TRANSCRIPTIONAL REGULATION OF THE HUMAN NONMUSCLE MYOSIN-II HEAVY CHAIN-A GENE - IDENTIFICATION OF 3 CLUSTERED CIS-ELEMENTS IN INTRON-1 WHICH MODULATE TRANSCRIPTION IN A CELL TYPE-DEPENDENT AND DIFFERENTIATIONSTATE-DEPENDENT MANNER
N. Beohar et S. Kawamoto, TRANSCRIPTIONAL REGULATION OF THE HUMAN NONMUSCLE MYOSIN-II HEAVY CHAIN-A GENE - IDENTIFICATION OF 3 CLUSTERED CIS-ELEMENTS IN INTRON-1 WHICH MODULATE TRANSCRIPTION IN A CELL TYPE-DEPENDENT AND DIFFERENTIATIONSTATE-DEPENDENT MANNER, The Journal of biological chemistry, 273(15), 1998, pp. 9168-9178
In an attempt to identify cis-acting elements for transcriptional regu
lation of the human nonmuscle myosin II heavy chain (MHC)-A gene, the
region extending 20 kilobases (kb) upstream and 40 kb downstream from
the transcription start sites, which includes the entire 37-kb intron
1, was examined. Using transient transfection analysis of luciferase r
eporter constructs, a 100-base pair (bp) region (N2d) in intron 1, loc
ated 23 kb downstream from the transcriptional start sites, has been f
ound to activate transcription in a cell type-and differentiation stat
e-dependent manner, Maximum activity (similar to 20-fold) is seen in N
IH 3T3 fibroblasts and intermediate activity (7-fold) in proliferating
and undifferentiated C2C12 myoblasts, In contrast, this region is alm
ost inactive in terminally differentiated C2C12 myotubes, in which end
ogenous nonmuscle MHC-A expression is down-regulated, Gel mobility shi
ft assays and methylation interference analyses were performed using N
IH 3T3 nuclear extracts to determine the protein-binding elements for
transcription factors, Three binding elements have been identified wit
hin the N2d region, Antibody-supershift experiments, as well as compet
ition experiments using consensus binding sequences for specific trans
cription factors, revealed that the most 5'-element, C (GGGAGGGGCC) is
recognized specifically and exclusively by Sp1 and Sp3 transcriptiona
l factors, Element C is immediately followed by a novel element, A (GT
GACCC), A third element, F (GTGTCAGGTG), which contains an E-box, is l
ocated 50 bp 3' to element A. Element F can be recognized partially by
upstream stimulatory factors, USF1 and/or USF2, Transfection studies
with luciferase reporter constructs which include mutations in all thr
ee elements in various combinations demonstrate that the A and C bindi
ng factors cooperatively activate transcriptional activity in NIH 3T3
cells, The F binding factor shows an additive effect on transcription.