Prox1 directly regulates the genes encoding the structural proteins
α-actinin, N-RAP and zyxin. (A) The sarcomere and
sarcomere-related protein genes Actn2 (sarcomeric α-actinin),
Nrap and Zyx were identified as potential downstream targets
of Prox1 by ChIP-on-chip. For each locus, the genomic region
immunoprecipitated by anti-Prox1 is indicated by a yellow box, the closest
gene is labelled and the degree of conservation shown. Conservation patterns
are based on phastCons scores
(http://genome.ucsc.edu).
(B) EMSAs with in vitro translated (IVT) Prox1 and
32P-labelled oligonucleotides (60 bp) identified from each of the
Actn2, Nrap and Zyx putative Prox1-bound elements (see Fig.
S9 in the supplementary material) isolated via the ChIP-on-chip shown in A. A
10-fold excess of unlabelled oligonucleotide was used in competitive assays as
evidence of specific binding (lanes C). (C) EMSAs with nuclear extracts
from mouse P19Cl6 cell lysates either untransfected (lanes 1-3) or transfected
with Flag-Prox1 (lanes 4-6) and 32P-labelled elements as in B.
Lanes 1 and 4 are lysate alone, lanes 2 and 5 are lysate plus an anti-Flag
antibody, and lanes 3 and 6 are anti-Flag-alone controls. Note the evidence of
a supershift in lane 5 compared with lane 4 for each of the Actn2,
Nrap and Zyx elements (arrowheads), which is indicative of
specific binding by Flag-Prox1. The presence of a comparatively weak band in
lanes 1 and 2 in each case represents binding by endogenous Prox1, which is
expressed in P19Cl6 cells (data not shown). (D) In vitro transcription
assays demonstrate Prox1 transactivation of a luciferase reporter downstream
of the Actn2, Nrap and Zyx putative Prox1-binding elements
and minimal reporter. Note the significant activation by Prox1 of the
Actn2, Nrap and Zyx reporters. (E) qRT-PCR for
Nrap and Zyx confirms reduced expression of these factors in
a Prox1-deficient background, as was previously determined for
Actn2 (see Fig. 3O).
In D,E, data are presented as mean ± s.e.m.;
*P<0.05, **P<0.001,
***P<0.003,
****P<9×10-7.