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PubMed2003

From DNA structure to gene expression: mediators of nuclear compartmentalization and dynamics.

Eukaryotic genomes are functionally compartmentalized into chromatin domains by their attachment to a supporting structure that has traditionally been termed the nuclear matrix. Present evidence indicates the dynamics of this entity, which requires particular properties of the elements that mediate this kind of interaction. Above all, this is enabled by the so-called 'mass binding phenomenon' by which scaffold/matrix-attachment regions (S/MARs) reversibly associate with ubiquitous factors. Recent investigations and novel techniques have shown that these contacts can be altered by modulators as well as by specific interactions with the components of enhancers and locus control regions.

Chromatin↗
PubMed1996

Synthesis of human globin polypeptides mediated by recombinant adeno-associated virus vectors.

Adeno-associated virus, serotype 2 (AAV2)-based chimeric plasmids that harbored a near-full-length human alpha- or beta-globin cDNA were constructed. The cDNAs were spliced into an AAV plasmid, pAAV delta K, downstream from the viral P40 promoter, substituting the capsid gene region. The correctness of the insertion with regard to the transcription polarity was ascertained by both restriction enzyme analysis and DNA sequencing. One of the constructs, pAAVcHBBLCR, contained the erythroid-specific enhancer elements, the locus control region, HS1 and HS2, to ensure an efficient and tissue-specific gene expression. Use of a defective complementing helper, pAVXB (Dixit, M.; et al. Gene 1991, 104, 253-257.) and adenovirus 2 made it possible to prepare recombinant AAVs (rAAVs). Infection of human 293 cells (embryonal kidney cell line) with the resultant rAAV (AAVcHBB) and cotransfection of mouse erythroleukemia (MEL) cells with the beta-globin construct (pAAVcHBBLCR) and an alpha-globin construct (pAAVcHAB) triggered efficient synthesis of human globin polypeptides in the cells, as analyzed by biochemical and immunohistochemical means. The LCR made the construct respond to an inducer, N,N-hexamethylenebisacetamide, the amount of expressed human beta-globin reaching a similar level as the endogenous mouse beta-globin in MEL cells. Electrotransfection of mouse bone marrow hematopoietic stem/progenitor cells with the constructs dramatically increased the number of benzidine-positive cells in liquid suspension culture, indicating expression and synthesis of a human hemoglobin in these cells. Thus, the rAAV constructs may be useful for gene therapy of hemoglobinopathies.

Animals↗
PubMed2002

The solitary long terminal repeats of ERV-9 endogenous retrovirus are conserved during primate evolution and possess enhancer activities in embryonic and hematopoietic cells.

The solitary long terminal repeats (LTRs) of ERV-9 endogenous retrovirus contain the U3, R, and U5 regions but no internal viral genes. They are middle repetitive DNAs present at 2,000 to 4,000 copies in primate genomes. Sequence analyses of the 5" boundary area of the erythroid beta-globin locus control region (beta-LCR) and the intron of the embryonic axin gene show that a solitary ERV-9 LTR has been stably integrated in the respective loci for at least 15 million years in the higher primates from orangutan to human. Functional studies utilizing the green fluorescent protein (GFP) gene as the reporter in transfection experiments show that the U3 region of the LTRs possesses strong enhancer activity in embryonic cells of widely different tissue origins and in adult cells of blood lineages. In both the genomic LTRs of embryonic placental cells and erythroid K562 cells and transfected LTRs of recombinant GFP plasmids in K562 cells, the U3 enhancer activates synthesis of RNAs that are initiated from a specific site 25 bases downstream of the AATAAA (TATA) motif in the U3 promoter. A second AATAAA motif in the R region does not serve as the TATA box or as the polyadenylation signal. The LTR-initiated RNAs extend through the R and U5 regions into the downstream genomic DNA. The results suggest that the ERV-9 LTR-initiated transcription process may modulate transcription of the associated gene loci in embryonic and hematopoietic cells.

Animals↗
PubMed1997

Multiple regions of p45 NF-E2 are required for beta-globin gene expression in erythroid cells.

Regulated expression of genes in the beta-globin cluster depends upon sequences located between 5 and 20 kb upstream of the epsilon gene, known as the locus control region (LCR). beta-Globin expression in murine erythroleukemia (MEL) cells depends on NF-E2, a transcription factor which binds to enhancer sequences in the LCR. To gain insight into the mechanism of globin gene activation by NF-E2, an NF-E2 null MEL cell line was used to map regions of NF-E2 required for beta-globin expression. Within the transactivation domain, two discrete proline-rich regions were required for rescue of beta-globin expression. The first was located at the N-terminus of NF-E2, while the second was located N-terminal of the cap 'n collar (CNC) domain. Other proline-rich sequences were dispensable, indicating that proline content per se does not determine NF-E2 activity. Mutations within the conserved CNC domain markedly diminished rescue of beta-globin expression. This domain was required, in addition to the basic leucine zipper domain, for DNA binding activity. The requirement for discrete proline-rich sequences within the transactivation domain suggests that globin gene expression in MEL cells depends on specific interactions between NF-E2 and downstream effector molecules.

Amino Acid Sequence↗
PubMed1998

Erythroid Krüppel-like factor is essential for beta-globin gene expression even in absence of gene competition, but is not sufficient to induce the switch from gamma-globin to beta-globin gene expression.

Different genes in the beta-like globin locus are expressed at specific times during development. This is controlled, in part, by competition between the genes for activation by the locus control region. In mice, gene inactivation of the erythroid Krüppel-like factor (EKLF) transcription factor results in a lethal anemia due to a specific and substantial decrease in expression of the fetal/adult-stage-specific beta-globin gene. In transgenic mice carrying the complete human beta-globin locus, EKLF ablation not only impairs human beta-globin-gene expression but also results in increased expression of the human gamma-globin genes during the fetal/adult stages. Hence, it may appear that EKLF is a determining factor for the developmental switch from gamma-globin to beta-globin transcription. However, we show here that the function of EKLF for beta-globin-gene expression is necessary even in absence of gene competition. Moreover, EKLF is not developmental specific and is present and functional before the switch from gamma-globin to beta-globin-gene expression occurs. Thus, EKLF is not the primary factor that controls the switch. We suggest that autonomous repression of gamma-globin transcription that occurs during late fetal development is likely to be the initiating event that induces the switch.

Animals↗
PubMed2005

Chromatin domain activation via GATA-1 utilization of a small subset of dispersed GATA motifs within a broad chromosomal region.

Cis elements that mediate transcription factor binding are abundant within genomes, but the rules governing occupancy of such motifs in chromatin are not understood. The transcription factor GATA-1 that regulates red blood cell development binds with high affinity to GATA motifs, and initial studies suggest that these motifs are often unavailable for occupancy in chromatin. Whereas GATA-2 regulates the differentiation of all blood cell lineages via GATA motif binding, the specificity of GATA-2 chromatin occupancy has not been studied. We found that conditionally active GATA-1 (ER-GATA-1) and GATA-2 occupy only a small subset of the conserved GATA motifs within the murine beta-globin locus. Kinetic analyses in GATA-1-null cells indicated that ER-GATA-1 preferentially occupied GATA motifs at the locus control region (LCR), in which chromatin accessibility is largely GATA-1-independent. Subsequently, ER-GATA-1 increased promoter accessibility and occupied the betamajor promoter. ER-GATA-1 increased erythroid Krüppel-like factor and SWI/SNF chromatin remodeling complex occupancy at restricted LCR sites. These studies revealed three phases of beta-globin locus activation: GATA-1-independent establishment of specific chromatin structure features, GATA-1-dependent LCR complex assembly, and GATA-1-dependent promoter complex assembly. The differential utilization of dispersed GATA motifs therefore establishes spatial/temporal regulation and underlies the multistep activation mechanism.

Animals↗
PubMed1994

A 5' beta-globin matrix-attachment region and the polyoma enhancer together confer position-independent transcription.

Insertions of reporter constructs into the genome of higher eukaryotes typically lead to variegated gene expression due to position effects at the sites of integration. The 20-kb human beta-globin (beta Glb) locus control region (LCR) has been found to dampen these position effects when included in an expression vector. Several studies have indicated that much of the activity of the beta Glb-LCR resides in hypersensitive site II, which contains a strong enhancer. In this study, we have focused on the matrix-attachment region (MAR) at the 5' boundary of the beta Glb-LCR. We find that the beta Glb-MAR, by itself, has little effect on transcription of a reporter gene in stable transformants. However, when the beta Glb-MAR is linked in cis with the polyoma virus enhancer, the MAR-enhancer construct confers high levels of copy-dependent transcription that is independent of the chromosomal site of integration. These results suggest that the beta Glb-MAR may work synergistically with particular enhancer elements to dampen chromosomal position effects and ensure high-level expression.

Base Sequence↗
PubMed2003

Dynamic regulation of histone H3 methylated at lysine 79 within a tissue-specific chromatin domain.

Post-translational modifications of individual lysine residues of core histones can exert unique functional consequences. For example, methylation of histone H3 at lysine 79 (H3-meK79) has been implicated recently in gene silencing in Saccharomyces cerevisiae. However, the distribution and function of H3-meK79 in mammalian chromatin are not known. We found that H3-meK79 has a variable distribution within the murine beta-globin locus in adult erythroid cells, being preferentially enriched at the active betamajor gene. By contrast, acetylated H3 and H4 and H3 methylated at lysine 4 were enriched both at betamajor and at the upstream locus control region. H3-meK79 was also enriched at the active cad gene, whereas the transcriptionally inactive loci necdin and MyoD1 contained very little H3-meK79. As the pattern of H3-meK79 at the beta-globin locus differed between adult and embryonic erythroid cells, establishment and/or maintenance of H3-meK79 was developmentally dynamic. Genetic complementation analysis in null cells lacking the erythroid and megakaryocyte-specific transcription factor p45/NF-E2 showed that p45/NF-E2 preferentially establishes H3-meK79 at the betamajor promoter. These results support a model in which H3-meK79 is strongly enriched in mammalian chromatin at active genes but not uniformly throughout active chromatin domains. As H3-meK79 is highly regulated at the beta-globin locus, we propose that the murine ortholog of Disruptor of Telomeric Silencing-1-like (mDOT1L) methyltransferase, which synthesizes H3-meK79, regulates beta-globin transcription.

Animals↗
PubMed2005

Organization and expression of the human serpin gene cluster at 14q32.1.

The serpins are a superfamily of gene sequences that have been conserved through evolution. These genes encode protein products that perform a variety of functions in vivo, and their regulation differs among different cell types. About one-third of the serpin genes in the human genome are located at 14q32.1, and the serpin genes in this ~370 kb region are organized into discrete proximal, central, and distal subclusters of four, three, and four genes each. In this report we discuss the genomic organization of the 14q32.1 serpin gene cluster, and we summarize what is known about the regulation of each serpin gene in this region. An approach for studying locus-wide regulation of chromosomal serpin genes in situ is also described. Using this approach, specific mutations in the proximal serpin subcluster were prepared by homologous recombination. These mutant alleles define a serpin locus control region that regulates gene activity and chromatin structure of the entire proximal subcluster. Prospects for further analyses of this complex genomic domain are discussed.

Animals↗
PubMed2000

Nuclear localization and histone acetylation: a pathway for chromatin opening and transcriptional activation of the human beta-globin locus.

We have investigated the mechanism, structural correlates, and cis-acting elements involved in chromatin opening and gene activation, using the human beta-globin locus as a model. Full transcriptional activity of the human beta-globin locus requires the locus control region (LCR), composed of a series of nuclease hypersensitive sites located upstream of this globin gene cluster. Our previous analysis of naturally occurring and targeted LCR deletions revealed that chromatin opening and transcriptional activity in the endogenous beta-globin locus are dissociable and dependent on distinct cis-acting elements. We now report that general histone H3/H4 acetylation and relocation of the locus away from centromeric heterochromatin in the interphase nucleus are correlated and do not require the LCR. In contrast, LCR-dependent promoter activation is associated with localized histone H3 hyperacetylation at the LCR and the transcribed beta-globin-promoter and gene. On the basis of these results, we suggest a multistep model for gene activation; localization away from centromeric heterochromatin is required to achieve general hyperacetylation and an open chromatin structure of the locus, whereas a mechanism involving LCR/promoter histone H3 hyperacetylation is required for high-level transcription of the beta-globin genes.

Acetylation↗
PubMed1995

The erythroid Krüppel-like factor transactivation domain is a critical component for cell-specific inducibility of a beta-globin promoter.

Erythroid Krüppel-like factor (EKLF) is an erythroid cell-specific DNA-binding protein that activates transcription from the beta-globin CACCC element, a functionally important and evolutionarily conserved component of globin as well as other erythroid cell-specific promoters and enhancers. We have attempted to elucidate the molecular role of EKLF in erythrocyte-specific transcriptional activation. First, in vivo and in vitro analyses have been used to demonstrate that the level of activation by EKLF is dependent on the orientation and number of CACCC elements, that EKLF contains separable activation and DNA-binding domains, and that the EKLF proline-rich region is a potent activator in CV-1 cells when fused to a nonrelated DNA-binding module. Second, we have established a transient assay in murine erythroleukemia cells in which reproducible levels of a reporter can be induced when linked to a locus control region enhancer-beta-globin promoter and in which induction is abolished when the promoter CAC site is mutated to a GAL site. Third, we demonstrate that the EKLF transactivation region, when fused to the GAL DNA-binding domain, can restore inducibility to this mutated construct and that this inducibility exhibits activator-, promoter-, and cell-type specificity. These results demonstrate that EKLF provides a crucial transactivation function for globin expression and further reinforce the idea that EKLF is an important regulator of CACCC element-directed transcription in erythroid cells.

Animals↗
PubMed1998

Mechanisms of developmental regulation in globin loci.

Recent advances in the study of globin gene switching in the context of the complete locus have contributed greatly to our understanding of the mechanisms of developmental regulation. It has become clear that the interactions between the distant locus control region and the individual globin genes, as well as the trans-acting factors and physical parameters that affect these interactions, are crucial determinants in the developmental modulation of globin gene expression. This review concentrates on recent advances in the highly studied human beta-globin locus and will compare and contrast data from the human alpha-globin locus as well as the alpha and beta loci from other species where appropriate.

Animals↗
PubMed2004

Heme regulates the dynamic exchange of Bach1 and NF-E2-related factors in the Maf transcription factor network.

Small Maf proteins serve as dual-function transcription factors through an exchange of their heterodimerization partners. For example, as heterodimers with hematopoietic cell-specific p45 NF-E2 or NF-E2-related factors (Nrf), they activate the beta-globin or antioxidative stress enzyme heme oxygenase 1 (HO-1) genes, respectively. In contrast, together with Bach1, they repress these same genes. However, the signals leading to this partner exchange are not known. Using chromatin immunoprecipitation assays in NIH 3T3 cells, we show that heme, an inducer of ho-1, promotes displacement of Bach1 from the MafK-occupied ho-1 enhancers, which is followed by Nrf2 binding to these elements. Whereas histone H3 at the ho-1 enhancers and promoter is hyperacetylated irrespective of gene activity, exposure of cells to heme results in de novo hyperacetylation and hypermethylation of histone H3 in the transcribed region. These data indicate that, under normal conditions, the chromatin structure of ho-1 is in a preactivation state, but transcription is repressed by Bach1. Heme induces switching of Maf dimers, resulting in ho-1 expression. Heme also promotes displacement of Bach1 from the beta-globin locus control region without affecting MafK binding in murine erythroleukemia cells. Thus, heme functions as a signaling molecule for gene expression in higher eukaryotes.

Acetylation↗
PubMed1995

Identification of a major positive regulatory element located 5' to the human zeta-globin gene.

The function of the zeta-globin promoter was studied using a series of zeta-globin promoter deletion constructs to drive luciferase expression in transiently transfected human erythroleukemia cells. The promoters were used without enhancers, or with enhancers derived from the beta-globin locus control region and the alpha-globin HS-40 enhancer. When transfected into K562 cells, which express zeta-globin, comparable amounts of activity were obtained from the -557 and -417 zeta-luciferase constructs and the alpha-luciferase constructs when no enhancers or the alpha-globin locus enhancers were used. When the constructs were transfected into OCIM1 cells, which do not express zeta-globin, the zeta-globin promoters were at best 20% as active as the alpha-globin promoters. When sequences from -417 to -207 5' to the zeta-globin mRNA cap site were deleted, up to 95% of the zeta-globin promoter activity was lost in K562 cells. Reinsertion of these sequences into zeta-luciferase constructs missing the -417 to -207 region showed that the sequences lack classical enhancer activity. Point mutation of a GATA-1 site at -230 reduced promoter activity by 37%. Point mutation of a CCACC site at -240 had no effect. Electrophoretic mobility shift assays indicated that the -230 GATA-1 site has a relatively low affinity for GATA-1. These experiments show the presence of a strong positive-acting element, located between -417 and -207 bp 5' to the zeta-globin mRNA cap site, is necessary for high-level promoter activity in K562 cells. This element requires GATA-1 and additional unknown factors for maximal activity.

Base Sequence↗
PubMed1997

Genetic recombinations between c-myc and Igh mu as precursors for recombinations between c-myc and Igh alpha in BALB/c plasmacytomas.

In this paper we present evidence that primary plasmacytomas harboring a balanced chromosomal translocation t(12;15) may consist of a mosaic of neoplastic clones of tumor cells, in which some of the clones are derived from common progenitors by large additional deletions which alter or "remodel" the fine structure of the t(12;15) on the c-myc-deregulating chromosome. This finding is based on the molecular analysis of the t(12;15) in three plasmacytomas, TEPC 1194, PCT 4127 and PCT 4132, in which primary clones of tumor cells with recombinations between the Ig heavy-chain (Igh) mu locus (Igh mu) and c-myc were found to be related to secondary clones of tumor cells with recombinations between the Igh alpha locus (Igh alpha) and c-myc. Clonal relatedness was based on unique junction fragments between S mu and c-myc that were found to be identical in Igh mu/c-myc and Igh alpha/c-myc breakpoint regions. S mu/c-myc breakpoints with adjoining sequences can thus be used as clonotypic markers, providing molecular fingerprints for the evolution of precursor cells with Igh mu/c-myc recombinations into progenitor cells with Igh alpha/c-myc recombinations. We propose that aberrant isotype switch recombination or illegitimate rearrangement in switch regions converts Igh mu/c-myc rearrangements, which are found very rarely in established plasmacytomas, into Igh alpha/c-myc rearrangements, which are present in about 80% of plasmacytomas with t(12;15). Remodeling of the t(12;15) results in closer juxtaposition of c-myc to the 3'-C alpha locus control region and probably in enhanced transcription of c-myc. We suggest that remodeling of c-myc-deregulating rearrangements constitutes a novel progression mechanism in malignant B-cell development.

Animals↗
PubMed1996

Human trefoil peptides: genomic structure in 21q22.3 and coordinated expression.

Trefoil peptides are small secretory proteins characterized by three intrachain disulfide bonds forming the trefoil motif or P-domain. They are abundantly expressed on mucosal surfaces, especially of the gastrointestinal tract. In pathological conditions such as ulcers, metaplasia and neoplasia, their expression is upregulated. Three human trefoil peptides have been described: the estrogen-inducible pS2 protein, the spasmolytic protein and the intestinal trefoil factor. Recently, their role in the maintenance of surface integrity and ulcer healing was discussed. We already mapped the corresponding three genes (BCEI), SML1, TFF3) to the same genomic region (21q22.3). In this paper, we show that the three genes are clustered in a tandemly orientated fashion within 50 kb on a bacterial artificial chromosome (BAC) recombinant. This cluster is located adjacent to D21S19 and the locus order is cen-D21S212-TFF3-SML1-BCEI-D21S19-tel, whereas transcription of all three genes is directed towards the centromere. The gene structure of SML1 exhibits four exons, two of which encode the two separate trefoil motifs. TFF3 and BCEI, both containing one trefoil motif, are composed of three exons each, suggesting gene duplication and exon-shuffling events during evolution. The 5'-flanking region of SML1 was compared to the corresponding region of other trefoil genes. Two motifs with identical sequence and positions are shared between SML1 and BCEI, thus presenting possible targets for stomach-specific gene regulation. Two other motifs are shared within all known human and rat trefoil genes, suggesting a coordinated regulation and/or a common locus-controlling region. Using RT-PCR, a change in the pattern of trefoil gene expression is detected in tissue samples from normal gastric mucosa, hyperplastic polyps, gastric cancer, and gastric cancer cell lines, respectively.

Amino Acid Sequence↗
PubMed1993

An opportunistic promoter sharing regulatory sequences with either a muscle-specific or a ubiquitous promoter in the human aldolase A gene.

The human aldolase A gene is transcribed from three different promoters, pN, pM, and pH, all of which are clustered within a small 1.6-kbp DNA domain. pM, which is highly specific to adult skeletal muscle, lies in between pN and pH, which are ubiquitous but particularly active in heart and skeletal muscle. A ubiquitous enhancer, located just upstream of pH start sites, is necessary for the activity of both pH and pN in transient transfection assays. Using transgenic mice, we studied the sequence controlling the muscle-specific promoter pM and the relations between the three promoters and the ubiquitous enhancer. A 4.3-kbp fragment containing the three promoters and the ubiquitous enhancer showed an expression pattern consistent with that known in humans. In addition, while pH was active in both fast and slow skeletal muscles, pM was active only in fast muscle. pM activity was unaltered by the deletion of a 1.8-kbp region containing the ubiquitous enhancer and the pH promoter, whereas pN remained active only in fast skeletal muscle. These findings suggest that in fast skeletal muscle, a tissue-specific enhancer was acting on both pN and pM, whereas in other tissues, the ubiquitous enhancer was necessary for pN activity. Finally, a 2.6-kbp region containing the ubiquitous enhancer and only the pH promoter was sufficient to bring about high-level expression of pH in cardiac and skeletal muscle. Thus, while pH and pM function independently of each other, pN, remarkably, shares regulatory elements with each of them, depending on the tissue. Importantly, expression of the transgenes was independent of the integration site, as originally described for transgenes containing the beta-globin locus control region.

Animals↗
PubMed2006

Enhancer blocking by chicken beta-globin 5'-HS4: role of enhancer strength and insulator nucleosome depletion.

The 5'-HS4 chicken beta-globin insulator functions as a positional enhancer blocker on chromatinized episomes in human cells, blocking the HS2 enhancer of the human beta-globin locus control region from activating a downstream epsilon-globin gene. 5'-HS4 interrupted formation of a domain of histone H3 and H4 acetylation encompassing the 6-kb minilocus and inhibited transfer of RNA polymerase from the enhancer to the gene promoter. We found that the enhancer blocking phenotype was amplified when the insulated locus contained a weakened HS2 enhancer in which clustered point mutations eliminated interaction of the transcription factor GATA-1. The GATA-1 mutation compromised recruitment of histone acetyltransferases and RNA polymerase II to HS2. Enhancer blocking correlated with a significant depletion of nucleosomes in the core region of the insulator as revealed by micrococcal nuclease and DNase I digestion studies. Nucleosome depletion at 5'-HS4 was dependent on interaction of the insulator protein CCCTC-binding factor (CTCF) and was required for enhancer blocking. These findings provide evidence that a domain of active chromatin is formed by spreading from an enhancer to a target gene and can be blocked by a nucleosome-free gap in an insulator.

Animals↗