z********i
发帖数: 610 | 1 通过array找到了一个Notch的target gene，在细胞里面验证了，NICD下调，dnMAML上
调，我们猜想Hes1参与了调控，Hes1 cell line也验证了一下。然后克隆了target
gene promoter，预测发现了一个非常保守的Hes1 binding site，但是这个结果是WT
Hes1上调promoter，dnHes1不调节promoter activity，非常奇怪的结果。
想问一下Notch方面的专家，Hes1能直接上调gene 表达吗？
多谢了 | r*******y
发帖数: 48 | 2 Although more info regarding your experiments is needed, but basically,
1. it is possible that WT hes1 activated promoter but dnHes1 (dominant
negative?) did not; it only suggests that the endogenous Hes1 in the cell
line you tested was not invoved in maintaining the basal activity of that
promoter. Actually, it is not a bad observation; it suggests that hes1
activated this promoter via DNA binding, becaseu usually dnHes should have
no or little DNA binding activity.
2. However, if you test both Wt and dnHes1 on the promoter activity,
probably you will see the inhibition of dnHes1 on Wt hes1 function, but you
need to do dose-response curve.
3. you found a very conserved hes bidning element in that promoter by using
information tools, but that does not mean that Hes1 will bind it; you need
to provide the evidence showing that hes1 binds to it in both vivo and vitro
. Also, if you want to show that potential binding element was critical for
Hes1 to activate this promoter, you need to do mutagenesis studies. | z********i
发帖数: 610 | 3 Thanks very much for your reply.
Although more info regarding your experiments is needed, but basically,
1. it is possible that WT hes1 activated promoter but dnHes1 (dominant
negative?) did not; it only suggests that the endogenous Hes1 in the cell
line you tested was not invoved in maintaining the basal activity of that
promoter. Actually, it is not a bad observation; it suggests that hes1
activated this promoter via DNA binding, becaseu usually dnHes should have
no or little DNA binding activity.
想您说的那样，Hes1 DNA binding肯定起了一定的作用。
2. However, if you test both Wt and dnHes1 on the promoter activity,
probably you will see the inhibition of dnHes1 on Wt hes1 function, but you
need to do dose-response curve.
我没有做的很细致，但是我看到了dnHes1能antagonize WT Hes1 induced promoter
activity.
3. you found a very conserved hes bidning element in that promoter by using
information tools, but that does not mean that Hes1 will bind it; you need
to provide the evidence showing that hes1 binds to it in both vivo and vitro
. Also, if you want to show that potential binding element was critical for
Hes1 to activate this promoter, you need to do mutagenesis studies.
保守的序列我已经做了突变，但是仍然能看到WT Hes1的激活作用，因为有好几个
potential Hes1 binding sites，所以我不能排除其它的位点不起作用。我现在在做
truncation mutation，想找到Hes1 responsive element。
不好解释的地方是，在细胞里面过表达Hes1能repress gene expression,但是promoter
-luciferase assay确是相反的结果。
many thanks
you
【在 r*******y 的大作中提到】
: Although more info regarding your experiments is needed, but basically,
: 1. it is possible that WT hes1 activated promoter but dnHes1 (dominant
: negative?) did not; it only suggests that the endogenous Hes1 in the cell
: line you tested was not invoved in maintaining the basal activity of that
: promoter. Actually, it is not a bad observation; it suggests that hes1
: activated this promoter via DNA binding, becaseu usually dnHes should have
: no or little DNA binding activity.
: 2. However, if you test both Wt and dnHes1 on the promoter activity,
: probably you will see the inhibition of dnHes1 on Wt hes1 function, but you
: need to do dose-response curve.
| r*******y
发帖数: 48 | 4 1. 我没有做的很细致，但是我看到了dnHes1能antagonize WT Hes1 induced promoter
activity.
That is good;
2. 保守的序列我已经做了突变，但是仍然能看到WT Hes1的激活作用，因为有好几个
potential Hes1 binding sites，所以我不能排除其它的位点不起作用。我现在在做
truncation mutation，想找到Hes1 responsive element。
how long of this promoter you tested? mutation of that conserved binding
site resulted in how much percentage of reduction in its activation compared
with that of the WT promoter by Hes1? in most cases,it w ill not be only
one site that contributes 100% to that activation; however, if the mutated
promoter exhibited over 50% decrease in its activity induced by Hes1, I
believe it should be good enough. for sure you may always continue to search
for more responsive elements.
3. 不好解释的地方是，在细胞里面过表达Hes1能repress gene expression,但是
promoter-luciferase assay确是相反的结果。
Actually, it is quite common that the in vitro luciferase data contradicts
with the in vivo expression data. in such case, you need to go along with in
vivo data, and you need to look for the fragment of promoter/enhancer which
can faithfully reflect the in vivo expression data once cotransfected with
Hes1. most of time, the length of promoter/enhancer matters, and you
propably need to find a certain length of promoter that can be repressed by
Hes1. if you just go ahead to present your in vitro activation data and your
in vivo reprssion data in one paper, most of time it will get back to you;
for sure it depends on which journal you send it to. | z********i
发帖数: 610 | 5 Thanks so much for your suggestions.
刚才重新看了一下data，发现有一些变化，我以前忽视了。potential Hes1 binding
sites 突变之后，induction fold 重两倍变化了5倍变化为3.7倍。我在试试看是否能
的到稳定的结果。 |
|
