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Results for “Locus Control Region”

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PubMed2001

The role of the -50 region of the human gamma-globin gene in switching.

During the switch from human gamma- (fetal) to beta- (adult) globin gene expression, the gamma and beta genes are expressed competitively by an alternating transcription mechanism. The -50 region of the gamma gene promoter has been proposed to be responsible for the early competitive advantage of the gamma genes and to act as a stage selector element (SSE) in hemoglobin switching. We analyzed the effect of mutating the -50 region of the gamma gene in the presence of a competing beta gene in transgenic mice. This shows that the -50 region does not affect silencing of the beta gene in early development and does not act as a stage selector. However, it affects the ratio of gamma versus beta gene expression in the early, but not later, stages of fetal development. Interestingly, both the wild-type and mutant minilocus constructs show a higher frequency of alternate transcription than observed in the complete locus, suggesting that sequences normally present between the gamma and beta genes facilitate the interaction of the locus control region (LCR) and beta-globin gene in the complete locus.

Animals↗
PubMed1998

The dynamics of globin gene expression and gene therapy vectors.

The most important level of the regulation of the beta-globin genes is by activation of all of the genes by the locus control region (LCR). Part of the developmental regulation of the locus is achieved by competition of the genes for the interaction with the LCR. Although this level of gene regulation is quantitatively of less importance than the direct repression mechanism for the early genes, it has important implications and has provided an excellent assay to probe the regulation of transcription at the single cell level. The results of these studies indicate that the LCR interacts with individual globin genes and that LCR/gene interactions are dynamic with complexes forming and dissociating continually. We conclude that transcription only appears to take place while the LCR and gene interact and that the level of transcription is determined by the frequency and duration of such interaction rather than by changes in the rate of transcription of the promoters. This mechanism has clear implications for the design of vectors for the purpose of gene therapy.

Animals↗
PubMed2002

Regulation of the globin genes.

The a- and b-globin gene clusters are subject to several levels of regulation. They are expressed exclusively in the erythroid cells, only during defined periods of development and in a perfectly tuned way, assuring, at any stage of ontogeny, a correct balance in the availability of a- and b-globin chains for hemoglobin assembling. Such a tight control is dependent on regulatory regions of DNA located either in proximity or at great distances from the globin genes in a region characterized by the presence of several DNAse I hypersensitive sites and known as the Locus Control Region. All these sequences exert stimulatory, inhibitory or more complex activities by interacting with transcription factors that bridge these regions of DNA to the RNA polymerase machinery. Many of these factors have now been cloned and the corresponding mouse genes inactivated, shading new light on the metabolic pathways they control. It is increasingly recognized that such factors are organized into hierarchies according to the number of genes and circuits they regulate. Some genes such as GATA-1 and 2 are master regulators that act on large numbers of genes at early stage of differentiation whereas others, like EKLF, stand on the lowest step and control only single or limited number of genes at late stages of differentiation. We will review recent data gathered from expression studies in cell cultures, in transgenic or K.O. murine models as well as from a clinical settings. We will also discuss the development of novel theories on the regulation of the a- and b-globin genes and clusters.

Animals↗
PubMed2003

Chromatin folding and gene expression: new tools to reveal the spatial organization of genes.

An important aim in biology is to understand how gene expression is regulated in the context of chromatin. Much progress has been made towards cracking the 'histone code', which describes the composition and organization of chromatin at high resolution. At the lower resolution provided by microscopy, nuclear compartmentalization has been linked to the control of gene expression and silencing. I will review two new techniques able to reveal the three-dimensional organization of individual loci, providing a view of the folding of the chromatin fibre at an intermediate level of resolution. Carter and colleagues and Tolhuis and colleagues have used the new techniques to demonstrate direct physical contact between the locus control region (LCR) and expressed genes in the active murine beta-globin locus. The techniques will allow us to assess the role of locus organization when transcription is directed by distant regulatory elements. The new techniques (and their foreseeable descendants) will permit investigation of many genomic activities involving physical contact between separate regions of any genome. As such, they provide us with a new level of resolution at which to investigate the functional significance of chromatin organization as patterns of gene expression are initiated and modulated during development.

Animals↗
PubMed2002

LCR-regulated transgene expression levels depend on the Oct-1 site in the AT-rich region of beta -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.

AT Rich Sequence↗
PubMed1997

Transcriptional behavior of LCR enhancer elements integrated at the same chromosomal locus by recombinase-mediated cassette exchange.

Efficient integration of transgenes at preselected chromosomal locations was achieved in mammalian cells by recombinase-mediated-cassette-exchange (RMCE), a novel procedure that makes use of the CRE recombinase together with Lox sites bearing different spacer regions. We have applied RMCE to the study of the human beta-globin gene Locus Control Region by integrating at the same genetic locus in MEL cells, a LacZ gene driven by the human beta-globin promoter linked to HS2 and HS3 alone or in combination with HS4. Expression studies at the cell population level and in individual cells before and after induction of differentiation with hemin or DMSO show that the presence of these enhancers is associated with variegated patterns of expression. We were able to show that the LCR fragments tested act by controlling both the probability of expression and the rate of transcription of the linked beta-globin promoter. Both of these factors were also dependent on the state of differentiation of the MELc and on the presence of a second transcription unit located in cis. The ability to manipulate by RMCE constructs integrated into chromosomes should help in the creation of complex, rationally designed, artificial genetic loci.

Animals↗
PubMed2000

A DNase I hypersensitive site near the murine gamma1 switch region contributes to insertion site independence of transgenes and modulates the amount of transcripts induced by CD40 ligation.

Several cis-acting elements regulate the expression of germline transcripts of heavy chain constant region genes and their subsequent switch recombination. To study such elements in the murine gamma1 gene, we have utilized a transgenic approach. In this study we focused on a DNase I hypersensitive site (termed 'Site II') that lies about 2 kb 3' of the gamma1 promoter region and I exon, just 5' to the gamma1 switch region. We have reported that gamma1 transgenes with Site II display the characteristics of a locus control region (LCR) in that they are insertion site independent and copy number dependent. For the present study we prepared six lines of transgenic mice that have the promoter region and I exon, but lack Site II. Expression of RNA from gamma1 transgenes that lack Site II is not correlated with transgene copy number; expression is insertion site dependent. This result indicates that DNase hypersensitive Site II is an important part of the LCR-like elements in the murine gamma1 gene. RNA expression from the gamma1 transgenes that lack Site II is inducible by IL-4 and by CD40 ligation. However, the induction of transgenic RNA expression by CD40 ligation is greater than expected, suggesting that elements within Site II participate in negative regulation of the amount of germline transcripts after CD40 ligation.

Animals↗
PubMed2003

Insulators to improve expression of a 3(')IgH LCR-driven reporter gene in transgenic mouse models.

A locus control region (LCR) containing four transcriptional enhancers lies downstream of the IgH chain locus. We studied transgenes carrying a 3(')IgH LCR-driven GFP reporter gene for expression and B cell differentiation stage specificity. We also compared transgenes that were or were not flanked by two copies of the beta-globin HS4 insulator, an element defined by its ability to protect transgenes from the influences of surrounding genes at the insertion site. Results indicate that insulators are instrumental in sustaining GFP expression in GFP-3(')LCR transgenic mice when they were included. Flow cytometry experiments reported a strictly B cell specific GFP expression from pre-B cells in bone marrow to mature B cells in spleen. Despite addition of 5(')HS4 insulators to the GFP-3(')LCR construct, complete transgene silencing occurred in some transgenic lines and was systematically observed in ageing animals from all lines.

Animals↗
PubMed2002

Positive and negative transcriptional states of a variegating immunoglobulin heavy chain (IgH) locus are maintained by a cis-acting epigenetic mechanism.

Analyses of transgene expression have defined essential components of a locus control region (LCR) in the J(H)-C(mu) intron of the IgH locus. Targeted deletion of this LCR from the endogenous IgH locus of hybridoma cells results in variegated expression, i.e., cells can exist in two epigenetically inherited states in which the Ig(mu) H chain gene is either active or silent; the active or silent state is typically transmitted to progeny cells through many cell divisions. In principle, cells in the two states might differ either in their content of specific transcription factors or in a cis-acting feature of the IgH locus. To distinguish between these mechanisms, we generated LCR-deficient, recombinant cell lines in which the Ig(mu) H chain genes were distinguished by a silent mutation and fused cells in which the mu gene was active with cells in which mu was silent. Our analysis showed that both parental active and silent transcriptional states were preserved in the hybrid cell, i.e., that two alleles of the same gene in the same nucleus can exist in two different states of expression through many cell divisions. These results indicate that the expression of the LCR-deficient IgH locus is not fully determined by the cellular complement of transcription factors, but is also subject to a cis-acting, self-propagating, epigenetic mark. The methylation inhibitor, 5-azacytidine, reactivated IgH in cells in which this gene was silent, suggesting that methylation is part of the epigenetic mark that distinguishes silent from active transcriptional states.

Alleles↗
PubMed2006

CTCF-dependent chromatin insulator is linked to epigenetic remodeling.

Chromatin insulators are boundary elements between distinctly regulated, neighboring chromosomal domains, and they function by blocking the effects of nearby enhancers in a position-dependent manner. Here, we show that the SNF2-like chromodomain helicase protein CHD8 interacts with the insulator binding protein CTCF. Chromatin immunoprecipitation analysis revealed that CHD8 was present at known CTCF target sites, such as the differentially methylated region (DMR) of H19, the locus control region of beta-globin, and the promoter region of BRCA1 and c-myc genes. RNA interference-mediated knockdown of CHD8 significantly abolished the H19 DMR insulator activity that depends highly on CTCF, leading to reactivation of imprinted IGF2 from chromosome of maternal origin. Further, the lack of CHD8 affected CpG methylation and histone acetylation around the CTCF binding sites, adjacent to heterochromatin, of BRCA1 and c-myc genes. These findings provide insight into the role of CTCF-CHD8 complex in insulation and epigenetic regulation at active insulator sites.

Animals↗
PubMed2002

Artificial chromosome transgenesis in pigmentary research.

Pigmentary genes were among the first mammalian genes to be studied, mostly because of the obvious phenotypes associated with their mutations. In 1990, tyrosinase, encoding the limiting enzyme in the melanin synthesis pathway, was eventually assigned to the c (albino) locus by classical rescue experiments driven by functional constructs in transgenic mice. These pioneer reports triggered the study of the regulation of endogenous tyrosinase gene expression by combining different amounts of upstream regulatory and promoter regions and testing their function in vivo in transgenic animals. However, faithful and reproducible transgenic expression was not achieved until the entire tyrosinase expression domain was transferred to the germ-line of mice using artificial-chromosome-type transgenes. The use of these large tyrosinase transgenic constructs and the ease with which they could be manipulated in vitro enabled the discovery of previously unknown but fundamental regulatory regions, such as the tyrosinase locus control region (LCR), whose presence was required in order to guarantee position-independent and copy-number-dependent expression of tyrosinase transgenes, with an expression level, per copy, comparable to that of an endogenous wild-type allele. Subsequently, functional dissection of elements present within this LCR through the generation of new artificial-chromosome type tyrosinase transgenes has revealed the existence of different regulatory activities. The existence of some of these units had been suggested previously by standard-type transgenic analyses. In this review, we will discuss both independent approaches and conclude that optimal tyrosinase transgene expression requires the use of its complete expression domain.

Animals↗
PubMed1997

The human beta globin locus introduced by YAC transfer exhibits a specific and reproducible pattern of developmental regulation in transgenic mice.

The human beta globin locus spans an 80-kb chromosomal region encompassing both the five expressed globin genes and the cis-acting elements that direct their stage-specific expression during ontogeny. Sequences proximal to the genes and in the locus control region, 60 kb upstream of the adult beta globin gene, are required for developmental regulation. Transgenic studies have shown that altering the structural organization of the locus disrupts the normal pattern of globin gene regulation. Procedures for introducing yeast artificial chromosomes (YACs) containing large genetic loci now make it possible to define the sequences required for stage-restricted gene expression in constructs that preserve the integrity of the beta globin locus. We demonstrate that independent YAC transgenic lines exhibit remarkably similar patterns of globin gene expression during development. The switch from gamma to beta globin predominant expression occurs between day 11.5 and 12.5 of gestation, with no more than twofold differences in human beta globin mRNA levels between lines. Human beta globin mRNA levels were twofold to fourfold lower than that of mouse betamaj, revealing potentially significant differences in the regulatory sequences of the two loci. These findings provide an important basis for studying regulatory elements within the beta globin locus.

Adult↗
PubMed2000

Regulation of the promoter for human immunoglobulin gamma3 germ-line transcription and its interaction with the 3'alpha enhancer.

The mechanism underlying the differential regulation of switching to human IgG subclasses is still largely unknown. We demonstrate that the region upstream of the initiation sites for gamma3 germ-line (GL) transcripts contains a functional promoter which is synergistically induced by IL-4, antibody to CD40 and phorbol dibutyrate in transient transfection assays in the human DG75 cell line. Linker-scanning mutations identified multiple elements in the 3' half of the evolutionarily conserved sequence that are required for inducibility. Electrophoretic mobility shift assays showed that Stat6 and NF-kappaB p50 / p65 are induced after stimulation, and bind to specific sequence motifs within the promoter. Overexpression of Stat6, NF-kappaB p50 / p65 and C / EBPgamma synergistically induced the GL gamma3 promoter. Insertion of DNA segments from the human 3' IgH regions, which may function as a locus control region for switch recombination, greatly activated the promoter in an orientation-independent manner. Duplication of the enhancer fragments resulted in a further increase of promoter activity. The greater enhancement of the HS1,2 fragment from the 3' alpha1 rather than the alpha2 locus may suggest a mechanistic explanation for the differential expression of various isotypes.

B-Lymphocytes↗
PubMed2002

ChIPs of the beta-globin locus: unraveling gene regulation within an active domain.

Recent studies of beta-globin gene expression have concentrated on the analysis of factor binding and chromatin structure within the endogenous locus. These studies have more precisely defined the extent and nature of the active chromosomal domain and the elements that organize it. Surprisingly, the beta-globin locus control region (LCR), although critical for high-level gene expression, plays little role in the overall architecture of the active locus. Analysis of the effects of targeted deletion of the beta-globin LCR, along with emerging knowledge of the behavior of the erythroid transcription factor NF-E2, leads to a new perspective on factor binding and LCR function.

Acetylation↗
PubMed2001

Intergenic transcription in the human beta-globin gene cluster.

Our previous studies on nascent transcription across the human beta-globin gene cluster revealed the presence of intergenic transcripts in addition to the expected genic transcripts. We now show that transcription into the beta-globin locus control region (LCR) begins within an ERV9 endogenous retroviral long terminal repeat upstream of DNase I hypersensitive site 5. However, in a transgenic mouse, which has the human beta-globin LCR but lacks the ERV9 LTR, transcription begins upstream of the transgenic locus. We postulate that in this transgenic mouse nearby endogenous mouse promoters are activated by the LCR. Intergenic transcription is also detected across the whole transgenic globin gene locus independently of the stage of erythroid development. Intergenic transcription in the beta-globin cluster is erythroid specific; however, it can be induced in nonerythroid cells by several means: by transinduction with a plasmid transcribing part of the cluster, by exogenous addition of transcription factors, and by treatment with the histone deacetylase inhibitor trichostatin A.

5' Untranslated Regions↗
PubMed1998

Insertion of the IgH locus 3' regulatory palindrome in expression vectors warrants sure and efficient expression in stable B cell transfectants.

We have explored the effect of inserting 3' immunoglobulin heavy chain (IgH) locus transcriptional regulatory elements in stable expression vectors driven by a heavy chain variable gene promoter (pVH). A cassette was constructed, associating three enhancer elements from the palindromic part of the 3' IgH regulatory region, namely Calpha3'/hs3 reverse, alpha3'E/hs1-2, and hs3. As regard to stable expression, this cassette carried some features of a locus control region (LCR) and conferred expression to an associated cat reporter gene in the majority of B cells having integrated the transgene. The palindromic cassette was inserted in an expression vector carrying Ig light chain coding sequences. In this construct, transcription driven by a pVH promoter/Emu cassette upstream of the transcription initiation site was boosted by the palindromic cassette located downstream of the coding sequence. This potent expression plasmid mimicking the architecture of endogenous Ig loci, definitely manifested a potent stimulatory activity for stable transcription, outscoring conventional ubiquitous or B-cell specific expression vectors.

Animals↗
PubMed2002

Looping and interaction between hypersensitive sites in the active beta-globin locus.

Eukaryotic transcription can be regulated over tens or even hundreds of kilobases. We show that such long-range gene regulation in vivo involves spatial interactions between transcriptional elements, with intervening chromatin looping out. The spatial organization of a 200 kb region spanning the murine beta-globin locus was analyzed in expressing erythroid and nonexpressing brain tissue. In brain, the globin cluster adopts a seemingly linear conformation. In erythroid cells the hypersensitive sites of the locus control region (LCR), located 40-60 kb away from the active genes, come in close spatial proximity with these genes. The intervening chromatin with inactive globin genes loops out. Moreover, two distant hypersensitive regions participate in these interactions. We propose that clustering of regulatory elements is key to creating and maintaining active chromatin domains and regulating transcription.

Animals↗
PubMed2002

Replication initiation patterns in the beta-globin loci of totipotent and differentiated murine cells: evidence for multiple initiation regions.

The replication initiation pattern of the murine beta-globin locus was analyzed in totipotent embryonic stem cells and in differentiated cell lines. Initiation events in the murine beta-globin locus were detected in a region extending from the embryonic Ey gene to the adult betaminor gene, unlike the restricted initiation observed in the human locus. Totipotent and differentiated cells exhibited similar initiation patterns. Deletion of the region between the adult globin genes did not prevent initiation in the remainder of the locus, suggesting that the potential to initiate DNA replication was not contained exclusively within the primary sequence of the deleted region. In addition, a deletion encompassing the six identified 5' hypersensitive sites in the mouse locus control region had no effect on initiation from within the locus. As this deletion also did not affect the chromatin structure of the locus, we propose that the sequences determining both chromatin structure and replication initiation lie outside the hypersensitive sites removed by the deletion.

Animals↗