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Regulation of RNA polymerase III transcription by Id proteins and E47

Final Report Summary - ID AND POL III (Regulation of RNA polymerase III transcription by Id proteins and E47)

The aim of the IRG funded “ID and Pol III” project was to establish a link between Id, Id related protein, E47, and RNA polymerase (pol) III transcription. RNA Polymerase III (pol III) is exclusively responsible for transcribing short non-coding genes essential for protein synthesis, such as tRNA and 5sRNA. It is well documented that pol III transcripts are overexpressed in tumours and pol III activity is relevant to cancer. Inhibitor of Differentiation (Id) proteins are a subgroup of transcription factors containing a helix-loop-helix (HLH) dimerization domain. Id proteins bind and regulate several transcription factors that do not have bHLH dimerization motifs, such as RB and Ets, the ability of the Id family to counteract RB and the induction of cell cycle inhibitors by Ets, E12 and E47 allows them to promote cell cycle progression. E47 is a ubiquitous transcription factor that can inhibit cell proliferation and promote differentiation. It is antagonised by ID2, an oncogenic protein that binds E47 and inhibits its DNA-binding activity. Current literature on pol III transcription and Id class of transcription factors suggests that both are involved directly in cell proliferation and oncogenic transformation. We initially started looking for links between Id proteins and pol III transcription and obtained some exciting preliminary data. Budding from these data throughout the grant period we carried out the outlined research in the proposal and further consolidated the link between Id proteins, E47 and pol III activity. Work carried out in this proposal strongly suggests a direct involvement of Id2 and its binding partner E47 on pol III transcribed genes
This IRG grant proposal had two main aims. The first is to determine how Id and E47 proteins are recruited to pol III-transcribed genes and furthermore to show the mechanism of how they influence pol III transcription. The majority of objectives set forth in the grant proposal were achieved in the project and a great deal of work is done towards establishing how Id2 and E47 are recruited at tRNA genes, which are transcribed by RNA polymerase III. We have also determined if E47 and Id2 affect RNA Pol III transcription in a positive or negative manner. Below I am outlining, in a paper format, most of the findings that were obtained during this grant period

E47 is present at many Pol III-transcribed genes.
The consensus DNA sequence (CAGCTG) recognised by E47 can be found within 1kb of 56% of all tRNA genes in the human genome and within 250bp of 19%. This prompted us to conduct ChIP analyses in HeLa cells to look for E47 binding. As predicted, endogenous E47 was detected at a variety of tRNA genes. In contrast, only background binding was detected with a negative control antibody against the pol I-specific factor SL1. Evidence for selectivity is provided by the lack of E47 signal at the apolipoprotein E gene (APOE), an extra TFIIIC (ETC) site and a tRNALeu(TAA) gene.
Although it is selective, the presence of E47 at tRNA genes does not seem to require a nearby E-box, as binding was detected at three tRNA genes that are not within 250bp of even the loose E-box consensus (CANNTG). However it should be noted that as with all tRNAs there is at least one CANNTG motif located within a 1kb window. Conversely, there are matches to the E-box motif near the tRNA81Leu (TAA) gene, where E47 was not detected. In contrast to others, this tRNALeu gene is not occupied by pol III, TFIIIB or TFIIIC in HeLa cells. This led us to consider the possibility that even though E47 could potentially bind at E-boxes, the presence of pol III and/or its associated factors may dictate whether E47 is present at tRNA genes.
We hypothesised that E47 is brought and maintained at tRNA gene by the members of the pol III transcription machinery. Indeed using a GST purified GST-E47 construct we could pull down BRF1, a member of the TFIIIB complex necessary for pol III transcription. The interaction between BRF1 and E47 was also observed in cells using whole cell protein extract from HeLa cells. Furthermore a slight interaction of E47 and other members of the pol III transcription machinery were seen in vivo. Together with the ChIP data these in vitro and in vivo immunoprecipitations put E47 at the pol III transcription machinery on tRNA genes, enriching the number and variety of transcription factors acting to regulate tRNA production.
E47 represses Pol III-transcribed genes by affecting recruitment of pol III transcription factors.
Next, we wondered if E47 presence has an effect on pol III transcription. To test for a role in regulating pol III output, RNAi was used to deplete HeLa cells of endogenous E47. A substantial decrease in E47 protein was found to raise expression of three different tRNA messages and 5S rRNA by ~1.5-fold. This increase in transcript levels cannot be explained by changes in abundance of pol III transcription factors due to effects of E2A knockdown.
Although indirect effects cannot be excluded in such experiments, since E47 is found at tRNA genes in vivo we believe that E47 is acting in a direct manner in regulating pol III transcription.
A common way of affecting pol III transcription is by affecting loading of different members of the pol III machinery at tRNA genes. Since knocking down E47 had a positive effect on tRNA transcription we decided to look at loading of BRF1 and RPC 155, a subunit of pol III holoenzyme. Upon down regulation of E47 protein levels we see a ~1.5-fold increase in tRNA gene loading of BRF1 and the RPC155 subunit of pol III at three different tRNAs. The effect is specific, as occupancy of histone H3 shows little or no change. Given these data we conclude that endogenous E47 is a repressor of tRNA and 5S rRNA gene transcription in HeLa cells.
Histone acetyltransferase activity has been shown to promote gene occupancy by BRF1 and pol III in vivo, without affecting TFIIIC loading. Indeed, the presence of acetylated histones correlates strongly with gene occupancy by pol III in mammalian genomes. As E47 has been shown to bind to histone deacetylases (HDACs), we hypothesized that these may contribute to its negative effect on pol III transcription.
In support of this, endogenous HDAC1 and HDAC2 were detected by ChIP at tRNA and 5S rRNA genes, and E47 depletion reduced detection of both HDACs at these genes. In contrast, occupancy of these loci by c-MYC and the acetyltransferases GCN5 and p300 was increased by E47 knockdown. As expected, levels of histone acetylation at the tRNA and 5S rRNA genes increased in parallel with HDAC release and the recruitment of acetyltransferases. These data suggest that E47 can exert a repressive effect at these sites by decreasing histone acetylation and hence gene usage by BRF1 and pol III. A counterintuitive feature of this model is that E47 discourages promoter occupancy by BRF1, despite possible recruitment by BRF1, as conferred from immunoprecipitation experiments. To explain this, we envisage a dynamic cycle, where E47 is attracted to promoters by BRF1, but then alters the chromatin environment in a manner that destabilises the complex and encourages its release. ID proteins, that antagonise E47, may counteract its inhibitory effect in this context.
ID2 associates with pol III-transcribed genes and stimulates their expression.
As E47 forms stable heterodimers with ID proteins, we tested for the presence of the latter at pol III-transcribed genes. Endogenous ID2 was detected by ChIP at active tRNA genes, but not at the inactive tRNA81Leu (TAA) locus. Id2, just like all ID family members does not contain a DNA binding domain, and as such we believe that our ChIP experiments do not detect a direct DNA binding event perse but the mere presence of ID2 at tRNA genes, possibly via protein-protein interactions. Indeed ID2 signal at tRNA genes is very week, compared to that observed for E47.
An attractive model of recruiting ID2 to tRNA genes could be via its binding partner E47. To test this, we depleted E47 by RNAi and found reduced ID2 occupancy of tRNA and 5S rRNA genes, despite elevated crosslinking of BRF1. We therefore propose that ID2 recruitment to pol III promoters is facilitated by the presence of its high-affinity binding partner E47. In order to position ID2 at tRNA genes with pol III transcription machinery we performed in vitro and in vivo immunoprecipitation experiments. We noticed an interaction of ID2 with BRF1 both in vitro and in vivo. The interactions between ID2 and BRF1 might also contribute to maintain ID2 at tRNA genes, but seem insufficient to compensate for a reduction in E47. Furthermore ID2, just like E47, can be co-immunoprecipitated from HeLa cells with pol III, TFIIIB and TFIIIC, without the need for overexpression. This suggests the existence of one or more complexes involving these endogenous proteins.
To assess how ID2 influences pol III transcription, we depleted it from HeLa cells by RNAi using constructs from two different companies. Upon ID2 depletion we saw a mild down-regulation of pol III transcription as seen by the reduction in levels of three different tRNAs tested. On the other hand up-regulation of ID2, using an adenoviral vector in HeLa cells, has the opposite effect and up-regulates tRNA and 5S rRNA transcription. These data suggest that ID2 is a positive regulator of pol III transcription. Similar to E47 we decided to see if knocking down ID2 has any effects on assembling of the pol III machinery at tRNA genes. Reducing ID2 protein levels resulted in decreased occupancy by BRF1 and pol III at tRNA and 5S rRNA genes, the opposite of what is seen following depletion of E47. This drop in occupancy of BRF1 and Pol III could explain the fall in tRNA levels after RNAi directed against ID2. Thus, ID2 and E47 have opposite effects on the levels of pol III products, similar to their antagonism in regulating many pol II-transcribed genes.
All together data from our experiments carried out in this proposal points towards a model where members of the RNA pol III transcription machinery, more specifically BRF1 and/ RPC155 could bring E47 in the vicinity of tRNA genes when the cellular output is diminished, such as for example in case of nutrient starvation. Presence of E47 at tRNA genes recruits histone deacetylase complexes that via deacetylating histone tails bring about a repressive chromatin environment at the pol III transcribed genes. In a more favourable environment cells start to grow, proliferate or become more metabolically active. According to our model, at this time, E47 recruits Id2 at the tRNA genes, this in turn helps to upregulate pol III transcription. This part of the model needs to be elucidated further as we also find that Brf1 and RPC155 are necessary to bring Id2 at tRNA genes. A possible explanation could be that interaction of ID2 with the members of the pol III machinery is necessary for its function to further increase the assembly of the pol III transcription machinery at tRNA genes. The increase in the presence of the pol III transcription machinery upon Id2 recruitment brings to a global increase in RNA pol III transcription.
Data generated from following the proposed research give invaluable insights on how pol III transcription is affected by E47 and ID proteins and point towards a possible mechanism. We hope that by the completing this work we are one step forward further in understanding how RNA pol III transcription is carried out and have unraveled a novel way to control RNA pol III transcription, via Id and E47 proteins. Since levels of both Id proteins and pol III transcription are attenuated during tumorigenesis work performed under this grant will point towards new possible connections between pathways that can influence tumorigenesis.