The structural and functional analysis of transcription factor E4bp4

Natural Killer (NK) cells are large granulocytic lymphocytes that have a key role in immune surveillance;
targeting host cells that are either virally infected, stressed or cancerous. Once activated NK cells can
release proinflammatory cytokines, cytotoxic granules and induce apoptosis of the target cell via death
receptors or antibody dependent cellular cytotoxicity. NK cell deficiencies and abnormalities caused by
drugs, an underlying health condition (e.g. X-linked SCID) or in genetically modified mice models reveal
patterns of persistent viral infections and increased risk of cancer development. The importance to study
the development, activation and function of NK cells aid in our understanding of NK cell related conditions
and enhance the development of NK cell based immunotherapy.
NK cells are derived from haematopoietic stem cells (HSCs) within the bone marrow. During HSC's
differentiation into common lymphoid progenitors and Natural Killer progenitors (CLP and NKP
respectively), one crucial transcription factor upregulated at this stage is E4bp4, a basic leucine zipper
(bZip) transcription factor. Knock out studies of E4bp4 in mice revealed normal levels of CLPs but a
decrease in NKPs, resulting in NK cell deficiency or abnormally functioning NK cells. However, not much
is known about the molecular mechanisms that regulate E4bp4 activity during early stage NK cell
commitment. E4bp4, also known as "Nuclear factor regulated by IL-3" (Nfil3), is a 60kDa transcription factor
found to exhibit both transcriptional repressor and activator roles. Although there has been no solved 3-
dimensional structure for E4bp4, there is an N-terminal bZip domain and C-terminal repression domain
identified through functional assays of truncated E4bp4. With the limited data on E4bp4 tertiary structure,
the next stage is to use bioinformatical tools and experimental techniques to predict and solve the structure
of the domains or hopefully, the entire protein.
Previous work conducted by Kostrzewski et al., (manuscript submitted) showed that the E4bp4 protein also
undergoes post-translational modifications (PTMs), with the study focusing on SUMOylation and
phosphorylation. These PTMs were shown to negatively regulate E4bp4 activity. Once the sites were
mutated, E4bp4 was limited in its ability to enable NK cell differentiation from a mixed population of
progenitor cells. Current interests include observing if other PTMs, such as N-linked glycosylation, can be
identified on E4bp4 and if they also have a regulatory role on E4bp4 activity.
My long term goal during this PhD is to continue to study E4bp4 on both a structural and functional aspect.
I aim to try and solve regions, if not all of the proteins structure and observe how changes in its expression
and transcriptional activity affect the development and function of NK cells.
1)Characterise the structure of E4bp4 and its two functional domains using X-ray crystallography.
X-ray crystallography involves the process of generating protein crystals whereby accelerated X-ray
beams shot and diffracted from the crystals result in a diffraction patterns which can be analysed
produce 3D template of the protein. I will first clone the full length human E4bp4 gene, the bZip domain
and the repression domain into bacterial expression vectors and induce protein expression for
purification. A selection of co-factors, ligands or protein based interacting partners will also be included
in the screens to encourage order in the target proteins structure for crystal formation.
2)Determine if the E4bp4 protein is glycosylated using mass spectrometry.
The amino acid sequence of E4bp4 revealed 7 potential N-linked glycosylation sites. Mammalian 293T
cells will be transfected with pCMV expression vectors encoding FLAG-tagged E4bp4, which will be isolated

and purified using affinity chromatography. The protein sample will then be trypsinised and partially
deglycosylated to b