Human Beta-globin Locus Control Region Research Articles

Mechanisms of Pol II Recruitment to the Human Beta-Globin Locus Control Region Super-Enhancer

The human ꞵ-globin gene locus consists of five genes that encode subunits of hemoglobin. The locus control region (LCR) regulates these genes, acting as a super-enhancer to promote high-level of β-globin expression in erythroid cells. The LCR, like other super-enhancers, is known to recruit transcription complexes and generate non-coding enhancer RNA (eRNA). Past research has suggested that super-enhancers form phase-separated transcription initiation domains that concentrate RNA polymerase II (Pol II) and transfer Pol II to target gene promoters during transient looping interactions. The aim of this project is to examine the role of TFIIH, involved transcription initiation, in enhancer RNA production and Pol II transfer in the globin gene locus. The LCR was immobilized on magnetic beads and incubated with erythroid nuclear protein extracts. Proteins not bound were washed away and bound proteins were subjected to western blotting experiments. The data show that TFIIH is bound at the LCR and thus may be involved in eRNA production. Cut & Run experiments were performed to examine the localization of TFIIH in the globin gene locus in intact cells. In the future, the laboratory plans to deplete TFIIH and examine production of eRNA and Pol II recruitment at the LCR.

ErythRED, a hESC line enabling identification of erythroid cells

A human embryonic stem cell (hESC) line that enabled globin-expressing cells to be easily recognized would facilitate optimization of erythroid differentiation in vitro and aid in the identification of hESC-derived erythroid cells in transplanted animals. We describe a genetically modified hESC line, ErythRED, in which expression of RFP, controlled by regulatory sequences from the human beta-globin locus control region, is restricted to maturing erythroid cells.

Expression of green fluorescent protein under the regulation of human locus control region elements HS2 and HS3 in transgenic mice

Human beta-globin locus control region (LCR) is composed of five DNase I hypersensitive sites (HSs). We previously demonstrated that when HS2 and HS3 were constructed together in one vector and integrated into one position in transgenic mice, the two cis-elements showed a marked synergy in regulating the spatial and temporal expression of beta-globin transgene. This study is to investigate whether these two elements still show a synergy or superposition when they are not located in one construct. HS2/GFP and HS3/GFP transgenic mice, with the transgene encoding green fluorescent protein (GFP) driven by beta-globin promoter and under the control of HS2 (HS2/GFP mice) and HS3 (HS3/GFP mice), were mated to generate double-heterozygotes (HS2/GFP + HS3/GFP mice). The expression and integration status of GFP genes in transgenic mice were analyzed. Our data showed that there was no difference in the percentages of GFP positive cells in peripheral blood between double-heterozygotes and their corresponding founders (HS2/GFP or HS3/GFP). Interestingly, it was observed that exogenous GFP genes were highly expressed in another transgenic mouse pedigree with HS2 element integrated twice into the same chromosome at two different loci. However, when one site was lost in some offspring because of recombination, the percentages of GFP positive cells in peripheral blood as well as in some erythroid tissues were decreased obviously. These results demonstrated that HS2 and HS3 elements would not show any synergy or superposition if they were located on different chromosomes in transgenic mice, and that HS2 element containing the core and flanking sequences might not be able to overcome the position effects and control position dependent expression of linked genes in transgenic mice.

Improved Human β-globin Expression from Self-inactivating Lentiviral Vectors Carrying the Chicken Hypersensitive Site-4 (cHS4) Insulator Element

Effective gene therapy for beta-thalassemia major (beta-TM) requires consistent, high expression of human beta-globin (hbeta-globin) in red blood cells (RBCs). Several groups have now shown that lentiviral (LV) vectors stably transmit the hbeta/hgamma-globin genes and large elements of the locus control region, resulting in correction of the murine thalassemia intermedia (TI) phenotype and survival of mice with the TM phenotype. However, current LVs show variable hbeta/hgamma-globin expression and require a high number of vector copies/cell for a therapeutic effect. To address this, we designed LVs flanked by the chicken hypersensitive site-4 (cHS4) chromatin insulator element and compared them with their "un-insulated" counterparts. We observed a consistent twofold-higher hbeta expression from insulated vectors in single-copy mouse erythroleukemia cell clones, an increase that resulted from reduced position effect variegation (PEV) and increased probability of expression from individual integrants. This effect was confirmed in vivo: an approximately twofold increase in hbeta expression was seen in the RBC progeny of murine hematopoietic stem cells, with significantly higher numbers of hbeta-expressing cells in individual secondary spleen colony-forming units. In summary, cHS4-insulated hbeta-globin LVs showed distinct chromatin barrier activity, resulting in higher, consistent hbeta expression. These studies have important implications for vector design for clinical trials for gene therapy for hemoglobinopathies.

Nucleosome and transcription activator antagonism at human β-globin locus control region DNase I hypersensitive sites

Locus control regions are regulatory elements that activate distant genes and typically consist of several DNase I hypersensitive sites coincident with clusters of transcription activator binding sites. To what extent nucleosomes and activators occupy these sites together or exclusively has not been extensively studied in vivo. We analyzed the chromatin structure of human β-globin locus control region hypersensitive sites in erythroid cells expressing embryonic and fetal globin genes. Nucleosomes were variably depleted at hypersensitive sites HS1-HS4 and at HS5 which flanks the 5′ of the locus. In lieu of nucleosomes, activators were differentially associated with these sites. Erythroid–specific GATA-1 resided at HS1, HS2 and HS4 but the NF-E2 hetero-dimer was limited to HS2 where nucleosomes were most severely depleted. Histones H3 and H4 were hyperacetylated and H3 was di-methylated at K4 across the LCR, however, the H3 K4 MLL methyltransferase component Ash2L and histone acetyltransferases CBP and p300 occupied essentially only HS2 and the NF-E2 motif in HS2 was required for Ash2L recruitment. Our results indicate that each hypersensitive site in the human β-globin LCR has distinct structural features and suggest that HS2 plays a pivotal role in LCR organization at embryonic and fetal stages of globin gene expression.

Enhancer Blocking by Chicken β-Globin 5′-HS4: ROLE OF ENHANCER STRENGTH AND INSULATOR NUCLEOSOME DEPLETION

The 5'-HS4 chicken beta-globin insulator functions as a positional enhancer blocker on chromatinized episomes in human cells, blocking the HS2 enhancer of the human beta-globin locus control region from activating a downstream epsilon-globin gene. 5'-HS4 interrupted formation of a domain of histone H3 and H4 acetylation encompassing the 6-kb minilocus and inhibited transfer of RNA polymerase from the enhancer to the gene promoter. We found that the enhancer blocking phenotype was amplified when the insulated locus contained a weakened HS2 enhancer in which clustered point mutations eliminated interaction of the transcription factor GATA-1. The GATA-1 mutation compromised recruitment of histone acetyltransferases and RNA polymerase II to HS2. Enhancer blocking correlated with a significant depletion of nucleosomes in the core region of the insulator as revealed by micrococcal nuclease and DNase I digestion studies. Nucleosome depletion at 5'-HS4 was dependent on interaction of the insulator protein CCCTC-binding factor (CTCF) and was required for enhancer blocking. These findings provide evidence that a domain of active chromatin is formed by spreading from an enhancer to a target gene and can be blocked by a nucleosome-free gap in an insulator.

An insulator blocks spreading of histone acetylation and interferes with RNA polymerase II transfer between an enhancer and gene.

We studied the mechanism by which an insulator interrupts enhancer signaling to a gene using stably replicated chromatin templates containing the human beta-globin locus control region HS2 enhancer and a target globin gene. The chicken beta-globin 5' HS4 (cHS4) insulator acted as a positional enhancer blocker, inhibiting promoter remodeling and transcription activation only when placed between the enhancer and gene. Enhancer blocking by cHS4 reduced histone hyperacetylation across a zone extending from the enhancer to the gene and inhibited recruitment of CBP and p300 to HS2. Enhancer blocking also led to accumulation of RNA polymerase II at HS2 and within cHS4, accompanied by its diminution at the gene promoter. The enhancer blocking effects were completely attributable to the CTCF binding site in cHS4. These findings provide experimental evidence for the involvement of spreading in establishment of a broad zone of histone modification by an enhancer, as well as for blocking by an insulator of the transfer of RNA polymerase II from an enhancer to a promoter.

Chromatin structure at the flanking regions of the human beta-globin locus control region DNase I hypersensitive site-2: proposed nucleosome positioning by DNA-binding proteins including GATA-1

The human beta-globin locus control region DNase I hypersensitive site-2 (LCR HS-2) is erythroid-specific and is located 10.9 kb upstream of the epsilon-globin gene. Most studies have only examined the core region of HS-2. However, previous studies in this laboratory indicate that positioned nucleosomes are present at the 5′- and 3′-flanking regions of HS-2. In addition, footprints were observed that indicated the involvement of DNA-binding proteins in positioning the nucleosome cores. A consensus GATA-1 site exists in the region of the 3′-footprint. In this study, using an electrophoretic mobility shift assay (EMSA) and DNase I footprinting, we confirmed that GATA-1 binds in vitro at the 3′-end of HS-2. An additional GATA-1 site was found to bind GATA-1 in vitro at a site positioned 40 bp upstream. At the 5′-end of HS-2, DNase I footprinting revealed a series of footprints showing a marked correlation with the in vivo footprints. EMSA indicated the presence of several erythroid-specific complexes in this region including GATA-1 binding. Sequence alignment for 12 mammalian species in HS-2 confirmed that the highest conservation to be in the HS-2 core. However, a second level of conservation extends from the core to the sites of the proposed positioning proteins at the HS-2 flanking regions, before declining rapidly. This indicates the importance of the HS-2 flanking regions and supports the proposal of nucleosome positioning proteins in these regions.

Human beta-globin locus control region HS5 contains CTCF- and developmental stage-dependent enhancer-blocking activity in erythroid cells.

The human beta-globin locus contains five developmentally regulated beta-type globin genes. All five genes depend on the locus control region (LCR), located at the 5' end of the locus, for abundant globin gene transcription. The LCR is composed of five DNase I-hypersensitive sites (HSs), at least a subset of which appear to cooperate to form a holocomplex in activating genes within the locus. We previously tested the requirement for proper LCR polarity by inverting it in human beta-globin yeast artificial chromosome transgenic mice and observed reduced expression of all the beta-type globin genes regardless of developmental stage. This phenotype clearly demonstrated an orientation-dependent activity of the LCR, although the mechanistic basis for the observed activity was obscure. Here, we describe genetic evidence demonstrating that human HS5 includes enhancer-blocking (insulator) activity that is both CTCF and developmental stage dependent. Curiously, we also observed an attenuating activity in HS5 that was specific to the epsilon-globin gene at the primitive stage and was independent of the HS5 CTCF binding site. These observations demonstrate that the phenotype observed in the LCR-inverted locus was in part attributable to placing the HS5 insulator between the LCR HS enhancers (HS1 to HS4) and the promoter of the beta-globin gene.

Hairpin-duplex equilibrium reflected in the A-->B transition in an undecamer quasi-palindrome present in the locus control region of the human beta-globin gene cluster.

Our recent work on an A-->G single nucleotide polymorphism (SNP) at the quasi-palindromic sequence d(TGGGG[A/G]CCCCA) of HS4 of the human beta-globin locus control region in an Indian population showed a significant association between the G allele and the occurrence of beta-thalassemia. Using UV-thermal denaturation, gel assay, circular dichroism (CD) and nuclease digestion experiments we have demonstrated that the undecamer quasi- palindromic sequence d(TGGGGACCCCA) (HPA11) and its reported polymorphic (SNP) version d(TGG GGGCCCCA) (HPG11) exist in hairpin-duplex equilibria. The biphasic nature of the melting profiles for both the oligonucleotides persisted at low as well as high salt concentrations. The HPG11 hairpin showed a higher T(m) than HPA11. The presence of unimolecular and bimolecular species was also shown by non-denaturating gel electrophoresis experiments. The CD spectra of both oligonucleotides showed features of the A- as well as B-type conformations and, moreover, exhibited a concentration dependence. The disappearance of the 265 nm positive CD signal in an oligomer concentration-dependent manner is indicative of an A-->B transition. The results give unprecedented insight into the in vitro structure of the quasi-palindromic sequence and provide the first report in which a hairpin-duplex equilibrium has been correlated with an A-->B interconversion of DNA. The nuclease-dependent degradation suggests that HPG11 is more resistant to nuclease than HPA11. Multiple sequence alignment of the HS4 region of the beta-globin gene cluster from different organisms revealed that this quasi-palindromic stretch is unique to Homo sapiens. We propose that quasi-palindromic sequences may form stable mini- hairpins or cruciforms in the HS4 region and might play a role in regulating beta-globin gene expression by affecting the binding of transcription factors.

The ERV-9 LTR enhancer is not blocked by the HS5 insulator and synthesizes through the HS5 site non-coding, long RNAs that regulate LTR enhancer function.

A solitary long terminal repeat (LTR) of ERV-9 human endogenous retrovirus is located upstream of the HS5 site in the human beta-globin locus control region and possesses unique enhancer activity in erythroid K562 cells. In cells transfected with plasmid LTR-HS5-epsilonp-GFP, the LTR enhancer activates the GFP reporter gene and is not blocked by the interposed HS5 site, which has been reported to have insulator function. The LTR enhancer initiates synthesis of long RNAs from the LTR promoter through the intervening HS5 site into the epsilon-globin promoter and the GFP gene. Synthesis of the sense, long LTR RNAs is correlated with high level synthesis of GFP mRNA from the epsilon-globin promoter. Mutations of the LTR promoter and/or the epsilon-globin promoter show that (i) the LTR enhancer can autonomously initiate synthesis of LTR RNAs independent of the promoters and (ii) the LTR RNAs are not processed into GFP mRNA or translated into GFP. However, reversing the orientation of the LTR in plasmid (LTR)rev-HS5-epsilonp-GFP, thus reversing the direction of synthesis of LTR RNAs in the antisense direction away from the epsilon-globin promoter and GFP gene drastically reduces the level of GFP mRNA and thus LTR enhancer function. The results suggest that the LTR-assembled transcription machinery in synthesizing non-coding, LTR RNAs can reach the downstream epsilon-globin promoter to activate transcription of the GFP gene.

Interaction of NF-E2 in the Human β-Globin Locus Control Region before Chromatin Remodeling

When transcription is initiated under repressive conditions, such as when chromatin are packed together, binding followed by the functioning of key components in the transcriptional apparatus should be appropriately facilitated in the chromatin architecture. We provide evidence that the erythroid-specific enhancer- binding protein NF-E2 interacts with the cognate motif at DNase I-hypersensitive site 2 of the human beta-globin locus control region in a repressive state. The nucleosome containing the NF-E2-binding site showed characteristic rotational and translational phases in vitro. The binding site had less affinity to the histone octamers than nearby regions while showing greater accessibility to DNase I and micrococcal nuclease. Furthermore, the motif was recognized by the exogenous NF-E2 protein expressed in HeLa cells, which have a repressive state of chromatin at the beta-globin locus, as shown by ligation-mediated PCR and chromatin immunoprecipitation assay. These lines of evidence indicate that NF-E2 interacts with the cognate motif on the nucleosome before chromatin is remodeled.

The interaction between the human beta-globin locus control region and nuclear matrix.

Our previous study showed that hydroxyurea (Hu) could induce HEL cells to express human beta-globin gene. However the molecular mechanisms by which the expression of beta-globin gene is activated and regulated are poorly understood. Here we show that the binding patterns between the core DNA sequences (HS2 core sequence -10681 approximately -10971 bp, HS3 core sequence -14991 approximately -14716 bp and HS4 core sequence -18586 approximately -18306 bp) of DNase I hypersensitive sites in the human beta-globin LCR and nuclear matrix proteins isolated from Hu induced and uninduced HEL cells are quite different. Results demonstrated that nuclear matrix proteins might play important roles in regulating the expression of human beta-like globin genes through their interaction with HSs (HS2, HS3 and HS4 core sequences) in the LCR. Moreover, the results obtained from the in vitro DNA-matrix binding assay showed that the core DNA sequences of DNase I hypersensitive sites (HS2, HS3 and HS4) were unable to bind to the nuclear matrix isolated from uninduced HEL cells; in addition, HS2 core DNA sequence was capable of binding to the nuclear matrix prepared from Hu-induced HEL cells, while both HS3 and HS4 core DNA sequences could not do so. Results indicated that the HS2 core DNA sequence may be a functional MAR (matrix attachment region). We suggest that the HS2 core DNA sequence binding to the nuclear matrix in Hu-induced HEL cells may open the structure of chromatin to make the LCR accessible to the promoter of beta-globin gene and to promote its transcription.

Evidence that DNase I hypersensitive site 5 of the human beta-globin locus control region functions as a chromosomal insulator in transgenic mice.

We have previously reported that DNase I hypersensitive site 5 (5'HS5) of the human beta-globin locus control region functions as a chromatin insulator in stable transfection assays. In this report we show that a 3.2 kb DNA fragment containing the entire 5'HS5 region can protect a position-sensitive (A)gamma-globin gene against position effects in transgenic mice. Bracketing is required for function of 5'HS5 as an insulator. The 5'HS5 insulator operates in adult as well as in embryonic murine erythroid cells. The insulator has no significant stimulatory effects of its own. These results indicate that 5'HS5 can function as a chromatin insulator in vivo.

Study of the single nucleotide polymorphism (SNP) at the palindromic sequence of hypersensitive site (HS)4 of the human beta-globin locus control region (LCR) in Indian population.

LCR, a genetic regulatory element, was examined in beta-thalassemia patients who do not show any mutation in the beta-globin genes. We sequenced LCR-HS2, HS3, and HS4 in samples from 16 such patients from the Indian population and found only one SNP A-G in the inverted repeat in HS4. A significant association was observed between the G allele and occurrence of beta-thalassemia by Fisher's exact test. The AG and GG genotypes showed higher relative risk as compared to the AA genotype. We also observed linkage disequilibrium between the A/G polymorphism and the AT-rich motif of the LCR HS2 region, suggesting that the G allele could be an evolutionarily new mutation in the study population.

Enhancer activity of HS2 of the human beta-LCR is modulated by distance from the key nucleosome.

A class of curved DNA appears universally in eukaryotic genomic DNA at an average distance of approximately 680 bp and shows nucleosome positioning activity by having high affinity for histone core particles in an orientation- and position-dependent manner. Here, we report that the enhancer activity at DNase I hypersensitive site 2 (HS2) of the human beta-globin locus control region (beta-LCR) can be modulated by the curved DNA located at a distance of two nucleosomes from HS2 and that the nucleosome at the curved DNA regulates nearby nucleosome phases as a key nucleosome. Erythroid-specific nucleosome phases which caused deviation of the NF-E2 (p18-p45 dimer) binding site from the nucleosome dyad axis were over-represented when the distance between the key nucleosome and HS2 exceeded 80 bp longer than the original length. At this state, enhancer activity was approximately 50% of that in the original construct, presumably due to reduced binding of transcription factors.

A (GATA)(7) motif located in the 5' boundary area of the human beta-globin locus control region exhibits silencer activity in erythroid cells.

A 40-bp DNA, consisting of seven tandem GATA repeats, is located near the HS5 site in the 5' boundary area of the locus control region (LCR) of human beta-globin gene. This (GATA)(7) motif, named 5a, exhibits silencer activity in erythroid cells. In transfected, recombinant plasmids containing the chloramphenicol acetyltransferase (CAT) reporter gene, 5a repressed the activity of the cis-linked housekeeping phosphoglycerate kinase (pgk) promoter; 5a also repressed the activity of the cis-linked HS2 enhancer regardless of whether the CAT gene was driven by the pgk or the epsilon-globin promoter. Repression by 5a was most severe when 5a was spliced upstream of HS2 at a distance of less than 200 bases from the HS2 enhancer core. The silencer activity of 5a was independent of whether the component GATA motifs were in head to tail orientation as in the wild type 5a or in head to head or tail to tail orientation as in a mutant 5a. Band shift experiments show that the GATA-1 protein binds to both 5a and the mutant 5a and forms a large protein complex. Together, the results suggest that GATA-1 bound at 5a is a strong, proximal repressor of HS2 enhancer activity.

Expression of beta-globin in primary erythroid progenitors of beta-thalassemia patients using an SV40-based gene delivery system.

SV40-based vectors are very efficient in gene delivery into human hematopoietic cells. In the present work, we investigated the expression of constructs carrying the human beta-globin gene that were delivered as beta-globin pseudovirions. Expression studies were performed by RNA analysis of primary human erythroid progenitors cultivated from peripheral blood of beta(0)-thalassemia patients who are unable to produce normal beta-globin RNA. This erythroid culture system recapitulates in vitro the process of growth, differentiation, and maturation of authentic erythroid precursors. The progenitors were induced to differentiate by the addition of erythropoietin (EPO). Five days later, the cells were infected with pseudovirions containing the normal beta-globin gene, and RNA was harvested on day 8. The results showed significant levels of normal beta-globin gene mRNA. A small DNA fragment derived from the 5'-region of the HSII element of the human beta-globin locus control region (LCR) enhanced expression of the linked beta-globin gene 20-30-fold. Normal beta-globin mRNA expression was in direct correlation to the multiplicity of infection. These studies suggest the potential feasibility of using the beta-globin delivery system for gene therapy of beta-thalassemia.

Long range interaction of cis-DNA elements mediated by architectural transcription factor Bach1.

A central question in vertebrate transcriptional regulation is how cis-regulatory modules, including enhancers, silencers and promoters, communicate with each other over long distances to mandate proper gene expression. In order to address this question we analysed protein/DNA interactions in the human beta-globin locus control region (LCR). One of the many proteins that are potentially implicated in LCR function is Bach1. Bach1 possesses a basic leucine zipper (bZip) domain, as well as a BTB/POZ domain that has been shown to be involved in the regulation of chromatin structure. Bach1 forms heterodimers with small Maf proteins through its leucine zipper and binds to Maf recognition elements (MARE). Using atomic force microscopy we visualized large looped DNA structures between MAREs located in different regulatory elements within the human beta-globin LCR that were mediated by Bach1/MafK heterodimers. The formation of these DNA loops required the Bach1 BTB/POZ protein interaction domain. Furthermore, in transfection studies we found that Bach1 repressed the enhancer activity of the LCR in a BTB/POZ domain-dependent manner. Our results suggest that Bach1 and other BTB/POZ transcription factors may represent a class of nuclear architectural proteins that mediate long range interactions between cis-regulatory elements in order to regulate gene expression.

The polyoma virus enhancer cannot substitute for DNase I core hypersensitive sites 2-4 in the human beta-globin LCR.

The polyoma virus enhancer (PyE) is capable of conferring integration position-independent expression to linked genes in stably transfected erythroid cells after joining to DNase I hypersensitive site (HS) 5 of the human beta-globin locus control region (LCR). In attempting to separate the chromatin opening activity of the LCR from its enhancer activity and to investigate contributions of the individual HS core elements to LCR function, the human beta-globin LCR HS2, HS3 and HS4 core elements were replaced with the PyE within the context of a yeast artificial chromosome (YAC) bearing the whole locus. We show here that, in contrast to its function in cultured cells, the PyE is unable to replace HS core element function in vivo. We found that the PyE substitution mutant LCR is unable to provide either chromatin opening or transcriptional potentiating activity at any erythroid developmental stage in transgenic mice. These data provide direct evidence that the human beta-globin LCR core elements specify unique functions that cannot be replaced by a ubiquitous enhancer activity.