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DNase I hypersensitivity assays and phylogenetic analyses previously led to the identification of multiple enhancers in the Th2 cytokine cluster. As described in this issue of Immunity, Lee et al. have now used a transgenic mouse assay to identify a locus control region (LCR) that supports integration site-independent, copy number-dependent transcription of Il4 and Il13.
In order to verify the genetic factors influencing the clinical expression of beta-thalassemia we have studied 292 Italian patients, 165 with thalassemia intermedia and 127 with thalassemia major. The beta-globin gene mutations were defined in all cases. The number of alpha-globin genes and the integrity of specific control regions of the beta-globin cluster--gamma promoters and beta-Locus Control Region (beta-LCR)--were studied in selected cases. Homozygosity for mild mutations (group I) accounts for 24% of the intermedia patients and it is not represented among major patients. Forty-four percent of intermedia patients had combinations of mild/severe (group II) mutations and 32% had homozygosity or double heterozygosity for severe mutations (group III). Seventy-six percent of patients with thalassemia major were classified in group III and 24% in group II. Deletion type-alpha3.7 thalassemia, assessed in a part of the cases, was found in 5% of thalassemia major and 19.5% of intermedia patients in groups II and III. Structural analysis of gamma promoters and beta-LCR HS2 and HS4 regions, carried out in order to look for alterations associated with Hb F increase, did not reveal new mutations. Only rare polymorphic changes were observed at the HS2 and HS4 level. The -158G gamma C T change was found with an increased incidence in intermedia patients in groups II and III. A subset of 10 beta-thalassemia heterozygotes with mild intermedia phenotype resulted from coinheritance of a triplicated alpha-locus. We have been unable to find a molecular basis for the benign clinical course in approximately 20% of patients with thalassemia intermedia. Other genetic or acquired factors must be hypothesized which ameliorate the clinical condition.
Four transcriptional enhancers lie downstream of the immunoglobulin heavy chain locus: Calpha3'/hs3a, hs1,2, hs3b, and hs4. Although individually weak, these elements have strong transcriptional synergies when combined and they altogether behave as a locus control region. Previous knockout experiments in the 3' region have shown that both hs3a and hs1,2 are dispensable for normal expression and rearrangement of the IgH locus but that their replacement with a transcribed neo gene severely affects class switch recombination. Here we show that even in the absence of a neo gene, joint deletion of the last two 3' enhancers, hs3b and hs4, severely impairs germline transcription and class switching to most isotypes and may in addition affect mu gene expression in resting B cells.
Trophoblast, the only fetal tissue in direct contact with maternal cells, fails to express the polymorphic HLA class I molecules HLA-A and -B, but does express the nonpolymorphic class I molecule HLA-G. It is thought that HLA-G may provide some of the functions of a class I molecule without stimulating maternal immune rejection of the fetal semiallograft. As a first step in identifying the cis-acting DNA regulatory elements involved in the control of class I expression by extraembryonic tissue, several types of transgenic mice were produced. Two HLA-G genomic fragments were used, 5.7 and 6.0 kb in length. These included the entire HLA-G coding region, 1 kb of 3' flanking sequence, and 1.2 or 1.4 kb of 5' flanking sequence, respectively. A hybrid transgene, HLA-A2/G, was produced by replacing the 5' flanking sequence, first exon, and early first intron of HLA-G with the corresponding elements of HLA-A. Comparison of transgene mRNA expression patterns seen in HLA-A2/G and HLA-G transgenic mice suggests that 5' flanking sequences are largely responsible for the differing patterns of expression typical of the classical class I and HLA-G genes. Studies comparing the extraembryonic HLA-G expression levels of founder embryos transgenic for either the 5.7- or 6.0-kb HLA-G transgene showed that the 6.0-kb transgene directed HLA-G expression far more efficiently than did the 5.7-kb HLA-G transgene, producing extraembryonic HLA-G mRNA levels similar to those seen in human extraembryonic tissues. The results of these studies suggest that the 250-bp fragment present at the extreme 5' end of the 6.0-kb HLA-G transgene and absent from the 5.7-kb HLA-G transgene contains an important positive regulatory element. This 250-bp fragment lies further upstream than any of the previously documented class I regulatory regions and may function as a locus control region.
The human beta-globin locus is home to five genes that are regulated in a tissue-specific and developmental stage-specific manner. While the exact mode of expression remains somewhat enigmatic, a significant effort has been focused at the locus control region (LCR). The LCR is marked by five DNase I-hypersensitive sites (HS) approximately 15 kb upstream of the epsilon-globin gene. Nuclear matrix-associated regions (MARs) organize chromatin into functional domains and at least one of the HS appears bound to the nuclear matrix. We have employed an in vivo based PCR MAR assay to investigate the role of MAR-mediated regulation of the beta-globin locus. This was facilitated with a novel reaction efficiency based quantitative real-time PCR analysis software tool, Target Analysis Quantification. Using a log-linear regression strategy, discordances were eliminated. This allowed us to reliably estimate the relative amount of initial template associated with the nuclear matrix at 15 unique regions spanning the beta-globin locus in both non-expressing and expressing cell lines. A dynamic association dependent on expression status was revealed both at the LCR/5'HS region and within the second intron of the beta-globin gene. These results provide the first evidence that nuclear matrix association dynamically mediates the looping of the beta-globin locus to achieve transcriptional control.
To further our understanding of the regulation of vertebrate globin loci, we have isolated cosmids containing alpha- and beta-globin genes from the pufferfish Fugu rubripes. By DNA fluorescence in situ hybridization (FISH) analysis, we show that Fugu contains 2 distinct hemoglobin loci situated on separate chromosomes. One locus contains only alpha-globin genes (alpha-locus), whereas the other also contains a beta-globin gene (alpha beta-locus). This is the first poikilothermic species analyzed in which the physical linkage of the alpha- and beta-globin genes has been uncoupled, supporting a model in which the separation of the alpha- and beta-globin loci has occurred through duplication of a locus containing both types of genes. Surveys for transcription factor binding sites and DNaseI hypersensitive site mapping of the Fugu alpha beta-locus suggest that a strong distal locus control region regulating the activity of the globin genes, as found in mammalian beta-globin clusters, may not be present in the Fugu alpha beta-locus. Searching the human and mouse genome databases with the genes surrounding the pufferfish hemoglobin loci reveals that homologues of some of these genes are proximal to cytoglobin, a recently described novel member of the globin family. This provides evidence that duplication of the globin loci has occurred several times during evolution, resulting in the 5 human globin loci known to date, each encoding proteins with specific functions in specific cell types.
Transgenic rats with the 130 kb bacterial artificial chromosome construct bLA, including the alpha-lactalbumin gene, had position-independent and copy number-dependent expression, which confirmed previous experiments using the 210 kb yeast artificial construct, yLALBA. To identify elements that confer a position effect, we compared the yLALBA and bLA sequences. yLALBA was chimeric. A common 32 kb region was identified and the total nucleotide sequence was determined. We previously analyzed transgenic rats using polymerase chain reaction to compare the integrity and expression of the transgenes. The -6 to +9 kb region is considered to be necessary for position-independent expression. Transgenic rats lacking the -3.4 to -0.85 kb region had a severe position effect. This 2.5 kb region contains two DNaseI hypersensitive sites at -1.0 and -2.8 kb. The 2.5 kb region is proposed to be a locus control region of the human alpha-lactalbumin gene.
The liver is one of the prime targets for gene therapy, and the correction of defects in a variety of clotting factor genes is one of the main goals of liver-directed therapies. The use of transcriptional regulatory elements derived from these genes may provide for the optimal expression of transduced genes. We have applied our knowledge of the promoter structure of the clotting Factor IX gene to design optimized expression vectors for use in gene therapy. The activity of the proximal promoter has been augmented by the introduction of a multimerized upstream site which we have previously shown to be a prime regulator of the pro- moter. Introduction of this element increases promoter activity at least 20-fold over the proximal promoter alone when assayed in the human liver cell line Hep G2. This optimized promoter is significantly more active than the SV40 enhancer/early promoter. The expression of the optimized Factor IX promoter is also more persistent in the short term. The inclusion of a liver-specific locus control region, derived from the apolipoprotein E/C locus, did not further augment expression levels. These Factor IX vectors also exhibit a high degree of tissue specificity, as measured by transfection into breast and muscle cell lines.
Recent studies of nuclear organization have shown an apparent correlation between the localization of genes within the interphase nucleus and their transcriptional status. In several instances, actively transcribed gene loci have been found significantly looped away from their respective chromosome territories (CTs), presumably as a result of their expression. Here, we show evidence that extrusion of a gene locus from a CT by itself is not necessarily indicative of transcriptional activity, but also can reflect a poised state for activation. We found the murine and a wild-type human beta-globin locus looped away from their CTs at a high frequency only in a proerythroblast cell background, prior to the activation of globin transcription. Conversely, a mutant allele lacking the locus control region (LCR), which is required for high-level globin expression, was mostly coincident with the CT. The LCR may thus be responsible for the localization of the globin locus prior to activation. Replacement of the LCR with a B-cell-specific regulatory element, while also extruding the globin locus, brought it closer to the repressive centromeric heterochromatin compartment. We therefore suggest that the looping of gene loci from their CTs may reflect poised and repressed states, as well as the previously documented transcriptionally active state.
Transgenic experiments in vertebrates often involve the insertion of tandem multiple-copy arrays at single sites. For many transgenes, expression is unpredictable from site to site, a phenomenon usually attributed to a repressive environment caused by nearby sequences. However, an alternative explanation comes from evidence that transgene repeat arrays in flies condense into heterochromatin, suggesting that low levels of expression in vertebrate transgene arrays might result from interactions between repeats within the array. A recent experiment using transgenic mouse lines demonstrates that reduction in copy number of silenced transgenes within an array leads to a striking increase in expression, demonstrating that silencing is intrinsic to the array, and is not attributable to position effects of nearby sequences. This work calls into question functions that have been attributed to vertebrate locus control regions and boundaries, and draws attention to the notion that repeat-induced gene silencing is a system for protection of eukaryotic genomes against threatening sequence elements.
One function of lineage-restricted transcription factors may be to control the formation of tissue-specific chromatin domains. In erythroid cells, the beta-globin gene cluster undergoes developmentally regulated hyperacetylation of histones at the active globin genes and the locus control region (LCR). However, it is unknown which transcription factor(s) governs the establishment of this erythroid-specific chromatin domain. We measured histone acetylation at the beta-globin locus in the erythroid cell line G1E, which is deficient for the essential hematopoietic transcription factor GATA-1. Restoration of GATA-1 activity in G1E cells led to a substantial increase in acetylation of histones H3 and H4 at the beta-globin promoter and the LCR. Time course experiments showed that histone acetylation occurred rapidly after GATA-1 activation and coincided with globin gene expression, indicating that the effects of GATA-1 are direct. Moreover, increases in histone acetylation correlated with occupancy of GATA-1 and the acetyltransferase CBP at the locus in vivo. Together, these results suggest that GATA-1 and its cofactor CBP are essential for the formation of an erythroid-specific acetylation pattern that is permissive for high levels of gene expression.
By sequencing regions flanking the beta-globin gene complex in mouse (Hbbc) and human (HBBC), we have shown that the beta-globin gene cluster is surrounded by a larger cluster of olfactory receptor genes (ORGs). To facilitate sequence comparisons and to investigate the regulation of ORG expression, we have mapped 5' sequences of mRNA from olfactory epithelium encoding beta-globin-proximal ORGs. We have found that several of these genes contain multiple noncoding exons that can be alternatively spliced. Surprisingly, the only common motifs found in the promoters of these genes are a "TATA" box and a purine-rich motif. Sequence comparisons between human and mouse reveal that most of the conserved regions are confined to the coding regions and transcription units of the genes themselves, but a few blocks of conserved sequence also are found outside of ORG transcription units. The possible influence of beta-globin regulatory sequences on ORG expression in olfactory epithelium was tested in mice containing a deletion of the endogenous beta-globin locus control region, but no change in expression of the neighboring ORGs was detected. We evaluate the implications of these results for possible mechanisms of regulation of ORG transcription.
To characterize the protein-DNA interactions important for the developmental control of the human beta-globin locus, we analyzed by in vivo dimethyl sulfate footprinting erythroid cells expressing either the fetal or the adult globin developmental program. In the locus control region (LCR) of the beta-globin locus, in vivo footprints on NF-E2 (or AP-1) and GATA-1 motifs remained the same regardless of whether the fetal or the adult globin genes are expressed. In contrast, in vivo footprints on GT (CACCC) motifs differed between the cells expressing the fetal or the adult globin program. In promoter regions, the actively transcribed genes demonstrated extensive and consistent footprints over the canonical elements, such as CACCC and CCAAT motifs. The adult globin expressing cells displayed more extensive footprints than the fetal globin expressing cells in the 3' regulatory sequences of both the Agamma- and the beta-globin genes, suggesting a role of these 3' elements in beta-globin gene expression. Our results suggest that the bulk of protein-DNA interactions that underlies the developmental control of globin genes takes place in the gamma- and beta-globin gene promoters, and that GT motifs of the beta-globin locus LCR may play a role in the developmental regulation of human beta-globin gene expression, perhaps by increasing the probability of interaction of the LCR holocomplex with the fetal or the adult globin gene.
The human globin genes are among the most extensively characterized in the human genome, yet the details of the molecular events regulating normal human hemoglobin switching and the potential reactivation of fetal hemoglobin in adult hematopoietic cells remain elusive. Recent discoveries demonstrate physical interactions between the beta locus control region and the downstream structural gamma- and beta-globin genes, and with transcription factors and chromatin remodeling complexes. These interactions all play roles in globin gene expression and globin switching at the human beta-globin locus. If the molecular events in hemoglobin switching were better understood and fetal hemoglobin could be more fully reactivated in adult cells, the insights obtained might lead to new approaches to the therapy of sickle cell disease and beta thalassemia by identifying specific new targets for molecular therapies.
Expression of a construct integrated at different genomic locations often varies because of position effects that have been subcategorized as stable (decreased level of expression) and variegating (decreased proportion of expressing cells). It is well established that locus control regions (LCRs) generally overcome position effects in transgenes. However, whether stable and variegated position effects are equally overcome by an intact LCR has not been determined. We report that single-copy yeast artificial chromosome transgenes containing an unmodified human beta -globin locus were not subject to detectable stable position effects but did undergo mild to severe variegating position effects at three of the four non-centromeric integration sites tested. We also find that, at a given integration site, the distance and the orientation of the LCR relative to the regulated gene contributes to the likelihood of variegating position effects, and can affect the magnitude of its transcriptional enhancement. DNase I hypersensitive site (HSS) formation varies with the proportion of expressing cells, not the level of gene expression, suggesting that silencing of the transgene is associated with a lack of HSS formation in the LCR region. We conclude that transcriptional enhancement and variegating position effects are caused by fundamentally different but inter-dependent mechanisms.
DNase I hypersensitive site 2 (HS 2) of the human beta-globin Locus Control Region (LCR) directs high level expression of the beta-globin gene located 50 kilobases downstream. Experiments in cultured cells and in transgenic mice demonstrate that duplicated AP1-like sites in HS 2 are required for this powerful enhancer activity. A cDNA clone encoding a basic, leucine-zipper protein that binds to these sites was isolated and designated Locus Control Region-Factor 1 (LCR-F1). This protein is a member of a new family of regulatory factors that contain a 63 amino acid 'CNC domain' overlapping the basic region. This domain is approximately 70% identical in the Drosophila Cap N Collar (CNC) protein, NF-E2 and LCR-F1. LCR-F1 transactivates an HS 2/gamma-globin reporter gene over 170-fold in transient transfection experiments specifically in erythroid cells. These results suggest that LCR-F1 may be a critical factor involved in LCR-mediated, human globin gene expression.
Biochemical analysis of the beta-globin gene function has led to the identification of several multi-protein complexes at the locus control region (LCR), insulator and promoters. This review briefly summarizes these multi-protein complexes and discusses their contribution towards the regulation of the beta-globin gene expression.
Transcription activation of a gene involves the ordered recruitment of components of the basal transcription machinery in concert with alterations in chromatin structure, including nucleosome remodeling and post-translational modification of histones. Enhancers and locus control regions (LCRs) that are remote from the genes they activate, recruit the complexes that carry out these alterations and, sometimes, recruit RNA polymerase II. The question of how these distant activators interact with their target genes has been of long-standing interest. Recent data indicate that LCRs mediate contact with their coordinate genes through the formation of domains of histone modification and of intra- and inter-chromosomal loops and that they might localize genes within nuclear regions that favor transcription.