessible to

gene-coding regions. The most prominent distal regulatory ORGANIZATION AND STRUCTURE element in the human b-globin locus is the locus control OF THE HUMAN b-G L O B I N L O C U S region (LCR), located from about 6 to 22 kb upstream of The five genes of the human b-globin locus are arranged in a the e-globin gene [2–4]. The LCR is composed of several linear array on chromosome 11 and are expressed in a domains that exhibit extremely high sensitivity to DNase I developmental stage-specific manner in erythroid cells in erythroid cells (called hypersensitive, or HS, sites), and is (Fig. 1) [1]. The e-globin gene is transcribed in the embry- required for high-level globin at all develop- onic yolk sac and located at the 5¢ end. After the switch in mental stages [5]. the site of hematopoiesis from the yolk sac to the fetal liver, The entire b-globin locus remains in an inactive DNase the e-gene is repressed and the two c-globin genes, located I-resistant chromatin conformation in cells in which the downstream of e, are activated. In a second switch, globin genes are not expressed. In erythroid cells, the entire completed shortly after birth, the bone marrow becomes locus shows a higher degree of sensitivity to DNase I, the major site of hematopoiesis, coincident with activation indicating that it is in a more open and accessible chromatin of the adult b-globin gene, while the c-globin genes become configuration [6]. Studies analyzing the human b-globin silenced. The d-globin gene is also activated in erythroid cells locus in transgenic mice have shown that sensitivity to derived from bone marrow hematopoiesis but is only DNase I in specific regions of the globin locus varies and expressed at levels less than 5% of that of the b-globin gene. depends on the developmental stage of erythropoiesis (yolk The complex program of transcriptional regulation sac, fetal liver, adult spleen) [7]. The LCR remains sensitive leading to the differentiation and developmental stage- to DNase I at all developmental stages, whereas sensitivity specific expression in the globin locus is mediated by DNA- to DNase I in the region containing the e-globin and regulatory sequences located both proximal and distal to the c-globin genes is higher in embryonic cells, and DNase I sensitivity in the region containing the d-globin and b-globin gene is higher in adult erythroid cells [7]. Correspondence to J. Bungert, Department of Biochemistry and This review focuses on the regulation of the human Molecular Biology, Gene Therapy Center, Center for Mammalian b-globin gene locus, and we would like to refer the reader to Genetics, College of Medicine, University of Florida, 1600 SW Archer another recent review that compares the regulation of Road, Gainesville, FL 32610, USA. Fax: + 352 392 2953, different complex gene loci [8]. Tel.: + 352 392 0121, E-mail: [email protected]fl.edu Abbreviations: LCR, locus control region; HS, hypersensitive; EKLF, DEVELOPMENTAL STAGE-SPECIFIC erythroid kru¨ ppel-like factor; MEL cells, murine erythroleukemia EXPRESSION OF THE GLOBIN GENES cells; ICD, interchromosomal domain; HLH, helix–loop–helix. (Received 15 November 2001, revised 16 January 2002, accepted The stage-specific activation and repression of the individual 21 January 2002) globin genes during development is regulated by various 1590 P. P. Levings and J. Bungert (Eur. J. Biochem. 269) Ó FEBS 2002

e- EKLF binding site leads to expression of the e-globin gene at the adult stage [15]. This observation indicates that repression of the e-globin gene at the definitive stage is in part due to proteins that interfere with the interaction of the transcriptional activator EKLF. There is also increasing evidence for the presence of stage- specific factors regulating the expression of the two c-globin genes. In particular, it has been shown that CACCC and CCAAT motifs are required for activation of the c-globin genes. The CACCC element is bound by members of the family of kru¨ ppel-like zinc finger (KLF) proteins [16]. Potential candidates for proteins acting through this element are EKLF, FKLF, FKLF-2, and BKLF [17]. The CCAAT box interacts with the heterotrimeric protein NF-Y Fig. 1. Diagrammatic representation of the human b-globin gene locus [18], which appears to play a role similar to EKLF and may (not drawn to scale). The five genes of the human b-globin gene locus recruit chromatin-remodeling activities to the c-globin gene are arranged in linear order reflecting their expression during develop- promoters at the fetal stage. ment. The LCR is represented as the sum of the five HS sites. It should The combined data demonstrate that stage-specific be noted that additional HS sites were mapped 5 to HS5 [95], but it ¢ factors interacting with individual globin gene promoters is currently not known whether these sites participate in globin gene play important roles in the regulation of local chromatin regulation or whether they are associated with the regulation of structure and stage-specific gene expression. genes located upstream of the globin locus. The HS core elements are Another important parameter regulating the stage-speci- 200–400 bp in size and separated from each other by more than 2 kb. During normal human development, the e-globin gene is expressed in fic activity of the globin genes is the relative position of the the first trimester in erythroid cells derived from yolk sac hematopoi- genes with respect to the LCR [19,20]. Inverting the genes esis. The c-globin genes are expressed in erythroid cells generated in the relative to the LCR leads to an inappropriate expression of fetal liver until around birth. The adult b-globin gene is expressed the adult b-globin gene at the embryonic stage and the around birth predominantly in cells derived from bone marrow absence of e-globin gene expression at all stages [21]. hematopoiesis. The expression pattern of the human globin genes is Although the mechanistic basis for the importance of gene somewhat different when analyzed in the context of transgenic mice order in the globin locus is not entirely clear, it is in [96], where the e-globin and c-globin genes are coexpressed in the agreement with the hypothesis that the genes in the globin embryonic yolk sac and the b-globin gene is expressed at high levels in locus are competitively regulated by the LCR [22,23] and fetal liver and circulating erythroid cells from bone marrow. suggests that repressors restrict the ability of the LCR to activate transcription of only one or two genes at specific developmental stages. These factors could either modulate mechanisms. First, genetic information governing the stage- the chromatin structure around the inactive genes [7] or specificity for all b-like globin genes is located in gene interact with globin gene promoters to prevent the interac- proximal regions. These elements represent transcription tion of a gene with the LCR in a developmental stage- factor-binding sites that recruit proteins or protein com- specific manner [15]. plexes in a stage-specific manner. Examples exist for the presence of both positive and negative acting factors that STRUCTURE AND FUNCTION turn genes on or off at a specific developmental stage [1]. OF THE LCR The most extensively studied stage-specific activator is EKLF (erythroid kru¨ ppel like factor), which is crucial for The overall organization of the LCR is conserved among human b-globin gene expression [9]. Gene-ablation studies several vertebrate species. The conservation of individual in mice have shown that EKLF deficiency leads to a specific factor-binding sites within the HS core elements implies that reduction in adult b-globin gene expression, with a these sites are important for LCR function [24]. However, concomitant increase in expression of the fetal genes [10– this by no means leads to the conclusion that transcription 12]. Associated with the dramatic decrease in adult b-globin factor-binding sites that are not conserved are functionally gene expression is a reduction in DNase I HS site formation irrelevant. Some of these nonconserved sites may mediate in the b-globin gene promoter as well as in LCR element novel functions acquired during evolution. For example, the HS3 [13]. These results demonstrate that EKLF is critically developmental pattern of globin gene expression in humans required for the expression of the adult b-globin gene and is quite different from that in mice (Fig. 1) [25]. suggest that EKLF may exert part of its function by Whereas almost all studies agree that the human b-globin changing chromatin structure. Indeed, Armstrong et al.[14] LCR is required for high-level transcription of all b-like showed that EKLF recruits chromatin-remodeling factors globin genes, the question of whether the LCR also to the adult b-globin promoter and that this remodeling regulates the chromatin structure over the whole locus is a activity was sufficient to activate b-globin gene expression in matter of debate. Deletion of the complete LCR from either an erythroid-specific manner in vitro. EKLF acts in a the murine or human locus does not appear to change the sequence-specific context to activate transcription of the overall general sensitivity to DNase I of the locus, indicating b-globin gene [15]. Although both the e-globin and b-globin that the LCR is not required for unfolding of higher-order gene promoters harbor binding sites for EKLF, only the chromatin structure [26–28]. Our understanding of the b-globin gene is expressed at definitive stages of erythro- structural basis for general DNase I sensitivity of chromatin poiesis. Disruption of direct repeat elements flanking the is limited. Loci permissive for transcription are within Ó FEBS 2002 Multistep model for locus control region function (Eur. J. Biochem. 269) 1591 domains of general DNase I sensitivity. However, the b-globin gene expression in b-thalassemic mice only in the presence of a DNase I-sensitive domain does not indicate presence of sequences flanking the LCR HS cores. Taken that all of the genes residing within the domain are together, the data suggest that the HS units interact with transcribed or even that they are permissive for transcription each other to generate an LCR holocomplex, formation of [28]. In this respect the LCR could be involved in regulating which is required for high-level b-globin gene expression. chromatin structure beyond the formation of a general The flanking sequences could be important in positioning DNase I-sensitive domain, for example by regulating the the HS core elements in ways that facilitate their interactions modification of histone tails (methylation, acetylation, [39]. phosphorylation) [29]. It is unquestionable that the LCR provides an open and LCR INTERACTING PROTEINS accessible chromatin structure at ectopic sites in transgenic assays [5]. Whether this is true for all chromosomal Knowledge about the proteins that interact with the LCR positions is not known, because there are no data available in vivo is very limited. Here we will focus on more recent that demonstrate LCRs function from within a defined results describing the activities of specific proteins or protein heterochromatic environment. However, globin gene complexes implicated in LCR function. For a more expression constructs reveal strong position-of-integration comprehensive summary of proteins interacting with regu- effects in transgenic assays in the absence of the LCR, latory sequences throughout the globin locus, we would like suggesting that at most sites the LCR is able to confer an to refer the reader to previous reviews [1,24]. accessible chromatin structure. It is important to understand The DNA sequence motifs that are most conserved that any model describing globin gene regulation must among different species are MARE (maf recognition address the LCR’s ability to open chromatin and enhance element) and GATA sequences in HS2, 3 and 4, KLF- globin gene expression at ectopic sites. binding sites in HS2 and HS3, and an E-box motif in HS2 Current models propose that the individual HS core [24]. MARE sequences are bound in vitro by a large number elements interact to form a higher-order structure, com- of different proteins that all heterodimerize with small maf monly referred to as the LCR holocomplex [30,31]. proteins [41]. Individual members of this family are Evidence supporting the holocomplex model came from characterized by the presence of leucine zipper motifs, the the genetic analysis of mutant LCRs in transgenic assays founding member being NF-E2 (p45) [42]. Other members [31–34]. Deletion of individual LCR HS elements in single- of this family also expressed in erythroid cells are Bach1, copy YAC transgenes led to strong reductions in globin NRF1 and NRF2 (NF-E2 related factor 1 and 2) [43–45]. gene expression and also impaired the formation of A variety of data suggest a pivotal role for p45 in LCR DNase I HS sites associated with the LCR and the globin function [42,46]. However, gene ablation studies have gene promoters. These data suggest that LCR HS site shown that erythropoiesis is not affected in mice lacking deletions render the LCR unable to protect from position- NF-E2 (p45), NRF1 or NRF2, suggesting functional of-integration effects in transgenic studies [32]. In contrast redundancy among the NF-E2 family members in erythroid with these findings, the consequence of deleting HS sites cells [47–49]. from the endogenous mouse locus on globin gene expression It should be noted that, although the NF-E2-like proteins is much milder and does not appear to affect the formation are all thought to interact with the same DNA-binding site, of remaining HS sites [35–37]. The different results from they are structurally different. Bach1 for example contains a studies of globin locus transgenes vs. endogenous loci could BTB/Poz domain and forms oligomers while bound to be explained in several ways [38]. First, the differences could DNA in vitro [50]. This observation prompted investigators solely be based on the observation that an incomplete LCR to analyze whether Bach1/small maf heterodimers could is not able to confer position-independent chromatin simultaneously bind to HS2, 3, and 4 and mediate the opening and gene expression in the globin locus at ectopic interaction between the core elements [51]. Using atomic sites. Secondly, differences in the size of the deleted force microscopy, it was shown that Bach1-containing fragments could result in different phenotypes. The most heterodimers could indeed cross-link HS sites in vitro, severe effects on globin gene expression were observed in indicating that proteins exist that bind to the LCR and are those transgenes in which only the 200–400-bp ÔcoreÕ able to mediate the interaction of HS sites. Importantly, this elements were deleted. All the experiments in the activity of Bach1 depends on the presence of the BTB/Poz endogenous murine globin locus removed the cores together domain. with the flanking sequences. Finally, it is possible that the The CACCC sites in HS2 and HS3 are probably bound endogenous murine globin locus contains sequences in in vivo by EKLF. First, transgenic mice containing the addition to the LCR that are able to provide an open human b-globin locus and lacking EKLF exhibit a reduc- chromatin configuration. tion in the formation of HS3 [13]. In addition, using the Recently, Hardison and colleagues analyzed the function Pin-Point assay, Lee et al. [52] demonstrated that EKLF of LCR HS sites in the presence or absence of the HS core binds to both HS2 and HS3 in vivo. Interestingly, the flanking sequences in murine erythroleukemia (MEL) cells binding of EKLF to HS3 is reduced in the absence of HS2, using recombination mediated cassette exchange [39]. At suggesting some form of communication between these two several fixed positions, the inclusion of the flanking elements [52]. sequences leads to a synergistic enhancement of expression The GATA sites are bound by either GATA-1 or by the combination of HS units, whereas combining the GATA-2, the only two members of the GATA family of core HS elements only additively enhanced reporter gene transcription factors known to be expressed in erythroid expression. Similarly, May et al. [40] showed that the cells [53]. GATA-1 is one of the earliest markers in red cell combination of HS2, 3, and 4 led to therapeutic levels of differentiation and is detectable in progenitor cells that do 1592 P. P. Levings and J. Bungert (Eur. J. Biochem. 269) Ó FEBS 2002 not yet express the globin genes [54]. Interestingly, LCR HS proteins responsible for HS site formation require a window sites are already detectable in these undifferentiated precur- of opportunity after replication to bind and then prevent the sor cells [55]. These results suggest that GATA-1 may be generation of repressive chromatin structure or do these involved in the regulation of chromatin structure at an early proteins recruit chromatin-remodeling activities that change stage of erythroid differentiation. the chromatin structure in a replication-independent man- The E-box in HS2 interacts with helix–loop–helix (HLH) ner? Experiments that indirectly addressed this issue were proteins in vitro, and both USF and Tal1 were shown to those in which investigators generated heterokaryons with interact with this element [56,57]. USF is a ubiquitously MEL cells, which represent definitive erythroid cells that expressed member of the HLH family of proteins and binds express the adult b-globin gene, and human K562 cells, to DNA as a heterodimer usually composed of USF1 and which represent primitive erythroid cells that express the USF2. USF has been implicated in the regulation of many e-globin gene [68]. These studies showed that trans-acting genes and normally acts as a transcriptional activator. factors in the MEL cells are able to activate transcription of However, it has also been reported to function through the human b-globin gene. Interestingly, the onset of initiator elements, in which case it mediates the recruitment b-globin gene expression in these experiments occurred of Pol II transcription complexes [58,59]. Tal1 is hemato- about 12 h after fusion. Because the globin locus replicates poietic specific and appears to function at an early step early in erythroid cells, these results could be interpreted to during the specification of hematopoietic progenitor cells mean that replication is required for trans-activation of the [60]. human b-globin genes in the heterokaryons. On the other Protein–protein interactions probably play important hand, this experiment could also lead to the interpretation roles in LCR function. We have already discussed the that the human locus can be activated by transcription multimerization of Bach/maf heterodimers. Other protein– factors and accessory proteins already present in the adult protein interactions known to occur among LCR-binding (MEL) erythroid cells. This mode of regulation would be proteins involve those between the GATA factors and similar to the induction of genes by hormone receptors [69]. between GATA factors and EKLF, LMO2/Tal1, and Sp1 However, differences in the two systems may exist, as the [61–63]. In addition, GATA-1, EKLF and NF-E2 (p45) globin locus is a developmentally regulated locus, the were shown to interact with coactivators and acetyltrans- expression of which changes as the cell differentiates. Genes ferase activities [64,65]. EKLF has also been demonstrated regulated by hormone and orphan receptors are transcribed to interact with members of the Swi/SNF family of in mature cells and their expression is regulated by external chromatin-remodeling complexes [14]. These results show stimuli, i.e. hormones. Obviously more studies are needed that most proteins binding to one LCR core element have that examine the relationship between replication and the potential to interact with proteins binding to another chromatin structure in the globin locus. For example, it LCR core HS site, which could initiate and stabilize an LCR would be interesting to examine the binding of chromatin holocomplex. In addition, the results also demonstrate that components and transcription factors during the cell cycle in LCR-interacting proteins recruit macromolecular com- erythroid cells. plexes involved in and histone acetylation. INTERGENIC TRANSCRIPTS IN THE GLOBIN LOCUS REPLICATION AND CHROMATIN In 1992, Tuan et al. [70] reported that long transcripts STRUCTURE initiate within LCR HS2 and proceed in a unidirectional The human b-globin locus replicates early in erythroid cells manner toward the globin genes. Further studies by the and late in nonerythroid cells. Earlier studies suggested that same group led to the startling observation that transcrip- the LCR regulates the timing and usage of an origin of tion always proceeds in the direction of a linked gene, replication located between the d-globin and b-globin gene independent from the orientation of HS2 [71]. This result [66]. This interpretation was based on the observation that a suggests some form of communication between the promo- large deletion in the human b-globin locus, starting ter and LCR HS2 in these experiments. Subsequent studies immediately upstream of HS1 and spanning about 30 kb, in the laboratories of Proudfoot [72] and Fraser [7] identified inactivates the entire globin locus [66]. The globin genes noncoding transcripts over the entire LCR and in between linked to this deletion are not transcribed, the locus becomes the globin gene coding regions. Interestingly, the pattern of late replicating, and remains in a DNase I-resistant and intergenic transcription during development appears to inaccessible configuration. However, recent analysis of the correlate with the pattern of general DNase I sensitivity [7]. consequence of a targeted deletion of the LCR demonstrates Mutations that delete the start site of the adult-specific that the LCR regulates neither the timing of replication in intergenic transcripts lead to a decrease in general DNase I the globin locus nor the usage of the replication origin [67]. sensitivity and b-globin gene transcription, suggesting that Thus, a putative element regulating replication timing in the intergenic transcription modulates the chromatin structure human b-globin locus must be located 5¢ to the LCR. of globin locus subdomains. Intergenic transcripts appear to An important question that has to be addressed is be generated in a cell-cycle-dependent manner, detectable whether activation of the globin locus and LCR function during early S-phase but predominantly present in G1 [7]. requires replication. During differentiation of erythroid These results provide evidence for the hypothesis that cells, the locus undergoes various transitions, the first of intergenic transcription is transient. Recently Plant et al. which is the formation of DNase I HS sites in the LCR [55]. [73] analyzed intergenic transcripts across the globin locus Does the formation of HS sites at this early stage in by nuclear run-on analysis and did not find any evidence for differentiation require replication? In other words, do the the stage-specific generation of these transcripts. The Ó FEBS 2002 Multistep model for locus control region function (Eur. J. Biochem. 269) 1593 discrepancy between this study and that of Gribnau et al.[7] ÔmatrixÕ-like structure, consisting of filamentous proteins, is not understood at the moment, but it is possible that at which is defined as the interchromosomal domain (ICD). certain stages of the cell cycle, the entire locus is transcribed Active gene loci are located at the surface of chromosomal for a short period of time. A subsequent step could then shut domains in direct contact with the ICD, whereas inactive off transcription in silenced domains, but reduced tran- loci are located away from the ICD within chromosomal scription could still be detectable by the more sensitive assay domains. It is proposed that macromolecular protein employed by Plant et al. [73]. complexes involved in chromatin remodeling, transcription, and splicing are enriched in the ICD, whereas single proteins or smaller protein complexes can diffuse into regions of the INSULATORS chromatin domains that are not in contact with the ICD. The chicken b-globin locus is flanked by elements The former ideas are based on indirect observations using which mark clear boundaries between active and inactive microscopy and fluorescent labeling. We can therefore only chromatin [74,75]. No such sequences have been conclu- describe the existence of chromosome territories and the sively identified in the human or murine globin locus, and it ICD as speculative at best. However, it is safe to say that appears that the DNase I-sensitive domain in these loci gene loci are located in specific regions of the nucleus and extend far 5¢ of the LCR and far 3¢ of the b-globin gene. that the relative position of these loci changes on activation. Recent experiments distinguish between insulator sequences If applied to the regulation of the globin genes, the ICD that block the action of an enhancer or and that of model could explain why deletion of the LCR in the boundary elements that separate open and closed chromatin endogenous human or murine globin loci silences globin domains [74]. The 5¢ most HS site of the chicken LCR, HS4, gene expression without altering the establishment of appears to harbor both activities [75]. In this sense it is quite DNase I and hyperacetylated chromatin. It is possible that possible that the human b-globin locus contains insulator transcription factors could gain access to the globin locus elements that restrict the action of the LCR to within and change higher-order chromatin structure, but that the specific domains. Some evidence suggests that HS5 may LCR is required to organize the globin locus in a way that it harbor insulator activity. First, HS5 harbors a binding site is located in close proximity to the ICD. The situation is for the protein CTCF, which is largely responsible for similar in concept to mechanisms described for the regula- insulator function of chicken HS4 [76]. Secondly, inversion tion of gene loci during differentiation of B-lymphocytes. of the entire LCR with respect to the genes reduces globin Fisher and colleagues [79] have shown that specific gene loci gene expression to less than 30% of wild-type levels [21]. relocate to inactive regions in the nucleus of cycling B-cells. Thirdly, an e-globin gene placed upstream of the LCR is not The relocation and inactivation is regulated by the DNA- transcribed [21]. Finally, HS5 was shown to exhibit binding protein Ikaros, which mediates the association of insulator activity in cell culture experiments [77]. gene loci with centromeric heterochromatin.

NUCLEAR LOCALIZATION A MULTISTEP MODEL FOR HUMAN b-GLOBIN GENE REGULATION Recent data suggest that enhancer and other regulatory elements affect the position of genes within the nucleus Step 1: generation of a highly accessible [78,79]. For example, it was shown that in the absence of LCR holocomplex an enhancer, the b-globin gene is located close to centromeric heterochromatin, an environment within the We propose that the first step towards activation of the nucleus that is incompatible with transcription [80]. In the globin genes during differentiation is the partial unfolding of presence of LCR element HS2, the b-globin gene localizes the chromatin structure containing the globin locus into a away from centromeric heterochromatin, suggesting that DNase I-sensitive domain (Fig. 2A). This step may or may activities associated with HS2 are able to relocate the not require replication. The initial unfolding of the transgene to a transcriptionally permissive nuclear region chromatin structure is mediated by the diffusion of eryth- [80]. This phenomenon has been most intensively analyzed roid-specific proteins into chromosomal domains that are in yeast, in which specific protein complexes appear to not permissive for transcription. These proteins bind to direct the location of genes into active or inactive regions sequences throughout the globin locus leading to the partial of the nucleus [81]. However, Milot et al.[32]showedthat unfolding and perhaps hyper-acetylation of the chromatin. a wild-type globin locus that integrated close to centro- If replication is required for globin locus activation, we meric heterochromatin was still active, suggesting that, in propose that erythroid-specific proteins bind to the globin the presence of the LCR, the globin locus is active even locus after DNA synthesis, prevent the formation of when situated close to a defined heterochromatic envi- repressive chromatin, and mark the locus by modification ronment. of histone tails. Recent advances in fluorescent labeling of chromatin as GATA factors may be involved in the initial step of well as three-dimensional fluorescent microscopy indicate globin locus activation, as their binding sequences are that chromosomes occupy distinct regions, or domains, located throughout the globin locus. In addition, GATA-1 within the cell nucleus [82]. These chromosome domains is one of the earliest markers of red cell differentiation [54] may be composed of up to 1 Mb of chromatin supported by and is known to associate with proteins containing histone the nuclear architecture and appear to contain loops of acetyltransferase activities. The partial unfolding into a about 50–200 kb of DNA possessing one or several gene DNase I-sensitive structure does not require activities loci that may or may not be co-regulated. The spaces associated with the LCR. This is shown by the fact that between these territories are believed to be occupied by a even in the absence of an intact LCR, the rest of the globin 1594 P. P. Levings and J. Bungert (Eur. J. Biochem. 269) Ó FEBS 2002

Fig. 2. Multistep model for human b-globin gene regulation. The model depicts four steps proposed to be involved in the regulation of chromatin structure and gene expression in the human b-globin locus. The model focuses on the regulation of the human globin locus in the context of transgenic mice, but it is assumed that the same principal mechanisms govern the correct expression of the b-globin genes during human development, except that the timing of expression of the genes is somewhat different (see Fig. 1). (A) Generation of a highly accessible LCR holocomplex. We propose that the initial events in activating the human globin gene locus during differentiation involves the partial unfolding of the chromatin structure into a DNase I-sensitive domain and the binding of protein complexes to the LCR HS sites. This will then generate the LCR holocomplex, the protein-mediated interaction of HS sites. (B) Recruitment of chromatin-remodeling, coactivator and transcription complexes. Once the LCR holocomplex is generated, the globin locus is relocated to an area of the nucleus enriched for macromolecular complexes involved in coactivation, chromatin remodeling (or modification of histone tails) and transcription. These complexes are recruited to the LCR, which provides a highly accessible platform for recruiting these activities. (C) Establishment of chromatin domains permissive for transcription. The macromolecular protein complexes recruited to the LCR will initially be used to establish chromatin domains that allow transcription of the genes. Specifically, we propose that the LCR recruits elongation-competent transcription complexes (or complexes that are rendered elongation competent at the LCR) that track along the DNA and modify the chromatin structure. This reorganization of the chromatin structure will render the promoters accessible for activating proteins and components of the preinitiation complex. Data published by Gribnau et al. [7] suggest that intergenic transcription and chromatin reorganization is stage-specific and restricted to the genes that are expressed either at the embryonic or adult stage. (D) Transfer of macromolecular protein complexes to individual globin gene promoters. Once active chromatin domains are established, the LCR recruits elongation-incompetent transcription complexes, which are transferred to the individual globin gene promoters present in the accessible chromatin domains. The polymerases are then rendered elongation-competent, possibly through phosphorylation of the C-terminal domain [88]. locus is rendered sensitive [28] and exhibits with heterochromatin, such as SUV39H1, M33, and BM-1, increased histone H4 acetylation [83]. We propose that all could be involved in regulating the accessibility and location subsequent steps require activities recruited to the LCR. It is of the globin locus [84]. possible that the initial invasion of the globin locus by The reorganization of the chromosomal domain, which erythroid factors could mark the locus for relocation to an renders the globin locus accessible to chromatin-remodeling, area that is close to the ICD. Proteins normally associated coactivator and transcription complexes present in the ICD, Ó FEBS 2002 Multistep model for locus control region function (Eur. J. Biochem. 269) 1595 is regulated by elements within the LCR. Once the locus of intergenic transcripts have been used to separate the becomes accessible to macromolecular complexes in the globin locus into developmental stage-specific chromatin ICD, protein complexes aggregate at the LCR HS core domains [7]. Although the exact mechanism by which the elements. In vitro experiments suggest that HS site forma- developmental patterns of chromatin structure and inter- tion occurs even in the absence of regular chromatin genic transcription are established is unknown, it is likely structure and may involve the generation of S1-sensitive that the recruitment of chromatin-modifying and transcrip- segments within the core HS sites [85]. Therefore, it is tion complexes to the LCR would initiate the processes proposed that protein complexes bind to the HS core sites involved (Fig. 2C). and bend or disturb the structure of the DNA. The There are three lines of evidence suggesting that formation of protein aggregates and the subsequent distur- intergenic transcription modifies the chromatin structure bance of DNA structure at the LCR HS core elements could within the globin locus subdomains. First, LCR transcripts lead to a highly accessible region in the b-globin locus. initiate both upstream or within the LCR and proceed in a The generation of an LCR holocomplex probably unidirectional manner toward the genes [7,71,72]. Sec- involves interactions between protein complexes at the ondly, deletion of a region containing the adult-specific different HS units including the cores and the flanking transcription initiation site leads to a decrease in general sequences. In early differentiation stages, NF-E2 sites may DNase I sensitivity within the subdomain and a decrease in be occupied by Bach1/maf heterodimers, which may expression of the adult b-globin gene [7]. Finally, it is facilitate interactions between HS sites, but may also hold feasible that chromatin-modifying activities associate with the LCR in an inactive configuration. Heme-mediated ÔpioneerÕ polymerase complexes at the LCR, which would inhibition of Bach1/maf binding at later stages of differen- initiate transcription and modify the chromatin structure of tiation would allow the binding of other members of the globin locus subdomains [87]. In vivo, nucleosomes in NF-E2 family of proteins [86]. transcribed regions of chromatin are unfolded exposing the cysteinyl-thiol groups of histone H3, and this unfolding was observed only in the presence of active transcription Step 2: recruitment of chromatin-remodeling [90,91]. Furthermore, these unfolded nucleosomes were and transcription complexes to the LCR associated with highly acetylated histones. The fact that Formation of the LCR holocomplex results in the massive reconstitution of nucleosomes with hyperacetylated disruption of chromatin structure and a high density of histones could not recapitulate the unfolded structure led DNA-bound proteins (Fig. 2B). The consequence of this the authors to conclude that acetylation was not a shift in structure is that activities that are normally requirement of nucleosome unfolding. More recently it associated with transcriptionally active chromatin will was found that histone acetylation was required to gravitate to the LCR. We propose that proteins bound to maintain the unfolded nucleosome structure that resulted the core HS sites, namely members of the NF-E2 family, from transcriptional elongation [92]. This result suggests GATA factors and EKLF, recruit chromatin-remodeling that transcription can modify the chromatin of an active complexes and coactivators. The recruitment of RNA gene domain so as to distinguish it from that of an polymerase II may involve HLH proteins as they have been accessible but otherwise inactive one [92]. shown to mediate transcription complex formation on Two models have been proposed to explain how the TATA-less genes [58,59]. LCR enhances globin gene transcription, the looping or Initially the LCR could recruit elongation-competent linking model [6,22]. According to the linking model, transcription complexes associated with chromatin-remode- activities recruited by the LCR would be transmitted to ling activities that would initiate the establishment of the globin genes through an array of proteins binding transcriptionally permissive chromatin domains within the along the DNA. The looping model proposes direct locus. Orphanides & Reinberg [87] have proposed the interactions between the LCR and individual genes with presence of ÔpioneerÕ polymerases which are involved in the intervening DNA looping out. The establishment of the modulation of chromatin structure. Such a ÔpioneerÕ transcriptionally permissive chromatin domains in the polymerase may be recruited to the LCR, associate with globin locus can be explained according to both models. chromatin-modifying activities, and track along the DNA It is possible that the LCR and segments of the adult- to modify the nucleosome structure of chromatin domains specific chromatin domain are in direct contact and that in the globin locus. Once active chromatin domains are transcription complexes and chromatin-modifying activit- established, the LCR could recruit elongation-incompetent ies are transferred by a looping mechanism. On the other transcription complexes. These complexes could then be hand, the observation that a certain fraction of adult cells delivered to individual globin gene promoters and would coexpress the c-globin and b-globin genes, which are then be rendered elongation-competent, possibly through located in different chromatin domains [7], could suggest phosphorylation of the C-terminal domain of RNA that at a certain stage, the whole locus is ÔopenÕ and that polymerase II [88]. the repression of the e/c-chromatin domain is a secondary process involving the deacetylation and inactivation of the embryonic domain. This idea is supported by the data of Step 3: establishment of chromatin domains Forsberg et al. [89] showing that the pattern of histone permissive for transcription acetylation across the globin locus varies during develop- Recent studies have shown that the b-globin locus under- ment. 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