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Fig. 5. General-activators of Hb9 in non-motoneurons. (A) Fragments of the
mouse Hb9 promoter were linked to the reporter nuclear lacZ
or GFP. (B-H) DNA constructs were electroporated into chick embryos and
reporter expression was detected using immunocytochemistry at HH stage 24. (B)
The 9.2 kb promoter of Hb9 drives motoneuron-specific gene
expression. (C) Deletion of the distal 5.6 kb BamHI fragment impairs
the motoneuron-specific activity leading to widespread ectopic reporter
expression. (D,E) Likewise, large 5' deletions to the NheI and
HindIII sites leaving only 1386 and 550 nucleotides of Hb9
sequences, respectively, continued to promote widespread ectopic reporter
expression. (F-H) The 2.5 kb distal element (–8129 to –5575) was
linked to three different proximal fragments of Hb9 as indicated in
A. 2.5 distal + 
NheI or HindIIII drove high
levels of GFP expression in motoneurons, but 2.5 distal + NI-HIII was less
active, suggesting the –550 proximal segment of Hb9 is necessary for
enhancer function. (I) General activators are predicted to interact with the
proximal (prox.) region of Hb9 and promote widespread transcription
of the gene. The enhancer of Hb9 (MNE) in
combination with general-activators drive motoneuron-specific expression.
Deletion of the proximal segment of Hb9 disrupts enhancer function.
(J) The nucleotide sequence of the proximal 550 nucleotide region of
Hb9 from mouse and human are aligned. The conserved sequences in both
species are marked in bold, and potential binding sites for activators are
boxed and labeled. This sequence lacks an obvious TATA box motif. The longest
cDNA extends to –195 in mouse Hb9 (–181 in human).
Because the precise start of transcription has not been mapped, we use the
start of translation as the reference point for the coordinates shown with the
sequences (see Materials and methods). (K) CV1 cells were transfected with
increasing concentrations of plasmids encoding E2F1 and/or Sp1 together with a
luciferase reporter linked to Hb9 (2.5
distal+NheI).
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