Functional sites in HS2 of the beta-globin LCR
Information on in vivo footprints and
site-directed mutations tested in transgenic mice and transfected cells
were combined to establish a set of experimentally-verified functional
sites in HS2 of the beta-globin locus control region (LCR). Additional
information on binding sites was also brought into the assignments. If
nucleotides identified as important by in vivo
footprints are part of a binding site for a protein, or
class of proteins, implicated as binding at that site (e.g. by
in vitro binding assays or mutagenesis), then the
entire consensus binding site for that protein was
included in the list of functional sites.
Region examined: 11240 11510
The functional nucleotides are:
- 11276 11304 non-conserved CCCGCC
- 11328 11401 5' to MARES, MAREs, conserved CACC, E8701, Bresnick site
- 11408 11413 non-consensus GATA
- 11416 11422 conserved "GATA" CTATCT
- 11449 11454 E8761
- 11474 11484 E box/USF (restrict to USF site + in vivo footprint)
The coordinates are for the sequence file starting 5' to HS5.
Coordinates in the GenBank file HUMHBB can be obtained by subtracting
2687.
References for in vivo footprints in HS2:
Data from Reddy and Shen 1991 (human), Ikuta and Kan 1991
(human), Reddy and Shen 1993 (mouse), Reddy et al 1994.
All data from Figures are included, even if the positions were not
mentioned in the text (this is particularly true for some data from
Reddy and Shen 1993 for the conserved CACC box and the E box).
Interestingly, some of these regions show up as results in Reddy et al.
1994.
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Ikuta, T., and Kan, Y. W. (1991). In vivo protein-DNA
interactions at the b-globin locus. Proc. Natl. Acad. Sci., USA
88, 10188-10192.
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Reddy, P. M. S., and Shen, C.-K. J. (1991). Protein-DNA interactions
in vivo of an erythroid-specific, human b-globin
locus enhancer. Proc. Natl. Acad. Sci., USA 88,
8676-8680.
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Reddy, P. M. S., and Shen, C.-K. J. (1993). Erythroid differentiation
of mouse erythroleukemia cells results in the reorganization of
protein-DNA complexes in the mouse b-globin promoter but not its distal
enhancer. Mol. Cell. Biol. 13, 1093-1103.
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Reddy, P. M. S., Stamatoyannopoulos, G., Papayannopoulou, T., and Shen,
C.-K. J. (1994). Genomic footprinting and sequencing of human
b-globin locus: Tissue specificity and cell line artifact. J. Biol. Chem.
269, 8287-8295.
References for mutagenesis studies:
Data are from Caterina et al. 1994 (transgenic mice), Lam
and Bresnick 1996, Elnitski et al. 1997 (transiently transfected K562
cells). Many other papers contain mutational analysis, but these three
papers implicate a fairly long region that covers the sites mutated by
lots of labs.
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Lam, L., and Bresnick, E. H. (1996). A novel DNA binding protein,
HS2NF5, interacts with a functionally important sequence of the human
b-globin locus control region. J. Biol. Chem. 271,
32421-32429.
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Caterina, J. J., Ciavatta, D. J., Donze, D., Behringer, R. R., and
Townes, T. M. (1994). Multiple elements in human b-globin
locus control region 5' HS2 are involved in enhancer activity and
position-independent transgene expression. Nucl. Acids Res.
22, 1006-1011.
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Elnitski, L., Miller, W., and Hardison, R. (1997). Conserved E boxes
function as part of the enhancer in hypersensitive site 2 of the
b-globin locus control region: Role of basic helix-loop-helix proteins.
J. Biol. Chem. 272, 369-378.