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5'HS5 of the human beta-globin locus control region is dispensable for the formation of the beta-globin active chromatin hub.

Chan PK, Wai A, Philipsen S, Tan-Un KC - PLoS ONE (2008)

Bottom Line: The results show that when 5'HS5 is deleted from the locus, both remote and internal regulatory elements are still able to interact with each other in a three-dimensional configuration termed the Active Chromatin Hub.Thus, binding of CTCF to 5'HS5 per se does not render it a functional border element.This is consistent with the previous data suggesting that CTCF has dual functionality.

View Article: PubMed Central - PubMed

Affiliation: Department of Zoology, Kadoorie Biological Science Building, The University of Hong Kong, Hong Kong Special Administrative Region (SAR), China.

ABSTRACT
Hypersensitive site 5 (5'HS5) of the beta-globin Locus Control Region functions as a developmental stage-specific border in erythroid cells. Here, we have analyzed the role of 5'HS5 in the three dimensional organization of the beta-gene locus using the Chromatin Conformation Capture (3C) technique. The results show that when 5'HS5 is deleted from the locus, both remote and internal regulatory elements are still able to interact with each other in a three-dimensional configuration termed the Active Chromatin Hub. Thus, the absence of 5'HS5 does not have an appreciable effect on the three dimensional organization of the beta-globin locus. This rules out models in which 5'HS5 nucleates interactions with remote and/or internal regulatory elements. We also determined the binding of CTCF, the only defined insulator protein in mammalian cells, to 5'HS5 by using chromatin immunoprecipitation (ChIP) assays. We detect low levels of CTCF binding to 5'HS5 in primitive erythroid cells, in which it functions as a border element. Surprisingly, we also observe binding levels of CTCF to 5'HS5 in definitive erythroid cells. Thus, binding of CTCF to 5'HS5 per se does not render it a functional border element. This is consistent with the previous data suggesting that CTCF has dual functionality.

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3C analysis of the PAC1B and PACΔ1B transgenic lines using a primer from the human β-globin gene in combination with primers from other parts of the locus.Fetal livers were collected from E14.5 embryos for this set of 3C experiments. (A) Representative examples of the PCR fragments resulting from the 3C experiments. (B) Histogram of the relative crosslinking efficiencies after quantitation and normalization. The histograms are the average of at least three separate experiments, with each PCR performed in triplicate.
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pone-0002134-g003: 3C analysis of the PAC1B and PACΔ1B transgenic lines using a primer from the human β-globin gene in combination with primers from other parts of the locus.Fetal livers were collected from E14.5 embryos for this set of 3C experiments. (A) Representative examples of the PCR fragments resulting from the 3C experiments. (B) Histogram of the relative crosslinking efficiencies after quantitation and normalization. The histograms are the average of at least three separate experiments, with each PCR performed in triplicate.

Mentions: To examine the possible consequences of deletion of 5′HS5 on ACH formation, we compared the 3D structures of the PAC1B and PACΔ1B transgenes in definitive erythroid cells isolated from 14.5 dpc fetal livers with the 3C technique (Fig. 3). The results show that the overall 3D of the β-globin locus has remained the same when 5′HS5 is deleted (PACΔ1B). However, the relative crosslinking frequency of the 5′HS6, the distal hypersensitive site located about 6 kb 5′ upstream to the HS5 [19], with the β-globin gene is increased by upon the removal of 5′HS5 (Fig. 3B, primer 418). This increase in association frequency with the ACH is presumably due to the closer proximity of 5′HS6 to the LCR after Cre-mediated excision of 5′HS5. 5′HS6 is located 800bp closer to the LCR in the PACΔ1B line. Besides, in the absent of HS5, the β-gene promoter interacts strongly with the A-γ promoter.


5'HS5 of the human beta-globin locus control region is dispensable for the formation of the beta-globin active chromatin hub.

Chan PK, Wai A, Philipsen S, Tan-Un KC - PLoS ONE (2008)

3C analysis of the PAC1B and PACΔ1B transgenic lines using a primer from the human β-globin gene in combination with primers from other parts of the locus.Fetal livers were collected from E14.5 embryos for this set of 3C experiments. (A) Representative examples of the PCR fragments resulting from the 3C experiments. (B) Histogram of the relative crosslinking efficiencies after quantitation and normalization. The histograms are the average of at least three separate experiments, with each PCR performed in triplicate.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2358975&req=5

pone-0002134-g003: 3C analysis of the PAC1B and PACΔ1B transgenic lines using a primer from the human β-globin gene in combination with primers from other parts of the locus.Fetal livers were collected from E14.5 embryos for this set of 3C experiments. (A) Representative examples of the PCR fragments resulting from the 3C experiments. (B) Histogram of the relative crosslinking efficiencies after quantitation and normalization. The histograms are the average of at least three separate experiments, with each PCR performed in triplicate.
Mentions: To examine the possible consequences of deletion of 5′HS5 on ACH formation, we compared the 3D structures of the PAC1B and PACΔ1B transgenes in definitive erythroid cells isolated from 14.5 dpc fetal livers with the 3C technique (Fig. 3). The results show that the overall 3D of the β-globin locus has remained the same when 5′HS5 is deleted (PACΔ1B). However, the relative crosslinking frequency of the 5′HS6, the distal hypersensitive site located about 6 kb 5′ upstream to the HS5 [19], with the β-globin gene is increased by upon the removal of 5′HS5 (Fig. 3B, primer 418). This increase in association frequency with the ACH is presumably due to the closer proximity of 5′HS6 to the LCR after Cre-mediated excision of 5′HS5. 5′HS6 is located 800bp closer to the LCR in the PACΔ1B line. Besides, in the absent of HS5, the β-gene promoter interacts strongly with the A-γ promoter.

Bottom Line: The results show that when 5'HS5 is deleted from the locus, both remote and internal regulatory elements are still able to interact with each other in a three-dimensional configuration termed the Active Chromatin Hub.Thus, binding of CTCF to 5'HS5 per se does not render it a functional border element.This is consistent with the previous data suggesting that CTCF has dual functionality.

View Article: PubMed Central - PubMed

Affiliation: Department of Zoology, Kadoorie Biological Science Building, The University of Hong Kong, Hong Kong Special Administrative Region (SAR), China.

ABSTRACT
Hypersensitive site 5 (5'HS5) of the beta-globin Locus Control Region functions as a developmental stage-specific border in erythroid cells. Here, we have analyzed the role of 5'HS5 in the three dimensional organization of the beta-gene locus using the Chromatin Conformation Capture (3C) technique. The results show that when 5'HS5 is deleted from the locus, both remote and internal regulatory elements are still able to interact with each other in a three-dimensional configuration termed the Active Chromatin Hub. Thus, the absence of 5'HS5 does not have an appreciable effect on the three dimensional organization of the beta-globin locus. This rules out models in which 5'HS5 nucleates interactions with remote and/or internal regulatory elements. We also determined the binding of CTCF, the only defined insulator protein in mammalian cells, to 5'HS5 by using chromatin immunoprecipitation (ChIP) assays. We detect low levels of CTCF binding to 5'HS5 in primitive erythroid cells, in which it functions as a border element. Surprisingly, we also observe binding levels of CTCF to 5'HS5 in definitive erythroid cells. Thus, binding of CTCF to 5'HS5 per se does not render it a functional border element. This is consistent with the previous data suggesting that CTCF has dual functionality.

Show MeSH
Related in: MedlinePlus