A central role for a single c-Myb binding site in a thymic locus control region.

Locus control regions (LCRs) are powerful assemblies of cis elements that organize the actions of cell-type-specific trans-acting factors. A 2.3-kb LCR in the human adenosine deaminase (ADA) gene first intron, which controls expression in thymocytes, is composed of a 200-bp enhancer domain and extended flanking sequences that facilitate activation from within chromatin. Prior analyses have demonstrated that the enhancer contains a 28-bp core region and local adjacent augmentative cis elements. We now show that the core contains a single critical c-Myb binding site. In both transiently cotransfected human cells and stable chromatin-integrated yeast cells, c-Myb strongly transactivated reporter constructs that contained polymerized core sequences. c-Myb protein was strongly evident in T lymphoblasts in which the enhancer was active and was localized within discrete nuclear structures. Fetal murine thymus exhibited a striking concordance of endogenous c-myb expression with that of mouse ADA and human ADA LCR-directed transgene expression. Point mutation of the c-Myb site within the intact 2.3-kb LCR severely attenuated enhancer activity in transfections and LCR activity in transgenic thymocytes. Within the context of a complex enhancer and LCR, c-Myb can act as an organizer of thymocyte-specific gene expression via a single binding site.     

Multiple elements in human beta-globin locus control region 5' HS 2 are involved in enhancer activity and position-independent, transgene expression.

The human beta-globin Locus Control Region (LCR) has two important activities. First, the LCR opens a 200 kb chromosomal domain containing the human epsilon-, gamma- and beta-globin genes and, secondly, these sequences function as a powerful enhancer of epsilon-, gamma- and beta-globin gene expression. Erythroid-specific, DNase I hypersensitive sites (HS) mark sequences that are critical for LCR activity. Previous experiments demonstrated that a 1.9 kb fragment containing the 5' HS 2 site confers position-independent expression in transgenic mice and enhances human beta-globin gene expression 100-fold. Further analysis of this region demonstrates that multiple sequences are required for maximal enhancer activity; deletion of SP1, NF-E2, GATA-1 or USF binding sites significantly decrease beta-globin gene expression. In contrast, no single site is required for position-independent transgene expression; all mice with site-specific mutations in 5' HS 2 express human beta-globin mRNA regardless of the site of transgene integration. Apparently, multiple combinations of protein binding sites in 5' HS 2 are sufficient to prevent chromosomal position effects that inhibit transgene expression.     

Functional and molecular analysis of the double-positive stage-specific CD8 enhancer E8III during thymocyte development

Several developmental stage-, subset-, and lineage-specific Cd8 cis-regulatory regions have been identified. These include the E8(III) enhancer, which directs expression in double-positive (DP) thymocytes, and E8(II), which is active in DP cells and CD8(+) T cells. Using a transgenic reporter expression assay, we identified a 285-bp core fragment of the E8(III) enhancer that retains activity in DP thymocytes. In vitro characterization of the core enhancer revealed five regulatory elements that are required for full enhancer activity, suggesting that multiple factors contribute to the developmental stage-specific activity. Furthermore, deletion of E8(III) in the mouse germline showed that this enhancer is required for nonvariegated expression of CD8 in DP thymocytes when E8(II) is also deleted. These results indicate that E8(III) is one of the cis-elements that contribute to the activation of the Cd8a and Cd8b gene complex during T cell development.     

An oligodendrocyte enhancer in a phylogenetically conserved intron region of the mammalian myelin gene Opalin

Opalin is a transmembrane protein detected specifically in mammalian oligodendrocytes. Opalin homologs are found only in mammals and not in the genome sequences of other animal classes. We first determined the nucleotide sequences of Opalin orthologs and their flanking regions derived from four prosimians, a group of primitive primates. A global comparison revealed that an evolutionarily conserved region exists in the first intron of Opalin. When the conserved domain was assayed for its enhancer activity in transgenic mice, oligodendrocyte-directed expression was observed. In an oligodendroglial cell line, Oli-neu, the conserved domain showed oligodendrocyte-directed expression. The conserved domain is composed of eight subdomains, some of which contain binding sites for Myt1 and cAMP-response element binding protein (CREB). Deletion analysis and cotransfection experiments revealed that the subdomains have critical roles in Opalin gene expression. Over-expression of Myt1, treatment of the cell with leukemia inhibitory factor (LIF), and cAMP analog (CREB activator) enhanced the expression of endogenous Opalin in Oli-neu cells and activated the oligodendrocyte enhancer. These results suggest that LIF, cAMP signaling cascades and Myt1 play significant roles in the differentiation of oligodendrocytes through their action on the Opalin oligodendrocyte enhancer.     

Evolution of lineage-specific functions in ancient cis-regulatory modules

Morphological evolution is driven both by coding sequence variation and by changes in regulatory sequences. However, how cis-regulatory modules (CRMs) evolve to generate entirely novel expression domains is largely unknown. Here, we reconstruct the evolutionary history of a lens enhancer located within a CRM that not only predates the lens, a vertebrate innovation, but bilaterian animals in general. Alignments of orthologous sequences from different deuterostomes sub-divide the CRM into a deeply conserved core and a more divergent flanking region. We demonstrate that all deuterostome flanking regions, including invertebrate sequences, activate gene expression in the zebrafish lens through the same ancient cluster of activator sites. However, levels of gene expression vary between species due to the presence of repressor motifs in flanking region and core. These repressor motifs are responsible for the relatively weak enhancer activity of tetrapod flanking regions. Ray-finned fish, however, have gained two additional lineage-specific activator motifs which in combination with the ancient cluster of activators and the core constitute a potent lens enhancer. The exploitation and modification of existing regulatory potential in flanking regions but not in the highly conserved core might represent a more general model for the emergence of novel regulatory functions in complex CRMs.     

The involvement of demethylation in the myeloid-specific function of the mouse M lysozyme gene downstream enhancer.

Lysozyme gene expression is a specific marker for the macrophage/granulocyte lineage of hematopoietic differentiation in mammals, its expression being gradually increased during maturation. Analysis of the mechanisms regulating mouse M lysozyme gene expression during myeloid differentiation revealed a complicated pattern of DNase I hypersensitive sites (HS sites) within the flanking regions of the gene. The HS-3 site, located in the 3'-flanking region of the gene, overlapped with an enhancer element, which is the only strong enhancer identified in the vicinity of the gene. We demonstrate a positive correlation between undermethylation of the entire 3'-flanking region, the appearance of the HS-3 site, and M lysozyme gene expression during in vitro differentiation of hematopoietic stem cells. We furthermore show that methylation of a single CpG site within the enhancer core element, only observed in immature macrophage cells in vivo, is sufficient to inhibit nuclear factor binding to this element in vitro and to inhibit its transactivation potential in DNA transfection experiments.     

Sequences flanking hypersensitive sites of the beta-globin locus control region are required for synergistic enhancement

The major distal regulatory sequence for the beta-globin gene locus, the locus control region (LCR), is composed of multiple hypersensitive sites (HSs). Different models for LCR function postulate that the HSs act either independently or synergistically. To test these possibilities, we have constructed a series of expression cassettes in which the gene encoding the enhanced green fluorescent protein (EGFP) is under the control of DNA fragments containing single and multiple HSs of the LCR. LCR DNA fragments containing only the minimal region needed for position-independent expression (HS cores) or containing cores plus flanking sequences (HS units) were compared to ascertain whether conserved sequences between the HS cores contributed to enhancement. Expression of these constructs was measured after targeted integration into three defined loci in murine erythroleukemia cells using recombinase-mediated cassette exchange. At all three marked loci, synergistic enhancement of expression was observed in cassettes containing a combination of HS2, HS3, and HS4 units. In contrast, HS2, HS3, and HS4 cores (without flanking sequences) give an activity equivalent to the sum of the activities of the individual HS cores. These data suggest a model in which an HS core plus flanking regions, bound by specific proteins, forms a structure needed for interaction with other HS units to confer strong enhancement by the LCR. The three targeted integration sites differ substantially in their permissivity for expression, but even the largest LCR construct tested could not overcome these position effects to confer equal expression at all three sites.     

Enhancer-blocking activity is associated with hypersensitive site V sequences in the human growth hormone locus control region

Activation of the human growth hormone gene (hGH-N) is linked to a locus control region (LCR) containing four (I-III, V) hypersensitive sites (HS). Pit-1 binding to HS I/II is required for efficient pituitary expression. However, inclusion of HS III and V, located about 28 and 32 kb upstream of the hGH-N gene, respectively, is also required for consistent hGH-N expression levels in vivo. HS V is referred to as a boundary for the hGH LCR, but no specific enhancer blocking or barrier function is reported. We examined a 547 bp fragment containing HS V sequences (nucleotides -32,718/-32,172 relative to hGH-N) for enhancer-blocking activity using a well-established transient gene transfer system and assessed these sequences for CCCTC binding factor (CTCF), which is linked to enhancer-blocking activity. The 547 bp HS V fragment decreased enhancer activity with a reverse-orientation preference when inserted between HS III enhancer sequences and a minimal thymidine kinase promoter (TKp). These sequences are associated with CTCF in human pituitary and nonpituitary chromatin. Enhancer-blocking activity with an orientation preference was further localized to a 45 bp sub-fragment, with evidence of CTCF and upstream binding factor 1 (USF1) binding; USF1 is linked more closely with barrier function. The presence of yin and yang 1 (Yy1) that cooperates with CTCF in the regulation of X-chromosome inactivation was also seen. A decrease in CTCF and Yy1 RNA levels was associated with a significant reduction in enhancer-blocking activity. Assessment of CpG-dinucleotides in the TKp indicates that the presence of HS V sequences are associated with an increased incidence of CpG-dinucleotide methylation of the GC box region. These data support association of CTCF and enhancer-blocking activity with HS V that is consistent with a role as a (LCR) boundary element and also implicates Yy1 in this process.