Locus Control Region Activity Research Articles

Mechanistic Insights into Forced Chromatin Looping Mediated Activation of Fetal Globin Gene Expression

The β-globin enhancer, called locus control region (LCR), is required for high level expression of all b-type globin genes. The LCR is in physical proximity with the genes it controls, with contacts shifting from embryonic (ε) to fetal (γ) and finally to adult (δ and β) globin gene promoters during development. In prior studies we showed that forced chromatin contacts between the LCR and the β-globin promoter led to transcriptional activation, suggesting that LCR-promoter looping causally underlies β-globin transcription (Deng et al. Cell 2012). In these studies, the transcription co-factor Ldb1 was tethered to the β-globin promoter using artificial zinc finger (ZF) DNA binding proteins, to trigger the promoter-LCR interaction. We subsequently showed that tethering Ldb1 to the promoters of developmentally silenced embryonic or fetal globin genes reactivated their expression in adult erythroblasts in a manner dependent on looped contacts with the LCR (Deng et al. Cell 2014). This work established a novel strategy to raise fetal globin expression in patients with sickle cell anemia.To examine mechanistically the effects of chromatin looping on gene expression we performed single molecule RNA FISH experiments to precisely measure transcription output at individual alleles before and after enforced LCR-γ-globin looping. The experiments were carried out in primary human erythroblasts, which produce elevated levels of γ-globin when exposed to culture conditions. Preliminary data suggest that the majority of transcripts emerge from the β-globin gene with a smaller fraction of transcripts coming from the γ-globin gene as expected. Among cells producing any type of globin primary transcripts, a significant fraction (25%-35%) of cells co-express γ- and β-globin. Importantly, γ- and β-globin are frequently transcribed from the same allele. Forced juxtaposition of the LCR and the γ-globin promoter increases the number of alleles expressing only γ-globin while reducing the number of alleles expressing only the β-globin gene. This result is consistent with the γ- and β-globin genes competing for LCR activity, and emphasizes the usefulness of this approach in the context of sickle cell anemia in which not only elevated levels of fetal hemoglobin but also reduction of the mutant hemoglobin are desirable. Surprisingly, however, the proportion of alleles co-expressing γ-globin and β-globin remains largely constant. We are testing whether co-expression from the same allele is LCR independent. Finally, our studies suggest that LCR-promoter contacts increase the probability of transcription of a given allele.We will also present work addressing the critical question as to how alteration of chromatin architecture overcomes the action of transcriptional repressive complexes, such as Bcl11a, which normally maintain embryonic and fetal globin genes in a repressed state throughout adulthood.In sum, our studies produce a deeper understanding of the interplay of chromatin architecture and gene expression in a system that holds great potential for therapeutic application in patients with hemoglobinopathies. DisclosuresNo relevant conflicts of interest to declare.

The chromatin "landscape" of a murine adult β-globin gene is unaffected by deletion of either the gene promoter or a downstream enhancer.

In mammals, the complex tissue- and developmental-specific expression of genes within the β-globin cluster is known to be subject to control by the gene promoters, by a locus control region (LCR) located upstream of the cluster, and by sequence elements located across the intergenic regions. Despite extensive investigation, however, the complement of sequences that is required for normal regulation of chromatin structure and gene expression within the cluster is not fully defined. To further elucidate regulation of the adult β-globin genes, we investigate the effects of two deletions engineered within the endogenous murine β-globin locus. First, we find that deletion of the β2-globin gene promoter, while eliminating β2-globin gene expression, results in no additional effects on chromatin structure or gene expression within the cluster. Notably, our observations are not consistent with competition among the β-globin genes for LCR activity. Second, we characterize a novel enhancer located 3′ of the β2-globin gene, but find that deletion of this sequence has no effect whatsoever on gene expression or chromatin structure. This observation highlights the difficulty in assigning function to enhancer sequences identified by the chromatin “landscape” or even by functional assays.

Transcriptional regulation of the Ikzf1 locus

AbstractIkaros is a critical regulator of lymphocyte development and homeostasis; thus, understanding its transcriptional regulation is important from both developmental and clinical perspectives. Using a mouse transgenic reporter approach, we functionally characterized a network of highly conserved cis-acting elements at the Ikzf1 locus. We attribute B-cell and myeloid but not T-cell specificity to the main Ikzf1 promoter. Although this promoter was unable to counter local chromatin silencing effects, each of the 6 highly conserved Ikzf1 intronic enhancers alleviated silencing. Working together, the Ikzf1 enhancers provided locus control region activity, allowing reporter expression in a position and copy-independent manner. Only 1 of the Ikzf1 enhancers was responsible for the progressive upregulation of Ikaros expression from hematopoietic stem cells to lymphoid-primed multipotent progenitors to T-cell precursors, which are stages of differentiation dependent on Ikaros for normal outcome. Thus, Ikzf1 is regulated by both epigenetic and transcriptional factors that target its enhancers in both redundant and specific fashions to provide an expression profile supportive of normal lymphoid lineage progression and homeostasis. Mutations in the Ikzf1 regulatory elements and their interacting factors are likely to have adverse effects on lymphopoiesis and contribute to leukemogenesis.

Complete TCR-α Gene Locus Control Region Activity in T Cells Derived In Vitro from Embryonic Stem Cells

Locus control regions (LCRs) are cis-acting gene regulatory elements with the unique, integration site-independent ability to transfer the characteristics of their locus-of-origin's gene expression pattern to a linked transgene in mice. LCR activities have been discovered in numerous T cell lineage-expressed gene loci. These elements can be adapted to the design of stem cell gene therapy vectors that direct robust therapeutic gene expression to the T cell progeny of engineered stem cells. Currently, transgenic mice provide the only experimental approach that wholly supports all the critical aspects of LCR activity. In this study, we report the manifestation of all key features of mouse TCR-α gene LCR function in T cells derived in vitro from mouse embryonic stem cells. High-level, copy number-related TCR-α LCR-linked reporter gene expression levels are cell type restricted in this system, and upregulated during the expected stage transition of T cell development. We also report that de novo introduction of TCR-α LCR-linked transgenes into existing T cell lines yields incomplete LCR activity. These data indicate that establishing full TCR-α LCR activity requires critical molecular events occurring prior to final T lineage determination. This study also validates a novel, tractable, and more rapid approach for the study of LCR activity in T cells, and its translation to therapeutic genetic engineering.

DNase I Hypersensitive Site II of the Human Growth Hormone Locus Control Region Mediates an Essential and Distinct Long-range Enhancer Function

Locus control regions (LCRs) comprise sets of DNA elements capable of establishing autonomous chromatin domains that support robust and physiologically appropriate expression of target genes, often working over extensive distances. Human growth hormone (hGH-N) expression in the pituitary is under the regulation of a well characterized LCR containing four DNase I hypersensitive sites (HSs). The two pituitary-specific HS, HSI and HSII, are located 14.5 and 15.5 kb 5' to the hGH-N promoter. HSI is essential for activation of hGH-N during pituitary development and for sustaining robust activity in the adult. To determine whether the closely linked HSII has a role in hGH-N expression, it was deleted from a previously validated hGH/P1 transgene. Analysis of three independent hGH/P1(ΔHSII) transgenic mouse lines revealed that this deletion had no adverse effect on the formation of HSI, yet resulted in a substantial loss (70%) in hGH-N mRNA expression. This loss of expression was accompanied by a corresponding reduction in recruitment of the pituitary-specific transcription factor Pit-1 to the hGH-N promoter and a selective decrease in promoter occupancy of the elongation-linked isoform of RNA polymerase II. Sufficiency of HSI and HSII in LCR activity was explored by establishing two additional sets of mouse transgenic lines in which DNA segments containing these HS were positioned within the λ phage genome. In this "neutral" DNA context, HSII was required for the recruitment of HAT activity. These data establish HSII as a nonredundant component of the hGH LCR essential for establishment of robust levels of hGH-N gene expression.

POU1F1-Mediated Activation of hGH-N by Deoxyribonuclease I Hypersensitive Site II of the Human Growth Hormone Locus Control Region

The human growth hormone gene (hGH-N) is regulated by a distal locus control region (LCR) composed of five deoxyribonuclease I hypersensitive sites (HSs). The region encompassing HSI and HSII contains the predominant pituitary somatotrope-specific hGH-N activation function of the LCR. This activity was attributed primarily to POU1F1 (Pit-1) elements at HSI, as linkage to HSI was sufficient for properly regulated hGH-N expression in transgenic mice, while HSII alone had no activity. However, the presence of HSII in conjunction with HSI further enhanced hGH-N transgene expression, indicating additional determinants of pituitary hGH-N activation in the HSII region, but limitations of transgenic models and previous ex vivo systems have prevented the characterization of HSII. In the present study, we employ a novel minichromosome model of the hGH-N regulatory domain and show that HSII confers robust POU1F1-dependent activation of hGH-N in this system. This effect was accompanied by POU1F1-dependent histone acetylation and methylation throughout the minichromosome LCR/hGH-N domain. A series of in vitro DNA binding experiments revealed that POU1F1 binds to multiple sites at HSII, consistent with a direct role in HSII function. Remarkably, POU1F1 binding was localized in part to the 3′ untranslated region of a primate-specific LINE-1 (long interspersed nuclear element 1) retrotransposon, suggesting that its insertion during primate evolution may have conferred function to the HSII region in the context of pituitary GH gene regulation. These observations clarify the function of HSII, expanding the role of POU1F1 in hGH LCR activity, and provide insight on the molecular evolution of the LCR.

Ectopic T Cell Receptor-α Locus Control Region Activity in B Cells Is Suppressed by Direct Linkage to Two Flanking Genes at Once

The molecular mechanisms regulating the activity of the TCRα gene are required for the production of the circulating T cell repertoire. Elements of the mouse TCRα locus control region (LCR) play a role in these processes. We previously reported that TCRα LCR DNA supports a gene expression pattern that mimics proper thymus-stage, TCRα gene-like developmental regulation. It also produces transcription of linked reporter genes in peripheral T cells. However, TCRα LCR-driven transgenes display ectopic transcription in B cells in multiple reporter gene systems. The reasons for this important deviation from the normal TCRα gene regulation pattern are unclear. In its natural locus, two genes flank the TCRα LCR, TCRα (upstream) and Dad1 (downstream). We investigated the significance of this gene arrangement to TCRα LCR activity by examining transgenic mice bearing a construct where the LCR was flanked by two separate reporter genes. Surprisingly, the presence of a second, distinct, reporter gene downstream of the LCR virtually eliminated the ectopic B cell expression of the upstream reporter observed in earlier studies. Downstream reporter gene activity was unaffected by the presence of a second gene upstream of the LCR. Our findings indicate that a gene arrangement in which the TCRα LCR is flanked by two distinct transcription units helps to restrict its activity, selectively, on its 5′-flanking gene, the natural TCRα gene position with respect to the LCR. Consistent with these findings, a TCRα/Dad1 locus bacterial artificial chromosome dual-reporter construct did not display the ectopic upstream (TCRα) reporter expression in B cells previously reported for single TCRα transgenes.

Desmin-regulated Lentiviral Vectors for Skeletal Muscle Gene Transfer

Lentiviral vectors (LVs) are highly attractive as a gene therapy agent as they are able to stably integrate their genomes in both dividing and nondividing cells and, in principle, provide long-term therapeutic benefit. However, their performance in skeletal muscle in adult animals has, to date, been disappointing. In order to gain clearer insight into their utility in this tissue type, we have conducted an extensive quantitative comparison of constitutive and muscle-specific promoter activities in skeletal muscle and nonmuscle systems following LV delivery in cell lines and neonatal mice. Our data show that LV delivery to hind leg skeletal muscle of neonatal mouse results in long-term transgene expression in adulthood. We find that the human desmin (DES) promoter/enhancer is the first muscle-specific control region to match the activity of the highly active constitutive human cytomegalovirus (hCMV) promoter/enhancer in skeletal muscle within a LV context both in vitro and in vivo. Furthermore, the DES promoter/enhancer provides six- to eightfold greater expression per viral copy than the muscle-specific human muscle creatine kinase (CKM) promoter/enhancer. DES also confers a more reproducible and tissue-specific transgene expression profile compared to CKM and is therefore a highly attractive regulatory element for use in muscle gene therapy vectors.

Expression of Pit-1 in Nonsomatotrope Cell Lines Induces Human Growth Hormone Locus Control Region Histone Modification and hGH-N Transcription

The POU domain transcription factor Pit-1 is expressed in somatotropes, lactotropes, and thyrotropes of the anterior pituitary. Pit-1 is essential for the establishment of these lineages during development and regulates the expression of genes encoding the peptide hormones secreted by each cell type, including the growth hormone gene expressed in somatotropes. In contrast to rodent growth hormone loci, the human growth hormone ( hGH) locus is regulated by a distal locus control region (LCR), which is required in cis for the proper expression of the hGH gene cluster in transgenic mice. The hGH LCR mediates a domain of histone acetylation targeted to the hGH locus that is associated with distal hGH-N activation, and the discrete determinants of this activity coincide with DNaseI hypersensitive site (HS) I of the LCR. The identification of three in vitro Pit-1 binding sites within the HS-I region suggested a model in which Pit-1 binding at HS-I initiates the chromatin modification mechanism associated with hGH LCR activity. To test this hypothesis directly and to determine whether Pit-1 expression is sufficient to confer hGH locus histone acetylation and activate hGH-N transcription from an inactive locus, we expressed Pit-1 in nonpituitary cell types. We show that Pit-1 expression established a domain of histone hyperacetylation at the LCR and hGH-N promoter in these cells similar to that observed in pituitary chromatin. This was accompanied by the activation of hGH-N transcription and an increase in intergenic and CD79b transcripts proximal to HS-I. These effects were coincident with Pit-1 occupancy at HS-I and the hGH-N promoter and were observed irrespective of the basal histone modification status of HS-I in the heterologous cell line. These findings are consistent with a role for Pit-1 as an initiating factor in hGH locus activation during somatotrope ontogeny, acting through binding sites at HS-I of the hGH LCR.

β-Globin LCR and Intron Elements Cooperate and Direct Spatial Reorganization for Gene Therapy

The Locus Control Region (LCR) requires intronic elements within β-globin transgenes to direct high level expression at all ectopic integration sites. However, these essential intronic elements cannot be transmitted through retrovirus vectors and their deletion may compromise the therapeutic potential for gene therapy. Here, we systematically regenerate functional β-globin intron 2 elements that rescue LCR activity directed by 5′HS3. Evaluation in transgenic mice demonstrates that an Oct-1 binding site and an enhancer in the intron cooperate to increase expression levels from LCR globin transgenes. Replacement of the intronic AT-rich region with the Igμ 3′MAR rescues LCR activity in single copy transgenic mice. Importantly, a combination of the Oct-1 site, Igμ 3′MAR and intronic enhancer in the BGT158 cassette directs more consistent levels of expression in transgenic mice. By introducing intron-modified transgenes into the same genomic integration site in erythroid cells, we show that BGT158 has the greatest transcriptional induction. 3D DNA FISH establishes that induction stimulates this small 5′HS3 containing transgene and the endogenous locus to spatially reorganize towards more central locations in erythroid nuclei. Electron Spectroscopic Imaging (ESI) of chromatin fibers demonstrates that ultrastructural heterochromatin is primarily perinuclear and does not reorganize. Finally, we transmit intron-modified globin transgenes through insulated self-inactivating (SIN) lentivirus vectors into erythroid cells. We show efficient transfer and robust mRNA and protein expression by the BGT158 vector, and virus titer improvements mediated by the modified intron 2 in the presence of an LCR cassette composed of 5′HS2-4. Our results have important implications for the mechanism of LCR activity at ectopic integration sites. The modified transgenes are the first to transfer intronic elements that potentiate LCR activity and are designed to facilitate correction of hemoglobinopathies using single copy vectors.

CTCF-Independent, but Not CTCF-Dependent, Elements Significantly Contribute to TCR-α Locus Control Region Activity

The mouse TCRalpha/TCRdelta/Dad1 gene locus bears a locus control region (LCR) that drives high-level, position-independent, thymic transgene expression in chromatin. It achieves this through DNA sequences that enhance transcription and protect transgene expression from integration site-dependent position effects. The former activity maps to a classical enhancer region (Ealpha). In contrast, the elements supporting the latter capacity that suppresses position effects are incompletely understood. Such elements likely play important roles in their native locus and may resemble insulator/boundary sequences. Insulators support enhancer blocking and/or chromatin barrier activity. Most vertebrate enhancer-blocking insulators are dependent on the CTCF transcription factor and its cognate DNA binding site. However, studies have also revealed CTCF-independent enhancer blocking and barrier insulator activity in the vertebrate genome. The TCRalpha LCR contains a CTCF-dependent and multiple CTCF-independent enhancer-blocking regions whose roles in LCR activity are unknown. Using randomly integrated reporter transgenes in mice, we find that the CTCF region plays a very minor role in LCR function. In contrast, we report the in vivo function of two additional downstream elements located in the region of the LCR that supports CTCF-independent enhancer-blocking activity in cell culture. Internal deletion of either of these elements significantly impairs LCR activity. These results reveal that the position-effect suppression region of the TCRalpha LCR harbors an array of CTCF-independent, positive-acting gene regulatory elements, some of which share characteristics with barrier-type insulators. These elements may help manage the separate regulatory programs of the TCRalpha and Dad1 genes.

New Sequence of Strategies to Define Gene Transcriptional Territories in Lymphocytes: The Perforin Example (35.32)

Abstract Distal regulatory regions control transcription by promoter interactions that are limited to a territory to avoid non-target genes. Few loci are defined with the scrutiny needed to fully understand how a territory is established, maintained and fenced off. Here we approach the human perforin gene (PRF1). First, we transferred an epigenetically silenced PRF1 within its chromosome into a rodent CTL cell line. Afterward PRF1 expression was turned on equivalently to the endogenous mouse gene without perturbing bordering genes. Ultra long-range DHS assays showed epigenetic remodeling during chromosome transfer and delineated a 150 kb territory with 16 DHS sites for PRF1. When a corresponding BAC was introduced into the CTL, transgene expression proceeded with locus control region activity dependent on 4 distal DHS sites that also protected PRF1 from transcriptional perturbations by bordering genes. Finally, differentiation of transgenic ES cells showed the area contained all regulatory domains required for developmentally appropriate PRF1 expression during in vitro lymphopoiesis. Thus, we defined a new sequence of strategies that reliably characterizes autonomous transcriptional territories in lymphocytes.

CTCF‐independent, but not CTCF‐dependent, elements significantly contribute to the insulation capacity of a locus control region

Locus control regions (LCR) provide high-level, position-independent expression to a transgene in chromatin. They achieve this through DNA sequences supporting activities that enhance transcription and somehow protect transgene expression from integration site-dependent position effects. The former activity has been mapped to classical enhancer DNA sequences of most LCRs. In contrast, the elements supporting the latter capacity that suppresses position effects are less well understood. Insulator/Boundary sequences are thought to play a role in separating regulatory influence in the genome and support enhancer blocking and/or chromatin barrier activity. The LCR in the mouse T cell receptor (TCR)- 1 □/Dad1 gene locus contains a CTCF-dependent and multiple CTCF-independent enhancer blocking regions whose roles in LCR activity are unknown. Using randomly integrated reporter transgenes in mice, we find that the CTCF region plays a very minor role in LCR function. In contrast, we report the in vivo function of two additional downstream CTCF-independent elements within the LCR. Internal deletion of either of these elements significantly impairs LCR activity. Our results reveal that the position effect suppression capacity of an LCR arises largely from an array of CTCF-independent elements, some of which share characteristics with barrier-type insulators. Support: NIH #AI-053050 and NSF #MCB-0236964

The TCRα Locus Control Region Specifies Thymic, But Not Peripheral, Patterns of TCRα Gene Expression

The molecular mechanisms ensuring the ordered expression of TCR genes are critical for proper T cell development. The mouse TCR alpha-chain gene locus contains a cis-acting locus control region (LCR) that has been shown to direct integration site-independent, lymphoid organ-specific expression of transgenes in vivo. However, the fine cell type specificity and developmental timing of TCRalpha LCR activity are both still unknown. To address these questions, we established a transgenic reporter model of TCRalpha LCR function that allows for analysis of LCR activity in individual cells by the use of flow cytometry. In this study we report the activation of TCRalpha LCR activity at the CD4-CD8-CD25-CD44- stage of thymocyte development that coincides with the onset of endogenous TCRalpha gene rearrangement and expression. Surprisingly, TCRalpha LCR activity appears to decrease in peripheral T cells where TCRalpha mRNA is normally up-regulated. Furthermore, LCR-linked transgene activity is evident in gammadelta T cells and B cells. These data show that the LCR has all the elements required to reliably reproduce a developmentally correct TCRalpha-like expression pattern during thymic development and unexpectedly indicate that separate gene regulatory mechanisms are acting on the TCRalpha gene in peripheral T cells to ensure its high level and fine cell type-specific expression.

Human High Mobility Group Box Transcription Factor 1 Affects Thymocyte Development and Transgene Variegation

It has been shown previously that a human CD2 (hCD2) disabled locus control region (LCR) transgene is unable to establish an open chromatin configuration in all the T cells, and this leads to position effect variegation of the transgene. In this study we show that thymus-specific overexpression of human high mobility group box transcription factor 1 (HBP1), a transcription factor that binds a specific sequence within the hCD2 LCR, affects thymus cellularity as well as the number of CD8(+) thymocytes in two independent transgenic mouse lines and increases the proportion of T cells that fully activate the transgenic locus in hCD2 variegating mice in a sequence-specific dependent manner. This finding suggests that overexpression of HBP1 can affect lineage commitment and can relieve the suppressive influence of heterochromatin, allowing thymocytes to express the variegating target locus more efficiently. These effects could be the result of direct HBP1 action on LCR activity. Alternatively, the extra HBP1 molecules may sequester repressive elements away from the LCR, thus allowing transcription permissive states to form on the transgene locus.

Factors Binding a Non-classical Cis-element Prevent Heterochromatin Effects on Locus Control Region Activity

A locus control region (LCR) is a cis-acting gene-regulatory element capable of transferring the expression characteristics of its gene locus of origin to a linked transgene. Furthermore, it can do this independently of the site of integration in the genome of transgenic mice. Although most LCRs contain subelements with classical transcriptional enhancer function, key aspects of LCR activity are supported by cis-acting sequences devoid of the ability to act as direct transcriptional enhancers. Very few of these "non-enhancer" LCR components have been characterized. Consequently, the sequence requirements and molecular bases for their functions, as well as their roles in LCR activity, are poorly understood. We have investigated these questions using the LCR from the mouse T cell receptor (TCR) alpha/Dad1 gene locus. Here we focus on DNase hypersensitive site (HS) 6 of the TCRalpha LCR. HS6 does not support classical enhancer activity, yet has gene regulatory activity in an in vivo chromatin context. We have identified three in vivo occupied factor-binding sites within HS6, two of which interact with Runx1 and Elf-1 factors. Deletion of these sites from the LCR impairs its activity in vivo. This mutation renders the transgene locus abnormally inaccessible in chromatin, preventing the normal function of other LCR subelements and reducing transgene mRNA levels. These data show these factor-binding sites are required for preventing heterochromatin formation and indicate that they function to maintain an active TCRalpha LCR assembly in vivo.

LCR-regulated transgene expression levels depend on the Oct-1 site in the AT-rich region of β-globin intron-2

Human beta-globin transgenes regulated by the locus control region (LCR) express at all integration sites in transgenic mice. For such LCR activity at ectopic sites, the 5'HS3 element requires the presence of the AT-rich region (ATR) in beta-globin intron-2. Here, we examine the dependence of 5'HS3 LCR activity on transcription factor binding sites in the ATR. In vitro DNaseI footprint analysis and electrophoretic mobility shift assays of the ATR identified an inverted double Gata-1 site composed of 2 noncanonical sequences (GATT and GATG) and an Oct-1 consensus site. Mutant Oct-1, Gata-1, or double mutant sites were created in the ATR of the BGT50 construct composed of a 5'HS3 beta/gamma-globin hybrid transgene. Transgenes with double mutant sites expressed at all sites of integration, but mean expression levels in transgenic mice were reduced from 64% per copy (BGT50) to 37% (P <.05). Mutation of the inverted double Gata-1 site had no effect at 61% per copy expression levels. In contrast, mutation of the Oct-1 site alone reduced per-copy expression levels to 31% (P <.05). We conclude that the ability of 5'HS3 to activate expression from all transgene integration sites is dependent on sequences in the ATR that are not bound at high affinity by transcription factors. In addition, the Oct-1 site in the ATR is required for high-level 5'HS3 beta/gamma-globin transgene expression and should be retained in LCRbeta-globin expression cassettes designed for gene therapy.

Specification of unique Pit-1 activity in the hGH locus control region.

The human GH (hGH) gene cluster is regulated by a remote 5' locus control region (LCR). HSI, an LCR component located 14.5 kb 5' to the hGH-N promoter, constitutes the primary determinant of high-level hGH-N activation in pituitary somatotropes. HSI encompasses an array of three binding sites for the pituitary-specific POU homeodomain factor Pit-1. In the present report we demonstrate that all three Pit-1 sites in the HSI array contribute to LCR activity in vivo. Furthermore, these three sites as a unit are fully sufficient for position-independent and somatotrope-restricted hGH-N transgene activation. In contrast, the hGH-N transgene is not activated by Pit-1 sites native to either the hGH-N or rat (r)GH gene promoters. These findings suggest that the structures of the Pit-1 binding sites at HSI specify distinct chromatin-dependent activities essential for LCR-mediated activation of hGH in the developing pituitary.

Function and factor interactions of a locus control region element in the mouse T cell receptor-alpha/Dad1 gene locus.

Locus control regions (LCRs) refer to cis-acting elements composed of several DNase I hypersensitive sites, which synergize to protect transgenes from integration-site dependent effects in a tissue-specific manner. LCRs have been identified in many immunologically important gene loci, including one between the TCRdelta/TCRalpha gene segments and the ubiquitously expressed Dad1 gene. Expression of a transgene under the control of all the LCR elements is T cell specific. However, a subfragment of this LCR is functional in a wide variety of tissues. How a ubiquitously active element can participate in tissue-restricted LCR activity is not clear. In this study, we localize the ubiquitously active sequences of the TCR-alpha LCR to an 800-bp region containing a prominent DNase hypersensitive site. In isolation, the activity in this region suppresses position effect transgene silencing in many tissues. A combination of in vivo footprint examination of this element in widely active transgene and EMSAs revealed tissue-unrestricted factor occupancy patterns and binding of several ubiquitously expressed transcription factors. In contrast, tissue-specific, differential protein occupancies at this element were observed in the endogenous locus or full-length LCR transgene. We identified tissue-restricted AML-1 and Elf-1 as proteins that potentially act via this element. These data demonstrate that a widely active LCR module can synergize with other LCR components to produce tissue-specific LCR activity through differential protein occupancy and function and provide evidence to support a role for this LCR module in the regulation of both TCR and Dad1 genes.

Locus control region activity by 5′HS3 requires a functional interaction with β-globin gene regulatory elements: expression of novel β/γ-globin hybrid transgenes

The human β-globin locus control region (LCR) contains chromatin opening and transcriptional enhancement activities that are important to include in β-globin gene therapy vectors. We previously used single-copy transgenic mice to map chromatin opening activity to the 5′HS3 LCR element. Here, we test novel hybrid globin genes to identify β-globin gene sequences that functionally interact with 5′HS3. First, we show that an 850-base pair (bp) 5′HS3 element activates high-level β-globin gene expression in fetal livers of 17 of 17 transgenic mice, including 3 single-copy animals, but fails to reproducibly activate Aγ-globin transgenes. To identify the β-globin gene sequences required for LCR activity by 5′HS3, we linked the 815-bp β-globin promoter to Aγ-globin coding sequences (BGT34), together with either the β-globin intron 2 (BGT35), the β-globin 3′ enhancer (BGT54), or both intron 2 and the 3′ enhancer (BGT50). Of these transgenes, only BGT50 reproducibly expresses Aγ-globin RNA (including 7 of 7 single-copy animals, averaging 71% per copy). Modifications to BGT50 show that LCR activity is detected after replacing the β-globin promoter with the 700-bp Aγ-globin promoter, but is abrogated when an AT-rich region is deleted from β-globin intron 2. We conclude that LCR activity by 5′HS3 on globin promoters requires the simultaneous presence of β-globin intron 2 sequences and the 260-bp 3′ β-globin enhancer. The BGT50 construct extends the utility of the 5′HS3 element to include erythroid expression of nonadult β-globin coding sequences in transgenic animals and its ability to express antisickling γ-globin coding sequences at single copy are ideal characteristics for a gene therapy cassette.