TC008142
Fly Ortholog:
Tat interactive protein 60kDA (Tip60)
Student Name:
Hunter Gazda and Connor Huelsman
School Name:
Miami University
Year of Study:
2018
Gene Information:
Tat Interactive Protein (TIP60) is lysine acetyltransferase. Tip60 is a member of a protein complex that as a whole performs histone acetylation and chromatin remodeling. Tip60 specifically acetylates histones H4 and H2A and the non-histone proteins: p53, c-Myc and androgen receptor. In addition, Tip60 is known to aid in chromatin packaging, DNA repair, DNA repair responses, and apoptosis.
NCBI Link:
iBeetle Link:
Tribolium Transcript: 5' UTR:
AATTATGCAATGGAAAAAAATCGTTATGGATATTGCGCATTATGTTTTTTGCGGTAGTTGTGGTGGTGAGGTTTGTTCGGCAATAATTACAATTTTTCCCCCCGAAATTGTGAAAAACAAGCAGAATCG
Tribolium Transcript: CDS:
ATGAAAGTGGAAATGGAGGAAATCGACGACAGCACAATTTGTGATTCTGTGAATTCGTTAGTGGAAGGATGCAGGCTCCCGGTTCACATGACCGGCACTTCCGACTGGCCTTTGGCCGAAATTATCAGTATTAAGGACGTGCAAGGCTCGAAATTGTACTACGTCCATTACGTTGATTTTAACAAGCGTTTGGACGAATGGGTGACCGAAGAATCGCTCGACACTCGTAAAGTCCACTTCCCGAGACGTGACGGCGGGAGTAACACTGGAGTCTCAACTCCGAAAAAAATTCACATTGGGGGCGGTGGTGCCATCGGTTCGGTATCAAGGCCCTCGAGCCCTGTGACAGCTGCCTCCGAACTTGTCAACGGCAGTGCTGTCCTAGCAGCAGCGTTGCAAAAACGCATAAATAGGAAACGAAAAGGGCCGTCTCTTGAGAGCGAAGACTCGCAAGATGGGCCCACACAGATGCCCCCAGGGCCGAGGCAGTCTGGGAGCATGGTGTCGCACCATGACGATGTGGTCACCAGGATGAAGAACATCGATATGATCGAGTTAGGGAAACACAGGATAAAGCCGTGGTATTTTGCGCCATATCCGCAAGAAATGGTCAACTTGAGCTGTATATACATCTGCGAGTTTTGCCTCAAGTACCGAAAGAGCAGGAAGTGTCTAGAACGCCACTTGGTCAAATGTAATTTGAGACACCCGCCCGGAAATGAGATTTATCGCAAGGAAAACGTCTCGTTTTTTGAAATTGACGGGCGGAAAAATAAAGTCTACGCCCAGAATTTGTGTTTGTTGGCGAAGCTGTTCCTCGATCATAAAACACTGTATTACGACACGGACCCGTTTTTGTTTTACGTAATGACCATTTTTGATAATAGAGGGTTTCACATTGTTGGCTATTTTTCGAAAGAGAAGGAATCAACGGAAGATTTTAACGTCGCGTGTATTTTAACCATGCCTCCGTATCAGCGAAAGGGTTACGGAAAATTACTAATCGAATTTAGTTATGAATTATCAAAGTTCGAAGGTAAAACGGGTTCTCCCGAAAAACCTCTCTCCGATTTGGGTTTGCTTTCGTATAGGAGTTACTGGGCACAAGCTATTTTAGAAATCTTAATATCAATGAAGCCAGTGGGTGATAACGAGAAGCCACAAATAACGATCAATGAAATTTGTGAGTTGACAAGTATTAAAAAAGAAGACGTCATCTCAACTTTACAAAACTTAAACTTAATCAATTATTACAAAGGCCAGTATATTATAACGTTAAATCAGGAAATTATGCAAAGTCACAAAAAAGCCATGGATAGTAGACTTATCCGAATCGACCCCAAGTGTCTGCATTGGACACCTAAAGATTGGGGCAAACGCGCCAAGTGGTAG
Tribolium Transcript: 3' UTR:
ATCAATAAATGATTTTTTACA
Tribolium Protein:
MKVEMEEIDDSTICDSVNSLVEGCRLPVHMTGTSDWPLAEIISIKDVQGSKLYYVHYVDFNKRLDEWVTEESLDTRKVHFPRRDGGSNTGVSTPKKIHIGGGGAIGSVSRPSSPVTAASELVNGSAVLAAALQKRINRKRKGPSLESEDSQDGPTQMPPGPRQSGSMVSHHDDVVTRMKNIDMIELGKHRIKPWYFAPYPQEMVNLSCIYICEFCLKYRKSRKCLERHLVKCNLRHPPGNEIYRKENVSFFEIDGRKNKVYAQNLCLLAKLFLDHKTLYYDTDPFLFYVMTIFDNRGFHIVGYFSKEKESTEDFNVACILTMPPYQRKGYGKLLIEFSYELSKFEGKTGSPEKPLSDLGLLSYRSYWAQAILEILISMKPVGDNEKPQITINEICELTSIKKEDVISTLQNLNLINYYKGQYIITLNQEIMQSHKKAMDSRLIRIDPKCLHWTPKDWGKRAKW
Forward Primer 1:
TGTGTTTGTTGGCGAAGCTG
Reverse Primer 1:
GCTTGTGCCCAGTAACTCCT
Forward Primer 2:
GGAGGAAATCGACGACAGCA
Reverse Primer 2:
CATCTGTGTGGGCCCATCTT
Gel Picture:
Cloned Fragment:
GGAGGAAATCGACGACAGCACAATTTGTGATTCTGTGAATTCGTTAGTGGAAGGATGCAGGCTCCCGGTTCACATGACCGGCACTTCCGACTGGCCTTTGGCCGAAATTATCAGTATTAAGGACGTGCAAGGCTCGAAATTGTACTACGTCCATTACGTTGATTTTAACAAGCGTTTGGACGAATGGGTGACCGAAGAATCGCTCGACACTCGTAAAGTCCACTTCCCGAGACGTGACGGCGGGAGTAACACTGGAGTCTCAACTCCGAAAAAAATTCACATTGGGGGCGGTGGTGCCATCGGTTCGGTATCAAGGCCCTCGAGCCCTGTGACAGCTGCCTCCGAACTTGTCAACGGCAGTGCTGTCCTAGCAGCAGCGTTGCAAAAACGCATAAATAGGAAACGAAAAGGGCCGTCTCTTGAGAGCGAAGACTCGCAAGATGGGCCCACACAGATG
Injection concentration:
1 µg/µl
Number Injected:
20
Number Survived to Adult:
12 (5 fully developed but died, 7 near adult but eclosion failure)
Number w/ RNAi Phenotype:
9 (7 eclosion failure, 2 with abnormal abdominal pigmentation)
Percent Penetrance:
75% of those that survived to near adult
Assay Description:
The beetles were observed under light microscope and high-resolution microscopy at regular intervals post RNAi injection to check for morphological abnormalities as well as indicators of early cancerous development such as abnormal pigmentation.
RNAi Phenotype Description:
Twelve beetles exhibited abnormal pigmentation of their wings. Seven of those twelve beetles also failed eclosion. Two of those twelve beetles also exhibited abnormal pigmentation of the lower abdominal region. Eight beetles did not survive past the pupae and larval stages (two died at larva stage and six died at pupae stage).
RNAi Phenotype/Assay Images:
Discussion:
Selective silencing of the gene Tat interactive protein (Tip60) with RNAi in Tribolium Casteneum resulted in some beetles failing eclosion, some developing abnormally pigmented wings, and some with abnormally pigmented spots on the lower abdominal region. Tip60 is a lysine histone acetyltransfrease known to aid in chromatin packaging, DNA repair, DNA repair responses, and apoptosis. As reported by Qi et al. the loss of function phenotype of Tip60 in Drosophila is that chromatin is not silenced (Qi, 2006). It has been found that TIP60 suppression in mouse cell lines results in lower DNA repair factors and DNA repair responses despite DNA damage (Bassi, 2016). Furthermore, TIP60 is essential for hypoxia inducible factor-A (HIF-A) dependent gene expressions and resulting vascular endothelial growth factor (VEGF) expression (Perez-Perri, 2016). VEGF’s are required for vasculature formation and in this way it can be assumed TIP60 is involved in normal growth and development processes. Because TIP60 is associated with chromatin packaging, DNA repair, DNA repair responses, and apoptosis it also likely plays a crucial role in suppressing tumorigenesis. Thus, the observed phenotype of failed eclosure can possibly be attributed to Tip60 suppression leading to inhibition of VEGF expression. The observed phenotypes of abnormally pigmented wings and abnormally pigmented spots of the lower abdominal region may be due to a combination of Tip60’s vital roles in chromatin packaging, DNA repair, DNA repair responses, and apoptosis. The abnormally pigmented spots could be an early indication of cancer formation due to Tip60 suppression. However, further analysis would need to be performed in order to identify early cancer characteristics.
References:
Bassi, C, et al. “The Acetyltransferase tip60 contributes to Mammary Tumorigenesis by Modulation DNA Repair.” Cell Death and Differentiation, vol. 23, 2016, pp. 1198-1208
Perez-Perri, Joel I., et al. Cell Reports, U.S. National Library of Medicine, 28 June 2016, www.ncbi.nlm.nih.gov/pmc/articles/PMC4957981/.
Qi, Dai, et al. “Drosophila Reptin and Other TIP60 Complex Components Promote Generation of Silent Chromatin.” Genetics, Genetics, 1 Sept. 2006, www.genetics.org/content/174/1/241.long.
Notebook:
Annotated Genbank File:
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