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R Hardison

Publications and source records attributed to R Hardison.

At least 19 recordsLinked to original sources
PubMed2001

Comparative genome analysis delimits a chromosomal domain and identifies key regulatory elements in the alpha globin cluster.

We have cloned, sequenced and annotated segments of DNA spanning the mouse, chicken and pufferfish alpha globin gene clusters and compared them with the corresponding region in man. This has defined a small segment ( approximately 135-155 kb) of synteny and conserved gene order, which may contain all of the elements required to fully regulate alpha globin gene expression from its natural chromosomal environment. Comparing human and mouse sequences using previously described methods failed to identify the known regulatory elements. However, refining these methods by ranking identity scores of non-coding sequences, we found conserved sequences including the previously characterized alpha globin major regulatory element. In chicken and pufferfish, regions that may correspond to this element were found by analysing the distribution of transcription factor binding sites. Regions identified in this way act as strong enhancer elements in expression assays. In addition to delimiting the alpha globin chromosomal domain, this study has enabled us to develop a more sensitive and accurate routine for identifying regulatory elements in the human genome.

Animals↗
PubMed2001

Characterization of a widely expressed gene (LUC7-LIKE; LUC7L) defining the centromeric boundary of the human alpha-globin domain.

We have identified the first gene lying on the centromeric side of the alpha-globin gene cluster on human 16p13.3. The gene, called 16pHQG;16 (HGMW-approved symbol LUC7L), is widely transcribed and lies in the opposite orientation with respect to the alpha-globin genes. This gene may represent a mammalian heterochromatic gene, encoding a putative RNA-binding protein similar to the yeast Luc7p subunit of the U1 snRNP splicing complex that is normally required for 5' splice site selection. To examine the role of the 16pHQG;16 gene in delimiting the extent of the alpha-globin regulatory domain, we mapped its mouse orthologue, which we found to lie on mouse chromosome 17, separated from the mouse alpha-cluster on chromosome 11. Establishing the full extent of the human 16pHQG;16 gene has allowed us to define the centromeric limit of the region of conserved synteny around the human alpha-globin cluster to within an 8-kb segment of chromosome 16.

Alternative Splicing↗
PubMed2000

Levels of GATA-1/GATA-2 transcription factors modulate expression of embryonic and fetal hemoglobins.

GATA transcription factors bind the consensus sequence WGATAR, present in the flanking regions of most erythroid specific genes. GATA-1 and GATA-2, coexpressed in erythroid cells, are important for expression of erythroid genes. To elucidate the role of specific GATA transcription factors on globin gene expression, we examined the human alpha- and beta-globin gene clusters for all GATA sites. Conserved GATA sites were found in each of the hypersensitive sites in both beta-and alpha clusters and in proximal regulatory regions of the zeta-, epsilon- and gamma-globin but not the alpha, delta or beta-globin genes. We then tested the effect of increasing levels of GATA-1 and GATA-2 on the expression of endogenous globin genes in human erythroid cells. Increasing GATA-1 levels in K562 cells decreased the levels of epsilon-globin mRNA but had no effect on the levels of expression of gamma, zeta or alpha-globin genes. Increasing GATA-2 levels increased epsilon-globin and gamma-globin transcripts. Increasing levels of GATA-1 also caused a decrease in the expression of endogenous GATA-2, while increased levels of GATA-2 had no effect on GATA-1 mRNA. Our results indicate a differential role of GATA-1 and -2 transcription factors on globin transcripts and suggest a correlation between the conservation of GATA sites in the regulatory regions and the ability of endogenous globin genes to respond to GATA transcription factors. They also suggest that quantitative changes in the levels of GATA-1 or GATA-2 can result in alterations of globin target gene expression and may participate in the ontogenic control of the globin genes.

Amino Acid Sequence↗
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

A negative cis-element regulates the level of enhancement by hypersensitive site 2 of the beta-globin locus control region.

The core of DNase hypersensitive site (HS) 2 from the beta-globin locus control region is a potent enhancer of globin gene expression. Although it has been considered to contain only positive cis-regulatory sequences, our study of the enhancement conferred by segments of HS2 in erythroid cells reveals a novel negative element. Individual cis-regulatory elements from HS2 such as E boxes or Maf-response elements produced as great or greater enhancement than the intact core in mouse erythroleukemia (MEL) cells, indicating the presence of negative elements within HS2. A deletion series through HS2 revealed negative elements at the 5' and 3' ends of the core. Analysis of constructs with and without the 5' negative element showed that the effect is exerted on the promoters of globin genes expressed at embryonic, fetal, or adult stages. The negative effect was observed in bipotential human cells (K562 and human erythroleukemia (HEL) cells), proerythroblastic mouse (MEL) cells, and normal adult human erythroid cells. The novel negative element also functions after stable integration into MEL chromosomes. Smaller deletions at the 5' end of the HS2 core map the negative element within a 20-base pair region containing two conserved sequences.

Adult↗
PubMed2000

PipMaker--a web server for aligning two genomic DNA sequences.

PipMaker (http://bio.cse.psu.edu) is a World-Wide Web site for comparing two long DNA sequences to identify conserved segments and for producing informative, high-resolution displays of the resulting alignments. One display is a percent identity plot (pip), which shows both the position in one sequence and the degree of similarity for each aligning segment between the two sequences in a compact and easily understandable form. Positions along the horizontal axis can be labeled with features such as exons of genes and repetitive elements, and colors can be used to clarify and enhance the display. The web site also provides a plot of the locations of those segments in both species (similar to a dot plot). PipMaker is appropriate for comparing genomic sequences from any two related species, although the types of information that can be inferred (e.g., protein-coding regions and cis-regulatory elements) depend on the level of conservation and the time and divergence rate since the separation of the species. Gene regulatory elements are often detectable as similar, noncoding sequences in species that diverged as much as 100-300 million years ago, such as humans and mice, Caenorhabditis elegans and C. briggsae, or Escherichia coli and Salmonella spp. PipMaker supports analysis of unfinished or "working draft" sequences by permitting one of the two sequences to be in unoriented and unordered contigs.

Animals↗
PubMed1999

Comparison of five methods for finding conserved sequences in multiple alignments of gene regulatory regions.

Conserved segments in DNA or protein sequences are strong candidates for functional elements and thus appropriate methods for computing them need to be developed and compared. We describe five methods and computer programs for finding highly conserved blocks within previously computed multiple alignments, primarily for DNA sequences. Two of the methods are already in common use; these are based on good column agreement and high information content. Three additional methods find blocks with minimal evolutionary change, blocks that differ in at most k positions per row from a known center sequence and blocks that differ in at most k positions per row from a center sequence that is unknown a priori. The center sequence in the latter two methods is a way to model potential binding sites for known or unknown proteins in DNA sequences. The efficacy of each method was evaluated by analysis of three extensively analyzed regulatory regions in mammalian beta-globin gene clusters and the control region of bacterial arabinose operons. Although all five methods have quite different theoretical underpinnings, they produce rather similar results on these data sets when their parameters are adjusted to best approximate the experimental data. The optimal parameters for the method based on information content varied little for different regulatory regions of the beta-globin gene cluster and hence may be extrapolated to many other regulatory regions. The programs based on maximum allowed mismatches per row have simple parameters whose values can be chosen a priori and thus they may be more useful than the other methods when calibration against known functional sites is not available.

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

A database of experimental results on globin gene expression.

Information on gene expression and regulation is expanding rapidly, and the complexity of the experimental design and data makes unique demands on databases to store the results. We describe a prototype database containing experimental results on the expression of mammalian beta-like globin genes, along with several query methods for accessing the information. The database has tables for DNA transfer experiments, protein-DNA binding results, and positions of DNase hypersensitive sites, which make extensive use of nested data structures. Comparison of data from various mammals is accomplished by providing a common coordinate system via a simultaneous alignment of matching DNA sequences. Interactive access to the database is available at a site called the Globin Gene Server on the World Wide Web (http://globin.cse. psu.edu). This software should be useful for any genetic system in which DNA sequence data are available.

Animals↗
PubMed1998

Multiple regulatory elements in the 5'-flanking sequence of the human epsilon-globin gene.

We have previously reported, on the basis of transfection experiments, the existence of a silencer element in the 5'-flanking region of the human embryonic (epsilon) globin gene, located at -270 base pairs 5' to the cap site, which provides negative regulation for this gene. Experiments in transgenic mice suggest the physiological importance of this epsilon-globin silencer, but also suggest that down-regulation of epsilon-globin gene expression may involve other negative elements flanking the epsilon-globin gene. We have now extended the analysis of epsilon-globin gene regulation to include the flanking region spanning up to 6 kilobase pairs 5' to the locus control region using reporter gene constructs with deletion mutations and transient transfection assays. We have identified and characterized other strong negative regulatory regions, as well as several positive regions that affect transcription activation. The negative regulatory regions at -3 kilobase pairs (epsilonNRA-I and epsilonNRA-II), flanked by a positive control element, has a strong effect on the epsilon-globin promoter both in erythroid K562 and nonerythroid HeLa cells and contains several binding sites for transcription factor GATA-1, as evidenced from DNA-protein binding assays. The GATA-1 sites within epsilonNRA-II are directly needed for negative control. Both epsilonNRA-I and epsilonNRA-II are active on a heterologous promoter and hence appear to act as transcription silencers. Another negative control region located at -1.7 kilobase pairs (epsilonNRB) does not exhibit general silencer activity as epsilonNRB does not affect transcription activity when used in conjunction with an epsilon-globin minimal promoter. The negative effect of epsilonNRB is erythroid specific, but not stage-specific as it can repress transcription activity in both K562 erythroid cells as well as in primary cultures of adult erythroid cells. Phylogenetic DNA sequence comparisons with other primate and other mammalian species show unusual degree of flanking sequence homology for the epsilon-globin gene, including in several of the regions identified in these functional and DNA-protein binding analyses, providing alternate evidence for their potential importance. We suggest that the down-regulation of epsilon-globin gene expression as development progresses involves complex, cooperative interactions of these negative regulatory elements, epsilonNRA-I/epsilonNRA-II, epsilonNRB, the epsilon-globin silencer and probably other negative and positive elements in the 5'-flanking region of the epsilon-globin gene.

Animals↗
PubMed1998

Hemoglobins from bacteria to man: evolution of different patterns of gene expression.

The discovery of hemoglobins in virtually all kingdoms of organisms has shown (1) that the ancestral gene for hemoglobin is ancient, and (2) that hemoglobins can serve additional functions besides transport of oxygen between tissues, ranging from intracellular oxygen transport to catalysis of redox reactions. These different functions of the hemoglobins illustrate the acquisition of new roles by a pre-existing structural gene, which requires changes not only in the coding regions but also in the regulatory elements of the genes. The evolution of different regulated functions within an ancient gene family allows an examination of the types of biosequence data that are informative for various types of issues. Alignment of amino acid sequences is informative for the phylogenetic relationships among the hemoglobins in bacteria, fungi, protists, plants and animals. Although many of these diverse hemoglobins are induced by low oxygen concentrations, to date none of the molecular mechanisms for their hypoxic induction shows common regulatory proteins; hence, a search for matches in non-coding DNA sequences would not be expected to be fruitful. Indeed, alignments of non-coding DNA sequences do not reveal significant matches even between mammalian alpha- and beta-globin gene clusters, which diverged approximately 450 million years ago and are still expressed in a coordinated and balanced manner. They are in very different genomic contexts that show pronounced differences in regulatory mechanisms. The alpha-globin gene is in constitutively active chromatin and is encompassed by a CpG island, which is a dominant determinant of its regulation, whereas the beta-globin gene is in A+T-rich genomic DNA. Non-coding sequence matches are not seen between avian and mammalian beta-globin gene clusters, which diverged approximately 250 million years ago, despite the fact that regulation of both gene clusters requires tissue-specific activation of a chromatin domain regulated by a locus control region. The cis-regulatory sequences needed for domain opening and enhancement do show common binding sites for transcription factors. In contrast, alignments of non-coding sequences from species representing multiple eutherian mammalian orders, some of which diverged as long as 135 million years ago, are reliable predictors of novel cis-regulatory elements, both proximal and distal to the genes. Examples include a potential target for the hematopoietic transcription factor TAL1.

Amino Acid Sequence↗
PubMed1997

Locus control regions of mammalian beta-globin gene clusters: combining phylogenetic analyses and experimental results to gain functional insights.

Locus control regions (LCRs) are cis-acting DNA segments needed for activation of an entire locus or gene cluster. They are operationally defined as DNA sequences needed to achieve a high level of gene expression regardless of the position of integration in transgenic mice or stably transfected cells. This review brings together the large amount of DNA sequence data from the beta-globin LCR with the vast amount of functional data obtained through the use of biochemical, cellular and transgenic experimental systems. Alignment of orthologous LCR sequences from five mammalian species locates numerous conserved regions, including previously identified cis-acting elements within the cores of nuclease hypersensitive sites (HSs) as well as conserved regions located between the HS cores. The distribution of these conserved sequences, combined with the effects of LCR fragments utilized in expression studies, shows that important sites are more widely distributed in the LCR than previously anticipated, especially in and around HS2 and HS3. We propose that the HS cores plus HS flanking DNAs comprise a 'unit' to which proteins bind and form an optimally functional structure. Multiple HS units (at least three: HS2, HS3 and HS4 cores plus flanking DNAs) together establish a chromatin structure that allows the proper developmental regulation of genes within the cluster.

Animals↗
PubMed1997

Phylogenetic footprinting of hypersensitive site 3 of the beta-globin locus control region.

Hypersensitive site 3 (HS3) of the beta-like globin locus control region has been implicated as an important regulator of the beta-like globin genes, but the trans factors that bind HS3 have only been partially characterized. Using a five-species alignment (human, galago, rabbit, goat, and mouse) that represents 370 million years of evolution, we have identified 24 phylogenetic footprints in the HS3 core and surrounding regions. Probes corresponding to the human sequence at each footprint have been used in binding studies to identify the nuclear factors that bind within and near these conserved sequence elements. Among the high-affinity interactions observed were several binding sites for proteins with repressor activity, including YY1, CCAAT displacement protein, and G1/G2 complexes (uncharacterized putative repressors) and several binding sites for the stage selector protein. To complement this analysis, orthologous galago sequences were also used to derive probes and the pattern of proteins binding to human and galago probes was compared. Binding interactions differing between these two species could be responsible for the different expression patterns shown by the two gamma genes (galago gamma is embryonic; human gamma is fetal). Alternatively, binding interactions that are conserved in the two species may be important in the regulation of common expression patterns (eg, repression of gamma in adult life).

Animals↗
PubMed1997

Conserved E boxes function as part of the enhancer in hypersensitive site 2 of the beta-globin locus control region. Role of basic helix-loop-helix proteins.

The human beta-globin gene cluster is regulated in part by a distal locus control region that is required for opening a chromatin domain in erythroid cells and enhancing expression of the beta-like globin genes at the correct developmental stages. One part of the locus control region, called hypersensitive site 2 (HS2), functions as a strong enhancer. Matches to the consensus binding sites for basic helix-loop-helix (bHLH) proteins (E boxes) are well conserved within the HS2 core. We show that mutations of the HS2 core that alter an invariant E box cause a 3.5-fold reduction in enhancement of expression of an epsilon-globin reporter gene in transiently transfected K562 cells, both before and after induction. Mutations of the HS2 core that alter a less-highly conserved E box cause a more modest reduction in enhancement. Footprint analysis shows binding of erythroid nuclear proteins in vitro to the invariant E box as well as an adjacent CAC/GTG box. Probes containing the E box regions form sequence-specific complexes with proteins from both K562 and MEL nuclear extracts; these are disrupted by the same mutations that decrease enhancement. Some of these latter complexes contain known bHLH proteins, as revealed by specific loss of individual complexes when treated with antibodies against TAL1 and USF. Interaction between the E boxes and the bHLH proteins, as well as other binding proteins, could account for the role of these sites in enhancement by HS2.

Base Sequence↗
PubMed1997

The complete sequences of the galago and rabbit beta-globin locus control regions: extended sequence and functional conservation outside the cores of DNase hypersensitive sites.

The locus control region (LCR) of mammalian beta-globin genes covers at least 17 kb at the 5' end of the gene cluster and has been implicated in chromatin domain opening, enhancement, and insulation from neighboring sequences. Functional dissection of the LCR has defined the minimal cores for four of the five major DNase hypersensitive sites (HSs) that mark this regulatory region. To examine fully the patterns of conserved sequences in the mammalian homologs to the beta-globin LCR, we determined the complete DNA sequence of the galago beta-globin LCR and completed previously unsequenced regions of the rabbit LCR. Simultaneous alignment of these sequences with the human, goat, and mouse LCRs revealed conserved sequences (phylogenetic footprints) detected using three largely independent methods. The most highly conserved segments are found both within the HS cores and in some but not all regions flanking the cores. These results argue for an extended pattern of well-conserved sequences, many of which lie outside the minimal cores, and we show that a key sequence required for domain opening by the region including HS3 maps about 1 kb 5' to the minimal core. Differential phylogenetic footprints, containing sequences conserved in nonhuman mammals but not in humans, are found primarily around HS3, consistent with some species-specific differences in function that may be important for differences in hemoglobin switching during development.

Animals↗
PubMed1996

Role of DNA sequences outside the cores of DNase hypersensitive sites (HSs) in functions of the beta-globin locus control region. Domain opening and synergism between HS2 and HS3.

The roles of each DNase hypersensitive site (HS), and the DNA sequences between them, in the activity of the locus control region of the mammalian beta-globin gene domain were examined by placing human and rabbit restriction fragments containing the cores of HS2, HS3, HS4, and HS5, along with varying amounts of flanking DNA, upstream of a hybrid epsilon-globin-luciferase reporter gene and testing for effects on expression both prior to and after integration into the chromosomes of K562 cells, a human erythroid cell line. Prior to integration, fragments containing HS2 enhanced expression to the greatest extent, and the modest enhancement by some fragments containing HS3 correlated with the presence of a well-conserved binding site for AP1/NFE2. The stronger effects of larger locus control region DNA fragments in clones of stably transfected cells indicates a role for sequences outside the HS cores after integration into the genome. The strong effect of a 1.9-kilobase HindIII fragment containing HS3 after, but not prior to, integration argues for the presence of a chromatin domain-opening activity. Use of a rabbit DNA fragment containing both HS2 and HS3 demonstrated a synergistic interaction between the two HSs when their natural context and spacing are preserved.

Animals↗