Nj. Wandersee et al., INTRONIC AND FLANKING SEQUENCES ARE REQUIRED TO SILENCE ENHANCEMENT OF AN EMBRYONIC BETA-TYPE GLOBIN GENE, Molecular and cellular biology, 16(1), 1996, pp. 236-246
In the course of studying regulatory elements that affect avian embryo
nic rho-globin gene expression, the multipotential hematopoietic cell
line K562 was transiently transfected with various rho-globin gene con
structs containing or lacking an avian erythroid enhancer element. Enh
anced levels of rho gene expression were seen from those constructs co
ntaining an enhancer element and minimal 5' or 3' flanking rho sequenc
es but were not seen from enhancer-containing constructs that included
extensive 5' and 3' flanking sequences. Deletion analysis localized 5
' and 3' ''enhancer-silencing elements'' to -2140 to -2000 and +1865 t
o +2180 relative to the mRNA cap site. A third element required for en
hancer silencing was identified within the second intron of the rho ge
ne. The treatment of K562 cells with hemin, which induces erythroid di
fferentiation, partially alleviated the enhancer-silencing effect. The
silencer elements were able to block enhancement from a murine erythr
oid enhancer, but not from a nonerythroid enhancer. Electrophoretic mo
bility shift assays demonstrated that the transcription factor YY1 is
able to bind both the 5' and 3' enhancer silencer elements; a point mu
tation of the single overlapping YY1/NF-Y binding site in the 3' eleme
nt completely abolished the enhancer-silencing effect. These results d
emonstrate a complex enhancer silencer that requires 5' flanking, intr
onic, and 3' flanking sequences for a single regulatory effect on a eu
karyotic gene.