scholarly journals Identification of a Novel Enhancer/Chromatin Opening Element Associated with High-Level γ-Globin Gene Expression

2018 ◽  
Vol 38 (19) ◽  
Author(s):  
Yong Shen ◽  
MacLean A. Bassett ◽  
Aishwarya Gurumurthy ◽  
Rukiye Nar ◽  
Isaac J. Knudson ◽  
...  
Keyword(s):  
Gene Expression ◽  
Transcription Factors ◽  
Globin Gene ◽  
Erythroid Cell ◽  
Globin Genes ◽  
Chromosome 11 ◽  
Erythroleukemia Cell ◽  
High Level ◽  
Globin Gene Expression

ABSTRACT The organization of the five β-type globin genes on chromosome 11 reflects the timing of expression during erythroid cell development, with the embryonic ε-globin gene being located at the 5′ end, followed by the two fetal γ-globin genes, and with the adult β- and δ-globin genes being located at the 3′ end. Here, we functionally characterized a DNase I-hypersensitive site (HS) located 4 kb upstream of the Gγ-globin gene (HBG-4kb HS). This site is occupied by transcription factors USF1, USF2, EGR1, MafK, and NF-E2 in the human erythroleukemia cell line K562 and exhibits histone modifications typical for enhancers. We generated a synthetic zinc finger (ZF) DNA-binding domain targeting the HBG-4kb HS (HBG-4kb ZF). The HBG-4kb ZF interacted with the target site in vitro and in the context of cells with a high affinity and specificity. Direct delivery of the HBG-4kb ZF to K562 and primary human erythroid cells caused a reduction in γ-globin gene expression which was associated with decreased binding of transcription factors and active histone marks at and downstream of the HS. The data demonstrate that the HBG-4kb HS is important for fetal globin production and suggest that it may act by opening chromatin in a directional manner.

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.

scholarly journals Regulatory factors specific for adult and embryonic globin genes may govern their expression in erythroleukemia cells

Blood ◽  
1985 ◽  
Vol 65 (3) ◽  
pp. 705-712 ◽  
Author(s):  
NP Anagnou ◽  
TY Yuan ◽  
E Lim ◽  
J Helder ◽  
S Wieder ◽  
...  
Keyword(s):  
Gene Expression ◽  
Human Chromosome ◽  
Globin Gene ◽  
Globin Genes ◽  
Chromosome 16 ◽  
Regulatory Factors ◽  
Erythroleukemia Cells ◽  
Erythroleukemia Cell ◽  
Globin Gene Expression ◽  
Mouse Erythroleukemia

Abstract In order to test if trans-acting regulatory factors specific for globin genes of the adult and embryonic stages of development exist in erythroid cells, transcriptionally active embryonic and adult globin genes on the same chromosome were transferred by cell fusion from the human leukemia cell K562 into phenotypically adult mouse erythroleukemia cells. Restriction-fragment-length polymorphisms of the K562 zeta (embryonic) globin genes were used to establish that all three copies of human chromosome 16 present in the K562 cell showed the same pattern of human globin gene expression after transfer to the mouse erythroleukemia cell. Adult (alpha) but not embryonic (zeta) human globin mRNA was detected in all nine of the independently derived mouse erythroleukemia hybrid cells, each of which contained human chromosome 16. Restriction endonuclease studies of the K562 alpha- and zeta-globin genes after transfer into the mouse erythroleukemia cell showed no evidence of rearrangements or deletions that could explain this loss of zeta-globin gene expression. These data suggest that regulation of globin gene expression in these erythroleukemia cells involves trans-acting regulatory factors specific for the adult and embryonic stages of development.

Transcriptional Regulation of Erythropoiesis.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. SCI-7-SCI-7
Author(s):  
Mitchell J. Weiss
Keyword(s):  
Gene Expression ◽  
Transcription Factors ◽  
Transcriptional Activation ◽  
Zinc Finger Protein ◽  
Conflicts Of Interest ◽  
Globin Gene ◽  
Globin Genes ◽  
Erythroid Development ◽  
Globin Gene Expression

Abstract Abstract SCI-7 Efforts to define the mechanisms of globin gene expression and transcriptional control of erythrocyte formation have provided key insights into our understanding of developmental hematopoiesis. Our group has focused on GATA-1, a zinc finger protein that was initially identified through its ability to bind a conserved cis element that regulates globin gene expression. GATA-1 is essential for erythroid development and mutations in the GATA1 gene are associated with human cytopenias and leukemia. Several general principles have emerged through studies to define the mechanisms of GATA-1 action. First, GATA-1 activates not only globin genes, but also virtually every gene that defines the erythroid phenotype. This observation sparked successful gene discovery efforts to identify new components of erythroid development and physiology. Second, GATA-1 also represses transcription through multiple mechanisms. This property may help to explain how GATA-1 regulates hematopoietic lineage commitment and also how GATA1 mutations contribute to cancer, since several directly repressed targets are proto-oncogenes. Third, GATA-1 regulates not only protein coding genes, but also microRNAs, which in turn, modulate erythropoiesis through post-transcriptional mechanisms. Fourth, GATA-1 interacts with other essential erythroid-specific and ubiquitous transcription factors. These protein interactions regulate gene expression by influencing chromatin modifications and controlling three-dimensional proximity between widely spaced DNA elements. Recently, we have combined transcriptome analysis with ChIP-chip and ChIP-seq studies to correlate in vivo occupancy of DNA by GATA-1 and other transcription factors with mRNA expression genome-wide in erythroid cells. These studies better elucidate how GATA-1 recognizes DNA, discriminates between transcriptional activation versus repression and interacts functionally with other nuclear proteins. I will review published and new aspects of our work in these areas. Disclosures No relevant conflicts of interest to declare.

Correct Function of the Locus Control Region May Require Passage Through a Nonerythroid Cellular Environment

Blood ◽  
1999 ◽  
Vol 93 (2) ◽  
pp. 703-712 ◽  
Author(s):  
George Vassilopoulos ◽  
Patrick A. Navas ◽  
Evangelia Skarpidi ◽  
Kenneth R. Peterson ◽  
Chris H. Lowrey ◽  
...  
Keyword(s):  
Gene Expression ◽  
Control Region ◽  
Globin Gene ◽  
Locus Control Region ◽  
Erythroid Cell ◽  
Globin Genes ◽  
Globin Mrna ◽  
L Cells ◽  
Correct Function ◽  
Globin Gene Expression

Abstract The function of the β-globin locus control region (LCR) has been studied both in cell lines and in transgenic mice. We have previously shown that when a 248-kb β-locus YAC was first microinjected into L-cells and then transferred into MEL cells by fusion, the YAC loci of the LxMEL hybrids displayed normal expression and developmental regulation.To test whether direct transfer of a β-globin locus (β-YAC) into MEL cells could be used for studies of the function of the LCR, a 155-kb β-YAC that encompasses the entire β-globin locus was used. This YAC was retrofitted with a PGK-neo selectable marker and with two I-PpoI sites at the vector arm-cloned insert junctions, allowing detection of the intact globin loci on a single I-PpoI fragment by pulsed field gel electrophoresis (PFGE). ThePpo-155 β-YAC was used to directly lipofect MEL 585 cells. In 7 β-YAC MEL clones with at least one intact copy of the YAC, the levels of total human globin mRNA (ie, ɛ + γ + β) per copy of integrated β-YAC varied more than 97-fold between clones. These results indicated that globin gene expression was strongly influenced by the position of integration of the β-YAC into the MEL cell genome and suggested that the LCR cannot function properly when the locus is directly transferred into an erythroid cell environment as naked β-YAC DNA. To test whether passage of the β-YAC through L-cells before transfer into MEL cells was the reason for the previously observed correct developmental regulation of human globin genes in the LxMEL hybrid cells, we transfected the YAC into L-cells by lipofection. Three clones carried the intact 144-kb I-PpoI fragment and transcribed the human globin genes with a fetal-like pattern. Subsequent transfer of the YAC of these L(β-YAC) clones into MEL cells by fusion resulted in LxMEL hybrids that synthesized human globin mRNA. The variation in human β-globin mRNA (ie, ɛ + γ + β) levels between hybrids was 2.5-fold, indicating that globin gene expression was independent of position of integration of the transgene, as expected for normal LCR function. The correct function of the LCR when the YAC is first transferred into the L-cell environment raises the possibility that normal activation of the LCR requires interaction with the transcriptional environment of an uncommitted, nonerythroid cell. We propose that the activation of the LCR may represent a multistep process initiated by the binding of ubiquitous transcription factors early during the differentiation of hematopoietic stem cells and completed with the binding of erythroid type of factors in the committed erythroid progenitors.

The intricacies of β-globin gene expression

1994 ◽  
Vol 72 (9-10) ◽  
pp. 377-380 ◽  
Author(s):  
Christi Andrin ◽  
Charlotte Spencer
Keyword(s):  
Gene Expression ◽  
Transcription Factors ◽  
Gene Locus ◽  
Developmental Regulation ◽  
Globin Gene ◽  
Globin Genes ◽  
Specific Expression ◽  
Complicated Process ◽  
The Individual ◽  
Globin Gene Expression

Gene expression is an extremely complicated process in which several mechanisms are involved. Owing to its developmental and tissue-specific expression, the β-globin gene is an excellent model for studying gene expression. β-Globin gene expression involves an interplay between several different mechanisms. Chromatin structure is thought to be altered by the locus control region (LCR) located far upstream of the β-globin gene locus. As well, multiple transcription factors come into play both in the LCR and in the individual promoters and enhancers of the β-globin genes. The interaction between these then allows for delicate regulation of β-globin gene expression. In the following review the elaborate system of β-globin gene expression will briefly be examined.Key words: β-globin, gene expression, chromatin, GATA-I, NF-E2, developmental regulation.

scholarly journals MiR-144 Regulates Hemoglobin Expression in Human Erythroid Cell Line

2020 ◽  
Vol 17 (11) ◽  
pp. 1221-1229
Author(s):  
Tipparat PENGLONG ◽  
Apisara SAENSUWANNA ◽  
Jitpanu KOCHAROENWAT ◽  
Wittawat BOORINTARAGOT ◽  
Suppanut FUPONGSIRIPHAN ◽  
...  
Keyword(s):  
Gene Expression ◽  
Cell Line ◽  
Target Gene ◽  
Globin Gene ◽  
Fetal Hemoglobin ◽  
Erythroid Cell ◽  
Globin Genes ◽  
Functional Studies ◽  
Genes Expression ◽  
Globin Gene Expression

The regulation of globin gene expression is significantly important to understand the pathogenesis of globin gene disorders. Recent findings have shown that microRNAs (miRNAs, miRs) play an important role in the regulation of globin gene expression. The miR-144 is an erythroid lineage-specific miRNA, in which its expression mediates NRF2 gene silencing and inhibits fetal hemoglobin expression. However, roles of miR-144 to other globin genes expression especially in ɑ-globin cluster remain unknown. This study, thus, examined the functional studies of miR-144 to globin gene expression in K562 human erythroid cell line. The results revealed that ɑ-globin and z-globin gene expression were silenced by the overexpressed miR-144 and that correlated with the reduced expression of KLF1- the suspected target gene. By contrast, transfection with miR-144 inhibitor reversed the silencing effect of miR-144. On the other hand, miR-144 had no effect to β-globin gene expression. Our results sustain the findings of the previous studies that the overexpression of miR-144 correlates with the repressing of NRF2 and 𝛄-globin gene expression. Taken together, our results suggest that miR-144 plays a key role in globin gene expression by silencing 𝛄-globin through NRF2 target mRNA and repressing adult ɑ-globin and embryonic z-globin gene expression possibly by targeting KLF1 gene.

Correct Function of the Locus Control Region May Require Passage Through a Nonerythroid Cellular Environment

Blood ◽  
1999 ◽  
Vol 93 (2) ◽  
pp. 703-712 ◽  
Author(s):  
George Vassilopoulos ◽  
Patrick A. Navas ◽  
Evangelia Skarpidi ◽  
Kenneth R. Peterson ◽  
Chris H. Lowrey ◽  
...  
Keyword(s):  
Gene Expression ◽  
Control Region ◽  
Globin Gene ◽  
Locus Control Region ◽  
Erythroid Cell ◽  
Globin Genes ◽  
Globin Mrna ◽  
L Cells ◽  
Correct Function ◽  
Globin Gene Expression

The function of the β-globin locus control region (LCR) has been studied both in cell lines and in transgenic mice. We have previously shown that when a 248-kb β-locus YAC was first microinjected into L-cells and then transferred into MEL cells by fusion, the YAC loci of the LxMEL hybrids displayed normal expression and developmental regulation.To test whether direct transfer of a β-globin locus (β-YAC) into MEL cells could be used for studies of the function of the LCR, a 155-kb β-YAC that encompasses the entire β-globin locus was used. This YAC was retrofitted with a PGK-neo selectable marker and with two I-PpoI sites at the vector arm-cloned insert junctions, allowing detection of the intact globin loci on a single I-PpoI fragment by pulsed field gel electrophoresis (PFGE). ThePpo-155 β-YAC was used to directly lipofect MEL 585 cells. In 7 β-YAC MEL clones with at least one intact copy of the YAC, the levels of total human globin mRNA (ie, ɛ + γ + β) per copy of integrated β-YAC varied more than 97-fold between clones. These results indicated that globin gene expression was strongly influenced by the position of integration of the β-YAC into the MEL cell genome and suggested that the LCR cannot function properly when the locus is directly transferred into an erythroid cell environment as naked β-YAC DNA. To test whether passage of the β-YAC through L-cells before transfer into MEL cells was the reason for the previously observed correct developmental regulation of human globin genes in the LxMEL hybrid cells, we transfected the YAC into L-cells by lipofection. Three clones carried the intact 144-kb I-PpoI fragment and transcribed the human globin genes with a fetal-like pattern. Subsequent transfer of the YAC of these L(β-YAC) clones into MEL cells by fusion resulted in LxMEL hybrids that synthesized human globin mRNA. The variation in human β-globin mRNA (ie, ɛ + γ + β) levels between hybrids was 2.5-fold, indicating that globin gene expression was independent of position of integration of the transgene, as expected for normal LCR function. The correct function of the LCR when the YAC is first transferred into the L-cell environment raises the possibility that normal activation of the LCR requires interaction with the transcriptional environment of an uncommitted, nonerythroid cell. We propose that the activation of the LCR may represent a multistep process initiated by the binding of ubiquitous transcription factors early during the differentiation of hematopoietic stem cells and completed with the binding of erythroid type of factors in the committed erythroid progenitors.

scholarly journals Induction of Human Fetal Globin Gene Expression by a Novel Erythroid Factor, NF-E4

2000 ◽  
Vol 20 (20) ◽  
pp. 7662-7672 ◽  
Author(s):  
Wenlai Zhou ◽  
David R. Clouston ◽  
Xi Wang ◽  
Loretta Cerruti ◽  
John M. Cunningham ◽  
...  
Keyword(s):  
Gene Expression ◽  
Fetal Liver ◽  
Globin Gene ◽  
K562 Cells ◽  
Erythroid Cell ◽  
Globin Genes ◽  
Erythroid Progenitors ◽  
Dimerization Domain ◽  
Preferential Expression ◽  
Globin Gene Expression

ABSTRACT The stage selector protein (SSP) is a heteromeric complex involved in preferential expression of the human γ-globin genes in fetal-erythroid cells. We have previously identified the ubiquitous transcription factor CP2 as a component of this complex. Using the protein dimerization domain of CP2 in a yeast two-hybrid screen, we have cloned a novel gene, NF-E4, encoding the tissue-restricted component of the SSP. NF-E4 and CP2 coimmunoprecipitate from extract derived from a fetal-erythroid cell line, and antiserum to NF-E4 ablates binding of the SSP to the γ promoter. NF-E4 is expressed in fetal liver, cord blood, and bone marrow and in the K562 and HEL cell lines, which constitutively express the fetal globin genes. Enforced expression of NF-E4 in K562 cells and primary erythroid progenitors induces endogenous fetal globin gene expression, suggesting a possible strategy for therapeutic intervention in the hemoglobinopathies.

scholarly journals Regulatory factors specific for adult and embryonic globin genes may govern their expression in erythroleukemia cells

Blood ◽  
1985 ◽  
Vol 65 (3) ◽  
pp. 705-712
Author(s):  
NP Anagnou ◽  
TY Yuan ◽  
E Lim ◽  
J Helder ◽  
S Wieder ◽  
...  
Keyword(s):  
Gene Expression ◽  
Human Chromosome ◽  
Globin Gene ◽  
Globin Genes ◽  
Chromosome 16 ◽  
Regulatory Factors ◽  
Erythroleukemia Cells ◽  
Erythroleukemia Cell ◽  
Globin Gene Expression ◽  
Mouse Erythroleukemia

In order to test if trans-acting regulatory factors specific for globin genes of the adult and embryonic stages of development exist in erythroid cells, transcriptionally active embryonic and adult globin genes on the same chromosome were transferred by cell fusion from the human leukemia cell K562 into phenotypically adult mouse erythroleukemia cells. Restriction-fragment-length polymorphisms of the K562 zeta (embryonic) globin genes were used to establish that all three copies of human chromosome 16 present in the K562 cell showed the same pattern of human globin gene expression after transfer to the mouse erythroleukemia cell. Adult (alpha) but not embryonic (zeta) human globin mRNA was detected in all nine of the independently derived mouse erythroleukemia hybrid cells, each of which contained human chromosome 16. Restriction endonuclease studies of the K562 alpha- and zeta-globin genes after transfer into the mouse erythroleukemia cell showed no evidence of rearrangements or deletions that could explain this loss of zeta-globin gene expression. These data suggest that regulation of globin gene expression in these erythroleukemia cells involves trans-acting regulatory factors specific for the adult and embryonic stages of development.

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