scholarly journals Active Chromatin Hub of the Mouse α-Globin Locus Forms in a Transcription Factory of Clustered Housekeeping Genes

2006 ◽  
Vol 26 (13) ◽  
pp. 5096-5105 ◽  
Author(s):  
Guo-Ling Zhou ◽  
Li Xin ◽  
Wei Song ◽  
Li-Jun Di ◽  
Guang Liu ◽  
...  
Keyword(s):  
Gene Expression ◽  
Fetal Liver ◽  
Globin Gene ◽  
Housekeeping Genes ◽  
Regulatory Elements ◽  
Gene Promoters ◽  
Globin Genes ◽  
Rna Pol Ii ◽  
Pol Ii ◽  
Transcription Factory

ABSTRACT RNA polymerases can be shared by a particular group of genes in a transcription “factory” in nuclei, where transcription may be coordinated in concert with the distribution of coexpressed genes in higher-eukaryote genomes. Moreover, gene expression can be modulated by regulatory elements working over a long distance. Here, we compared the conformation of a 130-kb chromatin region containing the mouse α-globin cluster and their flanking housekeeping genes in 14.5-day-postcoitum fetal liver and brain cells. The analysis of chromatin conformation showed that the active α1 and α2 globin genes and upstream regulatory elements are in close spatial proximity, indicating that looping may function in the transcriptional regulation of the mouse α-globin cluster. In fetal liver cells, the active α1 and α2 genes, but not the inactive ζ gene, colocalize with neighboring housekeeping genes C16orf33, C16orf8, MPG, and C16orf35. This is in sharp contrast with the mouse α-globin genes in nonexpressing cells, which are separated from the congregated housekeeping genes. A comparison of RNA polymerase II (Pol II) occupancies showed that active α1 and α2 gene promoters have a much higher RNA Pol II enrichment in liver than in brain. The RNA Pol II occupancy at the ζ gene promoter, which is specifically repressed during development, is much lower than that at the α1 and α2 promoters. Thus, the mouse α-globin gene cluster may be regulated through moving in or out active globin gene promoters and regulatory elements of a preexisting transcription factory in the nucleus, which is maintained by the flanking clustered housekeeping genes, to activate or inactivate α-globin gene expression.

Cooperative Silencing of Chicken ρ- and βH- Globin Gene Expression by Chicken β-Globin Regulatory Elements: Involvement of RNAi Mediated Repression.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 3627-3627
Author(s):  
Elliot M. Epner ◽  
Jin Wang ◽  
Jing Huang
Keyword(s):  
Gene Expression ◽  
Gene Silencing ◽  
Globin Gene ◽  
K562 Cells ◽  
Regulatory Elements ◽  
Gene Promoters ◽  
Globin Genes ◽  
Phenotypic Analysis ◽  
Pol Ii ◽  
Globin Gene Expression

Abstract The chicken β-globin locus represents a well characterized, model system where the relationship between chromatin structure, transcription and DNA replication can be studied. The locus contains several regulatory elements including an intergenic enhancer as well as upstream regulatory elements that may function either alone or in combination with the intergenic enhancer as an LCR. The availability of the recombination proficient chicken B cell line DT40 has allowed the introduction of mutations into the endogenous chicken β-globin locus and phenotypic analysis after microcell mediated chromosome transfer into human erythroleukemia (K562) cells. Using this system, we have introduced deletions in the chicken β-globin intergenic enhancer as well as 5′ HS 1,2, and 3. Expression of the embryonic ρ and fetal βH chicken globin genes were repressed by the intergenic enhancer, 5′ HS1, or 5′HS2. No ρ or βH globin gene expression was detected in K562 cells containing control chicken chromosomes, while ρ and βH mRNA were activated when the intergenic enhancer, 5′ HS1, or 5′HS2 were deleted. Chromatin immunoprecipitation (ChIP) experiments that assayed RNA polmerase II (pol II), GATA-1 and NF-E2 p45/ p18 binding at regulatory elements and gene promoters in targeted cell lines supported this hypothesis and suggested a potential role for 5′HS3 in gene activation. However, targeted deletion of 5′ HS3, unlike the other chicken β-globin regulatory elements, showed no transcriptional phenotype. Our results demonstrate the intergenic enhancer, 5′HS1, and 5′ HS2 function through a common silencing mechanism involving pol II, GATA-1, and NF-E2/P18. The recent demonstration of the involvement of Pol II in the synthesis of miRNA’s prompted us to investigate the role of miRNA’s in gene silencing in this system. A small miRNA was identified at the intergenic enhancer region. ChIP assays showed the binding of two components of the RISC (Dicer and Ago2) at the chicken globin regulatory elements. These results are consistent with the involvement of RISC and miRNA’s in gene silencing in this system.

Changes in Globin Gene Methylation and Covalent Histone Modifications of Chromatin Associated with the ε-, γ-, and β-Globin Promoters of the Baboon (P. Anubis) during Development.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 1206-1206
Author(s):  
Donald Lavelle ◽  
Kestas Vaitkus ◽  
Maria Hankewych ◽  
Mahipal Singh ◽  
Joseph DeSimone
Keyword(s):  
Gene Expression ◽  
Fetal Liver ◽  
Developmental Regulation ◽  
Globin Gene ◽  
Gene Promoters ◽  
Rna Pol Ii ◽  
Cpg Sites ◽  
Globin Promoter ◽  
Globin Gene Expression ◽  
Covalent Histone Modifications

Abstract The pattern of globin gene expression during development is conserved in all simian primates, but not in prosimians or other species. Therefore knowledge of the mechanisms regulating globin gene expression in animal models such as the baboon (P. anubis) is directly applicable to human. This investigation addressed the role of chromatin structure in developmental regulation of globin gene expression. DNA methylation of the ε- and γ-gene promoters and covalent histone modifications in chromatin associated with the ε- γ- and β-globin gene promoters have been investigated in 40d fetal primitive nucleated yolk sac-derived RBCs, and definitive erythroid precursor cells from fetal liver (40d to 56d), fetal BM (154d to 160d), and BM from phlebotomized adults. The methylation status of 3 CpG sites in the ε-globin promoter and 5 CpG sites in the γ-globin promoter was analyzed by sequencing 10 cloned PCR products of each sample following bisulfite modification. The ε-globin promoter was unmethylated in 40d primitive yolk-sac derived RBCs. Moderate methylation of the ε-globin promoter was observed in 40d fetal liver (33%: 50%) and was increased in fetal liver samples obtained 2 weeks later in gestation (54d: 76.6%, 56d: 79.1%) to levels observed in late term fetal BM ( 154d: 80%, 156d: 96.6%, 160 d: 93.1%) and adult BM (84.1%; n=2). Methylation of the γ-globin promoter was lowest in 40d primitive RBC (0%) and early fetal liver (40d: 3.1%, 54d: 0%, 56d: 7.1%) and was moderately increased in fetal BM (154d: 38.6%, 156d: 20%, 160d: 30%) compared to adult BM ( 67.3%; n=3). Levels of ac-H3, ac-H4, dimethyl H3 lys4 (H3-dimeK4), dimethyl H3 lys79 (H3-meK79), dimethyl H3 lys36 (H3-meK36), and RNA pol II bound to the ε-, γ-, and β-globin promoters were determined by immunoprecipitation of formaldehyde-fixed, sheared chromatin (ChIP) followed by real time PCR. The amount of RNA pol II, ac-H3, and ac-H4 associated with each globin promoter correlated with developmental-specific gene expression and differed from the pattern of H3-meK79 and H3-meK4 associated with these promoters during development. The amount of H3-meK79 and H3-dimeK4 bound to the the ε- and γ-globin promoters in 40d primitive RBC and fetal liver erythroid precursors (54 and 56d) was 5 times greater than to the β-globin promoter, while similar levels of each (< 2 fold difference) were associated with all three promoters in fetal and adult bone marrow cells. In contrast, the highest level of H3-meK36 was associated with developmentally silenced genes. The amount of H3-meK36 bound to the ε promoter was 2–3 fold higher than to the γ and β promoters in fetal liver (54 and 56d). Similar levels (<2 fold difference) of H3-meK36 were associated with the γ and ε promoters in late term fetal and adult BM and were 2–6 fold greater than bound to the β promoter. We conclude that the chromatin cofiguration of the β-globin locus undergoes distinctive changes associated with both gene activation and silencing during development. Changes in the levels of H3-dimeK4 and H3-meK79 may reflect generalized domain opening, while high levels of ac-H3 and ac-H4 are bound to the promoters of activated genes. In contrast, gene silencing is correlated with increased DNA methylation and enrichment of H3-meK36 bound to the promoters. Thus the baboon model offers unique opportunities to study developmental regulation of globin gene expression.

Human globin gene promoter sequences are sufficient for specific expression of a hybrid gene transfected into tissue culture cells

1987 ◽  
Vol 7 (1) ◽  
pp. 398-402
Author(s):  
T Rutherford ◽  
A W Nienhuis
Keyword(s):  
Gene Expression ◽  
Globin Gene ◽  
Gene Promoter ◽  
Gene Promoters ◽  
Globin Genes ◽  
Specific Expression ◽  
Tissue Specific ◽  
Promoter Sequences ◽  
Erythroleukemia Cells ◽  
Murine Erythroleukemia

The contribution of the human globin gene promoters to tissue-specific transcription was studied by using globin promoters to transcribe the neo (G418 resistance) gene. After transfection into different cell types, neo gene expression was assayed by scoring colony formation in the presence of G418. In K562 human erythroleukemia cells, which express fetal and embryonic globin genes but not the adult beta-globin gene, the neo gene was expressed strongly from a fetal gamma- or embryonic zeta-globin gene promoter but only weakly from the beta promoter. In murine erythroleukemia cells which express the endogenous mouse beta genes, the neo gene was strongly expressed from both beta and gamma promoters. In two nonerythroid cell lines, human HeLa cells and mouse 3T3 fibroblasts, the globin gene promoters did not allow neo gene expression. Globin-neo genes were integrated in the erythroleukemia cell genomes mostly as a single copy per cell and were transcribed from the appropriate globin gene cap site. We conclude that globin gene promoter sequences extending from -373 to +48 base pairs (bp) (relative to the cap site) for the beta gene, -385 to +34 bp for the gamma gene, and -555 to +38 bp for the zeta gene are sufficient for tissue-specific and perhaps developmentally specific transcription.

Endogenous Elevations of Short Chain Fatty Acids Up-Regulate Embryonic Globin Gene Expression during Primitive Erythropoiesis.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 1210-1210
Author(s):  
Lauren Sterner ◽  
Toru Miyazaki ◽  
Larry Swift ◽  
Ann Dean ◽  
Jane Little
Keyword(s):  
Gene Expression ◽  
Fatty Acids ◽  
Fetal Liver ◽  
Globin Gene ◽  
Short Chain Fatty Acids ◽  
Yolk Sac ◽  
Globin Genes ◽  
Wild Type ◽  
Alpha Type ◽  
Globin Gene Expression

Abstract We examined the effects of short chain fatty acids (SCFAs) on globin gene expression during development. We studied globin gene expression in transgenic mice that have endogenous elevations in the SCFA propionate due to a knockout (KO) of the gene for propionyl CoA carboxylase subunit A (PCCA, Miyazaki et al. JBC, 2001 Sep 21;276(38):35995–9). Serum propionate levels measured by gas chromatography were 2.5 to 3.6 mgms/ml in 2 adult PCCA KO mice and were undetectable in 2 wild type (wt) or heterozygous control adult mice. Embryonic PCCA KO offspring had propionate levels of 2.3 and 5.0 μgms/100 mgms of fetal liver, at day 16.5 (E16.5), while wt or heterozygotes at E14.5 had levels &lt;1 μgm/100 mgms. Analysis of expression from alpha (α), beta major (βmaj), embryonic beta-type epsilon-y (εy), embryonic beta-type beta H1 (βH1) and embryonic alpha-type zeta (ζ) globin genes plus 18S ribosomal RNA as a control was undertaken using real-time PCR with gene-specific primers and taqman probes. cDNA was reverse-transcribed from the mRNA of yolk sac (YS) and fetal liver of PCCA KO and wt progeny of more than one litter from timed pregnancies. Individual PCCA embryos at E10 (n=10), E12 (n=9), and E14 (n=7) were analyzed for globin gene expression, normalized to18S expression and were compared to age-matched wt embryos (n&gt;=4 for each time point). As expected, embryonic alpha- and beta-type globin gene expression (ζ and βH1 plus εy) predominated in E 10 YS, and definitive globin gene expression, α and βmaj, predominated in E12 or E14 fetal liver. Expression from embryonic alpha-type globin was calculated as normalized ζ/(ζ+α) and from embryonic beta-type globins as normalized (βH1+εy)/(βH1+εy+βmaj), see table. Embryonic globin gene expression was statistically significantly increased in PCCA KO E12 YS at 1.3 fold relative to wt ζ and in PCCA KO E14 YS at 1.8 fold and 2.1 fold relative to wt ζ or βH1 and εy respectively (p&lt;.05). No increase in embryonic globin mRNA was seen in adult PCCA KO animals. We conclude that elevations of SCFAs during normal murine development causes a persistence of both embryonic alpha-type and embryonic beta-type globin gene expression during primitive, but not definitive, erythropoiesis, suggesting that SCFAs cannot reactivate silenced murine embryonic globin genes in the absence of erythroid stress. Embryonic Globin Gene Expression in Mice with Endogenous Elevations of SCFAs % Expression PCCA KO wild type p value, t test E10 ζ Yolk Sac 53+/− 2 nd E10 βH1 & ε y Yolk Sac 99 +/− 0.3 nd E12 ζ Yolk Sac 32 +/− 3 25 +/− 1 p &lt; .05 E12 βH1 & ε y Yolk Sac 77 +/− 6 74 +/− 3 ns E14 ζ Yolk Sac 7 +/− 1.5 4 +/− 1.4 p &lt; .05 E14 βH1 & ε y Yolk Sac 13 +/− 6 6 +/− 0.5 p &lt; .05 E12 ζ Fetal Liver 11 +/− 4 9 +/− 2 ns E12 βH1 & ε y Fetal Liver 13 +/− 5 13+/− 3 ns E14 ζ Fetal Liver 1 +/− 0.4 0.7 +/− 0.2 ns E14 βH1 & εy Fetal Liver 6 +/− 1.8 4 +/− 1 ns

Context-Specific Roles for Erythroid Krüppel-Like Factor (EKLF) in Co-Ordinate High Level Expression of the Murine α- and β-Globin Genes.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 365-365 ◽  
Author(s):  
Valerie M. Jansen ◽  
Shaji Ramachandran ◽  
Aurelie Desgardin ◽  
Jin He ◽  
Vishwas Parekh ◽  
...  
Keyword(s):  
Gene Expression ◽  
Gene Transcription ◽  
Globin Gene ◽  
Erythroid Cell ◽  
Gene Promoters ◽  
Globin Genes ◽  
Context Specific ◽  
High Level ◽  
Kinetics Of ◽  
Globin Gene Expression

Abstract Binding of EKLF to the proximal promoter CACC motif is essential for high-level tissue-specific β-globin gene expression. More recent studies have demonstrated that EKLF regulates expression of other erythroid-specific genes, suggesting a broad role for EKLF in co-ordinating gene transcription in differentiating erythroblasts. Given these observations, we hypothesized that EKLF may play a role in synchronizing α- and β-globin gene expression. Supporting this model, studies of fetal erythroblasts derived from EKLF-null embryos revealed a 3-fold reduction in murine α-globin gene expression in fetal erythroblasts when compared to wild type littermate controls. A similar reduction in primary α-globin RNA transcripts was observed in these studies. To further examine the molecular consequences of EKLF function at the α- and β-globin genes in vivo, we utilized an erythroid cell line derived from EKLF null fetal liver cells. We have demonstrated previously that introduction into these cells of the wildtype EKLF cDNA, fused in frame with a mutant estrogen response element results in tamoxifen-dependent rescue of β-globin gene expression. Consistent with our observations in primary erythroblasts, α-globin gene expression is present in the absence of functional EKLF. However, with tamoxifen induction, we observed a 3–5 fold increase in α-globin gene transcription. Interestingly, the kinetics of the changes in transcription of the α- and β-gene transcripts were similar. Enhancement in α-gene transcription was associated with EKLF binding at the α- and β-globin promoters as determined by a quantitative chromatin immunoprecipitation (ChIP) assay. Interestingly, maximal EKLF binding and α-gene transcription was observed within 2 hours of tamoxifen induction. We hypothesized that the role of EKLF may differ function at the promoters, given that a basal level of α-globin gene expression occurs in absence of EKLF binding. Supporting this hypothesis, we observed sequential recruitment of p45NF-E2, RNA polymerase II (Pol II) and the co-activator CBP to the β-promoter with tamoxifen induction. No change in GATA-1 binding was observed. In contrast, p45NF-E2 does not bind to the α-promoter and the kinetics of GATA-1 and PolII association is unchanged after tamoxifen induction. Taken together, our results demonstrate that EKLF regulates the co-ordinate high-level transcription of the α- and β-globin genes, binding in a kinetically identical manner to the gene promoters. However, the effects of EKLF on transacting factor recruitment (and chromatin modification) differ between the promoters, consistent with the idea that EKLF acts in a context-specific manner to modulate gene transcription.

O-Glcnacylation Regulates γ-Globin Transcription

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 1020-1020
Author(s):  
Kenneth R Peterson ◽  
Zhen Zhang ◽  
Ee Phie Tan ◽  
Anish Potnis ◽  
Nathan Bushue ◽  
...  
Keyword(s):  
Gene Expression ◽  
Sodium Butyrate ◽  
Yeast Artificial Chromosome ◽  
Conflicts Of Interest ◽  
Fetal Liver ◽  
Globin Gene ◽  
Fetal Hemoglobin ◽  
Globin Genes ◽  
Globin Gene Expression ◽  
Dynamic Cycling

Abstract Patients with sickle cell disease (SCD), caused by mutation of the adult β-globin gene, are phenotypically normal if they carry compensatory mutations that result in continued expression of the fetal γ-globin genes, a condition termed hereditary persistence of fetal hemoglobin (HPFH). Thus, a logical clinical goal for treatment of SCD is to up-regulate γ-globin synthesis using compounds that are specific for increasing fetal hemoglobin (HbF) without pleiotropic effects on cellular homeostasis. Developmental regulation of the γ-globin genes is complex and normal silencing during the adult stage of erythropoiesis likely results from a combination of the loss of transcriptional activators and the gain of transcriptional repressor complexes. One mode of γ-globin silencing occurs at the GATA binding sites located at -566 or -567 relative to the Aγ-globin or Gγ-globin CAP sites respectively, and is mediated through the DNA binding moiety of GATA-1 and its recruitment of co-repressor partners, FOG-1 and Mi-2 (NuRD complex). Modifications of repressor complexes can regulate gene transcription; one such modification is O-GlcNAcylation. The O-GlcNAc post-translational modification is the attachment of a single N-acetyl-glucosamine moiety to either a serine or threonine residue on nuclear and cytoplasmic proteins. O-GlcNAc is added to proteins by O-GlcNAc transferase (OGT) and removed by O-GlcNAcase (OGA) in response to changes in extracellular signals and nutrients. A dynamic balance in protein levels also exists between these two enzymes; an increase or decrease of one results in a like compensatory change in the other. Thus, the rate of O-GlcNAc addition and removal is a dynamic cycling event that is exquisitely controlled for a given target molecule, which may offer a point of intervention in the turning off or on of gene expression. O-GlcNAcylation is involved in the regulation of many cellular processes such as stress response, cell cycle progression, and transcription. Potentially, O-GlcNAc plays a pivotal role in regulating transcription of the human γ-globin genes. We induced human erythroleukemia cell line K562 with sodium butyrate to differentiate toward the erythroid lineage and observed the expected increase of γ-globin gene expression. A robust increase of γ-globin gene expression was measured after pharmacological inhibition of OGA using Thiamet-G (TMG). Using chromatin immunoprecipitation (ChIP), we demonstrated that OGT and OGA are recruited to the -566 region of the Aγ-globin promoter, the same region occupied by the GATA-1-FOG-1-Mi-2 (NuRD) repressor complex. However, OGT recruitment to this region was decreased when O-GlcNAc levels were artificially elevated by OGA inhibition with TMG. When γ-globin expression was not induced, Mi-2 was modified with O-GlcNAc and interacted with both OGT and OGA. After induction, O-GlcNAcylation of Mi-2 was reduced and Mi2 no longer interacted with OGT. Stable K562 cells were generated in which OGA was knocked down using shRNA. Following induction of these cells with sodium butyrate, γ-globin gene expression was higher compared to control cells. These data suggest that the dynamic cycling of O-GlcNAc on the Mi-2 (NuRD) moiety contributes towards regulation of γ-globin transcription. Concurrent ChIP experiments in human β-globin locus yeast artificial chromosome (β-YAC) transgenic mice demonstrated that GATA-1, Mi2 and OGT were recruited to the -566 Aγ-globin GATA silencer site in day E18 fetal liver when γ-globin is repressed, but not in day E12 fetal liver when γ-globin is expressed. These data demonstrate that O-GlcNAc cycling is a novel mechanism regulating γ-globin gene expression and will provide new avenues to explore in how alterations in gene regulation lead to the onset, progression, and severity of hematological disease. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Analysis of (δβ)0 Thalassemia and HPFH Deletions Suggest a Hierarchy of Cis-Acting Elements Regulating Fetal Hemoglobin Gene Expression.

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 54-54 ◽  
Author(s):  
Heather L Edward ◽  
Tasha Morrison ◽  
Jacqueline N Milton ◽  
Hong-yuan Luo ◽  
Lance Davis ◽  
...  
Keyword(s):  
Gene Expression ◽  
Binding Site ◽  
Conflicts Of Interest ◽  
Globin Gene ◽  
Regression Tree ◽  
Fetal Hemoglobin ◽  
Regulatory Elements ◽  
Globin Genes ◽  
Cis Acting ◽  
Cart Analysis

Abstract Hereditary persistence of fetal hemoglobin (HPFH) and (δβ)0 thalassemia are caused by deletions within the β-globin gene (HBB) cluster that remove elements that affect the expression of the γ-globin genes (HBG2 and HBG1, or HBG). These deletions are of different lengths and have different 5’ and 3’ breakpoints. The phenotypes associated with heterozygous carriers of (δβ)0 thalassemia and HPFH deletions are differentiated by levels of 5-15% HbF distributed heterocellularly in the former and 15-30% HbF distributed pancellularly in the latter. We found a novel 588.6 kb deletion that removed both the 3.5 kb fragment 5’ to HBD that is deleted in Corfu β thalassemia and contains a BCL11A binding site, and the known cis-acting elements downstream of HBB. The proband with this deletion had a HbF of 5.4% (Morrison et al, Blood, 2014 abstract 3452). To study the relative importance of 5’ and 3’ regulatory elements in HBG expression we studied 209 cases culled from the literature and from our laboratory where the 3.5 kb element 5’ to HBD and enhancers 3’ to HBB were deleted and HBG remained intact. We used a backwards stepwise regression statistical analysis to determine which deleted elements had the greatest effect on HbF levels. The combination of the deletion of 3.5 kb intergenic region 5’ to HBD, the presence of the HPFH-1 “3D” enhancer juxtaposed to HBG, and the deletion of the 3’ HS1 region accounted for 66.7% of the HbF variation in heterozygotes for HPFH and (δβ)0-thalassemia deletions. The HPFH-1 “3D” enhancer juxtaposed to HBG— the main difference between HPFH-1 and 2 compared with Spanish (δβ)0-thalassemia—was associated with an increase in HbF of 20.78% (p<2e-16) after adjusting for the effects of the other 5’ and 3’ cis-acting elements. The next most significant factor was the deletion of the 3.5 kb fragment 5’ to HBD which resulted in an increase of 10.62% HbF after similar adjustments (p<2e-16); deletion of the 3’ HS1 region accounted for an increase in HbF of 5.25% (p<1.05e-5). The HPFH-3 and HPFH-6 enhancer regions each accounted for a less than 1% increase in HbF and were not significantly associated with HbF in this model. Among 194 individuals where both 5’ and some 3’ elements affecting γ-globin gene expression—excluding the “3D” enhancer—were deleted, HbF was 20±9.3%; in 13 cases where all 3’ enhancers—including the “3D” enhancer—were deleted, HbF was 6.8±3.7% (p=8.9e-07). To determine which combinations of cis-acting elements were associated with high and low HbF levels we performed a classification and regression tree (cART) analysis on HbF. The results of the regression tree (Figure) only included the deletion of the 5’ 3.5 kb fragment region, the presence of the HPFH-1 “3D” enhancer and the deletion of the 3’ HS1 region and were consistent with the results of the backwards selection model. The absence of the 5’ 3.5 kb fragment 5’ to HBD combined with the presence of the HPFH-1 “3D” enhancer was associated with the highest average HbF of 27.02%. The absence of the 3.5 kb fragment 5’ to HBD combined with the absence of the HPFH-1 “3D” enhancer was associated with the lowest average HbF of 6.82%.The 588.6 kb deletion is the largest deletion reported in the HBB cluster that leaves the γ-globin genes intact, and the second to remove both the BCL11A binding site and all known 3’ enhancer elements. By studying deletions in the HBBgene cluster we have further defined the hierarchy of cis-acting elements that modulate HbF levels in adults and suggest a paramount role of the distal “3D” enhancer. Figure 1 Figure 1. Disclosures No relevant conflicts of interest to declare.

scholarly journals The Human β Globin Locus Introduced by YAC Transfer Exhibits a Specific and Reproducible Pattern of Developmental Regulation in Transgenic Mice

Blood ◽  
1997 ◽  
Vol 90 (11) ◽  
pp. 4602-4609 ◽  
Author(s):  
Susanna Porcu ◽  
Michael Kitamura ◽  
Ewa Witkowska ◽  
Zemin Zhang ◽  
Annick Mutero ◽  
...  
Keyword(s):  
Gene Expression ◽  
Developmental Regulation ◽  
Globin Gene ◽  
Regulatory Elements ◽  
Regulatory Sequences ◽  
Mrna Levels ◽  
Globin Genes ◽  
Globin Mrna ◽  
Specific Expression ◽  
Artificial Chromosomes

Abstract The human β globin locus spans an 80-kb chromosomal region encompassing both the five expressed globin genes and the cis-acting elements that direct their stage-specific expression during ontogeny. Sequences proximal to the genes and in the locus control region, 60 kb upstream of the adult β globin gene, are required for developmental regulation. Transgenic studies have shown that altering the structural organization of the locus disrupts the normal pattern of globin gene regulation. Procedures for introducing yeast artificial chromosomes (YACs) containing large genetic loci now make it possible to define the sequences required for stage-restricted gene expression in constructs that preserve the integrity of the β globin locus. We demonstrate that independent YAC transgenic lines exhibit remarkably similar patterns of globin gene expression during development. The switch from γ to β globin predominant expression occurs between day 11.5 and 12.5 of gestation, with no more than twofold differences in human β globin mRNA levels between lines. Human β globin mRNA levels were twofold to fourfold lower than that of mouse βmaj, revealing potentially significant differences in the regulatory sequences of the two loci. These findings provide an important basis for studying regulatory elements within the β globin locus.

Role of gene order in developmental control of human gamma- and beta-globin gene expression

1993 ◽  
Vol 13 (8) ◽  
pp. 4836-4843
Author(s):  
K R Peterson ◽  
G Stamatoyannopoulos
Keyword(s):  
Gene Expression ◽  
Gene Order ◽  
Developmental Regulation ◽  
Globin Gene ◽  
Gene Promoters ◽  
Globin Genes ◽  
Beta Globin ◽  
Gamma Globin ◽  
Globin Gene Expression ◽  
Beta Gene

To determine the effect of gene order on globin gene developmental regulation, we produced transgenic mice containing two tandemly arranged gamma- or beta-globin or gamma beta- and beta gamma-globin genes linked to a 2.5-kb cassette containing sequences of the locus control region (LCR). Analysis of constructs containing two identical gamma or beta genes assessed the effect of gene order on globin gene expression, while analysis of constructs containing tandemly arranged gamma and beta genes assessed any additional effects of the trans-acting environment. When two gamma genes were tandemly linked to the LCR, expression from the proximal gamma gene was three- to fourfold higher than expression from the distal gamma gene, and the ratio of proximal to distal gene expression remained unchanged throughout development. Similarly, when two beta genes were tandemly linked to the LCR, the proximal beta gene was predominantly expressed throughout development. These results indicate that proximity to LCR increases gene expression, perhaps by influencing the frequency of interaction between the LCR and globin gene promoters. An arrangement where the gamma gene was proximal and the beta gene distal to the LCR resulted in predominant gamma-gene expression in the embryo. When the order was reversed and the gamma gene was placed distally to the LCR, gamma-gene expression in the embryo was still up to threefold higher than expression of the LCR-proximal beta gene. These findings suggest that the embryonic trans-acting environment interacts preferentially with the gamma genes irrespective of their order or proximity to the LCR. We conclude that promoter competition rather than gene order plays the major role in globin gene switching.

Differences in the Pattern of Histone H3 (K4) and (K27) Methylation throughout the β-Globin Gene Locus in Baboon Fetal Liver and Adult Bone Marrow Erythroblasts Pre- and Post-Decitabine Treatment.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 1584-1584
Author(s):  
Janet Chin ◽  
Donald Lavelle ◽  
Kestis Vaitkus ◽  
Maria Hankewych ◽  
Joseph DeSimone
Keyword(s):  
Gene Expression ◽  
Intergenic Region ◽  
Gene Locus ◽  
Fetal Liver ◽  
Globin Gene ◽  
Histone H3 ◽  
Globin Genes ◽  
Alu Sequence ◽  
Adult Bone ◽  
Globin Gene Expression

Abstract Understanding the role of chromatin structure in specifying the pattern of β-like globin gene expression during development would be important in the design of future pharmacologic therapies to increase fetal hemoglobin in patients with sickle cell disease and β-thalassemia. The baboon is an important experimental animal model to study the regulation of globin gene expression because the structure of the β-globin gene complex and developmental pattern of globin gene expression are similar to man, and HbF levels are greatly increased in baboons treated with the DNA methyltransferase inhibitor decitabine (5-aza-2′-deoxycytidine). To investigate the relationship between chromatin structure, DNA methylation, and globin gene regulation, the distribution of acetyl histone H3 (ac-H3), acetyl histone H4 (ac-H4), histone H3 (K4) dimethyl and trimethyl, and histone H3 (K27) dimethyl throughout the β-globin gene locus was determined in purified primary erythroblasts from baboon fetal liver (FL), and adult bone marrow (BM) pre- and post-decitabine treatment. Analysis was performed by chromatin immunoprecipitation (ChIP) of formaldehyde-fixed chromatin followed by real time PCR using 18 primer sets spanning the baboon β-globin gene locus from the 5′ region of the ε-globin gene to the β-globin gene. Comparison of the pattern of ac-H3 and ac-H4 suggested the presence of three subdomains of chromatin within the β-globin locus characterized by different levels of histone acetylation that exhibited a differential response to decitabine treatment. Histone H3 (K4) dimethyl was relatively enriched in the region containing the ε- and γ-globin genes and in the γ-β intergenic region 5′ to the duplicated Alu sequence in FL. Levels associated with the ε-, γ-, and γ-globin genes in adult BM were similar and relatively unaffected by decitabine treatment. In contrast, high levels of histone H3 (K4) trimethylation and pol II distribution were associated with the promoters and transcribed regions of active genes. Differences in the levels of H3 (K4) trimethylation and pol II associated with individual genes were well correlated with differences in their relative levels of expression in FL and adult BM pre- and post-decitabine treatment. The level of histone H3 (K4) trimethyl associated with the promoter of the developmentally inactive ε-globin gene was very low and not enriched compared to inactive necdin gene or the γ-β intergenic regon in adult BM suggesting that the ε-globin gene is not maintained in a “poised” transcriptional state by the presence of the histone H3 (K4) trimethyl mark near the ε-globin promoter. The pattern of histone H3 (K27) dimethyl differed in FL and adult BM. Levels of H3 (K27) dimethyl associated with the ε- and γ-globin genes in FL were 2–4 fold less than near the duplicated Alu sequence in the γ-β intergenic region, while levels were 4–10 fold higher near the ε- and γ-globin genes and γ-β intergenic region compared to the promoter and transcribed region of the β-globin gene in adult BM. Reactivation of γ-globin expression following decitabine treatment was associated with a relative decrease in the level of H3 (K27) dimethyl near the γ-globin gene. Increased H3 (K27) methylation in regions surrounding the silenced ε- and γ-globin genes suggests that the polycomb group (PcG) protein EZH2, a histone H3 (K27) methyltransferase, may be involved in globin gene silencing.

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