Enhancer-blocking Elements Research Articles

Erythroid Specificity and Safety of Globin-Encoding Lentiviral Vectors

The beta-thalassemias and sickle cell disease are the most common monogenic inherited blood disorders, both arising from mutations affecting the beta-globin locus. Recent clinical trials utilizing lentiviral-mediated beta-globin gene transfer in autologous CD34+ cells have shown encouraging results in patients with non-beta-zero thalassemias and sickle cell anemia. A case report of insertional mutagenesis in a beta-thalassemia patient resulted in a prolonged clonal expansion that eventually regressed without progressing to leukemic transformation. Nonetheless, this occurrence and the need to insert multiple vector copies per cell when using globin vectors that provide insufficient globin expression to achieve curative responses from a single integrated copy per cell, raise some questions about the erythroid specificity and safety of globin vectors. We thus found it imperative to investigate globin vector expression in hematopoietic progenitors and in non-erythroid cells. To this end, we investigated what regulatory elements, including locus control region (LCR) hypersensitive sites (HS) and others, achieve the highest beta-globin expression per vector copy while at the same time minimizing non-erythroid transcriptional activity. We developed an invivo assay to track the enhancer/promoter activity of different HS elements in hematopoietic progenitor and differentiated cell subsets. We designed lentiviral vectors expressing hrGFP under the control of a short β-globin promoter (137bp) controlled by a set of LCR HS elements and/or a novel biliverdin (BLV) enhancer. Analysis of hrGFP expression in bone marrow cells derived from C57BL6 mice transplanted with transduced syngeneic bone marrow cells revealed that many of the vectors encompassing elements thought to encode erythroid-specific elements were in fact expressed in long term-HSC (LT-HSC), short term-HSC (ST-HSC), multipotent progenitors (MPP), pre-GM, Granulocyte/macrophage progenitors (GMP) and megakaryocyte progenitors (MKP). These findings confirmed that ectopically integrated erythroid regulatory elements can serve as transcriptional enhancers in non-erythroid cells. This transcriptional leakiness was confirmed in therapeutic globin vectors harboring a combination of LCR elements. To avoid these effects, we flanked the globin transcription unit with a small human enhancer-blocking element called A1, kindly provided by the late George Stamatoyannopoulos (Liu, M. et al. 2015). The insulated vector showed markedly reduced non-erythroid expression in non-erythroid cells including ST-HSC, MPP, Pre-GM and GMP. Expression in mature erythroid cells was unchanged. Our findings underscore the benefits of selecting lineage-specific regulatory elements with the least lineage promiscuity for therapeutic vectors, with the eventual addition of enhancer-blocking elements. Based on these results, we are now investigating new globin expression vectors with optimal combination of HS elements. Such novel globin vectors should minimize the risk of oncogene trans-activation in hematopoietic progenitor cells and therefore improve the safety of globin gene therapy while providing lineage-specific high-level expression. Disclosures Sadelain: Mnemo: Patents & Royalties; Fate Therapeutics: Patents & Royalties, Research Funding; Atara: Patents & Royalties, Research Funding; Takeda: Patents & Royalties, Research Funding; Minerva: Other: Biotechnologies , Patents & Royalties.

The Mouse Immunoglobulin Heavy Chain V-D Intergenic Sequence Contains Insulators That May Regulate Ordered V(D)J Recombination

During immunoglobulin heavy chain (Igh) V(D)J recombination, D to J precedes V to DJ recombination in an ordered manner, controlled by differential chromatin accessibility of the V and DJ regions and essential for correct antibody assembly. However, with the exception of the intronic enhancer Emu, which regulates D to J recombination, cis-acting regulatory elements have not been identified. We have assembled the sequence of a strategically located 96-kb V-D intergenic region in the mouse Igh and analyzed its activity during lymphocyte development. We show that Emu-dependent D antisense transcription, proposed to open chromatin before D to J recombination, extends into the V-D region for more than 30 kb in B cells before, during, and after V(D)J recombination and in T cells but terminates 40 kb from the first V gene. Thus, subsequent V antisense transcription before V to DJ recombination is actively prevented and must be independently activated. To find cis-acting elements that regulate this differential chromatin opening, we identified six DNase I-hypersensitive sites (HSs) in the V-D region. One conserved HS upstream of the first D gene locally regulates D genes. Two further conserved HSs near the D region mark a sharp decrease in antisense transcription, and both HSs bind CTCF in vivo. Further, they both possess enhancer-blocking activity in vivo. Thus, we propose that they are enhancer-blocking insulators preventing Emu-dependent chromatin opening extending into the V region. Thus, they are the first elements identified that may control ordered V(D)J recombination and correct assembly of antibody genes.

Global analysis of the insulator binding protein CTCF in chromatin barrier regions reveals demarcation of active and repressive domains

Insulators are DNA elements that prevent inappropriate interactions between the neighboring regions of the genome. They can be functionally classified as either enhancer blockers or domain barriers. CTCF (CCCTC-binding factor) is the only known major insulator-binding protein in the vertebrates and has been shown to bind many enhancer-blocking elements. However, it is not clear whether it plays a role in chromatin domain barriers between active and repressive domains. Here, we used ChIP-seq to map the genome-wide binding sites of CTCF in three cell types and identified significant binding of CTCF to the boundaries of repressive chromatin domains marked by H3K27me3. Although we find an extensive overlapping of CTCF-binding sites across the three cell types, its association with the domain boundaries is cell-type-specific. We further show that the nucleosomes flanking CTCF-binding sites are well positioned. Interestingly, we found a complementary pattern between the repressive H3K27me3 and the active H2AK5ac regions, which are separated by CTCF. Our data indicate that CTCF may play important roles in the barrier activity of insulators, and this study provides a resource for further investigation of the CTCF function in organizing chromatin in the human genome.

Insulation of the Ubiquitous Rxrb Promoter from the Cartilage-specific Adjacent Gene, Col11a2

The retinoid X receptor beta gene (Rxrb) is located just upstream of the alpha2(XI) collagen chain gene (Col11a2) in a head-to-tail manner. However, the domain structures of these genes are unknown. Col11a2 is specifically expressed in cartilage. In the present study, we found Rxrb expression in various tissues with low expression in the cartilage. Col11a2 1st intron enhancer directed cartilage specific expression when linked to the heterologous promoter in transgenic mice. These results suggest the presence of enhancer-blocking elements that insulate Rxrb promoter from the Col11a2 enhancer. So far, most of insulators examined in vertebrates contain a binding site for CTCF. We found two possible CTCF-binding sites: one (11P) in the intergenic region between Rxrb and Col11a2 by electrophoretic mobility shift assays, and the other in the 4th intron of RXRB by data base search. To examine the function of these elements, we prepared bacterial artificial chromosome (BAC) transgene constructs containing a 142-kb genomic DNA insert with RXRB and COL11A2 sequences in the middle. Mutation of 11P significantly decreased the RXRB promoter activity in muscular cells and significantly increased expression levels of RXRB in chondrosarcoma cells. In transgenic mouse assays, the wild-type BAC transgene partly recapitulated endogenous Rxrb expression patterns. A 507-bp deletion mutation including 11P enhanced the cartilage-specific activity of the RXRB promoter in BAC transgenic mice. Chromatin immunoprecipitation analysis showed that CTCF was associated with RX4, but not with 11P. Our results showed that the intergenic sequence including 11P insulates Rxrb promoter from Col11a2 enhancer, possibly associating with unknown factors that recognize a motif similar to CTCF.

Characterization of Regulatory Elements in the Slc4a1 (Band 3) Locus To Create Safe and Effective Vectors for Globin Gene Therapy

Effective gene therapy for hemoglobin β-chain disorders, β-thalassemia and sickle cell disease, requires efficient, safe delivery of globin genes into hematopoietic stem cells (HSCs). Engraftment of ∼25% of HCSs expressing a globin gene at ∼20% the level of endogenous α-globin would be sufficient to improve both diseases. The β-globin promoter is inefficient, requiring sequences from the locus control region (LCR) to increase expression. LCR enhancers are included in globin gene therapy vectors, but unfortunately are prone to cryptic splicing and polyadenylation, resulting in low virus titer. In addition, the LCR enhancers are, in theory, capable of activating neighboring oncogenes. To improve safety and efficiency of globin vectors we have developed a novel strategy by fusing the γ-globin gene to LCR-independent, erythroid-specific promoters. Band 3/AE1 is an erythrocyte membrane protein expressed from the Slc4a1 gene. We have previously shown in transgenic mice that a 1750-bp Slc4a1 promoter linked to γ-globin gene (pSlc4a1/γ) and flanked by the chicken β-globin insulator 5′HS4 (ch5′HS4), which contains both barrier and enhancer-blocking elements, is capable of erythroid-specific, uniform γ-globin expression at therapeutic levels (∼19.8% α-globin/transgene copy). Without ch5′HS4, the pSlc4a1/γ gene was prone to silencing. Lentiviral vectors with two copies of ch5′HS4 either internal or in the Long Terminal Repeat cannot be produced at high titer. We hypothesized that flanking the pSlc4a1/γ-globin gene with distinct barrier elements would prevent recombination and gene silencing. Using this strategy, we developed first generation lentiviral vectors in which pSlc4a1/γ is flanked by combinations of the ch5′HS4 insulator and barrier elements we have identified in the ankyrin and α-spectrin loci. To test the effectiveness of these lentivirus vectors in mouse models, we pseudotyped each one with an ecotropic envelope. All three were produced at high titer (>1x106 infectious units/ml). We transduced primitive mouse hematopoietic progenitor cells and detected γ-globin mRNA in >20% of spleen foci at levels as high as 17% of endogenous α-globin. In mice repopulated with transduced stem and progenitor cells, 11–15% of peripheral blood erythrocytes were positive for γ-globin 8 to 21 weeks post-transplantation. To establish the safety of the Slc4a1 promoter we used a high throughput real-time PCR-based assay to identify DNaseI hypersensitive sites (HS) in a 119kb region including Slc4a1. We have identified 6 HS and tested each for enhancer and enhancer-blocking activity. One HS (−355 to −112) increases reporter gene expression in a position- and orientation-independent fashion consistent with the properties of an enhancer. A second HS (−112 to 0) is active in enhancer-blocking assays, and deletion analyses indicate that this region may also contain a transcriptional silencer. A third HS in intron 1 (+910 to +1581) displays enhancer-blocking activity. Three HS have no activity. We are testing a second generation of pSlc4a1/γ lentiviruses in which the Slc4a1 enhancer and silencer are deleted. A third generation of vectors flank pSlc4a1/γ upstream with ch5′HS4, and downstream with either the ankyrin or α-spectrin barrier elements plus the Slc4a1 intron 1 enhancer-blocker to prevent activation of neighboring genes. We hypothesize that these new vectors will allow safe expression of therapeutic levels of γ-globin.

CTCF Genomic Binding Sites in Drosophila and the Organisation of the Bithorax Complex

Insulator or enhancer-blocking elements are proposed to play an important role in the regulation of transcription by preventing inappropriate enhancer/promoter interaction. The zinc-finger protein CTCF is well studied in vertebrates as an enhancer blocking factor, but Drosophila CTCF has only been characterised recently. To date only one endogenous binding location for CTCF has been identified in the Drosophila genome, the Fab-8 insulator in the Abdominal-B locus in the Bithorax complex (BX-C). We carried out chromatin immunopurification coupled with genomic microarray analysis to identify CTCF binding sites within representative regions of the Drosophila genome, including the 3-Mb Adh region, the BX-C, and the Antennapedia complex. Location of in vivo CTCF binding within these regions enabled us to construct a robust CTCF binding-site consensus sequence. CTCF binding sites identified in the BX-C map precisely to the known insulator elements Mcp, Fab-6, and Fab-8. Other CTCF binding sites correlate with boundaries of regulatory domains allowing us to locate three additional presumptive insulator elements; “Fab-2,” “Fab-3,” and “Fab-4.” With the exception of Fab-7, our data indicate that CTCF is directly associated with all known or predicted insulators in the BX-C, suggesting that the functioning of these insulators involves a common CTCF-dependent mechanism. Comparison of the locations of the CTCF sites with characterised Polycomb target sites and histone modification provides support for the domain model of BX-C regulation.

Cloning and characterization of zebrafish CTCF: Developmental expression patterns, regulation of the promoter region, and evolutionary aspects of gene organization

CTCF is a nuclear phosphoprotein capable of using different subsets of its 11 Zn fingers (ZF) for sequence-specific binding to many dissimilar DNA CTCF-target sites. Such sites were identified in the genomic DNA of various multicellular organisms, in which the CTCF gene was cloned, including insects, birds, rodents, and primates. CTCF/DNA-complexes formed in vivo with different 50-bp-long sequences mediate diverse functions such as positive and negative regulation of promoters, and organization of all known enhancer-blocking elements (“chromatin insulators”) including constitutive and epigenetically regulated elements. Abnormal functions of certain CTCF sites are implicated in cancer and in epigenetic syndromes such as BWS and skewed X-inactivation. We describe here the cloning and characterization of the CTCF cDNA and promoter region from zebrafish, a valuable vertebrate model organism. The full-length zebrafish CTCF cDNA clone is 4244 bp in length with an open reading frame (ORF) of 2391 bp that encodes 797 amino acids. The zebrafish CTCF amino acid sequence shows high identity (up to 98% in the zinc finger region) with human CTCF, and perfect conservation of exon–intron organization. Southern blot analyses indicated that the zebrafish genome contains a single copy of the CTCF gene. In situ hybridization revealed the presence of zebrafish CTCF transcripts in all early stages of embryogenesis. Transfection assays with luciferase reporter-constructs identified a core promoter region within 146 bp immediately upstream of the transcriptional start site of zebrafish CTCF that is located at a highly conserved YY1/Initiator element.

Remote control of gene transcription

In this review, we look at the most recent studies of DNA elements that function over long genomic distances to regulate gene transcription and will discuss the mechanisms genes employ to overcome the positive and negative influences of their genomic neighbourhood in order to achieve accurate programmes of expression. Enhancer elements activate high levels of transcription of linked genes from distal locations. Recent technological advances have demonstrated chromatin loop interactions between enhancers and their target promoters. Moreover, there is increasing evidence that these dynamic interactions regulate the repositioning of genes to foci of active transcription within the nucleus. Enhancers have the potential to activate a number of neighbouring genes over a large chromosomal region, hence, their action must be restricted in order to prevent activation of non-target genes. This is achieved by specialized DNA sequences, termed enhancer blockers (or insulators), that interfere with an enhancer's ability to communicate with a target promoter when positioned between the two. Here, we summarize current models of enhancer blocking activity and discuss recent findings of how it can be dynamically regulated. It has become clear that enhancer blocking elements should not be considered only as structural elements on the periphery of gene loci, but as regulatory elements that are crucial to the outcome of gene expression. The transcription potential of a gene can also be susceptible to heterochromatic silencing originating from its chromatin environment. Insulator elements can act as barriers to the spread of heterochromatin. We discuss recent evidence supporting a number of non-exclusive mechanisms of barrier action, which mostly describe the modulation of chromatin structure or modification.

CTCF is conserved from Drosophila to humans and confers enhancer blocking of the Fab‐8 insulator

Eukaryotic transcriptional regulation often involves regulatory elements separated from the cognate genes by long distances, whereas appropriately positioned insulator or enhancer-blocking elements shield promoters from illegitimate enhancer action. Four proteins have been identified in Drosophila mediating enhancer blocking-Su(Hw), Zw5, BEAF32 and GAGA factor. In vertebrates, the single protein CTCF, with 11 highly conserved zinc fingers, confers enhancer blocking in all known chromatin insulators. Here, we characterize an orthologous CTCF factor in Drosophila with a similar domain structure, binding site specificity and transcriptional repression activity as in vertebrates. In addition, we demonstrate that one of the insulators (Fab-8) in the Drosophila Abdominal-B locus mediates enhancer blocking by dCTCF. Therefore, the enhancer-blocking protein CTCF and, most probably, the mechanism of enhancer blocking mediated by this remarkably versatile factor are conserved from Drosophila to humans.

CTCF-dependent enhancer blockers at the upstream region of the chicken alpha-globin gene domain.

The eukaryotic genome is partitioned into chromatin domains containing coding and intergenic regions. Insulators have been suggested to play a role in establishing and maintaining chromatin domains. Here we describe the identification and characterization of two separable enhancer blocking elements located in the 5' flanking region of the chicken alpha-globin domain, 11-16 kb upstream of the embryonic alpha-type pi gene in a DNA fragment harboring a MAR (matrix attachment region) element and three DNase I hypersensitive sites (HSs). The most upstream enhancer blocking element co-localizes with the MAR element and an erythroid-specific HS. The second enhancer blocking element roughly co-localizes with a constitutive HS. The third erythroid-specific HS present within the DNA fragment studied harbors a silencing, but not an enhancer blocking, activity. The 11 zinc-finger CCCTC-binding factor (CTCF), which plays an essential role in enhancer blocking activity in many previously characterized vertebrate insulators, is found to bind the two alpha-globin enhancer blocking elements. Detailed analysis has demonstrated that mutation of the CTCF binding site within the most upstream enhancer blocking element abolishes the enhancer blocking activity. The results are discussed with respect to special features of the tissue-specific alpha-globin gene domain located in a permanently open chromatin area.

Position-effect protection and enhancer blocking by the chicken beta-globin insulator are separable activities.

The 1.2-kb DNA sequence element (5'HS4) at the 5' end of the chicken beta-globin locus has the two defining properties of an insulator: it prevents an "external" enhancer from acting on a promoter when placed between them ("enhancer blocking") and acts as a barrier to chromosomal position effect (CPE) when it surrounds a stably integrated reporter. We previously reported that a single CTCF-binding site in 5'HS4 is necessary and sufficient for enhancer blocking. We show here that a 250-bp "core" element from within 5'HS4 is sufficient to confer protection against silencing of transgenes caused by CPE. Further dissection of the core reveals that 5'HS4 is a compound element in which it is possible to separate enhancer blocking and barrier activities. We demonstrate that full protection against CPE is conferred by mutant 5'HS4 sequences from which the CTCF-binding site has been deleted. In contrast, mutations of four other protein binding sites within 5'HS4 result in varying reductions in the ability to protect against CPE. We find that binding sites for CTCF are neither necessary nor sufficient for protection against CPE. Comparison of the properties of 5'HS4 with those of other CTCF-binding enhancer-blocking elements suggests that CPE protection is associated with maintenance of a high level of histone acetylation near the insulator, conferred by insulator binding-proteins other than CTCF.

Functional phosphorylation sites in the C-terminal region of the multivalent multifunctional transcriptional factor CTCF.

CTCF is a widely expressed and highly conserved multi-Zn-finger (ZF) nuclear factor. Binding to various CTCF target sites (CTSs) is mediated by combinatorial contributions of different ZFs. Different CTSs mediate distinct CTCF functions in transcriptional regulation, including promoter repression or activation and hormone-responsive gene silencing. In addition, the necessary and sufficient core sequences of diverse enhancer-blocking (insulator) elements, including CpG methylation-sensitive ones, have recently been pinpointed to CTSs. To determine whether a posttranslational modification may modulate CTCF functions, we studied CTCF phosphorylation. We demonstrated that most of the modifications that occur at the carboxy terminus in vivo can be reproduced in vitro with casein kinase II (CKII). Major modification sites map to four serines within the S(604)KKEDS(609)S(610)DS(612)E motif that is highly conserved in vertebrates. Specific mutations of these serines abrogate phosphorylation of CTCF in vivo and CKII-induced phosphorylation in vitro. In addition, we showed that completely preventing phosphorylation by substituting all serines within this site resulted in markedly enhanced repression of the CTS-bearing vertebrate c-myc promoters, but did not alter CTCF nuclear localization or in vitro DNA-binding characteristics assayed with c-myc CTSs. Moreover, these substitutions manifested a profound effect on negative cell growth regulation by wild-type CTCF. CKII may thus be responsible for attenuation of CTCF activity, either acting on its own or by providing the signal for phosphorylation by other kinases and for CTCF-interacting protein partners.

The Protein CTCF Is Required for the Enhancer Blocking Activity of Vertebrate Insulators

An insulator is a DNA sequence that can act as a barrier to the influences of neighboring cis-acting elements, preventing gene activation, for example, when located between an enhancer and a promoter. We have identified a 42 bp fragment of the chicken β-globin insulator that is both necessary and sufficient for enhancer blocking activity in human cells. We show that this sequence is the binding site for CTCF, a previously identified eleven–zinc finger DNA-binding protein that is highly conserved in vertebrates. CTCF sites are present in all of the vertebrate enhancer-blocking elements we have examined. We suggest that directional enhancer blocking by CTCF is a conserved component of gene regulation in vertebrates.

Enhancer-Blocking Activity Within the DNase I Hypersensitive Site 2 to 6 Region Between the TCR α and Dad1 Genes

AbstractAlthough tightly linked, the TCR α and δ genes are expressed specifically in T lymphocytes, whereas the Dad1 gene is ubiquitously expressed. Between TCR α and Dad1 are eight DNase I hypersensitive sites (HS). HS1 colocalizes with the TCR α enhancer (Eα) and is T cell-specific; HS2, -3, -4, -5, and -6 map downstream of HS1 and are tissue-nonspecific. The region spanning HS2–6 was reported to display chromatin-opening activity and to confer copy number-dependent and integration site-independent transgene expression in transgenic mice. Here, we demonstrate that HS2–6 also displays enhancer-blocking activity, as it can block an enhancer from activating a promoter when located between the two in a chromatin-integrated context, and can do so without repressing either the enhancer or the promoter. Multiple enhancer-blocking elements are arrayed across HS2–6. We show that HS2–6 by itself does not activate transcription in chromatin context, but can synergize with an enhancer when located upstream of an enhancer and promoter. We propose that HS2–6 primarily functions as an insulator or boundary element that may be critical for the autonomous regulation of the TCR α and Dad1 genes.