Biomedical subjects

Ann Dean

Publications and source records attributed to Ann Dean.

10 recordsLinked to original sources

PubMed2006

Distinctive signatures of histone methylation in transcribed coding and noncoding human beta-globin sequences.

The establishment of epigenetic marks, such as methylation on histone tails, is mechanistically linked to RNA polymerase II within active genes. To explore the interplay between these modifications in transcribed noncoding as well as coding sequences, we analyzed epigenetic modification and chromatin structure at high resolution across 300 kb of human chromosome 11, including the beta-globin locus which is extensively transcribed in intergenic regions. Monomethylated H3K4, K9, and K36 were broadly distributed, while hypermethylated forms appeared to different extents across the region in a manner reflecting transcriptional activity. The trimethylation of H3K4 and H3K9 correlated within the most highly transcribed sequences. The H3K36me3 mark was more broadly detected in transcribed coding and noncoding sequences, suggesting that K36me3 is a stable mark on sequences transcribed at any level. Most epigenetic and chromatin structural features did not undergo transitions at the presumed borders of the globin domain where the insulator factor CTCF interacts, raising questions about the function of the borders.

Acetylation↗

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↗

PubMed2005

On a chromosome far, far away: LCRs and gene expression.

Transcription activation of a gene involves the ordered recruitment of components of the basal transcription machinery in concert with alterations in chromatin structure, including nucleosome remodeling and post-translational modification of histones. Enhancers and locus control regions (LCRs) that are remote from the genes they activate, recruit the complexes that carry out these alterations and, sometimes, recruit RNA polymerase II. The question of how these distant activators interact with their target genes has been of long-standing interest. Recent data indicate that LCRs mediate contact with their coordinate genes through the formation of domains of histone modification and of intra- and inter-chromosomal loops and that they might localize genes within nuclear regions that favor transcription.

Animals↗

PubMed2005

Organizing the genome: enhancers and insulators.

Enhancers can activate their target genes over large linear distances. Insulators can delimit the influence of an enhancer to an appropriate target. There are a number of intertwined mechanisms by which the regulatory functions of enhancers and insulators might be carried out at the level of the chromatin fiber. Recent evidence suggests that both enhancers and insulators participate in higher-order organization of chromatin in the nucleus and in localization of their regulated sequences to both subnuclear structures and compartments. Novel experimental approaches are helping to reveal the mechanisms underlying nuclear organization of developmentally regulated genes.

Animals↗

PubMed2004

An insulator blocks spreading of histone acetylation and interferes with RNA polymerase II transfer between an enhancer and gene.

We studied the mechanism by which an insulator interrupts enhancer signaling to a gene using stably replicated chromatin templates containing the human beta-globin locus control region HS2 enhancer and a target globin gene. The chicken beta-globin 5' HS4 (cHS4) insulator acted as a positional enhancer blocker, inhibiting promoter remodeling and transcription activation only when placed between the enhancer and gene. Enhancer blocking by cHS4 reduced histone hyperacetylation across a zone extending from the enhancer to the gene and inhibited recruitment of CBP and p300 to HS2. Enhancer blocking also led to accumulation of RNA polymerase II at HS2 and within cHS4, accompanied by its diminution at the gene promoter. The enhancer blocking effects were completely attributable to the CTCF binding site in cHS4. These findings provide experimental evidence for the involvement of spreading in establishment of a broad zone of histone modification by an enhancer, as well as for blocking by an insulator of the transfer of RNA polymerase II from an enhancer to a promoter.

Acetylation↗

PubMed2004

Developmental stage differences in chromatin subdomains of the beta-globin locus.

The mammalian beta-globin loci each contain a family of developmentally expressed genes, and a far upstream regulatory element, the locus control region (LCR). In adult murine erythroid cells, the LCR and the transcribed beta-globin genes exist within domains of histone acetylation and RNA polymerase II (pol II) is associated with them. In contrast, the silent embryonic genes lie between these domains within hypoacetylated chromatin, and pol II is not found there. We used chromatin immunoprecipitation and real-time PCR to analyze histone modification and pol II recruitment to the globin locus in human erythroid K562 cells that express the embryonic epsilon-globin gene but not the adult beta-globin gene. H3 and H4 acetylation and H3 K4 methylation were continuous over a 17-kb region including the LCR and the active epsilon-globin gene. The level of modification varied directly with the transcription of the epsilon-globin gene. In contrast, this region in nonerythroid HeLa cells lacked these modifications and displayed instead widespread H3 K9 methylation. pol II was also detected continuously from the LCR to the epsilon-globin gene. These studies reveal several aspects of chromatin structure and pol II distribution that distinguish the globin locus at embryonic and adult stages and suggest that both enhancer looping and tracking mechanisms may contribute to LCR-promoter communication at different developmental stages.

Chromatin↗

PubMed2004

Chromatin remodelling and the interaction between enhancers and promoters in the beta-globin locus.

The beta-globin genes are among the most studied families of tissue-specific and developmentally regulated genes. Even before genome-wide sequencing was a reality, the sequences of extensive regions of the human, rabbit and mouse beta-globin loci were available through the individual efforts of several research groups. Many of the genomic approaches now being pursued in order to understand the control of gene expression were applied early to this gene family with success. Higher levels of regulation attributable to networks of transcription factor interaction, chromatin remodelling and enhancer-promoter communication remain as challenges in the post-genomic era.

Animals↗

PubMed2003

A major role for the TATA box in recruitment of chromatin modifying complexes to a globin gene promoter.

The developmentally regulated mammalian beta-globin genes are activated by a distant locus control region/enhancer. To understand the role of chromatin remodeling complexes in this activation, we used stably replicated chromatin templates, in which transcription activation of the human embryonic epsilon-globin gene depends on the tandem Maf-recognition elements (MAREs) within the beta-globin locus control region HS2 enhancer, to which the erythroid factor NF-E2 binds. The HS2 MAREs are required for nucleosome mobilization and histone hyperacetylation at the distant promoter. Nucleosome mobilization also requires the promoter TATA box, and is independent of histone hyperacetylation. In contrast, promoter hyperacetylation requires the promoter GATA-1, and CACC-factor activator motifs, as well as the TATA box. ChIP analysis reveals that NF-E2 is associated with the active epsilon-globin promoter, which lacks an NF-E2 binding sequence, in a TATA box and HS2/MARE-dependent fashion. NF-E2 association with the epsilon-globin promoter coincides with that of RNA polymerase II at both regulatory sites. The results emphasize MARE-TATA box interactions in the recruitment of complexes modifying promoter chromatin for transcription activation and imply close physical interaction between widely separated regulatory sequences mediated through these sites.

Acetylation↗

PubMed2003

Beta-globin locus control region HS2 and HS3 interact structurally and functionally.

The overall structure of the DNase I hypersensitive sites (HSs) that comprise the beta-globin locus control region (LCR) is highly conserved among mammals, implying that the HSs have conserved functions. However, it is not well understood how the LCR HSs, either individually or collectively, activate transcription. We analyzed the interactions of HS2, HS3 and HS4 with the human epsilon- and beta-globin genes in chromatinized episomes in fetal/embryonic K562 cells. Only HS2 activates transcription of the epsilon-globin gene, while all three HSs activate the beta-globin gene. HS3 stimulates the beta-globin gene constitutively, but HS2 and HS4 transactivation requires expression of the transcription factor EKLF, which is not present in K562 cells but is required for beta-globin expression in vivo. To begin addressing how the individual HSs may interact with one another in a complex, we linked the beta-globin gene to both the HS2 and HS3. HS2 and HS3 together resulted in synergistic stimulation of beta-globin transcription. Unexpectedly, mutated, inactive forms of HS2 impeded the activation of the beta-globin gene by HS3. Thus, there appear to be distinct interactions among the HSs and between the HSs and the globin genes. These preferential, non-exclusive interactions may underlie an important structural and functional cooperativity among the regulatory sequences of the beta-globin locus in vivo.

Binding Sites↗

PubMed2003

A human globin enhancer causes both discrete and widespread alterations in chromatin structure.

Gene activation requires alteration of chromatin structure to facilitate active transcription complex formation at a gene promoter. Nucleosome remodeling complexes and histone modifying complexes each play unique and interdependent roles in bringing about these changes. The role of distant enhancers in these structural alterations is not well understood. We studied nucleosome remodeling and covalent histone modification mediated by the beta-globin locus control region HS2 enhancer at nucleosome-level resolution throughout a 5.5-kb globin gene model locus in vivo in K562 cells. We compared the transcriptionally active locus to one in which HS2 was inactivated by mutations in the core NF-E2 sites. In contrast to inactive templates, nucleosomes were mobilized in discrete areas of the active locus, including the HS2 core and the proximal promoter. Large differences in restriction enzyme accessibility between the active and inactive templates were limited to the regions of nucleosome mobilization, which subsumed the DNase I hypersensitive sites. In contrast to this discrete pattern, histone H3 and H4 acetylation and H3 K4 methylation were elevated across the entire active locus, accompanied by depletion of linker histone H1. The coding region of the gene differed from the regulatory regions, demonstrating both nucleosome mobilization and histone hyperacetylation, but lacked differences in restriction enzyme accessibility between transcriptionally active and inactive genes. Thus, although the histone modification pattern we observe is consistent with the spreading of histone modifying activity from the distant enhancer, the pattern of nucleosome mobilization is more compatible with direct contact between an enhancer and promoter.

Acetylation↗