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Results for “Locus Control Region”

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PubMed1998

Studying the recruitment of Sp1 to the beta-globin promoter with an in vivo method: protein position identification with nuclease tail (PIN*POINT).

Transcription is thought to be regulated by recruitment of transcription factors, adaptors, and certain enzymes to cis-acting elements through protein-DNA interactions and protein-protein interactions. To better understand transcription, a method with the capability to detect in vivo recruitment of these individual proteins will be essential. Toward this end, we use a previously undescribed in vivo method that we term protein position identification with nuclease tail (PIN*POINT). In this method, a fusion protein composed of a chosen protein linked to a nonsequence-specific nuclease is expressed in vivo, and the binding of the protein to DNA is made detectable by the nuclease-induced cleavage near the binding site. In this article, we used the technique protein position identification with nuclease tail to study the effect of the beta-globin locus control region (LCR) and promoter elements on the recruitment of transcription factor Sp1 to the beta-globin promoter. We present evidence that the hypersensitive sites of the LCR synergistically enhance the recruitment of a multimeric Sp1 complex to the beta-globin promoter and that this may be accomplished by protein-protein interactions with proteins bound to the LCR, the upstream activator region, and, possibly, general transcription factors bound near the "TATA" box.

Base Sequence↗

PubMed2004

Onset and inheritance of abnormal epigenetic regulation in hematopoietic cells.

Abnormal epigenetic regulation of gene expression contributes significantly to a variety of human pathologies including cancer. Deletion of hypersensitive site 2 (HS2) at the human beta-globin locus control region can lead to abnormal epigenetic regulation of globin genes in transgenic mice. Here, two HS2-deleted transgenic mouse lines were used as model to demonstrate that heritable alteration of chromatin organization at the human beta-globin locus in multipotent hematopoietic progenitors contributes to the abnormal expression of the beta-globin gene in mature erythroid cells. This alteration is characterized by specific patterns of histone covalent modifications that are inherited during erythropoiesis and, moreover, is plastic because it can be reverted by transient treatment with the histone deacetylase inhibitor Trichostatin A. Altogether, our results indicate that aberrant epigenetic regulation can be detected and modified before tissue-specific gene transcription, a finding which may lead to novel strategies for the prevention of chromatin-related pathologies.

Acetylation↗

PubMed1996

Regulation of azurophil granule-associated serine protease genes during myelopoiesis.

During myelopoiesis, the primary or azurophil granules are synthesized at the promyelocyte stage of development. The biogenesis of these granules requires the coordinated regulation of a large number of genes that comprise the constituent proteins of the granules themselves. The genes encoding the granule-associated proteins appear to be regulated primarily at the level of gene transcription; that is, most of the granule genes appear to be transcriptionally activated at the beginning of the promyelocyte stage and are transcriptionally repressed at the transition to myelocytes. The cis-acting DNA elements and trans-regulatory factors that control the regulation of granule genes are just beginning to be defined. The granule-associated genes are dispersed throughout the genome; however, several of the serine protease genes expressed in these granules are tightly clustered and the tight clustering may play an important role for their regulation. Recent transgenic studies suggest that DNA elements required for myeloid-specific targeting may be located just upstream from or very near to the granule genes themselves. DNA elements similar to the globin locus control region will probably play a role in the expression of these genes, although locus control elements have not yet been defined for any azurophil granule genes. Future studies directed at defining these key regulatory elements will enhance our understanding of the molecular events that underlie myeloid development.

Azure Stains↗

PubMed2004

A conserved sequence upstream of the B29 (Ig beta, CD79b) gene interacts with YY1.

The human, murine, and rat B29 (Ig beta, CD79b) genes are highly conserved in sequence and organization and exhibit strict B cell-specific expression. In the human and rat genomes, the B29 gene is located between the skeletal muscle-specific Na-channel alpha subunit (SCN4A) gene and the pituitary-specific growth hormone (GH-N) gene. The human pituitary-specific GH-N gene is controlled by a tissue-specific locus control region (LCR) located just upstream of the B29 promoter that mediates tissue-specific enhancement, histone acetylation, and an open chromatin conformation across the B29 gene in growth hormone (GH)-expressing pituitary cells. Here we show that B29 mRNA is not detected in a GH-expressing pituitary cell line and that GH-N mRNA is not detected in B cells. This differential expression suggests that the B29 gene is insulated or otherwise protected from the regulatory influences of the closely proximal GH LCR. We searched available sequences upstream of the human, mouse, and rat B29 genes and found a highly conserved sequence that fulfills the criteria recently established for non-coding DNA elements potentially involved in gene control. This B29 conserved sequence (BCS) bound ubiquitously expressed nuclear protein complexes. DNase I protection analysis of the BCS revealed a central 'footprinted' core which was confirmed to bind the multifunctional transcription factor, YY1. However, neither the BCS nor the YY1-binding core motif exhibited silencer or enhancer activity in transient transfections or position-independent insulator activity in enhancer-blocking assays. Thus, the BCS may function as a tissue-specific LCR or position-dependent insulator specifically countering the influences of the 5' GH LCR and controlling B29 gene expression.

5' Flanking Region↗

PubMed2000

Regulation of germline promoters by the two human Ig heavy chain 3' alpha enhancers.

The human IgH 3' enhancers, located downstream of each of the two Calpha genes, modulate germline (GL) transcription of the IgH genes by influencing the activity of promoter-enhancer complexes upstream of the switch and intervening (I) regions. The regulation of GL alpha1 and alpha2 promoters by different human 3' enhancer fragments was investigated in cell lines representing various developmental stages. Both alpha1HS1,2 and alpha2HS1,2 fragments show equally strong enhancer activity on the GL alpha1 and alpha2 promoters in both orientations when transiently transfected into a number of mature B cell line (DG75, CL-01, and HS Sultan). However, there is no activity in a human pre-B cell line (NALM-6) nor a human T cell line (Jurkat). HS3 shows no enhancer activity by itself in any of the cell lines, whereas a modest effect is noted using HS4 in the three mature B cell lines. However, the combination of the alpha2HS3-HS1,2-HS4 fragments, which together form a potential locus control region, displays a markedly stronger enhancer activity than the individual fragments with a differential effect on the alpha1 and alpha2 promoters as compared with the gamma3 promoter. Our results suggest that the human GL alpha promoter may be regulated by two independent pathways. One pathway is induced by TGF-beta1 which directs IgA isotype switch through activation of the GL alpha promoter and no TGF-beta1-responsive elements are present in the different 3' enhancer fragments. The other route is through the human 3' enhancer regions that cis-up-regulate the GL alpha promoter activity in mature B cells.

3' Untranslated Regions↗

PubMed2000

Reporter constructs showing proper globin gene switching in cultured erythroid cell lines.

Recent advances in the study of globin gene switching in the context of complete gene locus have contributed greatly to our understanding of developmental regulation mechanism of globin gene expression. However, it is not clear yet whether the cluster is sufficient in proper gene switching when the globin genes are replaced with conventional reporter genes. Furthermore, even though erythroid-specific and ubiquitous transcription factors involved in erythroid-specific globin gene expression have been characterized and some plausible globin gene switching models have been suggested, any specific factor directly involved in globin gene switching is not yet identified. In this study, as an effort to further understand globin switching mechanism and to identify globin switching factors, we constructed reporter vectors by juxtaposing several putative regulatory elements in human beta-globin locus to conventional reporter genes and analyzed their stage-specific expression in erythroid cell lines. At the end, we demonstrated that gammabeta-type constructs, in which both gamma-type and beta-type globin reporter genes were linked in cis below beta-globin locus control region (LCR), show proper stage-specific reporter gene expression in erythroid cell lines and also recapitulate globin switching in cell hybrids.

Animals↗

PubMed1998

Chromatin interaction mechanism of transcriptional control in vivo.

We have used a kinetic analysis to distinguish possible mechanisms of activation of transcription of the different genes in the human beta globin locus. Based on in situ studies at the single-cell level we have previously suggested a dynamic mechanism of single genes alternately interacting with the locus control region (LCR) to activate transcription. However, those steady-state experiments did not allow a direct measurement of the dynamics of the mechanism and the presence of loci with in situ primary transcript signals from two beta-like genes in cis has left open the possibility that multiple genes in the locus could initiate transcription simultaneously. Kinetic assays involving removal of a block to transcription elongation in conjunction with RNA FISH show that multiple beta gene primary transcript signals in cis represent a transition between alternating transcriptional periods of single genes, supporting a dynamic interaction mechanism.

Animals↗

PubMed1998

Individual LCR hypersensitive sites cooperate to generate an open chromatin domain spanning the human beta-globin locus.

BACKGROUND: The human beta-globin locus control region (LCR) is composed of five DNase I hypersensitive (HS) sites located 5' to the multiple genes it regulates. The LCR has been shown to comprise, among other essential properties, an activity that is required for generating a chromatin structure which renders the entire beta-globin gene locus accessible to exogenous nucleases. This nuclease-sensitive state is generally believed to be reflective of the chromatin environment that is permissive for transcriptional activation of the globin genes. RESULTS: Here we show, in mice bearing intact YAC transgenes that encompass the whole human beta-globin locus, that the deletion of individual core LCR HS sites negatively affects the ability of the LCR to confer this open chromatin conformation throughout the locus, and when analysed in concert with the effect that these same mutations have on transcription, the data show that the chromatin opening activity is a necessary, but not sufficient, prerequisite for globin gene expression. The results also show that after deletion of individual hypersensitive sites, the mutated LCR is no longer able to provide an accessible chromatin environment that is independent from the site of YAC transgene integration. CONCLUSIONS: These experiments provide further evidence for the hypothesis that the HS sites must act cooperatively to fulfil the multiple functions that are attributable to the LCR.

Adult↗

PubMedDate not supplied

Activation of the human GH gene cluster: roles for targeted chromatin modification.

The cluster of genes encoding the human growth hormone (GH) contains an array of five highly related genes. From 5' to 3' these are: GHN, CSL (encoding chorionic somatomammotropin-like gene), CSA, GHV (encoding GH-variant gene) and CSB. These five genes are expressed in mutually exclusive tissue distributions, GHN in pituitary somatotropes and the remaining four genes in placental villous syncytiotrophoblasts. The onset of GH expression during development is dependent upon epigenetic modifications at the GH locus under the control of its distal locus control region (LCR). A clear understanding of these normal epigenetic controls on the expression of GH could lead to new insights into the development and treatment of isolated GH deficiency in children. This review focuses on the role of the LCR in histone hyperacetylation at the GH locus and subsequent effects on the tissue-specific activation of these genes.

Acetyltransferases↗

PubMed1999

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

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

Animals↗

PubMed2002

Chromatin and CD4, CD8A and CD8B gene expression during thymic differentiation.

The regulation of gene expression during thymocyte development provides an ideal experimental system to study lineage-commitment processes. In particular, expression of the CD4, CD8A and CD8B genes seems to correlate well with the cell-fate decisions that are taken by thymocytes, and elucidating the molecular mechanisms that underlie the differential expression of these genes could reveal key events in differentiation processes. Here, we review examples of how gene cis elements (such as promoters, enhancers and locus control regions) and trans elements (such as transcription factors, chromatin-remodelling complexes and histone-modification enzymes) come together to orchestrate a finely tuned sequence of events that results in the complex pattern of CD4, CD8A and CD8B gene expression that is observed during thymocyte development.

Animals↗

PubMed2001

Developmental regulation of DNA replication timing at the human beta globin locus.

The human beta globin locus replicates late in most cell types, but becomes early replicating in erythroid cells. Using FISH to map DNA replication timing around the endogenous beta globin locus and by applying a genetic approach in transgenic mice, we have demonstrated that both the late and early replication states are controlled by regulatory elements within the locus control region. These results also show that the pattern of replication timing is set up by mechanisms that work independently of gene transcription.

Animals↗

PubMed2003

Mutation of a transcriptional motif of a distant regulatory element reduces the expression of embryonic and fetal globin genes.

High-level beta-globin gene expression is dependent on the presence of the locus control region (LCR), a powerful regulatory element physically characterized by five DNase I-hypersensitive sites (HS), designated HS1-HS5. Of these, HS3 contains seven GT motifs that are essential for its activity. One of the motifs, GT6, has been shown by in vivo footprinting to display the largest difference in signal between fetal and adult globin expressing cells. We assessed the contribution of GT6 on the downstream globin gene expression by mutating this motif in a 248 kb beta-globin locus yeast artificial chromosome and measuring the activity of beta-globin genes in GT6m beta-YAC transgenic mice. Seven transgenic lines were established, three of which contained at least one intact copy of the beta-globin locus and were further investigated. The mutation of the GT6 motif reduced the expression of epsilon- and gamma-globin genes during embryonic erythropoiesis. During definitive erythropoiesis, gamma-globin gene expression was significantly reduced while beta-globin gene expression was virtually indistinguishable from wild-type controls. We conclude that the GT6 motif of hypersensitive site 3 of the LCR is required for normal epsilon- and gamma-globin gene expression during embryonic erythropoiesis and for gamma-globin gene expression during definitive erythropoiesis in the fetal liver. Our results provide evidence that mutations of single transcriptional motifs of distant regulatory elements can have profound effects on gene expression.

Animals↗

PubMed1993

V(D)J recombination in B cells is impaired but not blocked by targeted deletion of the immunoglobulin heavy chain intron enhancer.

We have assessed the importance of the immunoglobulin heavy chain (IgH) intron enhancer for recombination of variable gene segments (V, D and J) during B cell development. We generated chimeric mice with embryonic stem cells lacking the intron enhancer from one of their IgH loci. The IgH intron enhancer was substituted by a short oligonucleotide through homologous recombination using the 'Hit and Run' procedure. V(D)J recombination occurred less frequently on mutant alleles, but was not blocked completely. Quantitative polymerase chain reaction analyses demonstrated that 15-30% of the mutated loci in mature B cells were unrearranged, in striking contrast to the wild-type alleles. The remainder of the mutated loci underwent D-J (65-80%) as well as V-DJ rearrangements, although the latter were less frequent (3-6%). These results are in line with previous data which showed that the V(D)J recombination machinery is modulated through cis-regulatory elements within the intron enhancer. However, our data predict the existence of additional cis-regulatory element(s) which, together with the intron enhancer, are required to activate the V(D)J recombination machinery fully. Such cis-regulatory element(s) might function as an enhancer of recombination or as a locus control region regulating the accessibility of the IgH locus.

Animals↗

PubMed1992

Production of functional human hemoglobin in transgenic swine.

A construct containing the locus control region (LCR) from the human beta globin locus together with two copies of the human alpha 1 gene and a single copy of the human beta A gene was used to obtain three transgenic pigs. The transgenic pigs are healthy, not anemic, and grow at a rate comparable to non-transgenic littermates. All animals expressed the human genes. However, alpha globin was consistently expressed at higher levels than beta globin. Isolation of the human hemoglobin from both porcine hemoglobin and other non-hemoglobin proteins was accomplished by ion exchange chromatography. The purified porcine derived human hemoglobin exhibited an oxygen affinity similar to that of human derived human hemoglobin.

Animals↗

PubMed1991

Molecular characterization of severe hemophilia A suggests that about half the mutations are not within the coding regions and splice junctions of the factor VIII gene.

Hemophilia A is an X chromosome-linked disorder resulting from deficiency of factor VIII, an important protein in blood coagulation. A large number of disease-producing mutations have been reported in the factor VIII gene. However, a comprehensive analysis of the mutations has been difficult because of the large gene size, its many scattered exons, and the high frequency of de novo mutations. Recently, we have shown that nearly all mutations resulting in mild-to-moderate hemophilia A can be detected by PCR and denaturing gradient gel electrophoresis (DGGE). In this study, we attempted to discover the mutations causing severe hemophilia A by analyzing 47 unselected patients, 30 of whom had severe hemophilia and 17 of whom had mild-to-moderate disease. Using DGGE as a screening method, we analyzed 99% of the coding region, 94% of the splice junctions, the promoter region, and the polyadenylylation site of the gene. We found the mutation in 16 of 17 (94%) patients with mild-to-moderate disease but in only 16 of 30 (53%) patients with severe hemophilia A. Since DGGE after computer analysis appears to detect all mutations in a given fragment, the lower-than-expected yield of mutations in patients with severe disease is likely not due to failure of the detection method; it is probably due to the presence of mutations in DNA sequences outside the regions studied. Such sequences may include locus-controlling regions, other sequences within introns or outside the gene that are important for its expression, or another gene involved in factor VIII expression that is very closely linked to the factor VIII gene.

Amino Acid Sequence↗

PubMed1993

Locating well-conserved regions within a pairwise alignment.

Within a single alignment of two DNA sequences or two protein sequences, some regions may be much better conserved than others. Such strong conservation may reveal a region that possesses an important function. When alignments are so long that it is infeasible, or at least undesirable, to inspect them in complete detail, it is helpful to have an automatic process that computes information about the varying degree of conservation along the alignment and displays the information in a graphical representation that is readily assimilated. This paper presents methods for computing several such 'robustness measures' at each position of a given alignment. These methods are all very space-efficient; they use only space proportional to the sum of the two sequence lengths. To illustrate their effectiveness, one of the methods is used to locate particularly well-conserved regions in the beta-globin gene locus control region and in the 5' flank of the gamma-globin gene.

Algorithms↗

PubMed1999

Mechanisms of developmental control of transcription in the murine alpha- and beta-globin loci.

We have characterized mRNA expression and transcription of the mouse alpha- and beta-globin loci during development. S1 nuclease and primary transcript in situ hybridization analyses demonstrate that all seven murine globin genes (zeta, alpha1, alpha2, epsilony, betaH1, betamaj, and betamin) are transcribed during primitive erythropoiesis, however transcription of the zeta, epsilony, and betaH1 genes is restricted to the primitive erythroid lineage. Transcription of the betamaj and betamin genes in primitive cells is EKLF-dependent demonstrating EKLF activity in embryonic red cells. Novel kinetic analyses suggest that multigene expression in the beta locus occurs via alternating single-gene transcription whereas coinitiation cannot be ruled out in the alpha locus. Transcriptional activation of the individual murine beta genes in primitive cells correlates inversely with their distance from the locus control region, in contrast with the human beta locus in which the adult genes are only activated in definitive erythroid cells. The results suggest that the multigene expression mechanism of alternating transcription is evolutionarily conserved between mouse and human beta globin loci but that the timing of activation of the adult genes is altered, indicating important fundamental differences in globin gene switching.

Animals↗