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Lack of NF-E2 (p45) impairs the assembly of protein complexes at LCR HS2 and at the adult majglobin gene promoter. A, ChIP analysis of protein chromatin interactions in LCR HS2 and the adult maj-globin gene promoter in MEL cells incubated with or without 1.5% DMSO for 3 days. After cross-linking MEL cells with 1% formaldehyde, chromatin was isolated, fragmented by sonication, and subjected to immunoprecipitation with antibodies against NF-E2 (p45), MafK, USF1, USF2, CBP, and TFIIB. Reactions with the IgG antibody served as a negative control. The DNA was purified from the precipitate and subjected to qPCR using primers specific for LCR HS2 and the adult maj-globin gene promoter as indicated. Error bars, S.E. of three independent experiments (**, sample versus IgG, p 0.05; , sample versus IgG, 0.05 p 0.1; *, DMSO versus no DMSO, p 0.05; , DMSO versus no DMSO, 0.05 p 0.1). B, ChIP analysis of protein chromatin interactions in LCR HS2 and the adult maj-globin gene promoter in CB3 cells incubated with or without 1.5% DMSO for 3 days. DNA was isolated from immunoprecipitated material and analyzed as described in A. Error bars, S.E. of three independent experiments (symbols are as in A). C, comparative ChIP analysis of protein chromatin interactions in uninduced CB3, MEL, and CB3/NF-E2 cells. Cross-linked chromatin was precipitated with IgG or antibodies against NF-E2 (p45), USF1, or USF2, and DNA was analyzed as described in A. Error bars, S.E. of three independent experiments (*, CB3/NF-E2 versus CB3, p 0.05; , CB3/NF-E2 versus CB3, 0.05 p 0.1; **, CB3/NF-E2 versus MEL, p 0.05; , CB3/NF-E2 versus MEL, 0.05 p 0.1; ***, CB3/NF-E2 versus CB3 and MEL p 0.05).
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The human β-globin gene is expressed at high levels in erythroid cells and regulated by proximal and distal cis-acting DNA elements, including promoter, enhancer, and a locus control region (LCR). Transcription complexes are recruited
not only to the globin gene promoters but also to the LCR. Previous studies have implicated the ubiquitously expres...
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Developmental stage-specific expression of the β-type globin genes is regulated by many cis- and trans-acting components. The adult β-globin gene contains an E-box located 60 bp downstream of the transcription start site that
has been shown to bind transcription factor upstream stimulatory factor (USF) and to contribute to efficient in vitro transc...
Many questions remain about how close association of genes and distant enhancers occurs and how this is linked to transcription activation. In erythroid cells, lim domain binding 1 (LDB1) protein is recruited to the β-globin locus via LMO2 and is required for looping of the β-globin locus control region (LCR) to the active β-globin promoter. We sho...
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... Hence, we employed ChIP assays to analyze the role of IKAROS for transcription control of Cdkn1a and Tp53 genes, by studying the distribution of histone post-translational modifications. IKAROS synergizes with the PRC2 enzyme EZH2 [13,14] and with the transcription elongation licensing factor P-TEFb [18,61]. Thus, H3K27me3 (deposited by EZH2) as well as H3K4me3 and H3K79me2, which are typical marks of productive elongation [62], were studied to define the role of IKAROS as transcriptional repressor and/or activator. ...
The tumor suppressor IKAROS binds and represses multiple NOTCH target genes. For their induction upon NOTCH signaling, IKAROS is removed and replaced by NOTCH Intracellular Domain (NICD)-associated proteins. However, IKAROS remains associated to other NOTCH activated genes upon signaling and induction. Whether IKAROS could participate to the induction of this second group of NOTCH activated genes is unknown. We analyzed the combined effect of IKAROS abrogation and NOTCH signaling on the expression of NOTCH activated genes in erythroid cells. In IKAROS-deleted cells, we observed that many of these genes were either overexpressed or no longer responsive to NOTCH signaling. IKAROS is then required for the organization of bivalent chromatin and poised transcription of NOTCH activated genes belonging to either of the aforementioned groups. Furthermore, we show that IKAROS-dependent poised organization of the NOTCH target Cdkn1a is also required for its adequate induction upon genotoxic insults. These results highlight the critical role played by IKAROS in establishing bivalent chromatin and transcriptional poised state at target genes for their activation by NOTCH or other stress signals.
... This may suggest that circRNA could play a potential regulatory role in the progression of some diseases [3]. For example, circRNAs could bind to RNA polymerase II in a variety of ways to form complexes that could regulate the activity of RNA polymerase II and then affect parental gene transcription thereby [4]. According to the study of Abdelmohsen et al., circular PABPN1 (polyadenylate-binding nuclear protein 1) and HuR (human antigen R) can be extensively bound to form the HuR-circRNA complex, which competitively inhibits the binding of HuR and PABPN1 mRNA, lead-ing to a decrease in the PABPN1 translation, thus disrupting the normal metabolism of cells [5]. ...
Circular RNAs (circRNAs) play an extremely important regulatory role in the occurrence and development of various malignant tumors including papillary thyroid cancer (PTC). circFAT1(e2) is a new type of circRNA derived from exon 2 of the FAT1 gene, which is distributed in the cytoplasm and nucleus of PTC cells. However, so far, the role of circFAT1(e2) in PTC is still unclear. In this study, circFAT1(e2) was found to be highly expressed in PTC cell lines and tissues. circFAT1(e2) knockdown suppressed PTC cell growth, migration, and invasion. Also, circFAT1(e2) acted as a sponge for potential microRNAs (miRNAs) to modulate cancer progression. A potential miRNA target was discovered to be miR-873 which was targeted by circFAT1(e2) in PTC. The dual-luciferase assay conducted later also confirmed that there was indeed a direct interaction between circFAT1(e2) and miR-873. This study also confirmed that circFAT1(e2) inhibited the miR-873 expression and thus promoted the ZEB1 expression, thus affecting the proliferation, metastasis, and invasion of PTC cells. In conclusion, the results of this study indicated that circFAT1(e2) played a carcinogenic role by targeting the miR-873/ZEB1 axis to promote PTC invasion and metastasis, which might become a potential novel target for therapy of PTC.
... It is well known that GATA1, NF-E2, and USF are required to recruit Pol II to LCR HS2, a mechanism that involves formation of a multimeric complex with Pol II while bound to LCR HS2 (31)(32)(33)(34)(35). Therefore, we carried out ChIP assays to examine the effect of triplex formation on association of Pol II with LCR HS2. ...
Significance
RNA containing only pyrimidines, transcribed from genomic DNA, can form DNA∙RNA triple helices with other sites in the genome. We characterize an intronic sequence in the human β-globin gene that forms a triplex with an upstream regulatory site in the β-globin locus, causing displacement of transcription factors and down regulation of expression of the genes in the locus. Consequently, overexpression of a full-length β-globin transcript containing the intron results in decreased expression of members of the β-globin gene cluster. This is a cis -regulatory feedback mechanism in which overexpression of the β-globin gene could result in signals to down-regulate that expression. Balance in the expression of the many genes associated with hemoglobin biosynthesis is essential for erythroid cell function.
... Interestingly, both motifs are supported by the literature. Specifically, NFE2 has been shown to bind an AP-1-like motif as a heterodimer with MAF family members, such as MAFF and MAFK (33); and, in another context, NFE2 was characterised to belong to a complex including USF (34). We were able to corroborate these facts by using the MethMotif batch query module with all NFE2 binding sites in K562 and GM12878 cells (Supplementary Figure S2B). ...
Several recent studies have portrayed DNA methy-lation as a new player in the recruitment of transcription factors (TF) within chromatin, highlighting a need to connect TF binding sites (TFBS) with their respective DNA methylation profiles. However, current TFBS databases are restricted to DNA binding motif sequences. Here, we present MethMotif, a two-dimensional TFBS database that records TFBS position weight matrices along with cell type specific CpG methylation information computed from a combination of ChIP-seq and whole genome bisul-fite sequencing datasets. Integrating TFBS motifs with TFBS DNA methylation better portrays the features of DNA loci recognised by TFs. In particular, we found that DNA methylation patterns within TFBS can be cell specific (e.g. MAFF). Furthermore, for a given TF, different DNA methylation profiles are associated with different DNA binding motifs (e.g. REST). To date, MethMotif database records over 500 TF-BSs computed from over 2000 ChIP-seq datasets in 11 different cell types. MethMotif portal is accessible through an open source web interface (https: //bioinfo-csi.nus.edu.sg/methmotif) that allows users to intuitively explore the entire dataset and perform both single, and batch queries.
... Among the factors interacting with this element are EGR1, USF1, USF2, NF-E2, and MafK (Fig. 1B). The association of USF and NF-E2 transcription factors is interesting, as we have shown before that they interact with each other and are implicated in the recruitment of transcription complexes to the -globin gene locus (24). Indeed, RNA polymerase II (Pol II) is associated with the HBG-4kb HS, and global nuclear run-on of 5=-capped RNA (GRO-cap) sequencing data show that transcription initiates within this region and proceeds primarily in a unidirectional manner toward the G␥-globin gene. ...
... The binding sites for EGR1 and USF are located in close proximity (USF) or overlap (EGR1) the HBG-4kb ZF target site ( Fig. 2A). We thus focused our attention on these proteins as well as on the NF-E2 and MafK transcription factors, as they have been shown to interact with USF2 (24). We examined the binding of these proteins to the HS at kb Ϫ4 as well as to the G␥-globin gene promoter and a positive-control site, a regulatory element associated with the CDC27 gene. ...
... In K562 cells, the HBG-4kb HS associates with a variety of transcription factors, including the ubiquitously expressed USF1, USF2, and EGR1 transcription factors as well as the tissue-restricted transcription factor NF-E2. We previously demonstrated that USF2 and NF-E2 together mediate the association of transcription complexes with the LCR and the adult -globin gene promoter (24). Indeed, according to publicly available ChIP-seq and GRO-cap sequencing data, the HBG-4kb HS recruits Pol II and initiates transcription of noncoding RNA that primarily proceeds toward the ␥-globin genes. ...
The organization of the five β-type globin genes on chromosome 11 reflects the timing of expression during erythroid development, with the embryonic ε-globin gene located at the 5′end, followed by the two fetal γ-globin genes, and the adult β- and δ- globin genes at the 3′end. Here, we functionally characterized a DNase I hypersensitive site 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 DNA-binding domain (ZF) targeting the HBG-4kb HS. The HBG-4kb ZF interacted with the target site in vitro and in the context of cells with 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 site. The data demonstrate that the HBG-4kb HS site is important for fetal globin production and suggest that it may act by opening chromatin in a directional manner.
... There were only six bound sites in GM12878 (note that a bound site must pass the p-value threshold of 10 -5 for matching the NFE2 motif), while the NFE2 ChIP-seq peaks in this cell type were highly enriched in the motif of another TF, USF1 (96% peaks contained an USF1 site), suggesting that the ChIP-seq peaks arose from the interaction between the NFE2 and USF transcription factors. NFE2 has been shown to interact with USF to cooperatively regulate the β-globin gene in erythroid cells (35). The NFE2 ChIP-seq peaks in K562 cells on the other hand, were highly enriched in the NFE2 motif (75.98% of the 1991 peaks matched the NFE2 motif) and less enriched in the USF1 motif (15.59% peaks matched the USF1 motif). ...
Transcription factors play a key role in the regulation of gene expression. Hypersensitivity to DNase I cleavage has long been used to gauge the accessibility of genomic DNA for transcription factor binding and as an indicator of regulatory genomic locations. An increasing amount of ChIP-seq data on a large number of TFs is being generated, mostly in a small number of cell types. DNase-seq data are being produced for hundreds of cell types. We aimed to develop a computational method that could combine ChIP-seq and DNase-seq data to predict TF binding sites in a wide variety of cell types. We trained and tested a logistic regression model, called LR-DNase, to predict binding sites for a specific TF using seven features derived from DNase-seq and genomic sequence. We calculated the area under the precision-recall curve at a false discovery rate cutoff of 0.5 for the LR-DNase model, a number of logistic regression models with fewer features, and several existing state-of-the-art TF binding prediction methods. The LR-DNase model outperformed existing unsupervised and supervised methods. Additionally, for many TFs, a model that uses only two features, DNase-seq reads and motif score, was sufficient to match the performance of the best existing methods.
... EPO signaling is essential for the survival of erythroid progenitors (14). Transcription nuclear factor-erythroid 2 (NF-E2) is a target for RUNX1 which is essential for the regulation of erythroid, megakaryocytic maturation and differentiation as well as globin expression (15). RUNX1 and NF-E2 upregulation is not specific for MPNs, but is also seen in polycythemic disorders with enhanced HIF signaling (16). ...
The aim of the present study was to investigate regulatory relationships among hypoxia-inducible factor-1α (HIF-1α), microRNA and erythroid transcription factors. K562 cells were transfected with HIF-1α knockout or with overexpression lentivirus of plasmid (MOI 10). The cells were divided into 3 groups: the negative control, overexpressing and interference groups. The cells were cultured under normoxia and hypoxia. Expression of miR-17*, miR-363 and miR-574-5p in the three groups was determined by quantitative PCR. Expression levels of erythroid transcription factor mRNAs such as GATA-1/GATA-2 and nuclear factor‑erythroid 2 (NF-E2) were measured using RT-qPCR while the protein expression was studied using western blot analysis. Under normoxia or hypoxia, the levels of miR-17*, miR-363 and miR‑574-5p in the overexpression group were higher than those in the other groups. Differences were statistically significant (P<0.05). Under hypoxia, the level of miR-363 in the interference group was less than that in the negative control group and difference was statistically significant (P<0.05). The level of GATA-1 mRNA in the overexpression group was higher than that in the negative control group, however, in the interference group the level was lower than that in the overexpression group under both normoxic and hypoxic conditions. The level of GATA-2 mRNA in the interference group was higher than that in other two groups under normoxic or hypoxic conditions. The NF-E2 mRNA was reversely related to GATA-2. The levels of HIF-1α, GATA-1 and NF-E2 mRNAs in the negative control under hypoxia were higher than those of normoxia. The level of HIF-1α mRNA in the overexpression group in hypoxia was lower than that in normoxia, while the GATA-1 and GATA-2 mRNA showed a reverse association. The levels of HIF-1α and GATA-2 mRNA in the interference group under hypoxia were higher compared to those of normoxia. Differences were statistically significant (P<0.05). Western blot results suggested that GATA-1, GATA-2 and NF-E2 protein expression correlated with changes in their respective mRNA transcription levels. The results therefore suggested that GATA-l and miR-363 were involved in the regulation of hematopoiesis via the HIF-1α pathway in K562 cells under hypoxic condition. The hsa-miR-17* and hsa-miR‑574-5p were not entirely dependent on HIF-1α, suggesting possible complex regulatory mechanisms involved in hypoxia.
... At least for the β-globin locus, it has been shown that NF-E2 is involved in the transfer of RNA Pol II from the LCR to the adult β-globin gene promoter [65]. Furthermore, data show that NF-E2 interacts with transcription factor USF2 (upstream stimulatory factor 2) and that both proteins are required for the recruitment of transcription complexes to the β-globin gene locus [66]. The genome-wide analysis of USF2 and NF-E2 DNase I footprints in erythroid cells suggest that NF-E2 directly binds to distal regulatory elements and indirectly to proximal promoter regions, likely mediated by USF2 [67]. ...
... USF is a heterodimer composed of USF1 and USF2 [56]. As mentioned before, previous studies have shown that USF interacts with NF-E2 and regulates recruitment of transcription complexes in the β-globin gene locus [66]. However, USF appears to be required at an early step during erythroid cell specification. ...
The red blood cell is specialized for gas transport from the lungs to the tissues and back. This specialized function requires the activation of genes encoding specific cell membrane proteins, enzymes for heme synthesis, and components of hemoglobin. The process of erythropoiesis is regulated by a complex interplay between cis-regulatory DNA elements, trans-acting proteins, and noncoding RNA. These activities mediate epigenetic as well as chromatin structure alterations and organize erythroid-specific gene loci within subdomains in the nucleus, leading to high-level transcription during differentiation of erythroid cells. At the same time that erythroid-specific genes are activated, a large number of genes that encode functions not required for the erythroid cells are repressed. The globin gene loci have been intensively studied over the last 30. years, and the discoveries have shaped understanding of cell-type-specific gene control and alterations in chromatin structure associated with activation and repression of gene loci. This chapter will focus on components of the red blood cells and how expression of these components is regulated during the process of erythropoiesis.
... The motif recognized by NFY was enriched near CpGs hypomethylated in fetal erythroblasts: NFY binds the HBG2 gene promoters to stimulate chromatin opening [44]. In adult erythroblasts, we detected an enrichment for the motif bound by NF-E2, a transcription factor important for erythroid maturation and HBB gene expression [45], as well as RUNX1, an important regulator of mammalian hematopoiesis that acts upstream of NF-E2 [46]. IRF2 was recently established as a transcriptional regulator of erythropoiesis that controls gene expression through adult erythroid enhancers [20]. ...
DNA methylation is an epigenetic modification that plays an important role during mammalian development. Around birth in humans, the main site of red blood cell production moves from the fetal liver to the bone marrow. DNA methylation changes at the β-globin locus and a switch from fetal to adult hemoglobin production characterize this transition. Understanding this globin switch may improve the treatment of patients with sickle cell disease and β-thalassemia, two of the most common Mendelian diseases in the world. The goal of our study was to describe and compare the genome-wide patterns of DNA methylation in fetal and adult human erythroblasts.
We used the Illumina HumanMethylation 450 k BeadChip to measure DNA methylation at 402,819 CpGs in ex vivo-differentiated erythroblasts from 12 fetal liver and 12 bone marrow CD34+ donors.
We identified 5,937 differentially methylated CpGs that overlap with erythroid enhancers and binding sites for erythropoiesis-related transcription factors. Combining this information with genome-wide association study results, we show that erythroid enhancers define particularly promising genomic regions to identify new genetic variants associated with fetal hemoglobin (HbF) levels in humans. Many differentially methylated CpGs are located near genes with unanticipated roles in red blood cell differentiation and proliferation. For some of these new candidate genes, we confirm the correlation between DNA methylation and gene expression levels in red blood cell progenitors. We also provide evidence that DNA methylation and genetic variation at the β-globin locus independently control globin gene expression in adult erythroblasts.
Our DNA methylome maps confirm the widespread dynamic changes in DNA methylation that occur during human erythropoiesis. These changes tend to happen near erythroid enhancers, further highlighting their importance in erythroid regulation and HbF production. Finally, DNA methylation may act independently of the transcription factor BCL11A to repress fetal hemoglobin production. This provides cues on strategies to more efficiently re-activate HbF production in sickle cell disease and β-thalassemia patients.
... Bach1 is a repressor that binds to MAREs in undifferentiated cells, whereas NF-E2 (p45) is an activator that binds to MAREs in differentiated erythroid cells (30,31). Expression of the small Maf subunits remains constant during erythroid cell differentiation, while Bach1 expression declines and expression of p45 increases (30,32). The ubiquitously expressed transcription factor USF interacts with NF-E2 and binds to E-box elements in the LCR and the adult -globin gene promoters (32,33). ...
... Expression of the small Maf subunits remains constant during erythroid cell differentiation, while Bach1 expression declines and expression of p45 increases (30,32). The ubiquitously expressed transcription factor USF interacts with NF-E2 and binds to E-box elements in the LCR and the adult -globin gene promoters (32,33). Previous studies have shown that NF-E2 is required for the recruitment of RNA polymerase II (Pol II) transcription complexes to the adult -globin gene promoter but not to the LCR (32,34). ...
... The ubiquitously expressed transcription factor USF interacts with NF-E2 and binds to E-box elements in the LCR and the adult -globin gene promoters (32,33). Previous studies have shown that NF-E2 is required for the recruitment of RNA polymerase II (Pol II) transcription complexes to the adult -globin gene promoter but not to the LCR (32,34). ...
Enhancers and promoters assemble protein complexes that ultimately regulate the recruitment and activity of RNA polymerases.
Previous work has shown that at least some enhancers form stable protein complexes leading to the formation of enhanceosomes.
We analyzed protein-DNA interactions in the murine β-globin gene locus using MAPit (methyltransferase accessibility protocol
for individual templates). The data show that a tandem MARE (Maf recognition element) in locus control region (LCR) hypersensitive
site 2 (HS2) reveals a remarkably high degree of occupancy during differentiation of mouse erythroleukemia cells. Most of
the other transcription factor binding sites in LCR HS2 or in the adult β-globin gene promoter regions exhibit low fractional
occupancy, suggesting highly dynamic protein-DNA interactions. Targeting of an artificial zinc finger DNA-binding domain (ZF-DBD)
to the HS2 tandem MARE caused reduction in the association of MARE binding proteins and transcription complexes at LCR HS2
and the adult βmaj-globin gene promoter but did not affect expression of the βmin-globin gene. The data demonstrate that a
stable MARE-associated footprint in LCR HS2 is important for the recruitment of transcription complexes to the adult βmaj-globin
gene promoter during erythroid cell differentiation.
