ALBERT

Description: Active gene promoters are associated with covalent histone modifications, such as hyperacetylation, which can modulate chromatin structure and stabilize binding of transcription factors that recognize these modifications. At the β-globin locus and several other loci, however, histone hyperacetylation extends beyond the promoter, over tens of kilobases; we term such patterns of histone modifications “hyperacetylated domains.” Little is known of either the mechanism by which these domains form or their function. Here, we show that domain formation within the murine β-globin locus occurs before either high-level gene expression or erythroid commitment. Analysis of β-globin alleles harboring deletions of promoters or the locus control region demonstrates that these sequences are not required for domain formation, suggesting the existence of additional regulatory sequences within the locus. Deletion of embryonic globin gene promoters, however, resulted in the formation of a hyperacetylated domain over these genes in definitive erythroid cells, where they are otherwise inactive. Finally, sequences within β-globin domains exhibit hyperacetylation in a context-dependent manner, and domains are maintained when transcriptional elongation is inhibited. These data narrow the range of possible mechanisms by which hyperacetylated domains form.

Description: The molecular basis for the hereditary persistence of fetal hemoglobin (HPFH) phenotype was studied in a Chinese individual who was heterozygous for a nondeletion form of A gamma-HPFH. Both allelic A gamma-globin genes were isolated by molecular cloning and subjected to nucleotide sequence analysis. One A gamma gene promoter showed a cytosine to thymine transition at position -196, whereas the other promoter was normal. This mutation at position -196 has now ben found in unrelated individuals with the A gamma-HPFH phenotype from Italy, Sardinia, and China, suggesting that it may have arisen independently. The implications of this mutation for models of fetal globin gene switching are discussed.

Description: The molecular basis for the hereditary persistence of fetal hemoglobin (HPFH) phenotype was studied in a Chinese individual who was heterozygous for a nondeletion form of A gamma-HPFH. Both allelic A gamma-globin genes were isolated by molecular cloning and subjected to nucleotide sequence analysis. One A gamma gene promoter showed a cytosine to thymine transition at position -196, whereas the other promoter was normal. This mutation at position -196 has now ben found in unrelated individuals with the A gamma-HPFH phenotype from Italy, Sardinia, and China, suggesting that it may have arisen independently. The implications of this mutation for models of fetal globin gene switching are discussed.

Description: Mammalian β-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 β-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 β-globin regulation.

Description: The mammalian β-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 β-globin LCR stimulates transcription of the linked β-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 β-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.

Description: The activity of the β-globin locus is regulated by the locus control region (LCR) which in humans and mice is comprised of six DNaseI hypersensitive sites (HSs) located upstream of the β-like globin genes. Hispanic thalassemia, a naturally occurring deletion of the LCR plus 25kb upstream results in the failure to activate the β-globin locus at the levels of chromatin structure, transcription and replication. In order to examine how the HSs interact to regulate the endogenous β-globin locus, we have utilized homologous recombination for the mutational analysis of the endogenous murine β-globin LCR in embryonic stem cells, followed by the generation of mice. Previously we reported that deletion of the endogenous LCR by homologous recombination (ΔLCR) does not completely silence expression of the β-like genes, and has no measurable effect on nuclease sensitivity, promoter hypersensitive site formation or core histone hyperacetylation. Thus the LCR provides a necessary enhancer-like activity. In addition, while loss of the LCR leads to only a slight decrease in pre-initiation complex formation and Pol II binding to the promoter, there is a significant decrease in downstream polymerase and this correlates with a basal level of ser-5 phosphorylation of Pol II. To determine if the decrease in downstream Pol II observed along Δthe LCR allele is due to decreased release of polymerase from the promoter, or downstream polymerase pausing, KMnO4 in vivo foot-printing was done. Comparison of the 5′ end of WT and ΔLCR β-maj globin genes reveal similar patterns consistent with pol II pausing on both alleles, and suggesting that the LCR stimulates elongation by releasing promoter proximal paused polymerases. To further characterize LCR mediated activation and determine what characteristics vary with the level of transcription we have continued our analysis of mice with ΔLCR allele expressing at 1–4% of wild-type (WT), and mice with a deletion of HS 2 and 3 (Δ23) expressing at 30% of WT. The Δ23 allele was chosen as it demonstrates an intermediate transcriptional phenotype and is one of several double HS deletions that demonstrate that the LCR HSs contribute additively to globin gene transcription. To determine if histone modifications other than acetylation vary with LCR mutations and is associated with the level of transcription, the state of K4-tri and K79-di histone methylation was assayed along exon 3 of the β-maj gene of WT, Δ23 and ΔLCR alleles. While in some systems enrichment of methylation of K4 and K79 are associated with a permissive or activated state, we find increases in histone methylation with deletion of the LCR, and an intermediate degree of methylation with deletion of HS 2 and 3. Thus, surprisingly, increasing levels of transcription correlate with decreases in histone methylation. Finally, as enhancers increase the probability rather than the rate of expression we have analyzed WT, Δ23 and ΔLCR alleles with single allele transcription assays. RT-PCR reveals all Δ 23 and ΔLCR alleles express mRNA at 30 and 4% of WT respectively, consistent with bulk RNA analysis and suggesting that individual alleles are not permanently silenced. In contrast, primary transcript RNA FISH analysis demonstrates that the mutant alleles are less likely to be expressed, thus LCR HSs may affect the probability as well rate of transcription.

Description: Abstract 349 The human and mouse β-globin loci share a conserved structure in which the locus control region (LCR) and genes are flanked by three CTCF bound DNaseI hypersensitive sites (HSs); 3‘HS1 downstream, and 5‘HS5 and human and mouse orthologues HS-111 or HS-62 upstream. In mice HS-62 and 3‘HS1 delineate a DNase sensitive domain. During erythroid differentiation, high-level expression of the β-globin locus is associated with LCR-dependent re-localization of the locus from the nuclear periphery to the nucleoplasm, where it associates with foci of serine-phosphorylated PolII deemed transcription factories (TFs). To investigate the relationships among chromatin structure, nuclear localization and β-globin expression during human erythropoiesis, CD34 progenitor cells were differentiated and analyzed by ChIP-array, primary transcript FISH, immuno-FISH, and chromatin conformation capture, carbon copy (5C). Localization of the β-locus away from the nuclear periphery and to TFs, and detection of β-nascent transcripts are rare events at day 4 (proerythroblasts), whereas by day 15 (polychromatic erythroblasts), nearly all loci are centrally located, associated with TFs and actively expressing. Three megabase profiles of complementary active and repressive histone marks (H3 lysine 4 di-methylation (DiK4) and H3 lysine 27 tri-methylation) reveal that DiK4 is enriched in the LCR and adult genes in undifferentiated CD34 cells and nears maximal enrichment by day 4. Thus the chromatin landscape is set up prior to erythroid commitment and is increased at day 4, but shows little change with activation. These profiles also reveal a previously un-described 257kb domain spanning from HS-111 to +146 relative to the ε-gene cap, with CTCF bound at its boundaries. 5C analysis reveals a high linkage frequency between the LCR and β-gene at day 4, prior to β-gene activation. Thus proximity may be necessary, but is not sufficient for high-level expression. In addition, the LCR and adult genes have frequent contact with surrounding regions, but interactions are sharply demarcated by HS-111 and +146, linking the above histone modification domain and 5C structure. The flanking regions HS-111, 3‘HS1 and +146 associate with the LCR and genes in an active chromatin hub (ACH)-like structure. By combining 5C with the Integrated Modeling Platform, a high-resolution three-dimensional (3D) model of chromatin structure was generated and revealed that the CTCF containing flanking regions, HS-111, 5'HS5, 3‘HS1 and +146 are in proximity and anchor loops of the intervening regions. The LCR and β-gene lie in close proximity (

Description: Mammalian β-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 β-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 β-globin regulation.

Description: We have investigated the relationships among nuclear positioning, association with RNA polymerase II (PolII) and expression of the murine β-globin locus during erythroid differentiation, as well as the role of the locus control region (LCR) in these processes. Fetal liver cells from wildtype and LCR-deletion mouse strains were stained with a panel of antibodies, and flow cytometry was used to define and isolate cells from four stages of erythropoiesis spanning pro-erythroblasts (stage 1) to orthochromatic normoblasts and nucleated RBC (stage 4). DNA FISH analyses reveal that with increasing erythroid maturation the β-globin locus is less likely to be located in the nuclear periphery. Immuno-FISH demonstrates that PolII speckles initially are spread diffusely throughout the nucleus, with the exception of extreme periphery. With erythroid maturation the number of speckles decline and become more centrally located. Combined DNA FISH /PolII immuno-staining reveals an increase in co-localization of PolII and β-globin alleles during erythroid maturation. These results are consistent with a model in which the locus is more likely to be in the nuclear periphery and away from PolII foci prior to activation, and with maturation, the locus re-locates more centrally and associates with PolII speckles, leading to β-globin activation. To investigate this model, α and β-globin primary transcript FISH were performed and revealed that while β activation lags behind α, both start at stage 2 and increase with maturation. Notably, activation occurs prior to the increased localization of the locus away from the periphery during maturation, suggesting that localization of the locus away from the periphery may be secondary to the redistribution of PolII during erythroid maturation. Analysis of sorted erythroid cells from mice with a targeted deletion of the LCR reveals less co-localization of the β-globin locus and PolII speckles and loss of re-localization away from the periphery during maturation. Moreover, primary transcript FISH analysis of sorted cells from mice lacking the LCR and those carrying a combined deletion of HS 2 and 3 suggests that the LCR affects the likelihood that an allele is expressed, as well as the amount of transcript generated during periods of expression. Taken together our results suggest that the LCR plays a role in locating to, or stabilization of, interactions between the locus and PolII speckles, increasing the probability of β-globin expression, after which the LCR also affects the rate of transcription.

Description: The locus control region (LCR) was thought to be necessary and sufficient for establishing and maintaining an open β-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 β-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 β-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 β-globin locus.