1. Non-canonical NF-B signaling and ETS1/2 cooperatively drive C250T mutant TERT promoter activation Li et al. (Nature Cell Biology, 2015)
Manraj Gill
April 6th, 2016
Undergraduate Biology Journal Club

2. Preventing the loss of genetic information
SOMATIC CELLS
REPRODUCTIVE CELLS
CELLULAR CLOCK
Maintain majority of genetic information
Shuffling of genetic information facilitated when linear!
Starting off from the point of view of a cell… in the case of “regular cells” in the body, i.e. somatic cells and not sperm/egg cells, the important idea is that genetic information must be passed on to the new cells after cell division in the process of giving rise to or maintaining tissues. And, so, this information must remain largely intact and complete. You don’t want new cells to gradually lose this information and thereby not be “identical” to neighboring and ancestral cells!
However, in the case of organismal reproduction, some variance has proven to be necessary because it leads to minute differences which evolution can act on. This impact which genetic information is more frequently passed on and a genetically diverse offspring can be better suited for survival in constantly changing conditions. To facilitate this diversity, and shuffling genetic information between individuals, a linear and non-circular carrier of genetic information, eukaryotic chromosomes, evolved since it served to be more efficient in recombining.
But this evolution of linear genetic information threatens the ability of cells to retain the majority of this information because cells cannot effectively copy information in the ends. To prevent this loss, the cell evolved to develop a clock which allows it to roughly measure the number of times it has divided so that it knows to stop dividing when vital information would be at risk!
“genetically identical”
genetically diverse offspring
Stops dividing when
information is at risk!

3. The Cellular Clock Telomeres human Stem Cells Time / DivisionsTR
human Stem Cells
Time / Divisions
Telomere Length
TERT
human Somatic Cells
Tumor Cells
The cellular clock is in the form of a stretch of repetitive DNA sequence, called telomeres, at the ends of linear eukaryotic chromosomes. The telomeric length allows the cell to know whether or not its genetic information will be at risk when it divides. Every cell division, a portion of the telomeres is lost due to the DNA replication machinery’s inability to fully replicate the sequence in the terminal regions of each chromosome.
Telomeres serve as buffer sequences at the ends to prevent loss of genetic information on the chromosome. Telomere shortening to critically small lengths over time due to cell divisions serves as a checkpoint for replicative senescence or cellular death because any further replication would threaten genetic stability.
In stem cells, however, the length of the ends is maintained at a constant level. This makes sense because these cells have the potential of differentiating into all cell types in an organism and need to constantly give rise to new cell types. But the length can no longer be maintained at a constant level once this “stem-ness” is lost.
Additionally, it has been observed that cancerous cells develop as a result of re-lengthening of these ends after the terminal length had been reached.
The questions that my project addresses relate to the process by which this length is regulated. More specifically, what allows for a sustained telomere length in hESCs and what happens mechanistically to prevent this maintenance once the stem cell has differentiated.
However, the telomerase enzyme can restore some lost telomeric sequence and increase the length of the telomere. In normal somatic cells, telomerase activity is absent because the transcription of the protein component of telomerase, TERT, is down regulated. However, cells with an unlimited potential to replicate, such as stem cells sustaining TERT expression.
Telomerase Enzyme

4. Genome Wide Associate Studies (GWAS) observed three frequent non-coding mutations in the TERT promoter that are all associated with specific types of tumors

5. These three individual mutations that increase transcription each lead to the generation of de novo (new) binding site on the promoter for an ETS (E26 Transformation-Specific) family transcription factor
G228A corresponds to -124 G>A
G250A corresponds to -146 G>A

7. TNFa (tumor necrosis factor- alpha): canonical pathway
NFκb (Nuclear Factor kappa-lightchain-enhancer of activated B cells) is a regulator of
immunity
stress responses
apoptosis
differentiation
TNFa (tumor necrosis factor- alpha):
canonical pathway
TWEAK (TNF-like weak inducer of apoptosis):
non-canonical pathway
In contrast, a subset of GBM cell lines exhibited TERT induction
following TWEAKexposure (Fig. 1a). Sequencing the TERT promoter
region of these cell lines unexpectedly led to a distinct segregation of
these lines that induce TERT after TWEAKstimulation, based on their
TERT promoter mutation status (Fig. 1b). Upregulation of TERT in
GBM cells containing the C250T mutation correlated with a strong
induction of telomerase activity in C250T-mutant cells (Fig. 1c). It is
noteworthy that small changes in TERT transcription are sucient for
a significant increase in telomerase activity
These results suggest that although
similar levels of p52/RelB are activated in all GBM cells, only
the C250T-mutant TERT promoter is responsive to these dimers.
Oeckinghaus et al. Nature Immunology (2011)

8. Lentiviral knockdown of p52, RelB and NIK abolished
the induction of TERT in TWEAK-stimulated C250T GBM cells
Lentiviral knockdown of p52, RelB and NIK abolished the induction of TERT in TWEAK-stimulated C250T GBM cells

9. p52 binds to half-sites in vivo
We thus examined
whether p52 can bind half-site at the C250T TERT promoter using the
only available in vivo p52 ChIP-seq data fromlymphoblastoid B cells37.
Through de novomotif analysis3840,we identified the palindromic p52
motif from 12,239 p52 ChIP-seq peaks
In contrast,
11 base pairswith lower full-site binding anity regions tend to enrich
in the half-site binding regions (Fig. 2barea in rectangles)where only
one of the half-site GL or GR is low41. We therefore provide evidence
that p52 binds to half-sites in vivo.

12. These findings highlight the significant concordance be-
tween enhanced NIK expression and increased telomerase expression
in GBM tumours carrying the C250T mutation.