Molecular Evolution of the cancer-related Histone acetyltransferase p300 gene

Lindsay Tucker, Chandrakanth Emani

Department of Biology, Western Kentucky University, KY

Abstract. The purpose of this study is to decipher the molecular biological evolution of

the p300 gene which is crucial for multiple tumor suppressor pathways. Mutations in

p300 was shown to correlate with tumor progression during cancer. p300 has a role in

many cancers, with specific prevalence in liver cancers, colorectal cancers, lymphoma,

breast cancers, and lung cancers. The protein’s primary function is to act as a

transcriptional coactivator for multiple oncoproteins that work as tumor suppressors.

p300 is involved in a number of diverse biological functions including proliferation, cell

cycle regulation, apoptosis, differentiation and DNA damage response. In this study we

analyze FASTA sequences of p300 from a diverse array of life forms using

computational tools. Analysis was performed using bioinformatics software databases

NCBI and EXPASY. Conserved domains and evolutionary ancestors were identified

through Quick-BLAST and neighbor-joining phylogenetic trees. The initial BLAST

analysis has identified a hypothetical evolutionary ancestor, the southern two-toed sloth.

Keywords: Cancer, p300, Transcriptional coactivator, Histone acetyltransferase, p53, E1A

INTRODUCTION

p300 is a key cofactor in the proper functioning of tumor-suppressor proteins and has

been implicated in cancer and other diseases. Critical pathways such as the p53, TGF-β, and Rb-

E2F pathways require p300 cofactor activation to mediate the transcription of target genes. The

p300 gene has been mapped and was determined to be located on chromosome 22q13. The p300
protein has a bromodomain in its center, which is characteristic of transcriptional coactivators.

p300 and cyclic AMP response element-binding protein (CBP) are adenoviral E1A-binding

proteins involved in multiple cellular processes, and function as transcriptional co-factors and

histone acetyltransferases. p300 molecules lacking an intact E1A-binding site can bypass E1A

repression and restore to a significant extent the activity of the SV40 enhancer, even in the

presence of high levels of E1A protein. (Eckner et.al, 1994) p300 mutations in solid tumors

result in truncated p300 protein products or amino-acid substitutions in critical protein domains,

and these are often associated with inactivation of the second allele. (Iyer, Özdag & Caldas,

2004) p300 has been shown to correlate with outcomes of multiple diseases including colorectal

cancer, Menke-Hennekam syndrome 2, and Rubinstein-Taybi syndrome 2.

p53, an important tumor suppressor protein, is a gene that codes for a protein found in the

nucleus of all cells in the body that helps regulate normal cell growth and multiplication. It plays

a critical role in suppressing tumors by inhibiting the division and growth of cells whose DNA

has been damaged. p53 is stabilized by the binding of p300 to the oncoprotein E1A, suggesting

that p300 regulates p53 degradation. Rapid turnover of the tumor suppressor protein p53 requires

the MDM2 ubiquitin ligase, and both interact with p300-CBP transcriptional coactivators. p300

and CBP are also known to interact with the histone acetyltransferase P/CAF, which promotes

transcriptional activation. (Ogryzko, et.al, 1996) P/CAF competes with the E1A protein for

binding sites with p300/CBP. With p300 and CBP both possessing characteristics of tumor

suppressor proteins, when unable to interact with P/CAF due to interreference from E1A protein,

the genes encoding for p300 and CBP can be mutated in human cancer. (Scolnick. Et.al, 1997)

TGF- β or transforming growth factor-β is an important signaling pathway that controls

many cellular processes including proliferation, differentiation, apoptosis, epithelial-

mesenchymal transition, and migration. p300 is involved in the pathway after the SMADs

complex is translocated into the nucleus, where it acts as a regulator for transcription. Certain

viral pathogens have been shown to interfere with the ability of p300 to bind to SMADs complex

within the TGF- β pathway. In HTLV‐1 (Human T-lymphotropic virus 1) the viral factor Tax

inhibited SMAD/p300 complex formation by competitive interactions with both SMAD and

p300; and the viral factor HBZ increased SMAD/p300 complex formation by HBZ‐mediated

ternary complex formation. When the SMAD/p300 association is increased it enhances signaling

and induces Foxp3 expression in naive T cells, which allows the virus to convert infected T cells

into Treg cells. Treg cells are involved in tumor development and progression by inhibiting

antitumor immunity. Beyond involvement in cancers, other viral pathogens can cause other

infections when interrupting the function of p300. In EBV (Epstein–Barr virus) the viral factor

LMP-1 blocks SMAD/CBP‐p300 complex formation by the induction of NF‐κB that

competitively interacts with CBP‐p300. In SARS‐CoV (severe acute respiratory syndrome) the

viral factor N enhanced PAI‐1‐induced tissue fibrosis via SMAD3/p300 complex promotion. In

AV (Adenovirus) the viral factor E1A suppressed TGF‐β‐mediated cell growth arrest by

interacting with p300 and SMAD3. Human hepatocellular carcinoma cells infected with AV

were also reported to be insensitive to TGF‐β‐induced apoptosis. (Mirzaei & Faghihloo, 2018)

E2F1 Transcription factor and RB tumor suppressor have roles in both regulating gene

expression and facilitating gene repair at sites of damage. When DNA damage occurs, E2F1 is

able to recruit p300/CBP to deposit histone acetylation marks in flanking chromatin. When E2F1

is acetylated, it interacts with the bromodomain in p300. This pathway can be seen repairing

DNA damaged by UV. However, if a bromodomain inhibitor is introduced then p300 cannot be

recruited and E2F1 acetylation is blocked, preventing DNA damage repair. (Manickavinayaham,
Et.al, 2020) E2Fs are a family of transcription factors that contain conserved DNA binding

domains, which bind target promoters and regulate their expression. Deregulated E2F activity is

correlated with aberrant cell proliferation and in extreme cases, cell death. (Chen, Tsai, & Leone,

2009) The deregulation of E2F activity is often a result of the functional inactivation of RB1 in

various cancers.

This gene has also been identified as a co-activator of HIF1A (hypoxia-inducible factor 1

alpha), and therefore plays a role in the stimulation of hypoxia-induced genes, with one example

being VEGF. HIF1A is subject to regulatory hydroxylation, which can block the CBP/p300

interaction. (Dengler, Et.al, 2014) Defects in this gene are a cause of Rubinstein-Taybi syndrome

and may also play a role in epithelial cancer.

Competition between cellular transcription factors to bind the limiting p300/CBP has

been shown to be an important regulator of transcription and when this regulation is interrupted

it has the ability to cause diseases including cancer. By exploring the molecular evolution of the

p300 gene, methods can be researched that can manipulate the pathways in which p300

regulates; in hopes of further understanding and reducing tumor expression, and therefore cancer.

MATERIALS & METHODS

p300 Sequence Collection

Using NCBI (http://www.ncbi.nih.gov) the FASTA sequence of p300 was identified,

which was then entered into QuickBLAST to identify the sequences of other related organisms.
 Bioinformatic Analysis

The resulting sequences were inserted into the COBALT multiple alignment tool to determine

the conserved domains and produce a phylogenetic tree for the p300 gene. Further protein

analysis was done by using multiple ExPASY resources (http://expasy.org/tools). Using

Protparam, the characteristics of amino acids present in the human protein, most common

ancestor, most evolved, and a close relative were identified. The same sequences were analyzed

using ProtScale, which revealed the profile produced by the amino acids present in each

sequence. Using the human sequence, PeptideCutter was utilized to produce potential cleavage

sites and identify the enzymes that cut the protein. It also aided in the identification and location

of specific domains present in the protein. Using Scan PROSITE, the profiles of each domain

were generated by PROSITE motifs. After uploading the sequences into MEME

(http://www.Meme.nbcr.net/meme/meme/.html), five motifs were identified that were conserved

among all of the sequences. Applied parameters of MEME were set at: minimum width for each

motif, six; maximum width for each motif, fifty; maximum number of motifs to find five and

occurrence of each motif, zero or one per sequence. TMHMM

(http://www.cbs.dtu.dk/services/TMHMM) was utilized to predict the transmembrane helices in

the protein, however no results were found. Inter ProScan classified the p300 protein into the F-

Histone acetyltransferase Rtt109/CBP (IPR013178) family. KEGG revealed no entries have been

recorded for a pathway. An OMIM Report of p300 revealed gene functions and gene phenotype

relationships, which identified the clinical synopses involved in the p300 gene.
RESULTS.

Phylogeny

The p300 gene codes for a 2414 amino acid protein in homo sapiens. NCBI

QuickBLAST revealed the Choloepus didactylus (southern two-toed sloth) as the hypothetical

evolutionary ancestor and the Mandrillus leucophaeus (drill) as the most evolved. The

phylogenetic tree also revealed that Rhesus macaque (rhesus monkey) is a close primate relative

to humans in regards to the p300 gene.

Fig. 1
Phylogenetic tree
generated by the
neighbor joining
method on NCBI
blast

Biochemistry
 The homo sapien p300 protein is made of 2414 amino acids, with a molecular weight of

264160.60 according to results from ExPasy ProtParam. The sequence contains 178 negatively

charged residues and 208 positively charged residues. The composition of this protein is made up

of 837 polar residues and 1,195 nonpolar residues; it also contains 832 essential residues. This

protein has an instability index of 67.04, classifying it as unstable. There is no evidence of

transmembrane helices in this protein; and data showed that there is higher probability of

presence inside the membrane.

The Choloepus didactylus p300 protein is made of 2434 amino acids, with a molecular

weight of 265753.13. The sequence contains 179 negatively charged residues and 208 positively

charged residues. The composition of this protein is made up of 598 polar residues and 1,201

nonpolar residues; it also contains 839 essential residues.

 The Mandrillus leucophaeus p300 protein is made of 2416 amino acids, with a molecular

weight of 264501.94. The sequence contains 178 negatively charged residues and 208 positively

charged residues. The composition of this protein is made up of 591 polar residues and 1,191

nonpolar residues; it also contains 837 essential residues.

The Rhesus macaque p300 protein is made of 2414 amino acids, with a molecular weight

of 264269.70. The sequence contains 178 negatively charged residues and 208 positively

charged residues. The composition of this protein is made up of 832 polar residues and 903

nonpolar residues. This protein has an instability index of 66.99, classifying it as unstable.
 Conserved Domains

Fig. 2 Image of conserved domains placement in p300, generated by the COBALT multiple

alignment tool.

Domain Name Function

Important in its function as a transcriptional

coactivator. This domain is key for p300’s
Bromodomain interaction and regulation of E2F1; which is
important for gene expression regulation and
DNA repair.
The primary E1A binding site; the expression
of E1A significantly reduces tumorigenesis,
Zinc finger domain promotes cell death, and inhibits cancer cell
mobility. The binding of p300 and E1A
stabilizes p53, a known tumor suppressor
gene.
Key to the binding of p300 and CBP to the
CREB. It serves as a binding site for
KIX domain heterodimers that form between the
coactivator and specific transcription factors.
The KIX domain has two known binding sites
where CBP/p300 can bind.
A three-helix bundle with marked similarity
to the KIX domain. The SREBP family of
Arc105 or Med15 transcription activators uses the ARC105
subunit to activate target genes in the
regulation of cholesterol and fatty acid
homeostasis.
A paralog of CREB-binding protein and is
involved in E1A function in cell cycle
PHD finger progression and cellular differentiation. This
domain allows for p300 to serve as a scaffold
or bridge for transcription factors and other
components of the basal transcription
machinery. This facilitates chromatin
remodeling and activates gene transcription.
 TonB
CREB binding domain
atypical RING domain found in CREB-
binding protein and p300 histone
acetyltransferases
(HAT KAT) Histone acetylation protein
Zing finger (zz type)
MEME Results
Motif #1
E-value: 5.8e-2546
Periplasmic protein TonB links inner and
outer membranes (Cell wall/ membrane/
envelope biogenesis)
Can act as a dock for thyroid hormone and
retinoid receptors. Docking of these domains
is required for the recruitment of RNA
polymerase II and the basal transcription
machinery.
This ring domain contains only a single zinc
ion-binding cluster instead of the usual two;
instead of a second zinc atom, a network of
hydrophobic interactions stabilizes the
domain. The RING domain has an inhibitory
role. Disease mutations that disrupt RING
attachment lead to upregulation of HAT
activity.
Histone acetylation is required in many
cellular processes including transcription,
DNA repair, and chromatin assembly. This
family contains the fungal KAT11 protein
(previously known as RTT109) which is
required for H3K56 acetylation. Loss of
KAT11 results in the loss of H3K56
acetylation, both on bulk histone and on
chromatin.
Zinc finger present in dystrophin, CBP/p300
and many other proteins. The ZZ motif
coordinates one or two zinc ions and most
likely participates in ligand binding or
molecular scaffolding.
Sites: 45 Width: 50
Motif #2

E-value: 1.0e-2481 Sites: 45 Width: 50

Motif #3

E-value: 2.4e-2477 Sites: 45 Wifth:50

Motif #4

E-value: 5.6e-2415 Sites: 45 Width: 50

Motif #5

E-value: 1.0e-2388 Sites: 45 Width: 50

After comparing the motif sequences computed by MEME with the conserved domains

identified by using PeptideCutter, it was determined that Motif #1 is a part of the zinc finger (zz
type), Motif #2 and Motif #4 are a part of the HAT KAT (histone acetylation) domain, Motif #3

is a part of the bromodomain, and Motif #5 is a part of the atypical RING domain.

OMIM Report

Phenotype Phenotype
Location Phenotype Clinical Synopses MIM number Inheritance mapping key

22q13.2 Colorectal cancer, somatic 114500 3

Menke-Hennekam syndrome 2 618333 AD 3

Rubinstein-Taybi syndrome 2 613684 AD 3

Nucleotide Data: Exons, mRNA’s, & CDS

Exons

1. 4983..5489 12. 60228..60337 23. 78990..79057

2. 29578..30212 13. 61429..61566 24. 80840..80990

3. 38255..38431 14. 62152..62589 25. 81112..81258

4. 39878..40139 15. 64224..64403 26. 81894..82007

5. 42281..42394 16. 64597..64741 27. 82797..82962

6. 43779..44024 17. 67386..67504 28. 84890..85054

7. 48204..48297 18. 69560..69799 29. 86014..86175

8. 50044..50181 19. 70803..70891 30. 88638..88919

9. 52531..52648 20. 73033..73113 31. 89164..9246

10. 53439..53613 21. 75114..75170

11. 59130..59207 22. 76444..76521

mRNA

join(4983..5489,29578..30212,38255..38431,39878..40139,

42281..42394,43779..44024,48204..48297,50044..50181,

52531..52648,53439..53613,59130..59207,60228..60337,

61429..61566,62152..62589,64224..64403,64597..64741,

67386..67504,69560..69799,70803..70891,73033..73113,

75114..75170,76444..76521,78990..79057,80840..80990,

81112..81258,81894..82007,82797..82962,84890..85054,

86014..86175,88638..88919,89164..92468)

PRODUCT: E1A binding protein p300, transcript variant 1

CDS

join(5396..5489,29578..30212,38255..38431,39878..40139,

42281..42394,43779..44024,48204..48297,50044..50181,

52531..52648,53439..53613,59130..59207,60228..60337,

61429..61566,62152..62589,64224..64403,64597..64741,

67386..67504,69560..69799,70803..70891,73033..73113,

75114..75170,76444..76521,78990..79057,80840..80990,

81112..81258,81894..82007,82797..82962,84890..85054,
 86014..86175,88638..88919,89164..91347)

PRODUCT: histone acetyltransferase p300 isoform 1

Neighboring Genes

The gene present on the right of the p300 gene in the human genome is GENEL3MBTL

histone methyl-lysine binding protein 2. Its protein title is lethal (3) malignant brain tumor-like

protein. This gene has been implicated in regulating chromatin architecture. (Huang, Et.al, 2018)

The gene present on the left of the p300 gene in the human genome is RPS9P2 ribosomal

protein S9 pseudogene 2.

DISCUSSION

The phylogenetic tree revealed an important close primate relative for the p300 gene in

the rhesus macaque. In terms of amino acid composition of the protein, the rhesus macaque and

homo sapien are similar. Both are made up 2414 amino acid residues, with both having 208

positively charged residues and 178 negatively charged residues. Further research in the

expression of the p300 gene in rhesus macaques provided insight into how the domains

contribute to tumor progression in cancer.

Understanding the role of the p300’s conserved domains involvement in various

biological pathways can provide information on how dysregulation or mutations can promote

tumor growth. The bromodomain is important for p300’s function as a transcriptional

coactivator. This domain contributes to p300’s interaction and regulation of E2F1; which is

important for both gene expression regulation and DNA repair. Bromodomains present in

overexpressed proteins has been linked to important roles in the epigenetic landscape and in
cancer development. Research has revealed that specific targeting of this domain can be an

effective treatment for some cancers. Bromodomain inhibitors disrupt acetyltransferase activity,

which is crucial for the function of EP300/CBP activity. (Perez-Salvia & Esteller, 2017) The

development of bromodomain inhibitors has produced significant results in its treatment of

certain cancers including castration- resistant prostate cancer, leukemia, and carcinoma.

Another important p300 domain that has been linked to cancer is the zinc finger domain.

The zing finger domain works as transcriptional activators by interacting with co-activators and

is the primary E1A binding site on p300. The expression of E1A significantly reduces

tumorigenesis, promotes cell death, and inhibits cancer cell mobility. The binding of p300 and

E1A stabilizes p53, a known tumor suppressor gene. Therefore, when E1A is unable to bind to

the zinc finger domain, the tumor suppressing properties are interrupted.

The histone acetylation protein or HAT KAT domain is crucial for many cellular

processes including transcription, DNA repair, and chromatin assembly. A research study

revealed that pharmacological inhibition of p300 KAT activity prolongs S phase in the cell cycle.

KATs have been shown to participate in promoting cancer due to its relationship with the RING

domain. Disruption of the RING domain enhances p300 KAT activity, and many times

mutations in the RING occur when malignancies are present including melanoma, endometrial

and colorectal carcinoma. (Attar & Kurdistani, 2017) There is also a high amount of point

missense mutations that occur in the KAT domain. Further research could reveal how the high

frequency of mutation can contribute to the progression of cancer.

CONCLUSION

From the study it was determined p300 is present in various species, including the close

relative, Rhesus macaque. Moreover, the study also supports the function of the domains in the

p300 signaling pathway. Overall, the study could be beneficial in understanding the mechanisms

involved in p300 signaling and regulation; which greatly affects tumor suppression or

progression. Ultimately these findings can be applicable in preventing diseases and cancers

associated with dysregulation or mutations in p300.

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