Function of normal and mutated gamma-globin gene promoters in electroporated K562 erythroleukemia cells

We examined the importance of cis-acting regulatory elements of the human gamma-globin gene promoter in a cell line (K562) where this gene normally functions. A gamma-Globin promoter fragments were fused to the neomycin phosphotransferase (neoR) gene in a plasmid-based vector and transiently transfected by electroporation into K562 cells. Correctly initiated “A gamma-neo” transcripts were detected with an S1 nuclease protection assay that was internally controlled for transfection efficiency and RNA content. We first optimized the conditions for electroporation, and then determined input DNA concentrations that permitted study of gamma-promoter function in the linear range of the assay. We discovered that a gamma-globin promoter fragment extending from -299 to +36 (with respect to the transcription initiation site) was active in this transient transfection assay, and that the expression of this promoter was increased by the SV40 enhancer. Deletion of the gamma-globin promoter to position -199 did not significantly affect gamma-globin promoter function. However, deletion to -160 reduced gamma promoter strength to 70% that of control, deletion to position -130 to 19% that of control, and deletion to position -61 to 8.7% that of control. Three gamma-globin promoters containing mutations associated with hereditary persistence of fetal hemoglobin (-202 C----G, -196 C----T, and -117 G----A) were not overexpressed in the K562 cell environment, consistent with the hypothesis that these promoters are not overexpressed in fetal erythroblasts, only adult erythroid cells. This system will allow us to further dissect the roles of regulatory globin cis-acting DNA elements in fetal erythroid cells.

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Transcription start site analysis reveals widespread divergent transcription in D. melanogaster and core promoter-encoded enhancer activities

10.1101/221952 ◽

2017 ◽

Cited By ~ 4

Author(s):

Sarah Rennie ◽

Maria Dalby ◽

Marta Lloret-Llinares ◽

Stylianos Bakoulis ◽

Christian Dalager Vaagensø ◽

...

Keyword(s):

Transcription Initiation ◽

Core Promoter ◽

Regulatory Element ◽

Regulatory Elements ◽

Chromatin Interaction ◽

Promoter Strength ◽

Gene Promoters ◽

Core Promoters ◽

Promoter Architecture ◽

Regulatory Functions

ABSTRACTMammalian gene promoters and enhancers share many properties. They are composed of a unified promoter architecture of divergent transcripton initiation and gene promoters may exhibit enhancer function. However, it is currently unclear how expression strength of a regulatory element relates to its enhancer strength and if the unifying architecture is conserved across Metazoa. Here we investigate the transcription initiation landscape and its associated RNA decay in D. melanogaster. Surprisingly, we find that the majority of active gene-distal enhancers and a considerable fraction of gene promoters are divergently transcribed. We observe quantitative relationships between enhancer potential, expression level and core promoter strength, providing an explanation for indirectly related histone modifications that are reflecting expression levels. Lowly abundant unstable RNAs initiated from weak core promoters are key characteristics of gene-distal developmental enhancers, while the housekeeping enhancer strengths of gene promoters reflect their expression strengths. The different layers of regulation mediated by gene-distal enhancers and gene promoters are also reflected in chromatin interaction data. Our results suggest a unified promoter architecture of many D. melanogaster regulatory elements, that is universal across Metazoa, whose regulatory functions seem to be related to their core promoter elements.

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Upstream G gamma-globin and downstream beta-globin sequences required for stage-specific expression in transgenic mice

Molecular and Cellular Biology ◽

10.1128/mcb.7.11.4024-4029.1987 ◽

1987 ◽

Vol 7 (11) ◽

pp. 4024-4029

Author(s):

M Trudel ◽

J Magram ◽

L Bruckner ◽

F Costantini

Keyword(s):

Transgenic Mice ◽

Fusion Gene ◽

Globin Gene ◽

Regulatory Elements ◽

Globin Genes ◽

Beta Globin ◽

Erythroid Cells ◽

Base Pairs ◽

Beta Globin Gene ◽

Gamma Globin

The human G gamma-globin and beta-globin genes are expressed in erythroid cells at different stages of human development, and previous studies have shown that the two cloned genes are also expressed in a differential stage-specific manner in transgenic mice. The G gamma-globin gene is expressed only in murine embryonic erythroid cells, while the beta-globin gene is active only at the fetal and adult stages. In this study, we analyzed transgenic mice carrying a series of hybrid genes in which different upstream, intragenic, or downstream sequences were contributed by the beta-globin or G gamma-globin gene. We found that hybrid 5'G gamma/3'beta globin genes containing G gamma-globin sequences upstream from the initiation codon were expressed in embryonic erythroid cells at levels similar to those of an intact G gamma-globin transgene. In contrast, beta-globin upstream sequences were insufficient for expression of 5'beta/3'G gamma hybrid globin genes or a beta-globin-metallothionein fusion gene in adult erythroid cells. However, beta-globin downstream sequences, including 212 base pairs of exon III and 1,900 base pairs of 3'-flanking DNA, were able to activate a 5'G gamma/3'beta hybrid globin gene in fetal and adult erythroid cells. These experiments suggest that positive regulatory elements upstream from the G gamma-globin and downstream from the beta-globin gene are involved in the differential expression of the two genes during development.

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Sardinian G gamma-HPFH: a T----C substitution in a conserved "octamer" sequence in the G gamma-globin promoter

Blood ◽

10.1182/blood.v71.3.815.815 ◽

1988 ◽

Vol 71 (3) ◽

pp. 815-817 ◽

Cited By ~ 33

Author(s):

S Ottolenghi ◽

S Nicolis ◽

R Taramelli ◽

N Malgaretti ◽

R Mantovani ◽

...

Keyword(s):

Globin Gene ◽

Single Point ◽

Point Mutations ◽

Fetal Hemoglobin ◽

Regulatory Elements ◽

Globin Genes ◽

Gamma Globin ◽

New Type ◽

Hereditary Persistence ◽

Globin Promoter

Abstract A survey of hemoglobinopathies in Northern Sardinia allowed the identification of two subjects heterozygous for a new type of G gamma hereditary persistence of fetal hemoglobin (HPFH). The G gamma-globin gene from the HPFH chromosome shows the presence of a T----C substitution 175 nucleotides upstream of the CAP site, adding a new example of single-point mutations occurring in the promoter region of the gamma-globin genes and linked to HPFH phenotypes. In this case the mutation affects the 3′ end nucleotide of a conserved octamer sequence known to be present in other regulatory elements of several genes.

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Roles of fetal G gamma-globin promoter elements and the adult beta-globin 3' enhancer in the stage-specific expression of globin genes.

Molecular and Cellular Biology ◽

10.1128/mcb.10.3.1116 ◽

1990 ◽

Vol 10 (3) ◽

pp. 1116-1125 ◽

Cited By ~ 19

Author(s):

C Perez-Stable ◽

F Costantini

Keyword(s):

Blood Cells ◽

Developmental Regulation ◽

Globin Gene ◽

Upstream Region ◽

Globin Genes ◽

Beta Globin ◽

Erythroid Cells ◽

Cis Acting ◽

Beta Globin Gene ◽

Gamma Globin

The human fetal G gamma-globin and adult beta-globin genes are expressed in a tissue- and developmental stage-specific pattern in transgenic mice: the G gamma gene in embryonic cells and the beta gene in fetal and adult erythroid cells. Several of the cis-acting DNA sequences thought to be responsible for these patterns of expression are located 5' to the G gamma-globin gene and 3' to the beta-globin gene. To further define the locations and functional roles of these elements, we examined the effects of 5' truncations on the expression of the G gamma-globin gene, as well as the ability of G gamma-globin upstream sequences to alter the developmental regulation of a beta-globin gene, as well as the ability of G gamma-globin upstream sequences to alter the developmental regulation of a beta-globin gene. We found that sequences between -201 and -136 are essential for expression of the G gamma-globin gene, whereas those upstream of -201 have little effect on the level or tissue or stage specificity of G gamma-globin expression. The G gamma-globin upstream sequences from -201 to -136 were, furthermore, capable of activating a linked beta-globin gene in embryonic blood cells; however, a G gamma-globin fragment from -383 to -206 was similarly active in this assay, and the complete fragment from -383 to -136 was considerably more active than either of the smaller fragments, suggesting the presence of multiple cis-acting elements for embryonic blood cells. Our data also suggested the possibility of a negative regulatory element between -201 and -136. These results are discussed in relation to several DNA elements in the G gamma-globin upstream region, which have been shown to bind nuclear factors in erythroid cells. Finally, we observed that removal of the beta-globin 3'-flanking sequences, including the 3' enhancer, from the G gamma-globin upstream-beta-globin hybrid gene resulted in a 25-fold reduction in expression in embryonic blood cells. This suggests that the beta-globin 3' enhancer is potentially active at the embryonic stage and thus cannot be solely responsible for the fetal or adult specificity of the beta-globin gene.

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Roles of fetal G gamma-globin promoter elements and the adult beta-globin 3' enhancer in the stage-specific expression of globin genes

Molecular and Cellular Biology ◽

10.1128/mcb.10.3.1116-1125.1990 ◽

1990 ◽

Vol 10 (3) ◽

pp. 1116-1125

Author(s):

C Perez-Stable ◽

F Costantini

Keyword(s):

Blood Cells ◽

Developmental Regulation ◽

Globin Gene ◽

Upstream Region ◽

Globin Genes ◽

Beta Globin ◽

Erythroid Cells ◽

Cis Acting ◽

Beta Globin Gene ◽

Gamma Globin

The human fetal G gamma-globin and adult beta-globin genes are expressed in a tissue- and developmental stage-specific pattern in transgenic mice: the G gamma gene in embryonic cells and the beta gene in fetal and adult erythroid cells. Several of the cis-acting DNA sequences thought to be responsible for these patterns of expression are located 5' to the G gamma-globin gene and 3' to the beta-globin gene. To further define the locations and functional roles of these elements, we examined the effects of 5' truncations on the expression of the G gamma-globin gene, as well as the ability of G gamma-globin upstream sequences to alter the developmental regulation of a beta-globin gene, as well as the ability of G gamma-globin upstream sequences to alter the developmental regulation of a beta-globin gene. We found that sequences between -201 and -136 are essential for expression of the G gamma-globin gene, whereas those upstream of -201 have little effect on the level or tissue or stage specificity of G gamma-globin expression. The G gamma-globin upstream sequences from -201 to -136 were, furthermore, capable of activating a linked beta-globin gene in embryonic blood cells; however, a G gamma-globin fragment from -383 to -206 was similarly active in this assay, and the complete fragment from -383 to -136 was considerably more active than either of the smaller fragments, suggesting the presence of multiple cis-acting elements for embryonic blood cells. Our data also suggested the possibility of a negative regulatory element between -201 and -136. These results are discussed in relation to several DNA elements in the G gamma-globin upstream region, which have been shown to bind nuclear factors in erythroid cells. Finally, we observed that removal of the beta-globin 3'-flanking sequences, including the 3' enhancer, from the G gamma-globin upstream-beta-globin hybrid gene resulted in a 25-fold reduction in expression in embryonic blood cells. This suggests that the beta-globin 3' enhancer is potentially active at the embryonic stage and thus cannot be solely responsible for the fetal or adult specificity of the beta-globin gene.

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Sardinian G gamma-HPFH: a T----C substitution in a conserved "octamer" sequence in the G gamma-globin promoter

Blood ◽

10.1182/blood.v71.3.815.bloodjournal713815 ◽

1988 ◽

Vol 71 (3) ◽

pp. 815-817

Author(s):

S Ottolenghi ◽

S Nicolis ◽

R Taramelli ◽

N Malgaretti ◽

R Mantovani ◽

...

Keyword(s):

Globin Gene ◽

Single Point ◽

Point Mutations ◽

Fetal Hemoglobin ◽

Regulatory Elements ◽

Globin Genes ◽

Gamma Globin ◽

New Type ◽

Hereditary Persistence ◽

Globin Promoter

A survey of hemoglobinopathies in Northern Sardinia allowed the identification of two subjects heterozygous for a new type of G gamma hereditary persistence of fetal hemoglobin (HPFH). The G gamma-globin gene from the HPFH chromosome shows the presence of a T----C substitution 175 nucleotides upstream of the CAP site, adding a new example of single-point mutations occurring in the promoter region of the gamma-globin genes and linked to HPFH phenotypes. In this case the mutation affects the 3′ end nucleotide of a conserved octamer sequence known to be present in other regulatory elements of several genes.

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Gamma-Globin Gene Promoter Elements Required for Interaction With Globin Enhancers

Blood ◽

10.1182/blood.v91.1.309 ◽

1998 ◽

Vol 91 (1) ◽

pp. 309-318 ◽

Cited By ~ 17

Author(s):

Scott D. Langdon ◽

Russel E. Kaufman

Keyword(s):

Binding Sites ◽

Globin Gene ◽

Gene Promoter ◽

Regulatory Elements ◽

Site Directed Mutagenesis ◽

Luciferase Reporter ◽

Promoter Strength ◽

Gene Promoters ◽

Wild Type ◽

Luciferase Reporter Gene

Abstract Normal expression of the human β-globin domain genes is dependent on at least three types of regulatory elements located within the β-globin domain: the locus control region (LCR), globin enhancer elements (3′β and 3′Aγ), and the individual globin gene promoter and upstream regions. It has been postulated that regulation occurs through physical interactions between factors bound to these elements, which are located at considerable distances from each other. To identify the elements required for promoter-enhancer interactions from a distance, we have investigated the expression of the wild-type, truncated, and mutated γ-globin promoters linked to the 5′HS2 enhancer. We show that in K562 cells, 5′HS2 increases activity approximately 20-fold from both a wild-type and truncated (-135 → +25) γ promoter and that truncation or site-directed mutagenesis of the tandem CCAAT boxes eliminated the enhancement by 5′HS2. Mutation of the γ-globin gene promoter GATA-1 binding sites did not decrease either promoter strength or enhancement of activity by 5′HS2. To determine if enhanced expression of γ-globin gene promoters carrying mutations associated with hereditary persistence of fetal hemoglobin (HPFH) was due to greater interactions with enhancers, we linked these HPFH γ-globin gene promoters to 5′HS2 and demonstrated a twofold to threefold higher expression than the corresponding wild-type promoter plus enhancer in MEL cells. Addition of the Aγ-globin gene 3′ enhancer to a plasmid containing the γ-globin gene promoter and 5′HS2 did not further enhance promoter strength. Furthermore, we have demonstrated that the previously identified core 5′HS2 enhancer (46-bp tandem AP-1/NF-E2 sites) increased expression only when located 5′, but not 3′, to the γ-globin-luciferase reporter gene, suggesting that its enhancer effect is not by DNA looping. Our results suggest that CCAAT boxes, but not GATA or CACCC binding sites, are required for interaction between the γ-globin promoter and the LCR/5′HS2 and that regulatory elements in addition to the core enhancer may be required for the enhancer to act from a distance.

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Regulation of Globin Gene Expression and Erythroid Differentiation by Sp/KLF Factors.

Blood ◽

10.1182/blood.v106.11.4241.4241 ◽

2005 ◽

Vol 106 (11) ◽

pp. 4241-4241

Author(s):

Jie Hong ◽

George Stamatoyannopoulos ◽

Chao-Zhong Song

Keyword(s):

Gene Expression ◽

Globin Gene ◽

Expression System ◽

K562 Cells ◽

Erythroid Differentiation ◽

Regulatory Elements ◽

Mrna Levels ◽

Gene Promoters ◽

Gamma Globin ◽

Globin Gene Expression

Abstract Sp/Krüppel-like factor (KLF) family of proteins are characterized by the presence of three highly homologous Cys2His2 type zinc-fingers near the C-terminus that bind GC/CACCC boxes, which are one of the most common regulatory elements found in promoters of many cellular and viral genes. Currently, more than 20 members have been identified in the family. This family of factors plays important roles in cell growth, differentiation, development and homeostasis by regulating the expression of their target genes. The GC and GT/CACCC boxes in the globin gene promoters and the beta globin locus control region play an important role in the tissue- and developmental stage- specific expression of globin genes. We have carried out extensive studies to identify the KLF factors that regulate gamma globin expression and erythroid differentiation. Gene expression analysis revealed that most of the Sp/KLF factors are expressed, albeit at variable levels, in human fetal liver and adult blood cells. To determine the role of the Sp/KLF factors in gamma globin expression and erythroid differentiation, functional studies using systematic RNAi to knockdown selected Sp/KLF factors were performed. We used a lentiviral mediated siRNA expression system for specific silencing selected Sp/KLF factors. Effective knockdown of Sp/KLF factors was achieved as judged by a 70–90% decrease in their mRNA levels in the cells. Analyses of globin gene expression showed that the knockdown of some Sp/KLF factors resulted in changes in globin gene expression in K562 cells. We also observed that knockdown of specific Sp/KLF factors resulted in erythroid differentiation of K562 cells. These results suggest that specific Sp/KLF factors may play a role in regulation of globin gene expression and erythroid differentiation.

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Gamma-Globin Gene Promoter Elements Required for Interaction With Globin Enhancers

Blood ◽

10.1182/blood.v91.1.309.309_309_318 ◽

1998 ◽

Vol 91 (1) ◽

pp. 309-318

Author(s):

Scott D. Langdon ◽

Russel E. Kaufman

Keyword(s):

Binding Sites ◽

Globin Gene ◽

Gene Promoter ◽

Regulatory Elements ◽

Site Directed Mutagenesis ◽

Luciferase Reporter ◽

Promoter Strength ◽

Gene Promoters ◽

Wild Type ◽

Luciferase Reporter Gene

Normal expression of the human β-globin domain genes is dependent on at least three types of regulatory elements located within the β-globin domain: the locus control region (LCR), globin enhancer elements (3′β and 3′Aγ), and the individual globin gene promoter and upstream regions. It has been postulated that regulation occurs through physical interactions between factors bound to these elements, which are located at considerable distances from each other. To identify the elements required for promoter-enhancer interactions from a distance, we have investigated the expression of the wild-type, truncated, and mutated γ-globin promoters linked to the 5′HS2 enhancer. We show that in K562 cells, 5′HS2 increases activity approximately 20-fold from both a wild-type and truncated (-135 → +25) γ promoter and that truncation or site-directed mutagenesis of the tandem CCAAT boxes eliminated the enhancement by 5′HS2. Mutation of the γ-globin gene promoter GATA-1 binding sites did not decrease either promoter strength or enhancement of activity by 5′HS2. To determine if enhanced expression of γ-globin gene promoters carrying mutations associated with hereditary persistence of fetal hemoglobin (HPFH) was due to greater interactions with enhancers, we linked these HPFH γ-globin gene promoters to 5′HS2 and demonstrated a twofold to threefold higher expression than the corresponding wild-type promoter plus enhancer in MEL cells. Addition of the Aγ-globin gene 3′ enhancer to a plasmid containing the γ-globin gene promoter and 5′HS2 did not further enhance promoter strength. Furthermore, we have demonstrated that the previously identified core 5′HS2 enhancer (46-bp tandem AP-1/NF-E2 sites) increased expression only when located 5′, but not 3′, to the γ-globin-luciferase reporter gene, suggesting that its enhancer effect is not by DNA looping. Our results suggest that CCAAT boxes, but not GATA or CACCC binding sites, are required for interaction between the γ-globin promoter and the LCR/5′HS2 and that regulatory elements in addition to the core enhancer may be required for the enhancer to act from a distance.

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