Characterisation of the Hypoxic Responsive Element in the LIMD1 Promoter

5.3.1 The IR3 Region of the LIMD1 Promoter is required for Hypoxic Responsiveness.

This initial reporter experiment demonstrated that the LIMD1 promoter shows increased transcriptional activity in hypoxia (Figure 5.1). To map the precise region of the promoter responsible for this, the ten internal CpG Island deletion mutants that were described previously (Figure 3.2) were again utilised. The ten deletion mutants were transfected in U2OS cells, and the cells exposed to either normoxia or 24 hours hypoxia prior to lysis as previously described. The luciferase values were then double normalised. Firstly the values for the vector only plasmids were normalised to one, and then the normoxic values for each mutant were normalised to one. This resulted in clearer visualisation of the data, as the hypoxic values represented the fold induction in transcriptional up- regulation of the promoter in hypoxia compared to normoxia. Again the induction of hypoxia was confirmed by using the HRE driven luciferase plasmid internal control (data not shown).

The ten internal deletion mutants exhibited the same pattern of increases and decreases in transcriptional activity that were observed in the initial promoter analysis experiment (Figure 3.2 and Figure 5.3A). This pattern was mirrored when cells were exposed to hypoxia. The wild type promoter exhibited a 3.5 fold induction in activity in hypoxia (Figure 5.3), which was slightly higher than the fold inductions seen previously. However the internal control HRE driven luciferase plasmid also had a higher induction in hypoxia than in previous experiments, which validates the hypoxic promoter inductions observed. The

LIMD1 promoter mutants exhibited a 2-3 fold increase in promoter activity, even

the I 5 mutant that exhibited a 90% repression in normoxia showed increased transcriptional activity following hypoxic exposure. The only exception was the I 3 mutant. The I 3 mutant showed no significant increase in transcription following hypoxic induction, indicating this region most probably contained a functional HRE.

To more clearly assess the hypoxic inductions observed, for each deletion mutant, the luciferase values (that had already been normalised to the renilla values) were normalised to their respective matched normoxic value. The result of this was the value for normoxia was 1.0, and the hypoxic value was then the

the hypoxic response, showing the I 3 mutant had no significant increase in transcription in hypoxia. 0 0.5 1 1.5 2 2.5 3 3.5 4 Lu ci fe ra se V a lu e (N o rm a lis e d to N o rm o xi a) p>0.1 0 200 400 600 800 1000 1200 N H N H N H N H N H N H N H N H N H N H N H N H VO WT I I I I I I I I I I Lu ci fe ra se V a lu e (N o rm a lis e d to R en ill a ) p>0.1

A

B

5.3.2 Mutagenesis of the HRE or HIF1 Depletion Abolishes the Hypoxic Response of the LIMD1 Promoter

The hypoxic promoter analysis thus far had been carried out blind with respect to comparing the result to the in silico promoter screening. Experimentally the HRE within LIMD1 had been narrowed to IR3; this agreed exactly with one of the matches in the in silico screen, which also predicted a HRE element within IR3. The other two potential HRE elements predicted were 5’ to the CpG Island. However, as the I 3 deletion ablated the hypoxic responsiveness of the promoter (Figure 5.3), this implicated the in silico identified non-CpG Island HREs as being a false positive result.

To confirm the HRE within IR3 was functional and responsible for the increased promoter activity in hypoxia, site directed mutagenesis was used to mutate the HRE motif from CGTG to TACA. As the in silico analysis identified a total of three potential HREs, for completeness, the other two predicted HREs were also mutated in the same way. The HRE mutant plasmids were named delta ( ) HRE1, HRE2 and HRE3 in the order they appeared from the 5’ end of the promoter. Therefore HRE1 and 2 were the putative non-functional response elements that were outside the CpG Island, and HRE3 was within IR3 in the CpG Island. Similar to the hypothesis with PU.1 binding to the promoter, it was hypothesised that if HIF1 was binding to a HRE within the promoter region, then mutation of the consensus should prevent the binding interaction, and as such prevent the hypoxic response of the promoter.

In conjunction with the promoter HRE mutational analysis, another technique to confirm the hypoxic response would be to deplete HIF1 , the major hypoxic responsive transcriptional enhancer. HIF1 is only active in hypoxia due to proteasomal degradation under normoxic conditions as previously described. Therefore depletion of HIF1 should ablate any differences in transcriptional activation as a result of hypoxia. Plasmids encoding shRNA targeted against HIF1 or a negative control drosophila HIF1 (sh-HIF1 or sh-drosHIF1 respectively) were transfected into U2OS cells 24 hours prior to transfection with the mutated HRE promoter constructs ( HRE1-3). Cells were then exposed to either normoxia or 24 hours hypoxia prior to lysis and analysis of luciferase (Figure 5.4).

The wild type, HRE1 and HRE2 LIMD1 promoter constructs all exhibited a 2 fold increase in transcriptional activation in hypoxia with the control shRNA, showing that in U2OS cells there is no functional HRE at HRE1 or 2 (Figure 5.4).

However, the HRE3 with the hypothesis th

LIMD1 promoter. shR

ablation of the hyp promoter mutants sh (Figure 5.4). Of note activity in hypoxia, a however, these data promoter is mediated

Figure 5.4: shRNA knockdown of HIF1 or mutation of the HIF1 binding E3 promoter construct had no hypoxic inducti

is that this was the functional HRE within the Cp shRNA mediated depletion of endogenous hypoxic response as neither the wild type s showed a significant induction following h

note, HRE3 showed a significant decrease

a, an observation that at present cannot be e data show that the hypoxic responsivenes

ted byHIF1 , and requires the HRE within IR3

shRNA knockdown of HIF1 or mutation of the HIF1 binding uction, which agreed he CpG Island of the s HIF1 resulted in ype or HRE LIMD1 g hypoxic exposure se in transcriptional e explained. In sum ness of the LIMD1

IR3.

5.3.3 Depletion of Endogenous HIF1 Significantly Reduces Endogenous LIMD1 Protein.

To demonstrate the physiological significance of the hypoxic responsive element

within the LIMD1 promoter, the effect of depleting HIF1 on endogenous LIMD1

protein expression within U2OS cells was examined. It was hypothesised that if HIF1 activity was the main requirement for the hypoxic responsiveness of the

LIMD1 promoter then depletion of HIF1 should prevent an increase in LIMD1

proteins levels in hypoxia.

To test this hypothesis, endogenous HIF1 was depleted from U2OS cells using shRNA and LIMD1 protein levels assayed by Western blot following either normoxic or hypoxic exposure. Again as a negative control shRNA against Drosophila HIF1 was included. 24 hours post transfection cells were exposed to 1% oxygen for 24hours, and then lysed in RIPA supplemented with protease inhibitors and MG132 (a potent inhibitor of the 26S proteasome, used to prevent degradation of HIF1 following cell lysis). Expression levels of HIF1 and LIMD1 protein were then quantified by Western blot, with actin used as a loading control.

Knockdown of HIF1 in hypoxia resulted in a reduction in LIMD1 protein levels when compared to the control drosophila HIF1 depleted cell extracts (Figure 5.5). These data support the reporter assay data (Figure 5.1) showing that

LIMD1 driven transcription is up regulated in hypoxia and the HRE mutation

experiments which suggested HIF1 mediates the hypoxic response of the

LIMD1 promoter (Figure 5.4). The shRNA experiment also showed that a

depletion of the low levels of HIF1 in normoxia, also resulted in a decrease in LIMD1 protein (Figure 5.5).

Figure 5.5: shRNA kn protein levels. shRNA

transfected into U2OS c 16 hours, prior to lysis with monoclonal anti H independent experimen

The results obtaine knockdown experime of LIMD1 is depende these observations, the role of increase tensions (hypoxia).