REVIEW PAPER
Paper to be Reviewed:
Identification
and functional characterization of a novel binding site
on TNF-(alpha) promoter
Xiaoren Tang, Matthew J. Fenton, and Salomon Amar
Proceedings of the National Academy of Sciences, Volume 100, Number 7, April 1, 2003 4096-4101
Review
Tumor necrosis factor (TNF)-(alpha) is an endogenous pleiotropic cytokine that is stimulated in the process of inflammation. With respect to the inflammatory system, TNF-(alpha) can be both helpful and/ or detrimental, therefore it is important to be able to regulate its production through the TNF-(alpha) promoter.
There are 2 basic levels at which the TNF-(alpha) promoter can be regulated: transcriptional
and posttranscriptional. This paper focuses on regulation at the transcriptional
level. One of the transcriptional factors that bind within the TNF-(alpha) promoter
region is lipopolysaccharide (LPS)-induced TNF-(alpha) factor (LITAF). LPS induces
the production and secretion of TNF-(alpha) by stimulating monocytes and macrophages.
The functional role of LPS (in vivo) is unclear. While LPS is known to up-regulate
DNA-binding activity of inducible transcription factors, such as NF-(kappa)B,
it is also known to down-regulate the DNA-binding activity of some constitutive
transcription factors. One of the inducible transcription factors that LPS effects
is NF-(kappa)B, which may up-regulate TNF-(alpha) transcription in response to LPS
in some cancerous human cells, but the role of NF-(kappa)B is ambiguous. Another
transcription factor that might have a clearer role in the activation of hTNF-(alpha)
transcription is hLITAF, which mediates hTNF-V transcription. The main goal
of Tang et al. was to determine the mechanism of hLITAF and hTNF-(alpha) interaction.
In order to accomplish this goal, Tang et al. decided to go after the binding
site of the TNF-(alpha) promoter—first they identified the site via DNase I
footprinting, then they characterized it via different shift assays (Electrophoretic
Mobility Shift Assay, EMSA and Enzyme-Linked Immunosorbent Assay, ELISA).
The data obtained from footprinting revealed a protected region on hTNF-(alpha) DNA
by the protein hLITAF from nucleotides –515 to –511, a CTCCC motif
(Figure 2a, b). However, the footprinting was not run with whole hLITAF; the
protein was broken into restriction fragments, bound to a fusion protein, then
mixed with the hTNF-(alpha) DNA. Therefore, Tang et al. were able to determine the
specific sequence of hLITAF that bound to hTNF-(alpha) as well. From the data, one
can see that the only regions in amino acid sequence 1 to 228 were capable of
protecting the binding site of hTNF-(alpha). Therefore, the binding interaction between
hTNF-(alpha) DNA and hLITAF occurs on nucleotides –515 to –511 and somewhere
in the amino acid sequence 1 to 228, respectively (Figure 2a).
The EMSA (or band shift assay) was used to further establish the protein-DNA
interaction. In this assay, Tang et al. tested smaller regions of fragment 1-228
of hLITAF in order to be able to focus on a more precise region of hLITAF that
is binding to the hTNF-(alpha) DNA. Shifted DNA bands indicate binding: there were
2 detected, in regions 152 to 228 and 1 to 228 (Figure 3a). Therefore, the binding
region must be within the amino acid sequence 152 to 228. A similar, but even
more specific EMSA was run next: the data revealed binding in regions 152 to
228 and 1 to 228 again, but also in the region 152 to 228 with a deletion from
180 to 195 (Figure 3b). Therefore, the binding region must be within the amino
acid sequence 165 to 180.
The next step for Tang et al. was to make sure that the hLITAF sequence was
doing what they thought it was doing. Therefore, based on the amino acid sequence
of hLITAF, they constructed 3 peptides, inserted them into THP-1 cells, and
tested for TNF-(alpha) secretion. The 3 peptides were cleverly named peptides A, B,
and C. Secretion of TNF-(alpha) was tested at several different concentrations of
the 3 peptides. Peptides A and C did not have a significant effect on TNF-(alpha)
secretion, but peptide B induced the secretion of significantly more TNF-(alpha) than
a control peptide did (Figure 4). This corresponds well with the previous results
of the paper, because peptide B is found in amino acid sequence 165 to 180 of
hLITAF (Figure 1a).
The final goal of the paper was to determine if the region -515 to -511 in the
hTNF-(alpha) promoter was indeed responsible for hLITAF binding. Tang et al. cloned
4 different constructs, 2 of which had the CTCCC sequence of -515 to -511 deleted.
They transfected the constructs into THP-1 cells and stimulated them with either
LPS (for a positive control) or peptides A, B, or C. Then they detected TNF-(alpha)
promoter activity with a luciferase assay. Only TNF-(alpha) promoters with the CTCCC
sequence in tact showed above control activity; among those, the only cells
that showed close to wild-type activity were those stimulated with LPS or peptide
B (Figure 5a, b).
From these results, Tang et al. conclude that the binding sequence of hLITAF
to the hTNF-(alpha) promoter is CTCCC (in the -515 to -511 region of the DNA). They
also suggest that the hLITAF sequence that binds to hTNF-(alpha) is between amino
acids 165 and 180. These studies were all done in vitro, but Tang et al. hypothesize
that the DNA binding region of hLITAF (165-180) also functions in vivo to regulate
hTNF expression.
Criticism
First off, this paper deserves credit for using a functional approach to clarify the details of this mechanism. The most convincing results Tang et al. had was the EMSA in which they determined that the most likely binding sequence on hLITAF is amino acids 165 to 180 (Figure3a, b). There was one problem I had with the figure: lane 7 of figure 3b has a blot where it was expected, however, there seems to be another prominent (albeit significantly less prominent) band right below it. Tang et al. did not explain that shifted DNA band.
Another point of contention that I had was that while it is compelling that
the location of peptide B corresponds to the amino acid sequence 165 to 180,
I am not thoroughly convinced that this region of hLITAF is definitely the binding
site. While there is much evidence to support that the binding region is within
amino acids 152 and 228, and it is not in the region 180 to 195, Tang et al.
failed to test two critical regions: 152 to 165 and 195 to 228. I would be much
more convinced if they had experimented with those two regions in addition to
the other regions they tested.
Overall, the science was very clean and well-controlled. Each experiment had
both positive and negative controls, which made their results quite convincing
to me.
Future Areas of Research
Tang et al. themselves indicated that they would like to see how their theory works in the human body, therefore it would be interesting to find a way to conduct an experiment with the same goals in vivo. I would also conduct another EMSA including the previously mentioned fragments in the amino acid sequence 152 to 228 that were neglected in this study.
The next phase of this study should aim to further elucidate the mechanisms
of hLITAF/ hTNF-(alpha) interactions. One possible area of research could be
to figure out what else is going on in the hTNF-(alpha) promoter when it stimulates
hLITAF activity. Tang et al. have suggested that other transcription factors
such as NF-(kappa)B might be involved.
The main goal of my future research (if I were to do it) would be to determine
other regions of the hTNF-(alpha) promoter that are induced by LPS. I would
perform the same procedures that Tang et al. did, except I would use hNF-(kappa)B
instead of hLITAF. I would expect to find similar results to Tang et al., except
I would expect the binding region not to be within the nucleotide sequence –515
to –511.
Please send your questions, comments, or concerns to Suzy Rizi