Figure 1. Computer model of ZBTB7A. [1]
Figure 1. Computer model of ZBTB7A. [1]


This Project

This web page originated as an assignment in Emory University's Biology 142 lab course. Students were assigned proteins of interest and asked to research what is known about the protein and to examine whether the newly sequenced whale shark genome had evidence of an orthologous protein.


Background Information

ZBTB7A is commonly referred to as a zinc finger protein and is part of a family of POK, Pokemon, transcriptional factors proteins (Choi et al, 2008). ZBTB7A was first identified as a protein that bound to the sequence in the human immunodeficiency virus and was known to actively express the gene of that sequence (Zu et al, 2009). The gene encoding ZBTB7A is known as a proto-oncogene, a gene that can become an oncogene through mutations. This photo-oncogene is associated mostly with cellular development, cellular processes, and cell differentiation. Studies done through microarrays and polymerase chain reactions, ZBTB7A was found to be important in development of neurons (Zu et al, 2010). Overexpression or gene amplification of ZBTB7A can cause the inactivation p53 and can lead to devastating effects like lung cancer (Apostolopoulou, 2007). Also, recent discoveries have shown that ZBTB7A is actively expressed in several cancer tissues. This happens when the transcription of the Rb gene is repressed by the ZBTB7A gene. When the Rb gene is repressed, the cell cycle does not stop and cancerous cells can proliferate (Jeon et al, 2008). Therefore, ZBTB7A plays an important role in the development of cancerous cells and malignancy. ZBTB7A reduces apoptosis which further promotes cancerous cell growth (Zhang et al., 2011).


Methods

Retrieving Human Protein Sequence

Given the Ensembl protein ID, ENSP00000323679, a search for the full ZBTB7A amino acid sequence was performed through the Ensembl Genome Browser website [2]. The resulting sequence was recorded in FASTA format.

Analyzing Protein Sequence

Using the human amino acid sequence found, it was blasted against the Whale shark database on the Whale Shark Genome Galaxy website [3]. The top hit protein match’s FASTA sequence was then recorded and underwent a reciprocal blast in the NCBI human proteome database [4]. Top hits were identified using the lowest e-value, highest query cover, and highest percent identity. Other orthologs were then detected in a similar manner by blasting the human ZBTB7A sequence on the NCBI website against the databases of the zebra fish, elephant shark, mouse, and yeast. The whale shark protein sequence was also blasted against the same organisms. The resulting best hit orthologous sequences were recorded.

Creating Phylogenetic Tree

With the orthologous protein sequence of the human, whale shark, zebra fish, elephant shark, mouse, and yeast in FASTA format, they were entered into ClustalW [5] along with the whale shark and human protein sequences to compare the protein sequences and deduce phylogeny and speciation. From the polypeptide sequence comparison, a phylogenetic tree was created.


Results & Discussion

Protein Domains and Motifs

As seen in Figure 1, the blasts between the human ZBTB7A protein against elephant sharks, zebra fish, yeast, and mouse showed that there was a conservation of the protein domains BTB superfamily, two zinc finger domains, and three zinc finger double domains. The numerous zinc finger (Znf) domains are short amino acid sequences within the protein structures that are recur as protein motifs and are specialized in their functions (Krishna, S. et al, 2002). The blast results show that many of these protein motifs are conserved between the human ZBTB7A protein and the other species listed. Similarly, the reciprocal blasts of the whale shark against the other species returned a conserved domain of the BTB superfamily as well as one Znf domain. The conservation of a zinc finger domain between the whale shark and other species show that there is a definite structural similarity in a zinc finger within the tertiary structure of orthologous proteins.
Screen Shot 2015-04-13 at 9.57.36 PM.png
Figure 2. The figure above shows the protein domains resulting from the BLAST of the human ZBTB7A protein against the whale shark, elephant shark, zebra fish, yeast, and mouse database. The figure shows the BTB superfamily, along with two zinc finger protein domains and three zinc finger double domains.


Screen Shot 2015-04-13 at 10.04.30 PM.png
Figure 3. The figure above shows the protein domains found in the best hit protein that resulted from the BLAST of whale shark protein against the protein databases for humans, elephant sharks, zebra fish, yeast, and mice. The major protein domains include the BTB super family and the zinc finger double domain.


Orthologs

The top hits listed between the human ZBTB7A protein and various other species' proteome (Table 1) reveal very low e-values for all the alignments and generally high query cover as well (with the exception of yeast). The percent identities, however, are particularly low between the human and whale shark and elephant shark. The reciprocal blast of the whale shark sequence back against the human proteome revealed a 100% query cover and and very low e-value, however, with the ZBTB7A protein. Similar blasts of the whale shark protein against the databases of the other species also showed similar results. Again, the percent identities are relatively low at around 50% (apart from the elephant shark which is 74%). This suggests that the overall structure of the protein has been conserved but the individual amino acids within the polypeptide sequences has changed. The consistent top hit of the whale shark protein against other organisms are the ZBTB7A proteins. The whale shark protein is most related to the elephant shark in terms and the two most likely evolved from each other more recently than the other species. The e-value between the two is zero with a 100% query cover. Yeast does not fit within the data as the protein hits are unrelated to the Znf proteins.
Query
Database
Description
Query Cover
E Value
Identity
Human
Whale Shark
g43924.t1
71.92%
9e-129
51.67%
Human
Elephant Shark
zinc finger and BTB domain-containing protein 7A
95%
0.0
53%
Human
Zebra Fish
zinc finger and BTB domain-containing protein 7A isoform X2
99%
0.0
88%
Human
Yeast
Azf1p
21%
2e-21
34%
Human
Mouse
zinc finger and BTB domain-containing protein 7A isoform X3
99%
0.0
88%
Whale Shark
Human
zinc finger and BTB domain-containing protein 7A
100%
2e-126
50%
Whale Shark
Zebra Fish
zinc finger and BTB domain-containing protein 7A isoform X2
98%
6e-80
39%
Whale Shark
Mouse
zinc finger and BTB domain-containing protein 7A
100%
5e-122
49%
Whale Shark
Elephant Shark
zinc finger and BTB domain-containing protein 7A
100%
0.0
74%
Whale Shark
Yeast
Zap1p
10%
1e-06
49%
Table 1. Running BLAST searches of the human ZBTB7A protein against the whale shark, elephant shark, zebra fish, yeast, and mouse and running the reciprocal BLAST searches of the whale shark g43924.t1 protein against the same organisms' proteome gave the above results. The top hits that are recorded above were chosen based on the highest query cover and percent identification and the lowest e-value in each search.

Phylogeny

The phylogenetic tree generated from the comparison of the orthologous protein sequences of each organism listed above (Table 1) is shown in Figure 4. The phylogeny matches the prediction that the whale shark and elephant shark are the most related and experienced speciation most recently within the species being compared. Likewise, the human and mouse have the most similar ZBTB7A orthologous protein sequence. From Table 1, we the query coverage was 99% with a 88% percent identity as well as an e-value of zero. Tracing the phylogenetic tree, we can see that speciation between yeast as a unicellular eukaryote and all the multicellular species. From there, subsequent evolutionary events occurred to arrive at the modern human, mouse, whale shark, elephant shark, and zebra fish. From the species compared, the human protein is most related to the mouse and the whale shark protein is most related to the elephant shark.
Screen Shot 2015-04-13 at 9.46.00 PM.png
Figure 4. Running the orthologous protein sequences in ClustalW, the human RUNX3 protein was compared to the best hit protein sequences found in other species using BLAST. The comparison of the individual amino acid sequence generated the phylogenetic tree above.


Conclusion

Based on the data collected, we conclude that the whale most likely has the ZBTB7A protein. The initial BLAST between the human protein against the whale shark protein database came up with a fairly large query coverage of the human protein sequence with a value of 71.92% with a low e-value of 9e-129 but a low percent identity of 51.67%. The low e-value but low percent identity and 71.92% query cover would suggest that the two proteins share protein domains in the analyzed sequence between the human and the whale shark. With the reciprocal BLAST between the whale shark and the humans done, we found a 100% query coverage with a low e-value of 2e-126 and a percent identity of 50%. This confirms that the protein, although not the same in both organisms because the low percent identities, share similar structures. The common protein domain found included the BTB superfamily and one Znf protein motif. Similar BLASTs with other organisms (mouse, elephant shark, yeast, and zebrafish) showed that the same protein domains between the organisms are conserved. Most importantly, the BLAST between the whale shark and elephant shark led to the query coverage of 100%, e-value of 0.0, and percent identity of 74%. This leads us to conclude that the whale does have a ZBTB7A protein ortholog most similar to the protein found in elephant sharks. The conservation of protein alignment of almost, if not, the whole whale shark protein sequence with not only the elephant shark but also humans, mouse, and zebrafish suggest that the protein ZBTB7A should exhibit similar structures and function, especially with the conserved BTB domain, which controls transcriptional repression, and Znf motif. The same relationship can be traced through the phylogeny of the species' proteins analyzed (Figure 4). Depending on the exact function of the ZBTB7A protein in whale sharks, we could learn more about the role of ZBTB7A as an important transcription factor especially in the field of oncology and immunology. We would also better understand the evolution of the protein over time.


Works Cited

Apostolopoulou, K., Pateras, I., Evangelou, K., Tsantoulis, P., Liontos, M., Kittas, C., Tiniakos, D., Kotsinas, A., Cordon-Cardo, C. and Gorgoulis, V. (2007), Gene amplification is a relatively frequent event leading to ZBTB7A (Pokemon) overexpression in non-small cell lung cancer. The Journal of Pathology 213: 294–302.

Choi, W., Jeon, B., Yun, C., Kim, P., Kim, S., Choi, K., Kim, S., Hur, M (2009). Proto-oncogene FBI-1 Represses Transcription of p21CIP1 by Inhibition of Transcription Activation by p53 and Sp1. The Journal of Biological Chemistry 284: 12633-12644.

Jeon, B., Yoo, J., Choi, W., Lee, C., Yoon, H., Hur, M (2008). Proto-oncogene FBI-1 (Pokemon/ZBTB7A) Represses Transcription of the Tumor Suppressor Rb Gene via Binding Competition with Sp1 and Recruitment of Co-repressors. The Journal of Biological Chemistry (238): 33199 – 33210.

Krishna, S. Sri, Majumdar, Indraneel, & Grishin, Nick V. (2002). SURVEY AND SUMMARY: Structural classification of zinc fingers. Oxford University Press.

Zhang, Q., Tian, D., Xu, X (2011). Depletion of Pokemon Gene Inhibits Hepatocellular Carcinoma Cell Growth through Inhibition of H-ras. Onkologie 34: 526-531.

Zu, X., Yu, L., Sun, Y., Tian, J., Liu, F., Sun, Q., He, S., Sun, G., Luo, W., Jiang, Y (2010). Global mapping of ZBTB7A transcription factor binding sites in HepG2 cells. Cellular and Molecular Biology Letter 15: 260-271.

Zu, X., Yu, L., Su, Q., Liu, F., Wang, J., Xie, Z., Wang, Y., Xu, W., Jiang, Y (2009). SP1 enhances Zbtb7A gene expression via direct binding to GC box in HePG2 cells. BMC 2: 175.


References

1. ^ Protein Data Bank in Europe. (n.d.). Retrieved from http://www.ebi.ac.uk/pdbe-srv/view/entry/2if5/summary

2. ^ http://useast.ensembl.org/index.html

3. ^ http://whaleshark.georgiaaquarium.org/

4. ^ http://blast.ncbi.nlm.nih.gov/Blast.cgi

5. ^http://www.genome.jp/tools/clustalw/