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M A Bender

Publications and source records attributed to M A Bender.

At least 19 recordsLinked to original sources
PubMed2006

The locus control region is required for association of the murine beta-globin locus with engaged transcription factories during erythroid maturation.

We have examined the relationship between nuclear localization and transcriptional activity of the endogenous murine beta-globin locus during erythroid differentiation. Murine fetal liver cells were separated into distinct erythroid maturation stages by fluorescence-activated cell sorting, and the nuclear position of the locus was determined at each stage. We find that the beta-globin locus progressively moves away from the nuclear periphery with increasing maturation. Contrary to the prevailing notion that the nuclear periphery is a repressive compartment in mammalian cells, beta(major)-globin expression begins at the nuclear periphery prior to relocalization. However, relocation of the locus to the nuclear interior with maturation is accompanied by an increase in beta(major)-globin transcription. The distribution of nuclear polymerase II (Pol II) foci also changes with erythroid differentiation: Transcription factories decrease in number and contract toward the nuclear interior. Moreover, both efficient relocalization of the beta-globin locus from the periphery and its association with hyperphosphorylated Pol II transcription factories require the locus control region (LCR). These results suggest that the LCR-dependent association of the beta-globin locus with transcriptionally engaged Pol II foci provides the driving force for relocalization of the locus toward the nuclear interior during erythroid maturation.

Animals↗
PubMed2006

Flanking HS-62.5 and 3' HS1, and regions upstream of the LCR, are not required for beta-globin transcription.

The locus control region (LCR) was thought to be necessary and sufficient for establishing and maintaining an open beta-globin locus chromatin domain in the repressive environment of the developing erythrocyte. However, deletion of the LCR from the endogenous locus had no significant effect on chromatin structure and did not silence transcription. Thus, the cis-regulatory elements that confer the open domain remain unidentified. The conserved DNaseI hypersensitivity sites (HSs) HS-62.5 and 3'HS1 that flank the locus, and the region upstream of the LCR have been implicated in globin gene regulation. The flanking HSs bind CCCTC binding factor (CTCF) and are thought to interact with the LCR to form a "chromatin hub" involved in beta-globin gene activation. Hispanic thalassemia, a deletion of the LCR and 27 kb upstream, leads to heterochromatinization and silencing of the locus. Thus, the region upstream of the LCR deleted in Hispanic thalassemia (upstream Hispanic region [UHR]) may be required for expression. To determine the importance of the UHR and flanking HSs for beta-globin expression, we generated and analyzed mice with targeted deletions of these elements. We demonstrate deletion of these regions alone, and in combination, do not affect transcription, bringing into question current models for the regulation of the beta-globin locus.

5' Flanking Region↗
PubMed2005

Deletion of the core region of 5' HS2 of the mouse beta-globin locus control region reveals a distinct effect in comparison with human beta-globin transgenes.

The beta-globin locus control region (LCR) is a large DNA element that is required for high-level expression of beta-like globin genes from the endogenous mouse locus or in transgenic mice carrying the human beta-globin locus. The LCR encompasses 6 DNaseI hypersensitive sites (HSs) that bind transcription factors. These HSs each contain a core of a few hundred base pairs (bp) that has most of the functional activity and exhibits high interspecies sequence homology. Adjoining the cores are 500- to 1000-bp "flanks" with weaker functional activity and lower interspecies homology. Studies of human beta-globin transgenes and of the endogenous murine locus show that deletion of an entire HS (core plus flanks) moderately suppresses expression. However, human transgenes in which only individual HS core regions were deleted showed drastic loss of expression accompanied by changes in chromatin structure. To address these disparate results, we have deleted the core region of 5'HS2 from the endogenous murine beta-LCR. The phenotype was similar to that of the larger 5'HS2 deletion, with no apparent disruption of chromatin structure. These results demonstrate that the greater severity of HS core deletions in comparison to full HS deletions is not a general property of the beta-LCR.

Animals↗
PubMed2005

Proximity among distant regulatory elements at the beta-globin locus requires GATA-1 and FOG-1.

Recent evidence suggests that long-range enhancers and gene promoters are in close proximity, which might reflect the formation of chromatin loops. Here, we examined the mechanism for DNA looping at the beta-globin locus. By using chromosome conformation capture (3C), we show that the hematopoietic transcription factor GATA-1 and its cofactor FOG-1 are required for the physical interaction between the beta-globin locus control region (LCR) and the beta-major globin promoter. Kinetic studies reveal that GATA-1-induced loop formation correlates with the onset of beta-globin transcription and occurs independently of new protein synthesis. GATA-1 occupies the beta-major globin promoter normally in fetal liver erythroblasts from mice lacking the LCR, suggesting that GATA-1 binding to the promoter and LCR are independent events that occur prior to loop formation. Together, these data demonstrate that GATA-1 and FOG-1 are essential anchors for a tissue-specific chromatin loop, providing general insights into long-range enhancer function.

Animals↗
PubMed2003

The beta -globin locus control region (LCR) functions primarily by enhancing the transition from transcription initiation to elongation.

To investigate the molecular basis of beta-globin gene activation, we analyzed factor recruitment and histone modification at the adult beta-globin gene in wild-type (WT)/locus control region knockout (DeltaLCR) heterozygous mice and in murine erythroleukemia (MEL) cells. Although histone acetylation and methylation (Lys 4) are high before and after MEL differentiation, recruitment of the erythroid-specific activator NF-E2 to the promoter and preinitiation complex (PIC) assembly occur only after differentiation. We reported previously that targeted deletion of the LCR reduces beta-globin gene expression to 1%-4% of WT without affecting promoter histone acetylation. Here, we report that NF-E2 is recruited equally efficiently to the adult beta-globin promoters of the DeltaLCR and WT alleles. Moreover, the LCR deletion reduces PIC assembly only twofold, but has a dramatic effect on Ser 5 phosphorylation of RNA polymerase II and transcriptional elongation. Our results suggest at least three distinct stages in beta-globin gene activation: (1) an LCR-independent chromatin opening stage prior to NF-E2 recruitment to the promoter and PIC assembly; (2) an intermediate stage in which NF-E2 binding (LCR-independent) and PIC assembly (partially LCR-dependent) occur; and (3) an LCR-dependent fully active stage characterized by efficient pol II elongation. Thus, in its native location the LCR functions primarily downstream of activator recruitment and PIC assembly.

Animals↗
PubMed2003

A complex chromatin landscape revealed by patterns of nuclease sensitivity and histone modification within the mouse beta-globin locus.

In order to create an extended map of chromatin features within a mammalian multigene locus, we have determined the extent of nuclease sensitivity and the pattern of histone modifications associated with the mouse beta-globin genes in adult erythroid tissue. We show that the nuclease-sensitive domain encompasses the beta-globin genes along with several flanking olfactory receptor genes that are inactive in erythroid cells. We describe enhancer-blocking or boundary elements on either side of the locus that are bound in vivo by the transcription factor CTCF, but we found that they do not coincide with transitions in nuclease sensitivity flanking the locus or with patterns of histone modifications within it. In addition, histone hyperacetylation and dimethylation of histone H3 K4 are not uniform features of the nuclease-sensitive mouse beta-globin domain but rather define distinct subdomains within it. Our results reveal a complex chromatin landscape for the active beta-globin locus and illustrate the complexity of broad structural changes that accompany gene activation.

Animals↗
PubMed2001

Targeted deletion of 5'HS1 and 5'HS4 of the beta-globin locus control region reveals additive activity of the DNaseI hypersensitive sites.

The mammalian beta-globin locus is a multigenic, developmentally regulated, tissue-specific locus from which gene expression is regulated by a distal regulatory region, the locus control region (LCR). The functional mechanism by which the beta-globin LCR stimulates transcription of the linked beta-like globin genes remains unknown. The LCR is composed of a series of 5 DNaseI hypersensitive sites (5'HSs) that form in the nucleus of erythroid precursors. These HSs are conserved among mammals, bind transcription factors that also bind to other parts of the locus, and compose the functional components of the LCR. To test the hypothesis that individual HSs have unique properties, homologous recombination was used to construct 5 lines of mice with individual deletions of each of the 5'HSs of the endogenous murine beta-globin LCR. Here it is reported that deletion of 5'HS1 reduces expression of the linked genes by up to 24%, while deletion of 5'HS4 leads to reductions of up to 27%. These deletions do not perturb the normal stage-specific expression of genes from this multigenic locus. In conjunction with previous studies of deletions of the other HSs and studies of deletion of the entire LCR, it is concluded that (1) none of the 5'HSs is essential for nearly normal expression; (2) none of the HSs is required for proper developmental expression; and (3) the HSs do not appear to synergize either structurally or functionally, but rather form independently and appear to contribute additively to the overall expression from the locus.

Age Factors↗
PubMed2001

The murine beta-globin locus control region regulates the rate of transcription but not the hyperacetylation of histones at the active genes.

Locus control regions (LCRs) are defined by their ability to confer high-level tissue-specific expression to linked genes in transgenic assays. Previously, we reported that, at its native site, the murine beta-globin LCR is required for high-level beta-globin gene expression, but is not required to initiate an open chromatin conformation of the locus. To further investigate the mechanism of LCR-mediated transcriptional enhancement, we have analyzed allele-specific beta-globin expression and the pattern of histone acetylation in the presence and absence of the LCR. In single cells from mice heterozygous for a deletion of the LCR, beta-globin expression from the LCR-deleted allele is consistently low ( approximately 1-4% of wild type). Thus, the endogenous LCR enhances globin gene expression by increasing the rate of transcription from each linked allele rather than by increasing the probability of establishing transcription per se. Furthermore, in erythroid cells from mice homozygous for the highly expressing wild-type beta-globin locus, hyperacetylation of histones H3 and H4 is localized to the LCR and active genes. In mice homozygous for the LCR deletion reduced histone hyperacetylation is observed in LCR proximal sequences; however, deletion of the LCR has no effect on the localized hyperacetylation of the genes. Together, our results suggest that, in its native genomic context, the LCR follows the rate model of enhancer function, and that the developmentally specific hyperacetylation of the globin genes is independent of both the rate of transcription and the presence of the LCR.

Acetylation↗
PubMed2000

Comparative structural and functional analysis of the olfactory receptor genes flanking the human and mouse beta-globin gene clusters.

By sequencing regions flanking the beta-globin gene complex in mouse (Hbbc) and human (HBBC), we have shown that the beta-globin gene cluster is surrounded by a larger cluster of olfactory receptor genes (ORGs). To facilitate sequence comparisons and to investigate the regulation of ORG expression, we have mapped 5' sequences of mRNA from olfactory epithelium encoding beta-globin-proximal ORGs. We have found that several of these genes contain multiple noncoding exons that can be alternatively spliced. Surprisingly, the only common motifs found in the promoters of these genes are a "TATA" box and a purine-rich motif. Sequence comparisons between human and mouse reveal that most of the conserved regions are confined to the coding regions and transcription units of the genes themselves, but a few blocks of conserved sequence also are found outside of ORG transcription units. The possible influence of beta-globin regulatory sequences on ORG expression in olfactory epithelium was tested in mice containing a deletion of the endogenous beta-globin locus control region, but no change in expression of the neighboring ORGs was detected. We evaluate the implications of these results for possible mechanisms of regulation of ORG transcription.

Animals↗
PubMed2000

Independent formation of DnaseI hypersensitive sites in the murine beta-globin locus control region.

Mammalian beta-globin loci are composed of multiple orthologous genes whose expression is erythroid specific and developmentally regulated. The expression of these genes both from the endogenous locus and from transgenes is strongly influenced by a linked 15-kilobase region of clustered DNaseI hypersensitive sites (HSs) known as the locus control region (LCR). The LCR encompasses 5 major HSs, each of which is highly homologous among humans, mice, and other mammals. To analyze the function of individual HSs in the endogenous murine beta-globin LCR, we have used homologous recombination in embryonic stem cells to produce 5 mouse lines, each of which is deficient for 1 of these major HSs. In this report, we demonstrate that deletion of the conserved region of 5'HS 1, 2, 3, 4, or 5/6 abolishes HS formation at the deletion site but has no influence on the formation of the remaining HSs in the LCR. Therefore, in the endogenous murine locus, there is no dominant or initiating site whose formation must precede the formation of the other HSs. This is consistent with the idea that HSs form autonomously. We discuss the implications of these findings for current models of beta-globin regulation.

Animals↗
PubMed2000

Deletions within the mouse beta-globin locus control region preferentially reduce beta(min) globin gene expression.

The mouse beta-globin gene cluster is regulated, at least in part, by a locus control region (LCR) composed of several developmentally stable DNase I hypersensitive sites located upstream of the genes. In this report, we examine the level of expression of the beta(min) and beta(maj) genes in adult mice in which HS2, HS3, or HS5,6 has been either deleted or replaced by a selectable marker via homologous recombination in ES cells. Primer extension analysis of RNA extracted from circulating reticulocytes and HPLC analysis of globin chains from peripheral red blood cells revealed that all mutations that reduce the overall output of the locus preferentially decrease beta(min) expression over beta(maj). The implications of these findings for the mechanism by which the LCR controls expression of the beta(maj) and beta(min) promoters are discussed.

Animals↗
PubMed2000

Beta-globin gene switching and DNase I sensitivity of the endogenous beta-globin locus in mice do not require the locus control region.

We have generated mice with a targeted deletion of the beta-globin locus control region (LCR). Mice homozygous for the deletion die early in embryogenesis but can be rescued with a YAC containing the human beta-globin locus. After germline passage, deletion of the LCR leads to a severe reduction in expression of all mouse beta-like globin genes, but no alteration in the developmental specificity of expression. Furthermore, a DNase I-sensitive "open" chromatin conformation of the locus is established and maintained. Thus, the dominant role of the LCR in the native locus is to confer high-level transcription, and elements elsewhere in the locus are sufficient to establish and maintain an open conformation and to confer developmentally regulated globin gene expression.

Animals↗
PubMed1999

Conservation of sequence and structure flanking the mouse and human beta-globin loci: the beta-globin genes are embedded within an array of odorant receptor genes.

In mouse and human, the beta-globin genes reside in a linear array that is associated with a positive regulatory element located 5' to the genes known as the locus control region (LCR). The sequences of the mouse and human beta-globin LCRs are homologous, indicating conservation of an essential function in beta-globin gene regulation. We have sequenced regions flanking the beta-globin locus in both mouse and human and found that homology associated with the LCR is more extensive than previously known, making up a conserved block of approximately 40 kb. In addition, we have identified DNaseI-hypersensitive sites within the newly sequenced regions in both mouse and human, and these structural features also are conserved. Finally, we have found that both mouse and human beta-globin loci are embedded within an array of odorant receptor genes that are expressed in olfactory epithelium, and we also identify an olfactory receptor gene located 3' of the beta-globin locus in chicken. The data demonstrate an evolutionarily conserved genomic organization for the beta-globin locus and suggest a possible role for the beta-globin LCR in control of expression of these odorant receptor genes and/or the presence of mechanisms to separate regulatory signals in different tissues.

Animals↗
PubMed1999

Antihyperalgesic effects of infection with a preproenkephalin-encoding herpes virus.

To test the utility of gene therapeutic approaches for the treatment of pain, a recombinant herpes simplex virus, type 1, has been engineered to contain the cDNA for an opioid peptide precursor, human preproenkephalin, under control of the human cytomegalovirus promoter. This virus and a similar recombinant containing the Escherichia coli lacZ gene were applied to the abraded skin of the dorsal hindpaw of mice. After infection, the presence of beta-galactosidase in neuronal cell bodies of the relevant spinal ganglia (lacZ-containing virus) and of human proenkephalin (preproenkephalin-encoding virus) in the central terminals of these neurons indicated appropriate gene delivery and expression. Baseline foot withdrawal responses to noxious radiant heat mediated by Adelta and C fibers were similar in animals infected with proenkephalin-encoding and beta-galactosidase-encoding viruses. Sensitization of the foot withdrawal response after application of capsaicin (C fibers) or dimethyl sulfoxide (Adelta fibers) observed in control animals was reduced or eliminated in animals infected with the proenkephalin-encoding virus for at least 7 weeks postinfection. Hence, preproenkephalin cDNA delivery selectively blocked hyperalgesia without disrupting baseline sensory neurotransmission. This blockade of sensitization was reversed by administration of the opioid antagonist naloxone, apparently acting in the spinal cord. The results demonstrate that the function of sensory neurons can be selectively altered by viral delivery of a transgene. Because hyperalgesic mechanisms may be important in establishing and maintaining neuropathic and other chronic pain states, this approach may be useful for treatment of chronic pain and hyperalgesia in humans.

Animals↗
PubMed1999

Analysis of mammalian cis-regulatory DNA elements by homologous recombination.

The use of homologous recombination to modify and thereby functionally analyze cis-regulatory DNA elements in mammalian cells has become an important approach in mammalian gene expression research. We have emphasized the necessity of designing a system that allows the removal of selectable markers used in targeting and facilitates the further modification of the region under study. To perform these tasks, we presently favor making an initial HR-mediated replacement of the entire element under study with an active positive selectable marker in combination with either an inactive second positive selectable marker or an active negative selectable marker. The plug and socket system, in which an inactive selectable marker is complemented by HR, is the most dependable and well-characterized option for making secondary modifications. However, the double-replacement system has certain advantages, and the recently developed RMCE approach, which allows replacement of a negative selectable marker by site-specific recombinase-mediated insertion without using a positive selectable marker, will likely prove very valuable in future experiments. Each of the systems, or combinations thereof, should be considered in light of the specifics of any given experiment to select the most appropriate option. Although the emphasis of this article has been the analysis of cis-acting regulatory elements involved in transcription, these same approaches can be used to analyze other regulatory elements (e.g., origins of replication) and to make multiple subtle mutations in polypeptides.

Animals↗
PubMed1998

Description and targeted deletion of 5' hypersensitive site 5 and 6 of the mouse beta-globin locus control region.

The most upstream hypersensitive site (HS) of the beta-globin locus control region (LCR) in humans (5' HS 5) and chickens (5' HS 4) can act as an insulating element in some gain of function assays and may demarcate a beta-globin domain. We have mapped the most upstream HSs of the mouse beta-globin LCR and sequenced this region. We find that mice have a region homologous to human 5' HS 5 that is associated with a minor HS. In addition we map a unique HS upstream of 5' HS 5 and refer to this novel site as mouse 5' HS 6. We have also generated mice containing a targeted deletion of the region containing 5' HS 5 and 6. We find that after excision of the selectable marker in vivo, deletion of 5' HS 5 and 6 has a minimal effect on transcription and does not prevent formation of the remaining LCR HSs. Taken together these findings suggest that the most upstream HSs of the mouse beta-globin LCR are not necessary for maintaining the beta-globin locus in an active configuration or to protect it from a surrounding repressive chromatin environment.

Animals↗
PubMed1998

Reduced beta-globin gene expression in adult mice containing deletions of locus control region 5' HS-2 or 5' HS-3.

To gain insights into the functions of individual DNA'se hypersensitive sites within the beta globin locus control region (LCR), we deleted the endogenous 5' HS-2 and HS-3 regions from the mouse germline using homologous recombination techniques. We demonstrated that the deletion of either murine 5' HS-2 or 5' HS-3 reduced the expression of the embryonic epsilon y and beta h1 globin genes minimally in yolk sac-derived erythrocytes, but that both knockouts reduced the output of the adult beta (beta-Major + beta-Minor) globin genes by approximately 30% in adult erythrocytes. When the selectable marker PGK-Neo cassette was retained within either the HS-2 or HS-3 region, a much more severe reduction in globin gene expression was observed at all developmental stages. PGK-Neo was shown to be expressed in an erythroid-specific fashion when it was retained in the HS-3 position. These results show that neither 5' HS-2 nor HS-3 is required for the activity of embryonic globin genes, nor are these sites required for correct developmental switching. However, each site is required for approximately 30% of the total LCR activity associated with adult beta-globin gene expression in adult red blood cells. Each site therefore contains some non-redundant information that contributes to adult globin gene function.

Animals↗