Human Beta-globin Locus Research Articles

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.

The dynamics of globin gene expression and position effects.

We have used gene competition to study the regulation of the human beta-globin locus in transgenic mice as a model system of a multigene locus. The locus is regulated by the locus control region (LCR), which is required for the expression of all the genes. Analysis of the locus at the single-cell level shows that the LCR appears to interact directly with the genes via a looping mechanism. This interaction is monogenic, and the level of transcription is determined by the frequency and stability of LCR/gene complex formation. These parameters are dependent both on the distance between the LCR and gene(s), and the concentration of transcription factors in the nucleus. Disturbance of complex formation leads to position effects, particularly when the locus is integrated in a heterochromatic environment.

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.

Increased γ‐globin gene expression in β‐thalassemia intermedia patients correlates with a mutation in 3′HS1

We report a novel set of genetic markers in the DNaseI hypersensitive sites comprising the human beta-globin locus chromatin hub (CH), namely HS-111 and 3'HS1. The HS-111 (-21 G>A) and 3'HS1 (+179 C>T) transitions form CH haplotypes, which occur at different frequencies in beta-thalassemia intermedia and major patients and normal (nonthalassemic) individuals. We also show that the 3'HS1 (+179 C>T) variation results in a GATA-1 binding site and correlates with increased fetal hemoglobin production in beta-thalassemia intermedia patients. In contrast, the HS-111 (+126 G>A) transition, found in three normal chromosomes, is simply a rare polymorphism. We conclude that the CH haplotypes are useful genetic determinants for beta-thalassemia major and intermedia patients, while the 3'HS1 (+179 C>T) mutation may have functional consequences in gamma-globin genes expression.

Intergenic Transcription, Cell-Cycle and the Developmentally Regulated Epigenetic Profile of the Human Beta-Globin Locus

Several lines of evidence have established strong links between transcriptional activity and specific post-translation modifications of histones. Here we show using RNA FISH that in erythroid cells, intergenic transcription in the human β-globin locus occurs over a region of greater than 250 kb including several genes in the nearby olfactory receptor gene cluster. This entire region is transcribed during S phase of the cell cycle. However, within this region there are ∼20 kb sub-domains of high intergenic transcription that occurs outside of S phase. These sub-domains are developmentally regulated and enriched with high levels of active modifications primarily to histone H3. The sub-domains correspond to the β-globin locus control region, which is active at all developmental stages in erythroid cells, and the region flanking the developmentally regulated, active globin genes. These results correlate high levels of non-S phase intergenic transcription with domain-wide active histone modifications to histone H3.

Interactions between replication and chromatin condensation in the human beta-globin locus

Interactions between replication and chromatin condensation in the human beta-globin locus

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.

Chromosome Conformation Capture Carbon Copy (5C): a massively parallel solution for mapping interactions between genomic elements.

Physical interactions between genetic elements located throughout the genome play important roles in gene regulation and can be identified with the Chromosome Conformation Capture (3C) methodology. 3C converts physical chromatin interactions into specific ligation products, which are quantified individually by PCR. Here we present a high-throughput 3C approach, 3C-Carbon Copy (5C), that employs microarrays or quantitative DNA sequencing using 454-technology as detection methods. We applied 5C to analyze a 400-kb region containing the human beta-globin locus and a 100-kb conserved gene desert region. We validated 5C by detection of several previously identified looping interactions in the beta-globin locus. We also identified a new looping interaction in K562 cells between the beta-globin Locus Control Region and the gamma-beta-globin intergenic region. Interestingly, this region has been implicated in the control of developmental globin gene switching. 5C should be widely applicable for large-scale mapping of cis- and trans- interaction networks of genomic elements and for the study of higher-order chromosome structure.

Cooperative sequence modules determine replication initiation sites at the human β-globin locus

The human beta globin locus contains two adjacent replicators, each capable of initiating DNA replication when transferred from its native locus to ectopic sites. Here, we report a detailed analysis of the sequence requirements for replication initiation from these replicators. In both replicators, initiation required a combination of an asymmetric purine:pyrimidine sequence and several AT-rich stretches. Modules from the two replicators could combine to initiate replication. AT-rich sequences were essential for replicator activity: a low frequency of initiation was observed in DNA fragments that included a short stretch of AT-rich sequences, whereas inclusion of additional AT-rich stretches increased initiation efficiency. By contrast, replication initiated at a low level without the asymmetric purine:pyrimidine modules but they were required in synergy to achieve efficient initiation. These data support a combinatorial model for replicator activity and suggest that the initiation of DNA replication requires interaction between at least two distinct sequence modules.

Lineage-specific activators affect β-globin locus chromatin in multipotent hematopoietic progenitors

During development, the regulated expression of tissue-specific genes can be preceded by their potentiation, that is, by chromatin activation in progenitor cells. For example, the human beta-like globin genes are potentiated in a gene- and developmental-specific manner in hematopoietic progenitors. Developmental regulation of human beta-gene expression in erythroid cells is mostly determined by transcriptional activators; however, it is not clear how gene-specific potentiation is set in hematopoietic progenitors. Using human and transgenic multipotent hematopoietic progenitors, we demonstrate that human beta-globin locus activation is characterized by TBP, NF-E2, CBP and BRG1 recruitment at both the Locus Control Region and human beta-gene promoter. Our results further indicate that in hematopoietic progenitors, EKLF influences chromatin organization at the human beta-globin locus and is instrumental for human beta-gene potentiation. Thus, we show that lineage-specific transcriptional activators expressed at basal levels in progenitor cells can participate in gene potentiation.

Two types of precursor cells in a multipotential hematopoietic cell line.

The biochemistry of early stages of hematopoietic differentiation is difficult to study because only relatively small numbers of precursor cells are available. The murine EML cell line is a multipotential cell line that can be used to model some of these steps. We found that the lineage- EML precursor cells can be separated into two populations based on cell surface markers including CD34. Both populations contain similar levels of stem cell factor (SCF) receptor (c-Kit) but only the CD34+ population shows a growth response when treated with SCF. Conversely, the CD34- population will grow in the presence of the cytokine IL-3. The human beta-globin locus control region hypersensitive site 2 plays different roles on beta-globin transcription in the CD34+ and CD34- populations. The two populations are present in about equal amounts in culture, and the CD34+ population rapidly regenerates the mixed population when grown in the presence of SCF. We suggest that this system may mimic a normal developmental transition in hematopoiesis.

Synergistic and Additive Properties of the Beta-Globin Locus Control Region (LCR) Revealed by 5′HS3 Deletion Mutations: Implication for LCR Chromatin Architecture

Deletion of the 234-bp core element of the DNase I hypersensitive site 3 (5'HS3) of the locus control region (LCR) in the context of a human beta-globin locus yeast artificial chromosome (beta-YAC) results in profound effects on globin gene expression in transgenic mice. In contrast, deletion of a 2.3-kb 5'HS3 region, which includes the 234-bp core sequence, has a much milder phenotype. Here we report the effects of these deletions on chromatin structure in the beta-globin locus of adult erythroblasts. The 234-bp 5'HS3 deletion abolished histone acetylation throughout the beta-globin locus; recruitment of RNA polymerase II (pol II) to the LCR and beta-globin gene promoter was reduced to a basal level; and formation of all the 5' DNase I hypersensitive sites of the LCR was disrupted. The 2.3-kb 5'HS3 deletion mildly reduced the level of histone acetylation but did not change the profile across the whole locus; the 5' DNase I hypersensitive sites of the LCR were formed, but to a lesser extent; and recruitment of pol II was reduced, but only marginally. These data support the hypothesis that the LCR forms a specific chromatin structure and acts as a single entity. Based on these results we elaborate on a model of LCR chromatin architecture which accommodates the distinct phenotypes of the 5'HS3 and HS3 core deletions.

Site-specific Acetylation of the Fetal Globin Activator NF-E4 Prevents Its Ubiquitination and Regulates Its Interaction with the Histone Deacetylase, HDAC1

Acetylation provides one mechanism by which the functional diversity of individual transcription factors can be expanded. This is valuable in the setting of complex multigene loci that are regulated by a limited number of proteins, such as the human beta-globin locus. We have studied the role of acetylation in the regulation of the transcription factor NF-E4, a component of a protein complex that facilitates the preferential expression of the human gamma-globin genes in fetal erythroid cells. We have shown that NF-E4 interacts directly with, and serves as a substrate for, the acetyltransferase co-activator PCAF. Acetylation of NF-E4 is restricted to a single residue (Lys(43)) in the amino-terminal domain of the protein and results in two important functional consequences. Acetylation of NF-E4 prolongs the protein half-life by preventing ubiquitin-mediated degradation. This stabilization is PCAF-dependent, since enforced expression in fetal/erythroid cells of a mutant form of PCAF lacking the histone acetyltransferase domain (PCAFDeltaHAT) decreases NF-E4 stability. Acetylation of Lys(43) also reduces the interaction between NF-E4 and HDAC1, potentially maximizing the activating ability of the factor at the gamma-promoter. These results provide further demonstration that co-activators, such as PCAF, can influence individual transcription factor properties at multiple levels to alter their effects on gene expression.

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.

The Role of p22 NF-E4 in Human Globin Gene Switching

The human stage selector protein, a complex containing the ubiquitous transcription factor CP2 and the erythroid-specific factor p22 NF-E4, facilitates the interaction of the gamma-globin genes with the locus control region in fetal erythroid cells. Enforced expression of p22 NF-E4 in K562 cells and human cord blood progenitors increases fetal globin gene expression, and in progenitors, reduces beta-globin expression. To examine the role of NF-E4 in an in vivo model of hemoglobin switching, we enforced the expression of p22 NF-E4 in transgenic mice carrying the human beta-globin locus yeast artificial chromosome. Although murine erythropoiesis and globin gene expression is unaffected in these mice, the expression profile of the human globin genes is altered. All three transgenic lines displayed an increased gamma:beta-globin ratio in E12.5-14.5 fetal liver, resulting in a delay in the fetal/adult switch. At E12.5, this is primarily due to a reduction of beta-gene expression, whereas at E14.5, the increased gamma:beta ratio is due to enhanced gamma-gene expression. Despite this, the switch in globin subtype is fully completed in the adult bone marrow. These findings indicate that p22 NF-E4 is capable of influencing human globin gene expression in vivo but is incapable of overriding the intrinsic mechanisms governing gamma-gene silencing in this context.

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.

The mammalian beta globin origin of DNA replication.

Initiation of DNA replication is a tightly regulated process aimed to insure that the entire genome is replicated at the appropriate time during each cell cycle. In the human beta globin locus, replication initiates from a region between the two genes that encode the adult subunit of hemoglobin (the beta globin initiation region, or IR). Mammalian beta globin loci replicate early during the S phase of the cell cycle in pre erythroid cells, in which the beta-globin locus is present in a euchromatin form. However, in cells that do not express globin and in which the locus is heterochromatic, these same loci replicate during the later stages of S phase. Both early and late replication patterns utilize similar replication initiation regions. These features make the beta globin locus an attractive model for studying the determinants of replication sites and replication timing, as well as the correlation between gene expression and DNA replication. Two genomic domains are essential for initiation of DNA replication within the locus: the initiation region (IR), and a 40 kb region upstream of the globin gene cluster known as the locus control region (LCR). The IR meets the genetic requirements for a chromosomal replicator, since it can initiate DNA replication at ectopic sites. The LCR regulates transcriptional activity and chromatin structure, and may act as a determinant of replication timing. This review will summarize recent findings characterizing the sequence requirements for initiation of DNA replication in mammalian beta globin loci and will discuss the specific influence of the location and the chromosomal environment in regulating DNA replication at the beta globin IR.

Chromatin remodelling and the interaction between enhancers and promoters in the beta-globin locus.

The beta-globin genes are among the most studied families of tissue-specific and developmentally regulated genes. Even before genome-wide sequencing was a reality, the sequences of extensive regions of the human, rabbit and mouse beta-globin loci were available through the individual efforts of several research groups. Many of the genomic approaches now being pursued in order to understand the control of gene expression were applied early to this gene family with success. Higher levels of regulation attributable to networks of transcription factor interaction, chromatin remodelling and enhancer-promoter communication remain as challenges in the post-genomic era.

Rapid isolation of yeast genomic DNA: Bust n' Grab

BackgroundMutagenesis of yeast artificial chromosomes (YACs) often requires analysis of large numbers of yeast clones to obtain correctly targeted mutants. Conventional ways to isolate yeast genomic DNA utilize either glass beads or enzymatic digestion to disrupt yeast cell wall. Using small glass beads is messy, whereas enzymatic digestion of the cells is expensive when many samples need to be analyzed. We sought to develop an easier and faster protocol than the existing methods for obtaining yeast genomic DNA from liquid cultures or colonies on plates.ResultsRepeated freeze-thawing of cells in a lysis buffer was used to disrupt the cells and release genomic DNA. Cell lysis was followed by extraction with chloroform and ethanol precipitation of DNA. Two hundred ng – 3 μg of genomic DNA could be isolated from a 1.5 ml overnight liquid culture or from a large colony. Samples were either resuspended directly in a restriction enzyme/RNase coctail mixture for Southern blot hybridization or used for several PCR reactions. We demonstrated the utility of this method by showing an analysis of yeast clones containing a mutagenized human β-globin locus YAC.ConclusionAn efficient, inexpensive method for obtaining yeast genomic DNA from liquid cultures or directly from colonies was developed. This protocol circumvents the use of enzymes or glass beads, and therefore is cheaper and easier to perform when processing large numbers of samples.

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.