-
[show abstract]
[hide abstract]
ABSTRACT: The human embryonic, fetal and adult β-like globin genes provide a paradigm for tissue- and developmental stage-specific gene regulation. The fetal γ-globin gene is expressed in fetal erythroid cells but is repressed in adult erythroid cells. The molecular mechanism underlying this transcriptional switch during erythroid development is not completely understood. Here, we used a combination of in vitro and in vivo assays to dissect the molecular assemblies of the active and the repressed proximal γ-globin promoter complexes in K562 human erythroleukemia cell line and primary human fetal and adult erythroid cells. We found that the proximal γ-globin promoter complex is assembled by a developmentally regulated, general transcription activator NF-Y bound strongly at the tandem CCAAT motifs near the TATA box. NF-Y recruits to neighboring DNA motifs the developmentally regulated, erythroid transcription activator GATA-2 and general repressor BCL11A, which in turn recruit erythroid repressor GATA-1 and general repressor COUP-TFII to form respectively the NF-Y/GATA-2 transcription activator hub and the BCL11A/COUP-TFII/GATA-1 transcription repressor hub. Both the activator and the repressor hubs are present in both the active and the repressed γ-globin promoter complexes in fetal and adult erythroid cells. Through changes in their levels and respective interactions with the co-activators and co-repressors during erythroid development, the activator and the repressor hubs modulate erythroid- and developmental stage-specific transcription of γ-globin gene.
PLoS ONE 01/2012; 7(10):e47175. · 4.09 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: Retrotransposons including endogenous retroviruses and their solitary long terminal repeats (LTRs) compose >40% of the human genome. Many of them are located in intergenic regions far from genes. Whether these intergenic retrotransposons serve beneficial host functions is not known. Here we show that an LTR retrotransposon of ERV-9 human endogenous retrovirus located 40-70 kb upstream of the human fetal gamma- and adult beta-globin genes serves a long-range, host function. The ERV-9 LTR contains multiple CCAAT and GATA motifs and competitively recruits a high concentration of NF-Y and GATA-2 present in low abundance in adult erythroid cells to assemble an LTR/RNA polymerase II complex. The LTR complex transcribes intergenic RNAs unidirectionally through the intervening DNA to loop with and modulate transcription factor occupancies at the far downstream globin promoters, thereby modulating globin gene switching by a competitive mechanism.
Proceedings of the National Academy of Sciences 07/2010; 107(29):12992-7. · 9.68 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: In the human epsilon-globin gene locus, the HS2 enhancer in the Locus Control Region regulates transcription of the embryonic epsilon-globin gene located over 10 kb away. The mechanism of long-range HS2 enhancer function was not fully established. Here we show that the HS2 enhancer complex containing the enhancer DNA together with RNA polymerase II (pol II) and TBP tracks along the intervening DNA, synthesizing short, polyadenylated, intergenic RNAs to ultimately loop with the epsilon-globin promoter. Guided by this facilitated tracking and transcription mechanism, the HS2 enhancer delivers pol II and TBP to the cis-linked globin promoter to activate mRNA synthesis from the target gene. An insulator inserted in the intervening DNA between the enhancer and the promoter traps the enhancer DNA and the associated pol II and TBP at the insulator site, blocking mid-stream the facilitated tracking and transcription mechanism of the enhancer complex, thereby blocking long-range enhancer function.
Nucleic Acids Research 02/2007; 35(16):5532-44. · 8.03 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: The solitary ERV-9 long terminal repeat (LTR) located upstream of the HS5 site in the human beta-globin locus control region exhibits prominent enhancer activity in embryonic and erythroid cells. The LTR enhancer contains 14 tandemly repeated subunits with recurrent CCAAT, GTGGGGA, and GATA motifs. Here we showed that in erythroid K562 cells these DNA motifs bound the following three transcription factors: ubiquitous NF-Y and hematopoietic MZF1 and GATA-2. These factors and their target DNA motifs exhibited a hierarchy of DNA/protein and protein/protein binding affinities: NF-Y/CCAAT > NF-Y/GATA-2 > NF-Y/MZF1 > MZF1/GTGGGGA; GATA-2/GATA. Through protein/protein interactions, NF-Y bound at the CCAAT motif recruited MZF1 and GATA-2, but not Sp1 and GATA-1, and stabilized their binding to the neighboring GTGGGGA and GATA sites to assemble a novel LTR enhancer complex, NF-Y/MZF1/GATA-2. In the LTR-HS5-epsilonp-GFP plasmid integrated into K562 cells, mutation of the CCAAT motif in the LTR enhancer to abolish NF-Y binding inactivated the enhancer, closed down the chromatin structure of the epsilon-globin promoter, and silenced transcription of the green fluorescent protein gene. The results indicated that NF-Y bound at the CCAAT motifs assembled a robust LTR enhancer complex, which could act over the intervening DNA to remodel the chromatin structure and to stimulate the transcription of the downstream gene locus.
Journal of Biological Chemistry 11/2005; 280(42):35184-94. · 4.77 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: EMSA and footprinting analyses have revealed that the -489-- -414 bp and the -390-- -345 bp (designated DC and PC respectively) upstream of the Aspergillus nigerT21 glaA gene were bound by one protein factor in the A. nigerT21 whole cell extract. Both DC and PC contained CCAAT pentanucleotides. The functions of DC and PC in regulation of expression of glucoamylase (GLA) were studied. CCAAT pentanucleotides were replaced with CGTAA and the mutated DNA fragments DCm and PCm lost the binding activities of protein factors in vitro. In vivo when either DC or PC was mutated or the relative orientations between them were changed on the PglaA, the transcriptional activity of PglaA decreased to a basal level. Introduction of multi-copies of DC into the original site at the PglaA in A. nigerT21 decreased the expression of endogenous GLA expression and the exogenous reporter E. coli uidA gene introduced under the PglaA promoter, while having no effect on the uidA gene under the control of PgpdA. EMSA revealed that the levels of the specific DNA-binding protein factors in the transformants maintained the same meaning that introduction of multi-copies of DC caused the titration effect. AnghapC gene was cloned from A. nigerT21 cDNA and introduced into the DC multi-copied strains. The expression of AnghapC improved the expression of the endogenous GLA and the exogenous gene controlled by PglaA. These results showed that both the CCAAT pentanucleotides were necessary for DC and PC binding to the protein factors, and the simultaneous binding of DC and PC to the protein was necessary for promoting the transcriptional activity of PglaA. AngHapC was the specific positive trans-acting protein factor binding to DC.
Science in China Series C Life Sciences 05/2004; 47(2):139-47. · 1.61 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: Electromobility shift assay (EMSA) was used to scan 600 bp of 5' cis regulatory sequence of Aspergillus niger (A. niger) T21 glucoamylase gene (glaA) for binding by partially fractionated T21 protein extracted from starchinduced mycelia. In this process, one protein, AngCP, was detected to bind specifically to three regions covering -374 to -344, -484 to -414 and -580 to -540 relative to the glaA translational start codon. UV-crosslinking of DNA-protein complex showed that MW of AngCP was 10 ku. DNase I footprinting analysis demonstrated that AngCP specifically binds to two CCAAT containing sequences within the regions between -374 and -344 and -484 and -414 bp. And the region between -580 and -540 bp contains CCAAT similar box, CCTAT. The results indicated that AngCP is probably one of the members of CCAAT-binding protein families, which are generally involved in enhancement of gene expression in filamentous fungi. These findings suggested that AngCP should be a transcription activator for high-level expression of glaA gene.
Science in China Series C Life Sciences 11/2002; 45(5):527-37. · 1.61 Impact Factor
