Introduction

Oct-4 for Octamer–binding transcription factor 4, also known as POU5F1 (POU domain, class 5, transcription factor 1) is a 352 amino acid protein encoded by the POU5F1 gene. It is the earliest expressed gene known, and the gene is developmentally regulated during mammalian embryogenesis. The map position of oct 4 on mouse chromosome 17 is between Q and T region in the Major histocompatibility. The Oct-4 POU transcription factor is expressed in mammalian totipotent embryonic stem and germ cells. Oct-4 is the only factor that cannot be replace by an other factor of the POU family to induce pluripotency. His remplacement by a transcription factor cocktail does not lead to the successful derivation of induced pluripotent stem cells. POU (pronounced “pow”) transcription factor ( Pou stands for Pit-1, Oct, Unc 86) are DNA–binding proteins that are able to activate the transcription of genes bearing cis acting elements containing an octamer motif ATGCAAAT. POU factors possess the capability to form homodimers to octamer motif variants and heterodimers with other POU members and also with members of the Sox family through the HMF ( DNA-binding) domain. The POU domain is a bipartite domain present in all POU proteins. It consists of two subdomains, called the POU-specific domain and the POU homeodomain, connected by a flexible linker, variable in length. Flexibility of the linker enables the two subdomains to contact the DNA-binding site independently of each other. The other region of the protein show no specificity of structure or function.

Oct-4 structure

There are two different domains in the Oct-4 sequence : the POU-specific domain (POUs) located on the N-terminal subunit and the POU-homeodomain (POUhd)located in the C-terminal subunit of the sequence.

The first figure shows an alignment between different transcription factors such as Pit-1[1] , Oct-1 [2] or Oct-2 [3]. We can see that there are few conserved sites between each transcription factors. We can also see the organization of the Oct-4 structure : a POUs linked to an POUhd thanks to a linker which is actually an alpha helix.

Pou-specific domain

is located on the N-terminal site of the Oct-4 sequence and precisely from the 131th amino acid to the 205th. The POU-specific domain is able to bind autonomously the DNA with the binding consensus : [gAATAT(G/T)CA]. Thanks to a Helix-turn-helixspecific motif. As its name suggests, this pattern is composed of two α helix [4] by an amino acids sequence which corresponds to the turn. The second helix is the most involved in the DNA binding. Indeed, this helix is able to carry out hydrogen bonds and Van der Waals interactions with bases in the major groove of DNA. The first helix allows to stabilize the complex DNA-protein. In the POU specific domain of the transcription factor Oct-4, there are 2 HTH motifs linked by another alpha helix. The HTH motif is responsible of the DNA binding thanks to the first amino acid located in every helix in HTH. Indeed, this amino acid is a which is able to interact with the backbone phosphate and the adenine base in the DNA binding site. Thanks to bioinformatics tools, alignments were carried out. Very good alignment between the Oct-4 POU domain and the Lambda phage POU domain was thus discovered.

Linker

tethers the two POU subdomains and is hypervariable in both sequence and length. Thus the linker in Oct-4 contrary to the linker of the other member of the POU family like Oct-1 [5], is a 17 amino acid alpha-helix and is exposed to the protein surface. The Oct-4 linker functions as a protein-protein interaction interface and plays a highly important role during reprogramming. This alpha 5 helix interacts with helices alpha 2 and alpha 4 of POUs mostly by Van der Waals interactions, except for the hydrogen bond between of the linker. The interaction between Val 36 of POUs and the the carboxy-terminal of the helix alpha 5 of the linker plays an important role. In all known Oct-4 sequences are invariant but not conserved in other members of Oct family. Mutation in the linker of GLn81 by an Arg leads to a complete loss-of-function phenotype. Likewise mutation of Leu80 by an Ala abolished biological activity. It is deduced that this mutation disturbs an interaction surface with yet unknown additional factors. Overall mutation of the linker of the protein lead (Asn76, Asn 77, Asn 79) to significantly fewer function. The integrity of the linker is essential for successful reprogramming.

On the figure n°4, we can see the surrounding of the linker. There are different hydrogen bonds to save the tridimentional structure of OCT-4: the first hydrogen bond is located between .

The linker recruits key partners to the Oct 4 target genes and change in the sequence of the linker lead to the loss of Oct-4' reprogramming activity.

Pou-homeodomain

The (POUhd) is a very conserved 60 amino acid three helices alpha DNA-binding domain. We can find the homeodomain from the 223th to the 282th amino acid in the Oct-4 sequence. Its DNA sequence is called “homeobox”, and the genes are known as “Hox gene”. The DNA recognition helix (alpha 3) binds the DNA major groove while the amino-terminal tail binds the DNA minor groove. We can draw a parallel with oct1 and oct2 about the Homeodomain and admit that the ninth residue of the DNA-recognition helix is a cysteine that might have been conserved at this position to confer DNA binding specificity , and promote more relaxed DNA sequence recognition by POU domains. The recognition helix and the inter-helix loops are rich in arginine and lysine residues, which form hydrogen bonds with the DNA backbone.

Applications

In vivo

Oct-4 is able to create a complex with Sox-2 to express genes involved in the embryonic development such as YES1. Oct-4 is also involved in the creation of intestinal and skin cancer. Indeed, the overexpression of Oct-4 provokes the upregulation of B-catenin [6] transcription which inhibit the cellular differentiation.

In vitro

Oct-4, Sox2, c-Myc and Klf4 are the four factors involved in the cell reprogamming. Indeed, those four factors were used in the Yamanaka’s studies to reprogram mature cells in inducted pluripotent stem cells (iPS )and are thus also named “Yamanaka factors”. When they are in a mature cell, they can induce the expression of genes involved in the reprogramation of cells in an embryonic-state: cells which possess the same morphology and the same growth properties than embryonic stem cells. The unique structure of the linker domain between the POU-specific domain and the Pou-homeodomain gives Oct-4 a protein very important for the study of iPS cells .Indeed, the others transcription factors are recruited and a complex is created with the linker domain.

References

• [7]POU-domain transcription factors: pou-er-ful developmental regulators. M G Rosenfeld GENES DEV. 1991

• NCBI page : http://www.ncbi.nlm.nih.gov/protein/P20263.1

• [8]A unique Oct4 interface is crucial for reprogramming to pluripotency Daniel Esch,Juha Vahokoski NATURE CELL BIOLOGY (2013)

• [9]Embryonic Stem Cells: Similarities and Differences Between Human and Murine Embryonic Stem CellsSonja Koestenbauer1, Nicolas H. Zech, Herbert Juch AMERICAN JOURNAL OF REPRODUCTIVE IMMUNOLOGY 2006

• [10] A unique Oct4 interface is crucial for reprogramming to pluripotencyDaniel Esch, Juha Vahokoski, NATURE CELL BIOLOGY March 2013

• [11] The POU domain: versatility in transcriptional regulation by a flexible two-in-one DNA-binding domainWinship Herr 1 and Michele A. Cleary GENES & DEVELOPMENT 1995

• [12]The ubiquitous octamer-binding protein Oct-1 contains a POU domain with a homeobox subdomain Richard A. Sturm, Gokul DasGENES & DEVELOPMENT 1988

Contributors

MUKOBO Noëlla and WELLY Sarah