Recognition of DNase I hypersensitive sites in multiple cell lines

2009 ◽  
Vol 5 (4) ◽  
pp. 378 ◽  
Author(s):  
Wei Chen ◽  
Liaofu Luo ◽  
Lirong Zhang ◽  
Hao Lin
Keyword(s):  
Cell Lines ◽  
Dnase I ◽  
Dnase I Hypersensitive Sites ◽  
Hypersensitive Sites ◽  
Multiple Cell

scholarly journals Further characterization of cis-acting regulatory sequences in the genomic locus of the murine erythropoietin receptor: evidence for stage- specific regulation

Blood ◽  
1994 ◽  
Vol 83 (5) ◽  
pp. 1428-1435 ◽  
Author(s):  
H Youssoufian
Keyword(s):  
Cell Lines ◽  
Tissue Specificity ◽  
Specific Activity ◽  
Dnase I ◽  
Erythropoietin Receptor ◽  
Erythroid Cell ◽  
Regulatory Sequences ◽  
Dnase I Hypersensitive Sites ◽  
Hypersensitive Sites ◽  
Murine Erythroleukemia

Abstract Expression of the murine erythropoietin receptor (EpoR) gene was investigated in progenitor cell lines representing distinct stages of hematopoietic differentiation. In murine erythroid cell lines, the EpoR mRNA level was fivefold higher in the more mature murine erythroleukemia (MEL) cells than in CB-5 cells and very low in granulocyte/macrophage-like FDC-P1 cells. GATA-1 mRNA was present in equivalent levels in both erythroid cell lines, but at a low level in FDC-P1 cells. To account for the elevated levels of EpoR mRNA, the activity of the promoter and expression of DNase I hypersensitive sites were assessed as markers of transcriptional activity in various cell lines. Among a series of 5′ flanking restriction fragments linked to a reporter gene, a 83-bp fragment that includes binding sites for the transcription factors GATA-1 and Sp-1 gave low levels of erythroid- specific activity, and a 256-bp fragment that includes, in addition, two sites for the putative CACCC-binding protein gave the highest level of erythroid-specific transcription. DNase I footprinting showed binding of a constitutive factor to the proximal CACCC-binding site, and deletion or mutation of this site significantly reduced the overall expression while maintaining tissue-specificity. Three DNase I hypersensitive sites were detected in the 5′ flanking region of the EpoR gene, two of which were unique to MEL cells. These sites were situated over the promoter region and approximately 0.5 kb and 2.4 kb upstream of the transcriptional initiation sites. A 0.8-kb restriction fragment spanning the distal site caused approximately a four-fold rise in transcription from the endogenous or a heterologous promoter in MEL cells independent of its orientation and up to 1.5-fold rise in CB-5 cells, but it was inactive in COS-1 cells that were cotransfected with an expression plasmid encoding GATA-1. These results show that (1) basal activity as well as tissue specificity of the EpoR promoter can be accounted for by its interaction with GATA-1, and (2) upstream sites regulate the strength of the promoter. Expression of the distal DNase I hypersensitive site and the corresponding enhancer activity in MEL cells suggests a role for this element in stage-specific transcriptional control.

Albumin and alpha-fetoprotein gene transcription in rat hepatoma cell lines is correlated with specific DNA hypomethylation and altered chromatin structure in the 5' region

1987 ◽  
Vol 7 (5) ◽  
pp. 1856-1864
Author(s):  
I Tratner ◽  
J L Nahon ◽  
J M Sala-Trepat ◽  
A Venetianer
Keyword(s):  
Cell Lines ◽  
Chromatin Structure ◽  
Hepatoma Cell ◽  
Dnase I ◽  
Alpha Fetoprotein ◽  
Hepatoma Cell Lines ◽  
Dnase I Hypersensitive Sites ◽  
Albumin Gene ◽  
Hypersensitive Sites ◽  
Rat Hepatoma

We examined DNA methylation and DNase I hypersensitivity of the alpha-fetoprotein (AFP) and albumin gene region in hepatoma cell lines which showed drastic differences in the level of expression of these genes. We assayed for methylation of the CCGG sequences by using the restriction enzyme isoschizomers HpaII and MspI. We found two methylation sites located in the 5' region of the AFP gene and one in exon 1 of the albumin gene for which hypomethylation is correlated with gene expression. Another such site, located about 4,000 base pairs upstream from the AFP gene, seems to be correlated with the tissue specificity of the cells. DNase I-hypersensitive sites were mapped by using the indirect end-labeling technique with cloned genomic DNA probes. Three tissue-specific DNase I-hypersensitive sites were mapped in the 5' flanking region of the AFP gene when this gene was transcribed. Similarly, three tissue-specific DNase I-hypersensitive sites were detected upstream from the albumin gene in producing cell lines. In both cases, the most distal sites were maintained after cessation of gene activity and appear to be correlated with the potential expression of the gene. Interestingly, specific methylation sites are localized in the same DNA region as DNase I hypersensitive sites. This suggests that specific alterations of chromatin structure and changes in methylation pattern occur in specific critical regulatory regions upstream from the albumin and AFP genes in rat hepatoma cell lines.

scholarly journals Albumin and alpha-fetoprotein gene transcription in rat hepatoma cell lines is correlated with specific DNA hypomethylation and altered chromatin structure in the 5' region.

1987 ◽  
Vol 7 (5) ◽  
pp. 1856-1864 ◽  
Author(s):  
I Tratner ◽  
J L Nahon ◽  
J M Sala-Trepat ◽  
A Venetianer
Keyword(s):  
Cell Lines ◽  
Chromatin Structure ◽  
Hepatoma Cell ◽  
Dnase I ◽  
Alpha Fetoprotein ◽  
Hepatoma Cell Lines ◽  
Dnase I Hypersensitive Sites ◽  
Albumin Gene ◽  
Hypersensitive Sites ◽  
Rat Hepatoma

We examined DNA methylation and DNase I hypersensitivity of the alpha-fetoprotein (AFP) and albumin gene region in hepatoma cell lines which showed drastic differences in the level of expression of these genes. We assayed for methylation of the CCGG sequences by using the restriction enzyme isoschizomers HpaII and MspI. We found two methylation sites located in the 5' region of the AFP gene and one in exon 1 of the albumin gene for which hypomethylation is correlated with gene expression. Another such site, located about 4,000 base pairs upstream from the AFP gene, seems to be correlated with the tissue specificity of the cells. DNase I-hypersensitive sites were mapped by using the indirect end-labeling technique with cloned genomic DNA probes. Three tissue-specific DNase I-hypersensitive sites were mapped in the 5' flanking region of the AFP gene when this gene was transcribed. Similarly, three tissue-specific DNase I-hypersensitive sites were detected upstream from the albumin gene in producing cell lines. In both cases, the most distal sites were maintained after cessation of gene activity and appear to be correlated with the potential expression of the gene. Interestingly, specific methylation sites are localized in the same DNA region as DNase I hypersensitive sites. This suggests that specific alterations of chromatin structure and changes in methylation pattern occur in specific critical regulatory regions upstream from the albumin and AFP genes in rat hepatoma cell lines.

scholarly journals Further characterization of cis-acting regulatory sequences in the genomic locus of the murine erythropoietin receptor: evidence for stage- specific regulation

Blood ◽  
1994 ◽  
Vol 83 (5) ◽  
pp. 1428-1435
Author(s):  
H Youssoufian
Keyword(s):  
Cell Lines ◽  
Tissue Specificity ◽  
Specific Activity ◽  
Dnase I ◽  
Erythropoietin Receptor ◽  
Erythroid Cell ◽  
Regulatory Sequences ◽  
Dnase I Hypersensitive Sites ◽  
Hypersensitive Sites ◽  
Murine Erythroleukemia

Expression of the murine erythropoietin receptor (EpoR) gene was investigated in progenitor cell lines representing distinct stages of hematopoietic differentiation. In murine erythroid cell lines, the EpoR mRNA level was fivefold higher in the more mature murine erythroleukemia (MEL) cells than in CB-5 cells and very low in granulocyte/macrophage-like FDC-P1 cells. GATA-1 mRNA was present in equivalent levels in both erythroid cell lines, but at a low level in FDC-P1 cells. To account for the elevated levels of EpoR mRNA, the activity of the promoter and expression of DNase I hypersensitive sites were assessed as markers of transcriptional activity in various cell lines. Among a series of 5′ flanking restriction fragments linked to a reporter gene, a 83-bp fragment that includes binding sites for the transcription factors GATA-1 and Sp-1 gave low levels of erythroid- specific activity, and a 256-bp fragment that includes, in addition, two sites for the putative CACCC-binding protein gave the highest level of erythroid-specific transcription. DNase I footprinting showed binding of a constitutive factor to the proximal CACCC-binding site, and deletion or mutation of this site significantly reduced the overall expression while maintaining tissue-specificity. Three DNase I hypersensitive sites were detected in the 5′ flanking region of the EpoR gene, two of which were unique to MEL cells. These sites were situated over the promoter region and approximately 0.5 kb and 2.4 kb upstream of the transcriptional initiation sites. A 0.8-kb restriction fragment spanning the distal site caused approximately a four-fold rise in transcription from the endogenous or a heterologous promoter in MEL cells independent of its orientation and up to 1.5-fold rise in CB-5 cells, but it was inactive in COS-1 cells that were cotransfected with an expression plasmid encoding GATA-1. These results show that (1) basal activity as well as tissue specificity of the EpoR promoter can be accounted for by its interaction with GATA-1, and (2) upstream sites regulate the strength of the promoter. Expression of the distal DNase I hypersensitive site and the corresponding enhancer activity in MEL cells suggests a role for this element in stage-specific transcriptional control.

scholarly journals Distinct DNase-I hypersensitive sites are associated with TAL-1 transcription in erythroid and T-cell lines

Blood ◽  
1994 ◽  
Vol 84 (11) ◽  
pp. 3819-3827 ◽  
Author(s):  
K Leroy-Viard ◽  
MA Vinit ◽  
N Lecointe ◽  
D Mathieu-Mahul ◽  
PH Romeo
Keyword(s):  
T Cell ◽  
Cell Line ◽  
Cell Lines ◽  
Cpg Island ◽  
Regulatory Elements ◽  
Dnase I ◽  
Dnase I Hypersensitive Sites ◽  
Hypersensitive Sites ◽  
T Cell Lines ◽  
Megakaryocytic Cell

The tal-1 gene, frequently activated in human T-cell acute lymphoblastic leukemia (T-ALL), is expressed in the erythroid, megakaryocytic, and mast cell lineages during normal hematopoiesis. To gain further insight into the molecular mechanisms that control tal-1 expression, we investigated tal-1 chromatin structure in erythroid/megakaryocytic cell lines and in T-cell lines either with or without tal-1 rearrangements. Tal-1 transcription was shown to be monoallelic in Jurkat, a T-cell line that expresses tal-1 in the absence of apparent genomic alteration of the locus. Methylation studies indicated that the tal-152 GC-rich region behaves like a CpG island, hypomethylated in normal cells, and methylated de novo on transcriptionally inactive alleles in established cell lines. Five major DNase-I hypersensitive sites (HS) were mapped in the tal-1 locus. HS I, IV, and V were exclusively observed in the erythroid/megakaryocytic cell lines that express tal-1 from the promoters 1a and 1b. HS II was weak in hematopoietic cell lines, absent in Hela, and greatly enhanced in Jurkat, suggesting that this region might be implicated in the cis-activation of tal-1 promoter 1b in this cell line. HS III was weak in HEL and Jurkat, and greatly enhanced in DU528, a T-cell line that bears a t (1;14) and initiates tal-1 transcription within exon 4. These results suggest that distinct regulatory elements are associated with the use of the different tal-1 promoters.

Functional Characterization of the BCL11B Breakpoint Region Reveals Multiple Enhancers at Dnase-I Hypersensitive Sites Some of Which Control NK-Family Homeobox Genes in T-ALL Cells with t(5;14)(q35;q32).

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 845-845
Author(s):  
Stefan Nagel ◽  
Michaela Scherr ◽  
Klaus Hornischer ◽  
Alexander Kel ◽  
Maren Kaufmann ◽  
...  
Keyword(s):  
T Cells ◽  
Transcription Factor ◽  
Cell Lines ◽  
Lymphoblastic Leukemia ◽  
Functional Characterization ◽  
Homeobox Genes ◽  
Dnase I ◽  
Dependent Manner ◽  
Dnase I Hypersensitive Sites ◽  
Hypersensitive Sites

Abstract The zinc-finger transcription factor BCL11B (14q32) is a key translocation target in pediatric T-cell acute lymphoblastic leukemia (T-ALL) where it ectopically activates NK-family homeobox genes, chiefly TLX3, or NKX2-5, via the recurrent t(5;14)(q35;q32). However, the mechanism underlying leukemic activation by BCL11B remains obscure. Breakpoints at 14q32 are dispersed over about 1.2 Mbp downstream of BCL11B amid a non-coding genomic “desert”. Detailed cytogenetic analysis of T-ALL cell lines, including one carrying a complex double-insertion enabled the putative breakpoint target zone to be narrowed to ~300 kbp covering the central and distal parts of the 3′-BCL11B region. Interestingly, Crawford et al. (PNAS101: 992–7, 2004) have identified a series of DNase-I hypersensitive sites (DHS) in T-cells amid this genomic desert including a unique cluster near the most distal breakpoint to serve as candidate BCL11B transcriptional enhancers. Accordingly, we used the TRANSFAC database to identify multiple transcription factor (TF) binding site sequences nearby these DHS. To investigate the role of DHS in activating homeobox transcription in T-ALL, we designed double-stranded 26-mer oligos matching each of 9 DHS which were used to transfect a T-ALL cell line (PEER) in which NKX2-5 is inserted about 1 Mbp downstream of BCL11B and thereby activated. Such oligos may inhibit TF binding by directly blocking access to DHS or act as decoys to sequester TF. We found that oligos targeting DHS in the central and distal regions of 3′-BCL11B, close to the insertion junctions in PEER cells, were effective in down-regulating NKX2-5 transcription in a dose dependent manner. In contrast, oligos targeting DHS near 3′-BCL11B were able to down-regulate BCL11B but not NKX2-5. Treatments with neither mutated DHS oligos, nor other control oligos caused inhibition. Transcription of ESTs present at 3′-BCL11B was unaffected by DHS-oligo transfection indicating specific targeting of BCL11B or NKX2-5 by their respective DHS. Analysis of t(5;14) T-ALL cell lines by Halo-FISH, showed the selective homing of 3′-BCL11B chromatin to the nuclear matrix when juxtaposed with NKX2-5, whereas 3′-BCL11B chromatin present on non-participant chromosomes was extruded to surrounding halos inimical to transcription. Taken together, our data show the presence of multiple DHS covering the ~1.2 Mbp breakpoint dispersal region at 3′-BCL11B in T-ALL cells which appear to serve as enhancers of BCL11B or nearby translocated NK-family homeobox genes. These multiple enhancers may be required for the precise timing of BCL11B transcription which is critical to thymocyte development - a question which will be addressed by planned future studies. As well as revealing novel aspects underlying the transcriptional control of BCL11B in T-cells, our data highlight a potential therapeutic target in leukemia within “junk DNA”.

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